CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
CHONDRODYSPLASIA PUNCTATA II: A CASE WITH CLINICAL
CHARACTERISTICS AND NO MUTATION ON THE EBP GENE
A thesis submitted in partial fulfillment of the requirements
For the degree of Master of Science
In Biology
By
Osvaldo Larios
May 2014
The thesis of Osvaldo Larios is approved
Dr. Rheem Medh
Date
Dr. Louis Rubino
Date
Dr. Stan Metzenberg
Date
Dr. Aida Metzenberg, Chair
Date
California State University, Northridge
ii ACKNOWLEDGEMENTS
I would like to thank God for his blessings and His guidance all these years. Life has had its ups
and downs and He has always been there to lift me while showing me the light. Having said so, he has
placed numerous individuals in my life who have helped me keep a cheerful and optimistic lifestyle. To my
parents who devoted all their lives to their children for a better tomorrow, I would like to express my
thanks. Se que casi nunca lo dijo pero los amo mucho y en los momentos que me encuentran callado son
los quales que siento mi alma y amor en sus vidas. Les agradesco todo lo que an hecho por nosotros.
To my friends, mentors, and advisors. My time here at CSUN would not have been possible if it
was not for your words of wisdom and motivation. There is still plenty of space on this page so I would like
to name a few if not all. To Wilber Escorcia for placing the idea of the Master's in my head and always
reminding me how true of a friend you are. To Ishita Shah and Chintan Pathak for teaching many lab
techniques and having time to chill and relax while waiting for lab results. To Maria Cruz for being one of
my closest friends and putting up with my crazy emotional roller coasters (believe me, there were many
and she not only stood in the lines, but rode each one). To Ricardo Rosales for taken the time to show me
the ropes and being my first laboratory mentor. To past and current lab members: Edward Meltser, Cinthiya
Ather, Cecily Cullors, Prana Yenkosky, Surabhi Mulchandani, Sawona Biswas, Nikita Tripuraneni,
Harmanpreet Panesar, Samia Jaffar, Sonia Gutierrez, Ricardo Gutierrez, Andrea Cosco, Bansari Shah,
Daniel Thomas, Edward Martin, Azadouhi Rptchian, Yasemine Modarresi, Forogh Taghavifar, Anamica
Sood, Dona Cherian, William Salloom, Christine Chester, Jessica Moreno, Diana Cruz, and…
I would also like to thank certain programs and departments, which have my education a
possibility beyond my dreams. The Eductional Opportunity Program (EOP) has and will forever hold a
special place in my success and endeavors. EOP provided me with numerous resources and tools that
guided me and supported when the tough got tougher. To Jose Luis Vargas, Frankie Augustin, Elizabeth
Riegos, Shelly Thompson, Ramon Muniz, Marvin Villanueva, and Jina Gonzales, you all represent EOP
and would like to thank you for being not just my advisors, but also familia. The College of Science and
Mathematics Student Services Center/ EOP will always be my home away from home and I am fortunate to
have shared many smiles and happy moments with its staff. To Cherie Hawthorne, Marc Felix, John
Brown, Y.C. Tung, William Krohmer, and the Biology department for their assistance and wisdom.
Lastly, I would like to thank Dr. Aida Metzenberg and Dr. Stan Metzenberg for believing in my
potential when I am not know to make a great first impression in science. Dr. Metz, you have been one of
my greatest mentors and are more like a family member. Having no experience in laboratory and showing
no interest in research, I was taken under your guidance and you provided me with the opportunity to give
biology a second chance. You guided me since the beginning of my scientific experience here at CSUN and
presented me with new challenges through different projects and tasks. As I met each project, you allowed
me to develop my skills while establishing the foundation for my success. Thank you so much for believing
in me and guiding me through some of the most challenging stages of my adult life.
To my thesis committee, I would like to express my gratitude. You all were vital for this thesis
and I acquired much knowledge from each of you. Dr. Rubino, thank you for not just being one of my
committee members, but also a long-life mentor and advisor.
Last but not least, to my fellow AB540, DACA, and Dreams to Be Heard (D2BH) I would like to
say, “Si Se Puede y Undocument and Proud”. Hechelen ganas a los estudios y sepan que cuantan con mi
ayuda y apoyo.
iii TABLE OF CONTENTS
Signature page
ii
Acknowledgements
iii
List of figures
viii
List of tables
ix
Abstract
x
CHAPTER 1: INTRODUCTION
Chondrodysplasia Punctata (CD)
1
Chondrodysplasia Punctata Subtypes
1
Metabolic Pathway of CDP
4
Peroxisomal Disorders (PD)
6
RCDP Biochemical Pathway
7
X-linked Chondrodysplasia Punctata
8
Cholesterol Biosynthetic Pathway: Pre-Squalene
10
Cholesterol Biosynthetic Pathway: Post-Squalene
15
Cholesterol Biosynthetic Disorders: (CBD)
18
CBD Resulting from Exposure to Teratogens
24
CBD and Maternal Diseases
24
Emopamil Binding Protein (EBP)
26
Evolutionary Studies of EBP, ERG2p and Sigma 1 Receptor
28
Mutational Analysis of EBP
30
EBP. A transmembrane Protein
31
Cellular Localization of EBP
33
iv Lipid Droplets (LD) as Repositories
34
Lipid Rafts and Cellular Cholesterol Localization
36
EPB Function and Localization
37
Misdiagnosing CDPX2 with CHILD Syndrome
39
X-inactivation, Mosaicism and CDPX2
40
Subject of Study Medical History
43
Purpose of the Study
45
CHAPTER 2: MATERIALS AND METHODS
Amplification of the Exons
46
Purification of PCR Amplicons by Polyacrylamide gel
52
Insertion of Amplicon into pGEM3Z(+)
54
Transformation of Constructs
54
Purification of Plasmids
55
Confirmation of pGEM3Z-CDPX2-Exon Constructs by Agarose gel
55
CHAPTER 3: RESULTS
Molecular Construction and Confirmation of three pGEM3Z-EBP
Exon Constructs
56
Sequence Analysis of pGEM3Z-Exon2, pGEM3Z-Exon3&4,
pGEM3Z-Exon5
60
Sequence Analysis of Exon 2: Missequencing of Sample 23M and 24M
62
CHAPTER 4: DISCUSSION
CDPX2 Review
73
Summary of Results
74
Possible Phenocopy of EBP
76
v Peroxisomes, Endoplasmic Reticulum, defective enzymes, and
overlapping symptoms of CDPX2, CHILD and SLO
77
Lipid Droplets and possible role for Cholesterol Biosynthesis
80
SREBP Cleavage-Activating Protein
81
Importance of Peroxisomes in Pre-Squalene Pathway
82
Maternal Autoimmune Diseases and CDP
82
Medical History of Subject: First and Third trimester
84
Significance of the Study
85
References
86
Appendix A: 7.5% Polyacrylamide Gel
105
Appendix B: 50x TAE
106
Appendix C: S.O.C. Medium
107
Appendix D: Promega’s Wizard Plus SVMiniPreps DNA Purification vi 108
LIST OF FIGURES
Figure 1: Phenotype of X-linked dominant CDP
4
Figure 2: Compartmentalization of the cholesterol biosynthetic pathway
5
Figure 3: Steroid biosynthesis pathway
9
Figure 4: Enzymatic cascade of post-squalene synthesis
11
Figure 5: Cholesterol Biosynthetic Pathway and Sterol Intermediates
17
Figure 6: Cytogenetic and Molecular location of EBP
27
Figure 7: Diagram Illustrating the Emopamil Binding Protein
29
Figure 8: Genomic Sequence of the Emopamil Binding Protein
61
Figure 9: pGEM3Z(+) EBP Exon Constructs
69
Figure 10: Concentrations of each purified construct
71
Figure 11: Representative Sequences for all samples from each construct
Subjected to sequencing
72
Figure 12: Multiple Sequence Alignment of all Exon 2 Samples
76
Figure 13: Multiple Sequence Alignment of all Exon 3&4 Samples
78
Figure 14: Multiple Sequence Alignment of all Exon 5 Samples
81
Figure 15: Sample CDPX2-3_M13F Sequence
82
Figure 16: Possible Mutations of CDPX2-3_M13F
83
Figure 17: Possible Mutations of CDPX2-3_M13F and CDPX2-4_M13F 84
vii LIST OF TABLES
Table 1: Primers Used for Amplification and Sequencing
57
Table 2: Amino Acid Sequences for four Exon 2 samples
74
Table 3: Amino Acid Sequences for Exon 3 and Exon 4 samples
77
Table 4: Amino Acid Sequences for five Exon 5 samples
79
viii ABSTRACT
Chondrodysplasia Punctata II: A Case with Clinical Characteristics and No Mutation on
the EBP Gene
By
Osvaldo Larios
Master of Science
Biology
X-linked dominant Chondrodysplasia Punctata (CDPX2) also known as ConradiHunermann–Happle Syndrome is a rare human genetic disorder resulting from
disfunction of Emopamil Binding Protein (EBP). EBP is better known as Sterol
isomerase (SI),which it acts as the 2nd to last enzyme in the 30-step Cholesterol
biosynthetic pathway. SI converts ∆8-cholestenol to ∆7- cholestenol (lathostenol) and
zymostenol to ∆7, 24-cholestadenol. A disturbance of any of the 30 metabolic steps
result in the accumulation of bioactive precursors. At present, there are over 40 known
distinct mutations along the EBP gene, although there is no clear genotype-phenotype
correlation. These intermediates may have teratogens effects on a devolving fetus,
producing any of several birth defects. These include a variety of Chondrodysplasia
syndromes, such as CDPX1, CDPX2 and RCP. Trademark features of CDPX2 include
cataracts, stippling of the vertebrae and the ribs, ichthyosis, and prominent lines of
Blaschko. Individuals diagnosed with CDPX2 may exhibit a diverse series of phenotypes
and Biochemical and DNA analysis are the two methods to confirm the disorder. In this
study, I performed DNA analysis on an individual diagnosed with CDPX2 due to
phenotypic characteristics present at birth and persisting through adulthood. My findings
ix present no known mutation within EBP to neither explain nor validate the CDPX2
diagnosis. A close examination of current literature brings forth several possibilities to
explain a case where an individual shows clinical features, but does not have a mutated
EBP gene.
x CHAPTER 1: Introduction
Chondrodysplasia Punctata (CDP) is a disorder comprised of a large hereditary
heterogenous group of skeletal dysplasias; the condition is characterized by stippling of
the epiphyses, and is the abnormal accumulation of calcium on the ends of the long
bones. Stippling has been seen in several other disorders, such as Peroxisomal,
cholesterol metabolism, lysosomal storage, vitamin K metabolism disorders, and
chromosomal abnormalities such as trisomy 13, 18 and 21 (Poznanski et al., 1994).
Punctate epiphyses result from premature calcification of cartilage in the ends of the long
bones and the spine (Spranger et al., 1971; Wulfsberg et al., 1992; Poznanski et al.,
1994). Premature stippling represents delayed activity in the center of ossification;
decreasing bone growth and the angulation of the spine. Stippling also presents a
deficiency in cartilaginous tissue, which creates a cartilage-like texture in the skeletal
system (Theander and Pettersson 1978). The calcification of non-bony structures, such as
the nose and trachea, results in insufficient oxygen supply to the lungs, which is due to a
small nasal septum or a malformed trachea, respectively.
Chondrodysplasia Punctata Subtypes
There are 4-6 forms of CDP in which each shows several overlapping clinical
symptoms. These forms of CDP are inherited in distinctive modes, but they share similar
phenotypic symptoms. The clinical symptoms include but are not limited to: short
humeri, cataracts, itchthyosis (scaly skin), and dry, brittle hair as well as alopecia. Five of
these forms have been identified as either being inherited in an autosomal or X-linked
fashion. The most common form of CDP is the autosomal recessive disorder entitled
Zellweger syndrome. Zellweger syndrome is characterized by hypotonia (weak muscle
1 tone), seizures, hearing and vision loss and has a prevalence of 1 in 50,000 newborns
(OMIM 214100). Life-threatening problems associated with Zellweger syndrome include
failure of vital organs such as the heart, liver and kidneys. Skeletal abnormalities seen in
individuals with Zellweger syndrome include wide fontanels (distant space in between
the bones of the skull), CDP, high forehead, and broad nasal bridge. Zellweger syndrome
has been shown to be a lethal CDP disorder in both genders as newborns do not live past
their first year of life. Rhizomelic Chondrodysplasia Punctata (RCDP1; OMIM 215100)
is another autosomal recessive form of CDP with a prevalence of 1 in 100,000 newborns.
It is characterized by hypotonia, shortening of the long bones in the upper arms and legs
(rhizomelia), CDP, hypertelorism (widely spaced eyes), growth retardation, severe
intellectual disability, demineralization of the humerus and femur at childhood,
metaphyseal flaring (widening of the fingers), microcephaly (a small head), and
congenital cataracts (Gilbert et al., 1976; Heymans et al., 1985). RCDP dysplasias
shortens the lifespans of individuals who are in the early to late childhood stages. In
1990, Rittler et al. first described a new form of CDP, which is called Chondrodysplasia
Punctata Tibia-Metacarpal (CDPTM). They described CDPTM as the autosomal
dominant form of CDP, which is characterized by short metacarpals, including tibial
metacarpal, humeral-metacarpal, and mesomelic dysplasia (Rittler et al., 1990;
Borochowitz 1991, Burck 1982). In contrast to the rhizomelic dysplasia seen in RCDP,
mesomelic dysplasia results from shortening of the upper and lower limbs in association
with proximal humerus or femur stippling (Argo et al., 1996). Other characteristics of
CDPTM include midface hypoplasia, nasomaxillary hypoplasia, and short limbs (Silengo
et al., 1980). The gene for CDPTM, along with its protein’s biochemical role and
2 prevalence, remain unknown. A fourth type of CDP is Chondrodysplasia Punctata
Brachytelephalangic (BCDP), but its prevalence, gene, and biochemical pathway of the
protein remain unknown. A fifth type of CDP is the recessive X-linked form: X-linked
Chondrodysplasia Punctata 1 (CDPX1) with its prevalence being recorded solely based
on several dozen males in literature (OMIM 300180). CDPX1 was first described in
1976 by Sheffield, and then in 1996 by Franco et al. These investigators demonstrated
that a mutated ARSE gene gave rise to CDPX1. This gene was shown to be located at
Xp22.3. CDPX1 is said to have an X-linked recessive mode of inheritance, because it
appears only in males. CDPX1 is characterized by CDP, brachytelephalangy (shortening
of the phalanges), and nasomaxillary hypoplasia (the incomplete development of the
nasal and maxillar cavity) (Maroteaux, 1989). The stippling in CDPX1is primarily seen
in the bones of the ankles, toes, and fingers; however, this appendage idiosyncrasy
disappears in early childhood. Affected individuals with CDPX1 tend to have a normal
life expectancy, and they also exhibit normal intellectual development. Life-threatening
complications in CDPX1 may include stenosis (abnormal thickening) of the cartilage in
the airway, and breathing can become restricted. Furthermore, CDPX1 patients may
experience uncommon features, such as hearing loss, visual abnormalities and heart
defects (Nino et al. 2008). The sixth subtype, Conradi-Hunermann-Happle syndrome
(CDPX2; OMIM 302960) has a prevalence of 1 in 400,000 newborns and was first
described by Happle in 1979. CDPX2 is primarily seen in females and is clinically
characterized by cataracts, ichthyosis, coarse hair (thick and rough cranial hair), alopecia,
craniofacial defects, dwarfism, and to a lesser extent polydactyly (extra fingers). CDPX2
also includes stippling of the vertebrae and the ribs. Stippling may also be found in the
3 trachea, humerus, radius, ulna, femur, fibula or tibia in CDPX2. Kyphoscoliosis (the
abnormal curvature of the spine), and rhizomelia (shortening of the proximal long bones),
may result in short stature of CDPX2 patients (Figure 1. NIH Genetics Home Reference).
All six forms of CDP are caused by mutations in different genes, from intertwining
metabolic pathways.
Figure 1. (Left) Phenotype of X-linked dominant chondrodysplasia punctata patient , aged 3 6/12 years:
coarse hair, alopecia, short stature with rhizomesomelic shortening of the limbs, and severe kyphoscoliosis.
(Right) Lateral radiograph of the knee joint of Patient 2 at the same age, showing epiphyseal stippling.
Ikegawa et al., 2000
Metabolic Pathway of CDP
The metabolic pathways in the six forms of CDP described above include Glycan
biosynthesis and metabolism, carbohydrate metabolism, energy metabolism, amino acid
metabolism, nucleic acid metabolism, and lipid metabolism. For the most part, the sites
of these events occur within organelles such as peroxisomes and/or the endoplasmic
reticulumn (Figure 2). For example the anabolic reaction for the synthesis of
4 plasmologens occurs within peroxisomes. Plasmologens are ether phospholipids which
are located in cell membranes and are most abundant in myelin: the protective sheet
covering nerve cells. It has been noted that individuals with RCDP exhibit low levels of
plasmalogens, due to improperly functioning peroxisomes. Also, newborns with
Zellweger syndrome have been described with degenerative myelin. A common finding
in CDP is that they are distinctive syndromes with unique genes encoding specific
proteins that have similar effects and intertwining metabolic pathways.
Figure 2. Compartmentalization of the cholesterol biosynthetic pathway. Kovacs et al., 2007
5 Peroxisomal Disorders (PD)
To date, there are 25 human peroxisomal disorders that are best described by the
lack of normally functioning peroxisomes. They have been subdivided into two groups:
the Peroxisome Biogenesis Disorders (PBD), which involved multiple peroxisomal
metabolic abnormalities, and the second group, which is comprised of disorders
involving single peroxisomal metabolic enzymes (Gould et al., 2001 and Wanders et al.,
2001). In 1973, peroxisomes were first associated with human disorders when
Goldfischer et al. recognized that the kidney and liver tissues of individuals with
Zellweger syndrome were devoid of peroxisomes. Two decades later in 1993,
Shimozawa, et al. identified the first of twelve defective genes for Zellweger syndrome
thus connecting peroxisomes to Zellweger syndrome. Zellweger syndrome is one of the
four PBD. These twelve defective genes include PEX1-3, 5-6, 10, 12-14, 16, 19, and
PEX 26. PEX1is the culprit in over 70% of individual affected with Zellweger syndrome.
(Genetic Home Reference). The malformed proteins encoded by these 12 genes are
responsible for the inadequate levels of peroxins that occur in Zellweger syndrome
(Gould et al., 2000). Peroxins are essential for the proper biogenesis and normal activity
of peroxisomes, and this is because they import enzymes needed for several metabolic
pathways. These include those needed for Plasmalogen biosynthesis and Cholesterol
biosynthesis. Two other PBD include the lesser severe subtypes of Zellweger spectrum:
neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease. The last PBD
includes all three subtypes of RCDP.
RCDP consists of three sub-types: RCDP1, RCDP2, and RCDP3, with RCDP1
being the most common. These three RCDP subtypes consist of mutations in distinct
6 genes with pathways that are intertwined; the mutations in either of these genes results in
the poorly manufactured plasmalogens within ether lipid metabolic pathway. Individuals
diagnosed with RCDP experience significant loss of plasmalogens in the nervous,
immune, and cardiovascular systems. The defective RCDP1 protein is called peroxisomal
biogenesis factor 7 (PEX7). The PEX7 gene is located on the petite (p) arm of
chromosome 6 at position 23.3. PEX7 is part of a set of peroxisomal proteins (PEX) that
make up the peroxisomal PTS2 receptor. The PTS2 receptor is responsible for the
importation of enzymes into the cells’ peroxisomes.
RCDP Biochemical Pathway
Mutations in the glyceronephosphate O-acyltransferase (GNPAT), which is also
known as dihydroxyacetonephosphate acyltransferase (DHAPAT) in the
Glycerophospholipid metabolism pathway (GMP), results in RCDP2. GNPAT/DHAPAT
is encoded by the GNPAT/DHAPAT gene, which is located on the petite arm (p) of
chromosome 1 at position 42. GNPAT/DHAPAT is crucial for the conversion of
Glycerone phosphate to 1-Acylglycerone 3-phosphate (C4H6O7PR). 1-Acylglycerone 3phosphate is the metabolite needed for the production of plasmalogens in the ether lipid
metabolism pathway Any of the five known mutations within this gene result in a
shortage of GNPAT/DHAPAT, as well as the plasmalogens within the cells. In addition
to GNPAT/DHAPAT entering the peroxisomes via PEX, the enzyme alkylglycerone
phosphate synthase (AGPS) is the most crucial for the production of plasmalogens.
AGPS is encoded by the AGPS gene located on the long arm (q) of chromosome 2 at
position 31.2, and it has been determined that any of the three known mutations within
AGPS can yield a faulty AGPS; this is seen in individuals with RCDP3. AGPS is
7 involved in the ether lipid metabolism pathway, where C4H6O7PR is taken from GMP
and is broken down to produce plasmalogens (KEGG hsa00565). Although it is known
that the mutations within GNPAT/DHAPAT and AGPS impair proper function of
peroxisomes, little is known about the etiology of the science and the distinctive
symptoms arising from their defective proteins. Of the four known PBDs, RCDP1 is the
most well studied with its notable symptoms being shortening of the long bones and
intellectual impairment. In regards to CDP, CDPX2 has been extensively analyzed, and it
is the most well understood X-linked inherited subtype.
X-linked Chondrodysplasia Punctata
X-linked Chondrodysplasia puncata can be inherited in both a recessive and
dominant manner. In males, the recessive form results in moderate symptoms, whereas
the dominant variant results in severe complications that usually leads to an early death.
For CDPX1, the faulty gene is arylsulfatase E (ARSE), and it is located on the petite (p)
arm of the X chromosome at position 22.3 (Franco et al. 1995). ARSE encodes the
enzyme ARSE, which belongs to the group of sulfatases responsible for cartilage and
bone development. It is known that the ARSE enzyme is involved in bone growth and
density. The enzyme is located in the Golgi apparatus and may play a role in a
biochemical pathway involving vitamin K (Daniele et al., 1998; Franco et al., 1995; Rost
et al., 2004). However, the exact function of ARSE remains unknown (Nino et al. 2008).
The alleles causing CDPX2 are located in the Emopamil Binding Protein (EBP).
EBP is encoded by the EBP gene that is located on the petite (p) arm of the X
chromosome at position 11.23 (Schindelhauer et al., 1996). The EBP gene encodes 3Bhydroxysteroid-∆8,-∆7-isomerase (Sterol isomerase; Moebius, 1994). Sterol isomerase
8 has been determined to be one of the final enzymes in the steroid biosynthesis pathway
for the production of cholesterol (Figure 3).The EBP and the sterol isomerase differ in
their pattern of gene expression; however, the protein product of the EBP gene holds the
same function throughout an organism. Also, the expression of sterol isomerase in the
rats have been found to be liver-specific, whereas the EBP in guinea pigs was found
throughout many tissues. These include the liver, ileum, kidneys, adrenal gland, uterus
and gonads (Bae et al., 2001). While health problems in humans arise from high levels
of cholesterol, either obtained from the consumption of animal fats or de novo,
cholesterol is a vital structural component in all cell membranes (Nwokoro, 2001;
Herman, 2003). Cholesterol is necessary for proper embryonic development and has an
important role in cell function, such as the production of hormones and digestive acids
(Porter et al., 1996; Chiang et al., 1996; Clayton, 1998). Cholesterol is centralized for the
production of steroid compounds, such as estrogen.
Figure 3. Steroid biosynthesis pathway. Highlighted in red is the EBP found in the latter steps of the
cholesterol biosynthesis pathway. (Kyoto Encyclopedia of Genes and Genomes)
9 Cholesterol Biosynthetic Pathway: Pre-Squalene
The cholesterol biosynthetic pathway involves several organelles in the cell,
primarily the peroxisome and the endoplasmic reticulum (ER). In these two organelles, a
step-wise process is responsible for correct synthesis of the cholesterol needed for cell
function, but more importantly, for normal embryogenesis. The pathway is comprised of
an enzymatic cascade of approximately 30 reactions seperated into two sections; PreSqualene and Post-Squalene (Goldstein et al., 1990; Kelley and Herman, 2001; Gaylor et
al., 2002; Kovacs et al., 2002; Figure 4). The first four enzymatic reactions take place in
the peroxisomes, and the enzymes are transported to the peroxisome matrix by either the
Peroxisomal Targeting Signal-1 (PTS-1) and/or the Peroxisomal Targeting Signal-2
(PTS-2) (Subramani 1993). Fukao et al. (1990) described the first enzymatic reaction: the
conversion of acetyl-CoA to acetoacetyl-CoA, which is catalyzed by Acetoacetyl-CoA
thiolase (AA-CoA thiolase) and found to be a cytosolic enzyme. Further studies revealed
that peroxisomes can also synthesize Acetoacetyl-CoA from acetyl-CoA (Thompson et
al., 1990; Hovik et al., 1991). A later study revealed co-localization of the enzyme in the
mitochondria (mt AA-CoA thiolase) for cytosolic AA-CoA thiolase localization (Song et
al., 1994). Analysis for both AA-CoA thiolase entities in a double-labeled
immunofluorescence study showed a consensus PTS-1 at the C-terminus for the mt AACoA thiolase sequence along with an N-terminus mitochondrial targeting sequence
(Olivier et al., 2000). Thus, through the identified PTS-1, mt AA-CoA thiolase can be
transported into the peroxisomes, whereas the cytosolic AA-CoA thiolase may be ruled
out from importation into peroxisomes due to the absence of a peroxisomal targeting
sequence.
10 Figure 4. Enzymatic cascade of post-squalene synthesis. Kyoto Encyclopedia of Genes and Genomes.
In addition to these findings, Kovacs et al. (2007) determined that the Acetyl-CoA
originating from peroxisomes is preferentially channeled to the peroxisomes without
mixing with cytosolic acetyl-CoA. Most notably, the PTS of both human and rodent AACoA thiolase have been shown to be conserved (Fukao et al., 1990; Bonaldo et al., 1996).
The second enzymatic reaction in the cholesterol biosynthesis pathway is the
conversion of AA-CoA to hydroxy-3-methylglutaryl-CoA (HMG-CoA) via HMG-CoA
synthase (Ayte et al., 1990). As for AA-CoA thiolase, HMG-CoA synthase is encoded by
two genes, which produce a mitochondrial enzyme. HMG-CoA was originally believed
to be a cytosolic enzyme, but it is now known to be a peroxisomal enzyme. Through
11 immunoelectron microscopy, Olivier et al. (2000) showed that there is a significant level
of HMG-CoA synthase in the peroxisomal matrix, whereas little detection was recorded
in the cytosol as originally described by Ayte et al (1990). Furthermore, double-labeled
immunofluorescence revealed a superimposable pattern between the cytosolic HMG-CoA
synthase antibody with the peroxisomal antibody marker (Olivier et al., 2000).
Interestingly, analysis of both mitochondrial and peroxisomal HMG-CoA synthase by
Kovacs et al. (2002) revealed no consensus for PTS-1 nor PTS-2; however, cytosolic
HMG-CoA was shown to have a sequence similarity to PTS-2. Moreover, the PTS of
both human and rodent AA-CoA thiolase have been shown to be conserved across
evolution (Ayte et al., 1990b; Russ et al., 1992). It is noteworthy that HMG-CoA is used
as a form of energy in the cell. This is so due to the enzymatically release of HMG-CoA
via HMG-CoA lyase. HMG-CoA is then hydrolyzed to produce ketones as an energy
source during fasting (Mitchell et al., 2001).
The third enzymatic reaction is the reduction of HMG-CoA to mevalonic acid via
3-hydroxy-3-methyl- glutaryl CoA reductase (HMGR). HMGR was first described by
Luskey and Stevens (1985), and through several studies, including immunofluorescence,
immunoelectron microscopy, enzyme assays, and western blotting of subcellular
fractions, it has been shown to be co-localized with the ER in peroxisomes (Keller et al.,
1985, 1986; Engfelt et al., 1997; Kovacs et al., 2001). HMGR is an insoluble protein, and
it has been recognized as the rate-limiting step in the cholesterol biosynthesis pathway
(Herman 2003). Current studies have found HMGR that is localized to the peroxisomes
to be more resistant to inhibitory statins than HMGR that is localized to the ER. The
levels of the latter are less impacted by cholesterol-lowering drugs (Aboushadi et al.,
12 2000 and Weinhofer et al., 2006). In addition to the localization of HMGR, it has been
found that peroxisomal and ER HMGR have separate functions in isoprenoid
biosynthesis in which they have distinctive regulatory mechanisms from each other
(Aboushadi et al., 2000). These conclusions were reached after the observation of
different levels of statins in the peroxisomal and ER HMGR, along with the unaffected
rate of peroxisomal HMGR degradation observed in the presence of mevalonate
(Aboushadi et al., 2000 and Weinhofer et al., 2006).
Phosphorylation of mevalonic acid via Mevalonate Kinase (MvK) is the fourth
enzymatic reaction in the biosynthesis of cholesterol. While working with PragueDawley rats, it was shown conclusively that phosphorylation of mevalonic acid was
localized within the peroxisomes (Stamellos et al., 1992; Biardi et al., 1994). In the first
half of this fourth enzymatic reaction, Phosphomevalonate Kinase (PvK), Mevalonate
diphosphate decarboxylase (MPD) and Isopentenyl diphosphate isomerase (IPP
Isomerase) were shown to convert mevalonate into isopenetenyl disphospate, while PvK
presents a PTS-1 signaling sequence (SRL consensus sequence) at its C-terminus (Olivier
et al., 1999, 2000; Paton et al., 1997). Even though these five enzymes of pre-squalene
cholesterol biosynthesis were shown to be localized within the peroxisomes, in recent
investigations, the role of peroxisomal enzymes localization have been questioned.
Hogenboom et al. (2004a,b,c) revealed that all three enzymes (MvK, PvK, and MPD)
needed for the conversion of Mevalonate to IPP were not localized within the
peroxisomes in humans cells; however, they did contain a consensus sequence for PTS.
This study was contested by Kovacs et al. (2007); through the use of isotopic techniques,
data computations with isotopomer spectral analysis, immunofluorescence, and selective
13 permeabilization techniques. Kovacs and colleagues demonstrated that the pre-squalene
synthesis, including the enzymes necessary to convert Mevalonate to IPP, are localized to
peroxisomes; thus, their work nullified the earlier findings from Hogenboom (2004). The
second half in the conversion of Mevalonic acid to IPP is the decarboxylation of the
phosphorylated mevalonic acid.
At this stage in the pre-squalene biosynthesis of cholesterol, three condensation
reactions of IPP are catalyzed by farnesyl pyrophosphatase (FPPS) to produce farnesyl
pyrophosphate (FPP) (Krisans et al., 1996). FPPS is found in the peroxisomes, and is
transported into different cytosolic locations where farnesyl pyrophosphate (FPP) may be
generated as well. Nevertheless, FPP has been found to be localized predominantly to the
persoxisomes. Subcellular localization was shown by immunoelectron microscopy, and
immunoflurorescence studies of myc tagged FPPS, as well as by subcellular rat liver
fractionation (Krissans et al., 1994). However, FPPS was observed to be present in the
cytosol when CHO cells were made deficient in peroxisomes. This led to the conclusion
that there were two pools of FPPS one, which is retained within the peroxisomes, and a
second pool, which diffuses into the cytosol (Gupta et al., 1999). However, it is
interesting to note the following: although the PTS of human and rodent MvK, PMvK,
and IPP have been shown to be conserved, FPPS has been recently found to lack both
PTS-1 and PTS-2 (Tanaka et al., 1990; Schafer et al., 1992; Olivier et al., 1999;
Chambliss et al., 1996; Biardi et al., 1996; Xuan et al., 1994; Fukao et al., 1990; Bonaldo
et al., 1996; Kovacs et al., 2002). These findings are consistent with co-localization of
FPPase in both the peroxisomes and the cytosol. The next step is for two molecules of
FPP to condense into squalene via squalene synthase within the endoplasmic reticulum.
14 This is then followed by the reconfiguration of squalene into a close-ring hydrocarbon by
oxidosqualen cylcase and so forming Lanosterol. Last of all, pre-squalene biosynthesis of
cholesterol descends into the conversion of Lanosterol to Cholesterol in a series of 25
enzymatic reactions, which were originally believed to take place solely outside of the
peroxisomes (Fischer et al., 1991; Gaylor 2002).
Cholesterol Biosynthetic Pathway: Post-Squalene
The hypothesis that the last 25 reactions in cholesterol biosynthesis occur both in
the peroxisomes and in the ER is based on the findings of Appelkvist et al. (1990), which
revealed that dihydrolanosterol oxidase, delta14-sterol reductase, c-4-sterol demythylase,
and delta8-delta7-sterol isomerase are localized within peroxisomes. In addition to these
post-squalene enzymes, Hashimoto et al. (1994) showed that intermediates (4Methylcholest-7-en-3-ol and 4,4-Dimethylcholest-7-ene-3-ol) from the conversion of
Lanosterol to Cholesterol to accumulate within peroxisomes. Furthermore, cholesterol is
found to be synthesized at equivalent levels from dihydrolanosterol in isolated
peroxisomes as in isolated microsomes (Krisans et al., 1996).
Lanosterol, the first intermediate sterol, is known to be the compound which
proliferates into all steroid hormones. Steroid hormones include, but are not limited to,
natural steroid hormones and cholesterol, which is derived from 14-demethylation
between lanosterol and CYP51 (Herman et al., 2003). As presented in the Steroid
Biosynthesis Pathway, the last 25 enzymatic reactions occur in sequence and then the
pathway bifurcates in two directions. Emapomil Binding Protein (EBP) acts in both
directions as a 3 beta-hyroxysteroid D8, D7 isomerase at the penultimate step of the postsqualene cholesterol biosynthesis pathway (Derry et al., 1999, Braverman et al., 1999).
15 EBP converts Cholesta-8-en-3beta-ol (cholestenol) into Cholest-7-en-3 beta-ol
(Lathosterol) from one side of the bifurcation, and at the second branch of the bifurcation,
converts Zymosterol into Cholesta-7,24-dien-3beta-ol (Derry et al., 1999; see figure 3;
Steroid Biosynthesis Pathway, KEGG). This split in the pathway results in the production
of two key intermediates: 7-Dehydrocholesterol (7DHC) and Desmosterol. 7DHC and
Desmosterol are both products of EBP (Gaylor et al., 2002; Kelley et al., 2001). Lastly,
7-dehydrocholesterol reductase (DCHR7) reduces 7DHC into Cholesterol, whereas 24dehydrocholesterol reductase (DCHR24) reduces Desmosterol into Cholesterol.
Cholesterol serves as a vital component for the production of key biological products, and
acts as the precursor for steroid hormones, including, but not limited to, Aldosterone,
Progesterone, Cortisol, and Testosterone.
The natural steroid hormones derived from cholesterol are synthesized in the
adrenal gland and gonads, where they regulate stress response (cortisol), salt balance
(aldersterone), as well as sexual development and osteochonrosis (estradiol). In addition
to cholesterol’s role in the biosynthetic pathways of steroid hormones, the sterol
intermediates produced in the cholesterol biosynthesis pathway also serve as precursors
for the biosynthesis of several key biological products, including bile acids (Figure 5a).
16 Figure 5: A) Overview of the latter half of the Cholesterol biosynthetic pathway. Herman 2003. B)
Pathway for the synthesis of cholesterol and non-sterol isoprenes. Clayton 1998.
These biological products also include Heme A and ubiquinone, which are necessary for
the electron transport chain. Another product is Isopentenyl-tRNA, which is made in the
pre-squalene synthesis. Isopentenyl-tRNA is important for stabilizing the codonanticodon interaction and preventing the genetic code from being misread (Clayton et al.,
1998; Figure 5b). The organic compound, dolichol, is produced from FPP and is essential
for N-linked protein glycosylation (Goldstein et al., 1990). Furthermore, farnesyl and
geranylgeranyl groups, which are generated from the series of enzymatic reactions within
the phosphorylation of mevalonic acid, are important for cell signaling and differentiation
(Zhang et al., 1996). Both lipophilic molecules 4,4-dimethyl-5alpha-cholesta-8,14,24trien-3beta-ol and 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol, referred to as
meiosis-activing sterol (MAS), have been shown to accumulate in the ovaries and testes
17 across species, including but not limited to humans, mice, and rats (Byskov et al, 1995;
Wang et al., 2010).
Cholesterol Biosynthetic Disorders (CBD)
In spite of the vital role of these biological products, the abnormal levels of sterol
intermediates have been shown to slightly alter the function of specific proteins, which
disrupt normal biological processes. To begin, it has been found that the intermediate
lanosterol stimulates the ubiquitination and degradation of Hmgcr (HMG-CoA reductase)
more readily than cholesterol (Song et al., 2005). The alterations of proteins’ function
disrupting biological process are evident through the known cholesterol biosynthesis
disorders. These disorders are known to arise from the impairment of enzymatic activity
and accumulation of precursors/sterol intermediates within the cholesterol biosynthesis
pathway.
The first of these six disorders to be connected with cholesterol biosynthesis was
Smith-Lemil-Opitz syndrome (SLOS; OMIM 270400). SLOS is an autosomal recessive
disorder, with an incidence of 1 in 20,000-60,000 newborns due to a deficiency in 3 betahydroxysteroid delta7-reductase (7DHCR), the last enzyme of the cholesterol
biosynthesis pathway (Smith et al., 1964; Opitz et al., 1987, 1994; Irons et al., 1993; Tint
et al., 1994; Shefer et al., 1995; Genetics Home Reference). A defective 7DHCR results
from any of the 120 mutations found within 7Dhcr; (OMIM 270400). Human 7DHCR
was found to be localized to the ER membrane through immunofluoresence analysis in
COS7 cells (Holmer et al., 1998). SLOS has been characterized with severe
developmental malformations, including but not limited to microcephaly, cleft palate,
micrognathia and anteverted nares; furthermore, this disorder leads to intellectual
18 impairment, cardiac and intestinal complications (Smith et al., 1964; Tierney et al.,
2000). Through in-vitro studies of cultured rat embryos, 7DHC was observed to impair
the development of embryos, as shown by Gaoua et al. (1999). It has been found that the
phenotypic findings in SLOS mice were accountable due to a lack of cholesterol or the
accumulation of 7DHC (Porter et al., 2002). SLOS was shown to be exacerbated by the
increased breakdown of Hmgcr (HMG-CoA reductase) via the accumulation of 7DHC
(Fitzky et al., 2001). In addition to the disruptive effects of 7DHC on the cholesterol
pathway, impairment of LDL-cholesterol metabolism was noted to have resulted from
elevated levels of 7DHC fibroblasts from an individual with SLOS (Wassif et al., 2002).
The group proposed that the function of Sterol Sensing Domain (SSD)-containing
proteins in SLOS may be perturbed by 7DHC. This proposal was later supported by
Kovacs et al. (2009), who observed reduced transcripts of both SREBP CleavageActivating Protein (SCAP; a SSD-containing protein) and several SREBP gene family
members, including FASN, SREBP2, S1P-MBTPS1 and SQS1. Mutational analysis of 7dehydrocholesterol reductase (Dhcr) substantiated earlier findings that 7DHC
accumulated as cholesterol levels were found to be reduced in SLOS (Jira et al., 2003). In
addition to the detrimental side effects from 7DHC accumulation, the binding activity of
the serotonin 1A receptor has been shown to be impaired by the aggregation of 7DHC in
the hippocampal membranes (Singh et al., 2007). It has been found that 7DHC and 8dehyrdrocholesterol (8DHC) accounts for the unsaturation of both steroid hormone
precursors and bile acids (Shackleton et al., 1999, 2002; Honda et al., 1999).These
unsaturated steroids have yet to be shown to have antagonistic properties in cholesterol
19 biosynthesis; however, there is evidence that developmental processes may be affected in
their presence (Porter et al., 2002).
The inadequate levels of sterol intermediates have further elucidated the impact of
protein malfunction regarding two other cholesterol biosynthesis disorders; CDPX2 and
CHILD syndrome. Both CHILD and CDPX2 result in similar phenotypic manifestations
with some slight differences, such as lesion distribution. Even before the studies
connecting abnormal levels of 7DHC with SLOS, it was known that a cholesterol
intermediate served as the precursor for vitamin D synthesis (Kandutsch et al., 1960). The
inadequate levels of cholesterol/Vitamin D were described along with the clinical
manifestations of an individual with elevated levels of 8(9)-cholestenol, 8dehydrocholesterol, 7-dehydrocholesterol and who presented the symptoms consistent
with CDPX2 (Furtado et al., 2010). Another complication pertaining to sterol precursors
include congenital hemidysplasia with ichthyosiform erythroderma and limb defects
syndrome (CHILD), which arises from the accumulation of 4-Methylzymosterolcarboxylase occurring exclusively in females; 60 females have been diagnosed with
CHILD to date. CHILD syndrome results from mutations in the NADH steroid
dehydrogenase-like (NSDHL; 3ß hydroxysteroid dehydrogenase) gene found on Xq28
(Konig et al., 2000). CHILD syndrome, just as in CDPX2, has been reported to contain
CDP patterns, including but not limited to punctate calcifications of cartilaginous
structures and ichtyosioform dermatoses (Happle et al., 1980; Konig et al., 2002;
Hummel et al., 2003). Symmetrical and unilateral skin lesions have been proposed to
follow Blaschko lines in CHILD, as opposed to CDPX2 (Konig et al., 2000; Grange et
al., 2000). In addition to these findings, it has been reported that CHILD syndrome arises
20 in part by mosaicism and X-inactivation. This is to say that within cells, one of the two
X-chromosomes becomes inactivated. Depending upon whether these inactivated cells
carry the faulty NSDHL gene, several tissues will be unilaterally abnormal, while others
will be normal on the opposite side as seen in the distribution of the lines of Blaschko
(Happle 1985, 1987, 1993; Christiansen et al., 1984, Emami et al., 1992). The lines of
Blaschko in CHILD syndrome not only appear unilaterally, but persist through one’s
lifespan as opposed to CDPX2, which disappear soon after early childhood (Grange et al.,
2000; Herman 2003). In addition to skin abnormalities, it is noteworthy to mention that
such mosaicism in CHILD syndrome are more likely to occur on the right side as
opposed to the left side of the body (Happle et al., 1980; Hummel et al., 2003). Other
malformations in CHILD syndrome include epiphyseal stippling, central nervous system
(CNS) abnormalities, kidney and cardiac complication; all of these are found to occur on
the affected side. Recent studies have demonstrated that the same levels of abnormal
sterols, such as 8-dehydrocholesterol and 8(9)-cholestenol, were found in individuals
affected with CHILD syndrome as well as in individuals affected with CDPX2 (Grange
et al., 2000). Such plasma sterol analysis has been disputed and continues to be
problematic in defining CHILD syndrome and CDPX2 (Happle et al., 2000). Despite the
disagreement between laboratories as to the levels of cholestenol in CHILD syndrome
and CDPX2, the accumulation of specific sterols resulting from deficient metabolizing
enzymes in peroxisomes was first postulated due to peroxisomal abnormalities in
fibroblasts from individuals with CHILD syndrome (Emami et al., 1992). Authors of
further studies reported the collection of sterol precursors in fibroblast cultures of
individuals with CHILD syndrome along with the accumulation of such precursors within
21 peroxisomes (Hashimoto et al., 1994, 1998). Kelley and Herman (2001) demonstrated
that mutations in the EBP gene were seen in individuals affected with CHILD syndrome.
As opposed to slight differences in skin and skeletal systems, cataracts, which are only
seen in CDPX2, tend to be a key trademark for the disorder. Interestingly, there have
been cases in which individuals with CHILD syndrome show bilateral and symmetric
abnormalities and cases where individuals with CDPX2 presented with erythroderma or
unilateral ichthyosis as in CHILD syndrome (Fink-Puches et al., 1997; Kalter et al., 1989;
Corbi et al., 1998). In addition to SLOS, CHILD, and CDPX2, there are three other
cholesterol biosynthetic inborn errors to date. These include mevalonic aciduria
(mevolonate kinase; OMIM 251170), desmosterolosis (3beta-hydroxysteroid delta24reductase; OMIM 602398), and Greenberg dysplasia (OMIM 215140). From a 30
stepwise metabolic pathway, it is safe to assume that there may be several other inborn
errors due to improper levels of cholesterol just as the six known cholesterol biosynthetic
disorders described above. The unknown or uncharacterized disorders may very well
pertain to the accumulation of sterol intermediates during embryogenesis in both the presqualene and the post-squalene enzymatic reactions.
During embryogenesis, sterol intermediates from the Cholesterol biosynthesis
pathways result in detrimental outcomes such as abnormal development and phenotypic
anomalies as it is seen in all cholesterol biosynthesis disorders. The abnormal skeletal
manifestations seen in CPDPX2 were first suggested to be due to the accumulation of
cholesterol precursors resulted in the interference of the Hedgehog cholesterol-modified
protein (Porter et al., 1996; Farese and Herz 1998). Hedgehog proteins have been shown
to be involved in several developmental stages, including the orientation of limbs,
22 chondrogenesis, and the migration of cells around the notochord. This process forms
primordial cartilage vertebral bodies and stimulates enchondral growth (Kelley and
Herman, 2001). Proper functionality of Hedgehog protein is attributed to their
modifications through the addition of cholesterol (Mann et al., 2000; Ingham et al., 2001;
Jeong et al., 2002; Copper et al., 2003). The absence of the sonic hedgehog protein in
mice results in various malformations, including holoprosencephaly (Chiang et al., 1996).
Also, the sonic hedgehog has been described with a definitive role involving the left-right
axis determination during embryogenesis (Meyers and Martin 1999). In addition to
cholesterol as a key modifier for Hedgehog proteins, it is also known that metabolites,
such as the sterol intermediates already described in this thesis, are known to be
requirements for proper functioning of Hedgehog signaling pathways (Porter et al.,
1996). Nevertheless, it has been postulated that the abnormal sterols levels may explain
the unilateral presentations of CHILD syndrome due to the impaired function of the sonic
hedgehog protein (Beachy et al., 1997; Meyers and Martin, 1999; Nwokoro 2001). One
such abnormal sterol level arises from the deficient activity of 3-beta hydroxysteroid
dehydrogenase, which is involved in the synthesis of hormones such as cortisol,
aldosterone, androgens and estrogen. Further studies showed that, in addition to the
detrimental effects of levels of sterol intermediates, sterol depletion has also resulted in
the malfunctioning of the Hedgehog proteins in SLO, CHILD, and Greenberg dysplasia
(Farese et al., 1998; Beachy et al., 1997; Kelley and Herman 2001; Cooper et al., 2003;
Maurer et al., 2008).
23 CBD Resulting from Exposure to Teratogens
Despite the sterol intermediate accumulation studies, there have been reports
implicating specific drugs or substances (teratogens) known to interfere with fetal
development and resulting in neonatal malformations as seen in inborn errors of
cholesterol biosynthesis (Krakowiak et al., 2003). One such report implicated triparanol,
a anticholesteremic agent, as a teratogen with D24-reductase and sterol D8- isomerase
inhibitory properties which resulted in rats’ limb abnormalities when treated with the
drug during embryogenesis (Chevy et al., 2002). Chevy postulated that these effects
resulted from the accumulation of desmosterol, D8-cholesten-3b-ol and Zymosterol, and
not because of low levels of cholesterol. Resently, Triparanol has been associated as a
therapeutic treatment for various cancerous human cells by impeding tumor growth
through the deregulation of Hedgehog signaling proteins (Bi et al., 2012). Two other
teratogenic agents, AY9944 and BM 15766, when introduced to rats embryos, resulted in
the following malformation anomalies: axial skeletal defects, cleft palate, pituitary
agensis, holprosencephaly, and limb abnormalities (Roux et al., 1979; Repetto et al.,
1990; Batta and Salen 1998; Honda et al., 1996; Lanoue et al., 1997; Herman 2003).
Futhermore, Batta and Salen recorded similar skeletal abnormalities in SLO individuals
and rats when their embryos were introduced to AY9944. Thus, it may be evident enough
that exposure of a teratogen agent may be sufficient to bring forth abnormal phenotypic
characteristics identical to those of individuals with genetic disorders such as CDP.
CBD and Maternal Diseases
Other factors attributing to the manifestations of CDP besides exposure to
teratogens and one’s genetic makeup include maternal diseases. One such disease is the
24 autoimmune disease Systemic Lupus Erythematosus (SLE) which was first associated
with newborn CDP cases in 1993 (Curry et al., 1993). The first of these many studies was
described in a mother diagnosed with SLE after delivering fraternal twins in which the
male was born with CDP characteristics as oppose to the female, who at 15 months of
age presented normal development (Mansour et al., 1994). A similar case of post-delivery
diagnosis of SLE resulted in the birth of a male child with CDP from an AfricanAmerican mother. (Kelly et al., 1999). Another post-delivery case resulted in the birth of
a male showing stippling, dwarfness at birth and postnatally, and with mild
developmental delay (Shanske et al., 2007). Just as SLE Post-delivery diagnosed
mothers, there has also been cases in which mothers have been diagnosed with SLE
before pregnancy. One such case of SLE Pre-delivery diagnosed mother resulted in the
loss of both males within 24-36 weeks of gestation (Elcioglu and Hall 1998). A second
SLE pre-delivery diagnosed case resulted in the birth of a female infant, who developed
CDP at the mid-trimester and whose mother had SLE for 3 years prior to delivery
(Austin-Ward et al., 1998). In addition to mothers diagnosed with SLE either Postdelivery or Pre-delivery, a case presented itself with a mother with epilepsy and the
delivery of two CDP- effected males (Kozlowski et al., 2004). Along with mother’s
diagnosed with SLE and epilepsy, a recent study involving several cases with maternal
autoimmune conditions, revealed radiological fetal manifestation of CDP whose mothers
had either SLE, Mixed Connective Tissue Disease (MCTD), or scleroderma (Chitayat et
al., 2008). In Chitayat’s study, it was intriguingly found all newborns with normal
karyotypes, no mutations in Arse, and the ruling out of PBDs through biochemical
studies. Hence, it would be adviceable to monitor mothers for the development of
25 autoimmune diseases during pregnancy, in order to promote the health of the baby whose
genetic make up does not present a genetic disorder as CDP. Monitoring of infants could
also be conducted by analyzing their biochemical sterol profiles and screaning for
defective enzymes localized either in the peroxisomes, ER, or any other organelle.
Specifically, the monitoring of proper function of Hedgehog signaling proteins and the
level of exposure to teratogenic agents or environment factors which may disrupt normal
fetal development. As shown, babies whose mother’s have been diagnosed with an
autoimmune condition, could develop inborn errors like those of chestrol biosynethsis
disorders, including EBP and NSDHL or any of the Peroxins/enzymes known to affect
proper peroxisome activity.
All six enzymes pertaining to disorders involving the cholesterol (pre-squalene)
biosynthetic pathway have been elucidated to occur primarily in the cytosol. Unlike
inborn errors due to abnormal cholesterol biosynthesis, individuals affected with PBDs
have been reported to have inefficient activity of HMG-CoA reductase, MvK, MPD, IPP
and FPPS within their liver. Nevertheless, the post-squalene enzymes found in the ER
and mitochondrial revealed to be at normal enzymatic activity for these individual
(Krisans et al., 1994). Any perturbation of the cholesterol biosynthesis pathway presents
a build up of toxic byproducts; in addition, these disruptions cause an inhibition of key
enzymatic steps, interruption of normal protein activity such as the Hedgehog protein,
and the malformation of bone development that is seen in CDP- affected individuals.
Emopamil Binding Protein
The EBP gene is located on the petite (p) arm of the X chromosome in the region
11.22-23, which spans 7.0 Kbp of genomic DNA. The encoded gene is composed of five
26 exons spanning ~1.0Kb (Moebius et al., 1997; Derry et al, 1999; Traupe et al., 2000;
Figure 6a, 6b). Exons 2-5 are the regions that encode the protein, which is alternating
known as emopamil-binding protein (EBP), 3β-hydroxysteroid-Δ8,Δ7-isomerase, 3-betahydroxysteroid-∆8-∆7, isomerase; sterol ∆8-isomerase and cholestenol ∆-isomerase
(sterol isomerase; Hanner et al., 1995; Moebius et al., 1993, 1994; Silve et al., 1996). In
1995, Hanner found that EBP codes for a 230 amino acid polypeptide embedded in the
Endoplasmic Reticulum membrane (ER; Figure 7a, 7b).
Figure 6a: Cytogenetic and Molecular location of EBP. Genetics Home Reference
4981 gggccgcctt cttcgcttca ccattggctc gctccgtaag gcaagagaac ccactagggg atgagcccga actagggatg 5061 tgacagagcg cgagacccag cctaaagaga gcccggagcc agcgtgggag gccgctgccg tcgcgcgcct
5051 tggtgagtgc cctccacccg gcccctgctc
....
6901 ttctgtactt tctatttgtc caggtttttc tgttcctttt tttttttttt ttttaacttc ctgcctatac acacgcagcc atcagcccac
6991 aaagacatga ctaccaacgc gggccccttg cacccatact ggcctcagca cctaagactg gacaactttg tacctaatga
7071 ccgccccacc tggcatatac tggctggcct cttctctgtc acaggggtct tagtcgtgac cacatggctg ttgtcaggtc
7151 gtgctgcggt tgtcccattg gggacttggc ggcgactgtc cctgtgctgg tttgcagtgt gtgggttcat tcacctggtg
7231 atcgagggct ggttcgttct ctactacgaa gacctgcttg gagaccaagc cttcttatct caactctgtg agtcctgatt tctttcatat
.....
10201 ttcttttctt cagggaaaga gtatgccaag ggagacagcc gatacatcct gtaagtgttt gcctctgtca atggagactg
10281 gcattggttt tcggggggtg gtgagttggg gagcactaat gggctaacct gtaggaagag cacaccgata ccagtgtccc
10361 ctcatgcttt ctcctgcagg ggtgacaact tcacagtgtg catggaaacc atcacagctt gcctgtgggg accactcagc
10441 ctgtgggtgg tgatcgcctt tctccgccag catcccctcc gcttcattct acagcttgtg gtctctgtgg gtaaggaaag
10521 ggcactagag gggcactggg cactagaggg gttgatgggg
.....
27 11401 ttggcgaaag tgtccccttc ctcactgggg cttctccttc ccctcctgcc acccacaggc cagatctatg gggatgtgct
11481 ctacttcctg acagagcacc gcgacggatt ccagcacgga gagctgggcc accctctcta cttctggttt tactttgtct
11561 tcatgaatgc cctgtggctg gtgctgcctg gagtccttgt gcttgatgct gtgaagcacc tcactcatgc ccagagcacg
11641 ctggatgcca aggccacaaa agccaagagc aagaagaact gaggagtggt ggaccaggct cgaacactgg ccgaggagga
11721 gctctctgcc tgccagaaga gtctagtcct gctcccacag tttggaggga caaagctaat tgatctgtca cactcaggct
11801 catgggcagg cacaagaagg ggaataaagg ggctgtgtga aggcactgct gggagccatt agaacacaga tacaagagaa
11881 gccaggaggt ctatgatggt gacgattttt aaaatcagga aataaaagat cttgactcta a
Figure 6b. EBP DNA Sequence. Intronic regions are marked by black text. Exons 1-­‐5 are represented by Blue, Green, Magenta, Red, and Purple text, respectively. http://www.ncbi.nlm.nih.gov/nucleotide/169881268?report=genbank&log$=nucltop&blast_rank=1&RID=
PEPM517J01S
The protein has been shown to conduct two functions, serving as a high-affinity receptor
for anti-ischemic drugs such as tamoxifen and acting as a D8-D7 sterol isomerase which
converts Cholest8(9)-en-3beta-ol (8,9 choletenol) into cholest-7-en-3beta-ol(lathosterol)
(Moebius et al., 1994; Silve et al., 1996; Derry et al., 1999).
Evolutionary Studies of EBP, ERG2p and Sigma 1 Receptor
To further expand on the understanding of EBP and its dual role, studies have
investigated the evolutionary conserved regions of EBP, its isoenzyme ERG2p in fungi
and the Sigma 1 Receptor. Both EBP and ERG2p share homology and are both integral
proteins of the ER. Both bind to various chemicals while having the same catalytic
activity (Moebius et al., 1998; Laggner et al., 2005). As for the Sigma 1 Receptor protein,
which it’s a mammalian protein and its function remains unknown, studies have shown
having greater homology with ERG2p than EBP (Moebius et al., 1997; Bowen et al.,
2000). As little as 30% amino acid homology is seen between the Sigma 1 Receptor and
ERG2p (Moebius et al., 1997;). Furthermore, EBP differs structurally as it contains 4
transmembrane domains (TDs) versus 3 TDs observed in both the Sigma 1 Receptor and
ERG2 (Moebius et al., 1997). It has been demonstrated that human EBP is 78% and 73%
identical to the amino acids of mice and guinea pigs, respectively (Moebius et al., 1998).
28 Figure 7a: Diagram illustrating the Emopamil Binding Protein. Amino acids are represented by their
letter symbol. Red circles are found in the transmembrane domain; blue circles are found in the cytoplasm
or in the lumen of the endoplasmic reticulumn. Known mutations as of 2012 are represented by Red
highlights (nonsense) and green highlights (missense). Canueto et al., 2012
TTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAAVVPLGTWRRLS
LCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQLWKEYAKGDSRYILGDNFTVCMETITACLW
GPLSLWVVIAFLRQHPLRFILQLVVSVGQIYGDVLYFLTEHRDGFQHGELGHPLYFWFYFVFMNA
LWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSKKN
Figure 7b: EBP Amino Acid Sequence. Coding regions, Exons 2-5, are represented by Green, Magenta,
Red, and Purple text, respectively.
29 Mutational Analysis of EBP
Given that much of today’s knowledge about the detrimental affects of
intermediate sterols on the cholesterol biosynthetic pathway, mutational analysis on EBP
have further supported a malfunctioning EBP. A defective EBP results in the
accumulation of two cholesterol intermediates, 8(9)- cholestenol and 8dehydrocholesterol/7-dehydrocholesterol in plasma analyzed by gas chromatograph and
mass spectrometry (GC/MS) (Braverman et al., 1999; Kelley et al., 1999; DiPreta et al.,
2000; Has et al., 2002). These two intermediates have been shown to be useful for the
diagnosis of an individual with CDPX2. At present, there are 64 known mutations in the
EBP gene, with the first having been found in 1999 by Derry et al. and Braverman et al.
(HGMD; OMIM 300205). Also in 1999, Moebius et al. found that key amino acids in
EBP were essential for the isomerization: H77, E81, T126, N194, and W197. All 64
mutations lie within four exonic regions, with the majority being distributed between
exons 2 and 4; these include point mutations, deletions and insertions (Braverman et al.,
1999; Has et al., 2000; Feldmeyer et al., 2006). Further mutational analysis in 14
individuals has revealed that 12 of the 64 known mutations are reported as being
unclassified CDP due to a lack of correlation between phenotype and the mutation
(Ikegawa et al., 2000). Ikegawa further suggested that mutations, such as nonsense and
frameshift mutations, produce a truncated EBP resulting in the phenotypic typical of
CDPX2 (oculocutaneous, lesion/lines of Blaschko and skeletal anomalies). Ikegawa also
suggested that missense mutations resulted in atypical CDPX2 phenotypes in which most
phenotypic characteristics were nonmarked, such as cataracts formation, while still
retaining the abnormal sterol profiles resulting from a dysfunctional EBP. Sterol profiles
30 vary amongst mutations in EBP. Normal sterol levels are observed with missense
mutations, but not with nonsense or frameshift mutations (Ikegawa et al., 2000; Whittock
et al 2003; Canueto et al., 2011). Molecular studies provide evidence of high rates of
mutations in the sterol isomerase (Derry et al., 1999; Braverman et al., 1999; Metzenberg
et al., 1999). Lethal cases in which males born with CDPX2 or CHILD syndrome have
been reported, sporadically (Crovato and Rebora 1985: Tronnier et al., 1992; Happle et
al., 1995; Sutphen et al., 1995; Metzenberg et al., 1999; Aughton et al., 2003; Milunsky
et al., 2003). Thus, an impaired EBP may cause detrimental effects during the early
stages of development.
EBP, A Transmembrane Protein
EBP is an integral membrane protein with four transmembrane domains of the
Endoplasmic Reticulum. The ER is responsible for, but not limited to, the production of
proteins, facilitating protein folding, and steroid biosynthesis (Moebius et al., 1993). The
membrane proteins maintained in the ER contain defined sequence motifs within their
polypeptide chain which permit them to anchor themselves onto the ER membrane
(Teasdale et al., 1996). These membrane bound ER proteins amass and seclude
themselves from other proteins, which are destined for other cell compartments or a
secretory pathway (Wieland et al., 1999). Type 1 membrane bound ER proteins have
been described by the presence of their defined double-lysine sequence motif found at the
C-terminus on the cytosolic side (Cosson et al., 1994). For the EBP transmembrane
protein, it was found to contain a lysine-rich motif and noted as serving as an anchorage
point in the ER (Hanner et al., 1995; Anderson et al., 1999). As described previously, one
such lysine modification located at the C-terminus has been observed in several integral
31 proteins and described as the retrieval of type 1 integral membrane proteins into the ER
(Jackson et al., 1990, 1993). Lysine 3 and Lysine 5 at the C-terminus of
Oligosaccharyltransferase complex (OST48) have been demonstrated to direct
localization of the ER protein by COPI-mediated retrieval even after one was substituted
by argenine (Hardt et al., 2002). Neither a single substitution or the combination of both
Lysines by arginine residues failed to direct localization of the ER protein by the COPimediated retrival complex. It has been previously noted that the COPI complex contains a
KKXX C-terminal mortification, is sufficient as the signal sequence necessary for the
transport of ER membrane proteins from the Golgi (Teasdale et al., 1996; Anderson et al.,
1999; Letourneur et al., 1994). In contrasts to type 1-membrane proteins, type 2
membrane proteins contain arginine-rich motifs at the N-terminus in the cytosolic region,
which act as ER retention motifs (Schutze et al., 1994). Hardt also demonstrated that
simple charge interactions were not enough to retain a protein in the ER by showing that
OST48 localization was not affected even if the two lysines were replaced by histidines.
Four amino acid residues (K-D-E-L) in transmembrane proteins have been shown to
retain proteins in the ER membrane, whereas the absence of KDEL resulted in the
secretion of the protein (Pelham et al., 1988). The specific tetrapeptide, K-D-E-L was
characterized in soluble proteins at their C-terminus and this region is best known as a
mediating ER-residency (Lewis and Pelham 1992; Yamamoto et al., 2001). Furthermore,
several studies involving the ER identified the location of four transmembrane domains,
which are attached to either the C-Terminus or N-Terminus of the protein in the
cytoplasmic region of the ER (Hanner et al., 1995; Dussossoy et al., 1999; Has et al 2000;
Bae et al., 2001).
32 The four domains are conserved in humans and rodents. Six amino acid residues
(His77, Glu81, Glu123, Thr126, Asn194, Trp 197) in these four domains may present
themselves in the catalytic cleft of the human EBP (Bae et al., 2001 and Moebius et al.,
1999). There is a conserved cytoplasmic domain important for enzyme function located
between the second and third transmembrane domains of the rat sterol isomerase gene,
compared to the human homology (Moebius et al., 1999). Also, a highly conserved motif
with various binding sites lies along the rat’s amino acid residues for the sterol isomerase.
The rat’s sterol isomerase gene also contains of several serine and threonine residues,
which are thought to serve as phosphorylation sites for serine/threonin kinases (Grand,
1989; Pinna et al., 1990). The rat sequence contains two protein kinase C phosphorylation
sites, two casein kinase II phophorylation sites, and an amidation site (Kreil 1984 and
Bradbury and Smith, 1987).
Cellular Localization of EBP
It has been difficult to determine the subcellular location of the EBP. Goldstein et
al. (1990) suggested that all post-squalene enzymes, starting with the production of
Lanosterol, were localized in the ER; later in 1995, Hanner demonstrated EBP was
localized in the ER. A more recent study revealed that EBP colocalized in the nuclear
membrane (Dussossoy et al., 1999). Earlier studies demonstrated that the low
concentration of cholesterol in the ER resulted to high membrane permeability, and
showed that ER proteins are easily transported across the ER membrance (Lange and
Steck 1997; Bretscher and Munro, 1993). Lange and Steck as Bretscher and Munro’s
findings would contest the notion of a single localization for EBP and provide proof of its
colocalization as an important role for cholesterol levels. Also, the translocation of
33 proteins through the ER is shown to be regulated by cholesterol levels (Bretscher and
Munro, 1993; Sugii et al., 2003). 30 mammalian genes encoding enzymes in cholesterol
biosynthesis have been identified in the past two decades, but the exact location(s) of the
encoded enzymes continue to evade scientists (Moebius et al., 2000). The cholesterol
biosynthetic pathway is comprises of numerous enzymes and these enzymes along with
their precursors are found in several compartments of the cell. These compartments
include, but may not be limited to the mitochondria, peroxisomes, cytosol, and the ER.
(Reinhart et al., 1987).
Lipid Droplets (LD) as Repositories
It has been found that there are Lipid Droplets (LDs) on the surface of
unesterified cholesterol, which may play a role in several cholesterol biosynthetic events
(Kraemer et al., 1994; Prattes et al., 2000). LDs also known as adiposomes are organelles
rich in lipids that are found in all eukaryotes including plants and fungi, specific
prokaryotes; serving as reservoirs of lipids in mammalian adipocytes (Murphy and
Vance, 1999; Brown et al., 2001; Zweytick et al., 2000; Guo et al., 2009). LDs are
thought to reside at the ER and are considered to serve as depots for lipids since they are
apparently an extension of the ER (Zweytick et al., 2000). A more recent study found that
NSDHL, the faulty enzyme in CHILD syndrome and the fifth to last enzyme in the
biosynthesis of cholesterol, is found on the membranes of the ER, yet interestingly
accumulated on the surface of LDs (Caldas and Herman, 2003).
For the first time, one of these cholesterol biosynthesis cascading events was
associated with LDs, indicating that this is an important site for the regulation of
cholesterol biosynthesis. Further, regulation of cholesterol biosynthesis was localized to
34 the ER of liposomes, since each of the enzymes in this pathway contains a C-terminus
retrieval sequence, RKDK (Caldas et al., 2003). Another study regarding the location of
NSDHL, revealed that NSDHL ceased to be produced on the surface of LDs, but
redistributed itself back to the ER when CHO cells were treated with sterol-depleted
medium (Ohashi et al., 2003). Furthermore, Ohashi was able to reduce the conversion of
lanosterol to cholesterol while inducing production of LDs by increasing oleic acid levels
in the medium. Ohashi’s also observed NSDHL to be predominately localized on these
induced LDs. Ohashi’s findings were observed through immunofluorescence microscopy
and subcellular fractionation. Moreover, a decreased of sterol precursors such as C-28,
4,4-dimethyl-5alpha-cholesta-8-en-3beta-ol (C-29), and Lanosterol (C-30) to Cholesterol
(27) were recorded by Ohashi. The directing of NSDHL to LD further shows that little
biosynthesis of cholesterol occurs in the ER. Low concentrations of cholesterol gives rise
to increased permeability of many organelles’ membranes such as the ER. This
permeability property makes it feasible for protein translocations as described earlier by
both Bretscher and Lange. The distribution of cholesterol throughout the cell and within
membranes must be strictly regulated, but the mechanism remains unclear.
One study of the regulation of cellular cholesterol balance elucidated the role of
caveolin as a key component on the surface of LDs (Pol et al., 2001). Pol discovered a
decreased of cholesterol synthesis on both Baby Hamster Kidney (BHK) and COS-1 cells
when in the presence of caveolin. It has been postulated that given the colocalization of
NSDHL and the role of caveolin, LDs may provide a link between cholesterol
biosynthesis and a site of a regulatory mechanism affecting cellular cholesterol (Ohashi et
al., 2003). Further studies also suggest that synthesized cholesterol is scarcely dispersed
35 throughout the ER by the presence of a transport system involving caveolin. Caveolin has
been shown to remove cholesterol from LDs and mediate synthesized cholesterol to the
plasma membrane (Uittenbogaard et al., 1998; Fujimoto et al., 2001; Ostermeyer et al.,
2001; Pol et al., 2001). NSDHL is the first of thirty enzymes in the cholesterol presqualene biosynthetic pathway that has been shown to be located in LDs. In
Saccharomyces cerevisiae, three of its cholesterol biosynthesis enzymes (squalene
epoxidase, lanosterol synthase, sterol-D24-methyltransferase) have been recorded to be
localized in LDs (Zweytick et al., 2000; Milla et al., 2002; Mo et al., 2003).
Lipid Rafts and Cellular Cholesterol Localization
Other cell structures comprised of rich levels of cholesterol are lipid rafts (LRs),
which have been thought to play a role in cell membrane function as well as modulating
protein activity (Herman 2003; Pike et al., 2003). Moreover, the amount of cholesterol in
these LRs has been shown to affect thickness, elasticity and curvature of membranes
(Allende et al., 2004; Bacia et al., 2005). LRs have recently been demonstrated to have a
regulatory role for intramembrane proteolysis of transmembrane proteins (Pike 2005).
LRs have also been shown to be for the insertion of receptors, the signaling of
neurotrophins, and the secretion of neurotransmitters (Pike 2003; Korade et al., 2009).
The content of cholesterol on LRs is crucial for proper membrane excitability,
conductivity, and the movement of iontropic receptors such as the ionotropic gultamate
receptor which may play a role in Ca+2 influx (Korade and Kenworthy 2008; Kwaaitaal
et al., 2011). These LRs have also been shown to reduced DHCR7 levels resulting in the
accumulation of 7DHC in LRs, rather than cholesterol thus altering their structure (Keller
et al., 2004). Recalling that SLO results from DHCR7 deficiency and 7DHC breaksdown
36 HMGCR resulting in further exacerbating the synthesis of cholesterol. Perhaps these LDs
may help us understand the regulatory mechanism(s) for cholesterol biosynthesis since it
remains to be determined how cells maintain a low level of cholesterol in the ER, the site
of cholesterol biosynthesis (Ohashi et al., 2003). Moreover, this link between the role of
LDs and the regulator mechanism of cholesterol biosynthesis raises the question, if not
the doubt, whether other cholesterol biosynthesis enzymes may be located in other
organelles such as LDs.
EBP/Sterol Isomerase Function and Localization
There have been several studies involving the functional properties of EBP which
may elucidate its actual localization. Two studies that investigated the protein structure
suggested that EBP acts as a drug pump since it is similar to P-glycoprotein (Pawagi et
al., 1994; Hanner et al., 1995). P-glycoprotein is a multi-drug resistant protein in the
plasma membrane that transports various substrates into specific cells (Pawagi et al.,
1994). P-glycoprotein is in the blood-brain barrier, the blood-testis barrier, adrenal
glands, hepatocytes and intestinal epithelium cells. The aromatic amino acid residues of
P-glycoprotein, which are known to function as drug transporters, are highly conserved in
the transmembrane portion of EBP (Pawagi et al., 1994; Hanner et al., 1995;). A
colocalization attempt was conducted by Dussossoy et al. in 1999 using antibodies for the
N-Terminus of the EBP and for the Sigma 1 Receptor (SR-BP-1). Both the EBP and SRBP-1 are targets of SR31747A: a sigma receptor ligand. Their results revealed that EBP
colocalized with the SR-BP-1, and that both are associated with the ER as well as the
inner and outer nuclear membranes. Such findings support the work of Gerace, L. and
Foisner, R. (1997), along with the work of Pathak et al. (1986). These investigators
37 demonstrated that the nuclear envelope is a sub-compartment of the ER, and that the
membranes of these organelles carry the same transmembrane proteins. Unexpectedly,
the report noted that the biosynthesis of cholesterol occurs in the ER compartment
(Krisans, 1996).
Sterol isomerase is one of the latter enzymes required for producing cholesterol in
the steroid biosynthesis pathway (Krisans, S.K., 1996). More specifically, the enzyme
produces lathosterol: the precursor for cholesterol (Moebius et al., 1994). This enzyme
serves as a “pluripotent” molecule for construction steroid hormones. Several mutations
have been found in the EBP transmembrane domains of patients that present
heterozygous mutations for CDPX2. These changes include missense, nonsense,
frameshift, insertions and deletions mutations and are summarized in Table 1(Braverman
et al., 1999). The mutation E80K, has further elucidated the importance of E80, which
has been found crucial for the catalytic activity and inhibitor binding of EBP (Moebius et
al., 1999). Such findings as described above emphasize the importance for the proper
function of EBP through its transmembrane domains and cytoplasmic linkers.
There have been considerable findings associating pharmaceutical drugs and their
effects to the EBP binding domains. In one study, the findings reported a shared binding
domain within the EBP carrying the binding site for the already known emopamil ligand,
the breast cancer drug tamoxifen, and for the anti-fungal drug tridemorph (Moebius et al.,
1999). Their analyses were reaffirmed by showing decreased sterol isomerase activity by
208-fold in rats with a 230 amino acid residue and an 87% identity to their human
counterparts (Cho et al., 1998). Furthermore, EBP and tamoxifen were found to behave
non-competitively, and both were shown to have more then one inhibition site, as
38 tamoxifen was shown to affect expression of EBP (Cho et al., 1998 and Bae et al., 2001).
Along with the inhibition of sterol isomerase activity, the incorporation of mevalonate
(see above for the cholesterol biosynthesis pathway) into cholesterol was shown to
degenerate. Through these findings, the scientific community can better understand the
pharmaceutical mechanisms that interfere with the mechanisms behind the cholesterol
biosynthesis pathway. As a result, genetic diseases may be better treated.
Misdiagnosing CDPX2 with CHILD Syndrome
X-linked dominant Chondrodysplasia Punctata may be mistakenly diagnosed as
CHILD syndrome by biochemical analysis as well as through DNA sequencing. CDPX2
results from an insufficient level of cholesterol or accumulation of toxic byproducts in
cells due to a faulty EBP in the steroid biosynthetic pathways. Unfortunately, similar
inherited disorders also result in improper levels of cholesterol, which represents
difficulty in diagnosing CDPX2 phenotypically. CDPX2 maybe confused with
Congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD),
which has been reported in less than 100 individuals. CHILD has been diagnosed primary
in females like CDPX2, and as CDPX2, it has been found to be lethal for hemizygous
males (Happle 1995, Sutphen 1995, Has 2000). The two disorders affect the production
of two distinct cholesterogenic enzymes and are inherited in a X-linked dominant
manner. Both also present skin and skeletal abnormalities with the onset at birth (Caldas
and Herman 2003; Herman and Caldas 2003; Konig et al., 2000; Happle et al., 1980).
These skin abnormalities best known as the lines of Blaschko are present in both
disorders; the lines disappear in CDPX2 whereas in CHILD syndrome the lines remain
throughout the lifespan. Other characteristic differences between the lines of Blaschko in
39 these two disorders include their physical appearances. For CDPX2, the lines tend to be
V-shaped over the spine while forming an S-shape on both the anterior and lateral portion
of the trunk (Haber et al., 1952; Allansmith and Senz 1960; Pfister et al., 1969; Thiel et
al., 1969; Edidin et al., 1977; Paltzik et al.,1982; Happle et al., 1985). Furthermore, the
lines in CDPX2, which vary between affected individuals, may appear as whorls on the
abdomen whereas on the arms they are often perpendicular lines(Hopkins and Machacek
1941; Gruneberg et al., 1955; Lenz et al., 1961; Gordon and Gordon 1970; Iancu et al.,
1975; Happle et al., 1985). The lines of Blaschko for CDPX2 are asymmetrically and
bilateral, whereas they are symmetrical and unilateral in CHILD syndrome (Happle et al.,
1977, 1980, 1985; Mueller et al., 1985; Happle et al., 1987, 1993). Based on numerous
cases of both disorders and having found anomalies in two distinctive genes of the Xchromosome, it is concluded that X-inactivation plays a role in the phenotypic variation
of the lines of Blaschko.
X-inactivation, Mosaicism and CDPX2
X-inactivation is the process for which a definitive set of genes on the X
chromosomes are expressed and not duplicated in females due to the inactivation of one
of their X chromosomes. This was first hypothesized by Mary Lyon in her 1961
investigation “Gene action in the X-chromosome of the mouse” (Mus musculus L.). Her
work was based upon numerous previous studies from other leading investigators who
studied both mice and human disorders. Following Lyons’ hypothesis other investigators
such as Liane Russell, Jean Bangham, Ernest Beutler and Susumo Ohno had similar
findings and thus showed that Lyonization was responsible for the expression of one and
40 not two X chromosomes in females (Russell and Bangham 1961; Beutler et al., 1962;
Ohno 1967).
Since EBP was localized to the X chromosome by Hanner, it has been proposed
that it is subject to X-inactivation (Schindelhauer et al., 1996). This suggestion was later
confirmed through several molecular studies of EBP and females affected with CDPX2
(Braveman et al., 1999; Derry et al., 1999; Ikegawa et al., 2000; Traupe and Has 2000).
Moreover, Braverman and Herman demonstrated the difficulty of showing a clear
relationship between the genotype-phenotype in females affected with CDPX2 is due to
X-inactivation being random. This was also demonstrated by Ikewaga, described in his
2000 paper, in which the EBP sequence differed among three CDPX2 affected females
and their unaffected mothers through peripheral blood leukocyte analysis. Ikegawa
concluded that both the pattern and the timing of X-inactivation affected the severity of
the clinical phenotypes as much as or more than the mutation(s) in the gene. Random Xinactivation accounts for both the variety of phenotype manifestations in intra and
interfamilial cases of CDPX2, and also explains why the disorder is lethal in males
(Metzenberg et al., 1999; Aughton et al., 2003). Mosaicism is the phenomenon in which
the cells of an individual vary by having different genotypes. Random X-inactivation is
analogous to variable genotypes, because the expression of a gene is dependent upon its
activity state rather than on its presence in the cell. The lines of Blaschko are revealed
when there are two populations of cells resulting in a mosaic pattern due to random Xinactivation. One population of cells bear the active abnormal X chromosome that affects
the appearance of skin, whereas the second population of cells bear the active normal X
chromosome (Lenz 1975; Wettke-Schfifer and Kantner 1983). The phenotypes that result
41 from random X-inactivation may be mild or severe, depending upon the proportion of
wild type or mutant alleles that are expressed in each cell. If a mother is a gonadal
mosaic, her sons may be either wholly affected or wholly unaffected, and her
heterozygous daughters will range from being unaffected to being severely affected,
depending upon the degree of random X-inactivation. (Maurer et al., 2008). An example
of gonadal mosaicism was provided by a family with two CDPX2 affected females from
two unaffected parents. In this case, the disorder appeared to by sporadic, but because of
the presence of gonadal mosaicism, the risk of reoccurrence was higher than expected.
(Has et al., 2000). Gonadal mosaicism was further proposed by Haas in a second family
whose females were affected by CDPX2, yet had a grandmother with no detectable
mutation. This may best be interpreted as the grandmother having gonadal mosaicism,
and her daughters not being mosaic. In a recent study describing gonadal mosaicism, a
novel heterozygous missense EBP mutation was seen in an affected female, while no
mutation was found in gDNA from either parent (Morice-Picard et al., 2011). A mutation
was detected in the mother’s skin lesions, which is consistent with gonadal mosaicism. In
contrast to gonadal mosaicism, Metzenberg and Aughton independently demonstrated
two CDPX2 affected males with heterozygous EBP mutations and normal karyotypes as
being somatic mosaic. Although it is not always possible to explain inheritance patterns,
the mechanism of developmental defects of mutations in the CDPX2 gene is not yet
known.
Determination of the genotype-phenotype correlation in mutations of the CDPX2
gene is made difficult because of the confusing overlap in the types of skin lesions
presented. The pigment patterns observed in CDPX2 and those of Incontiuentia Pigmenti
42 (IP) are unquestionably similar (Schneidman and Snyder 1958; Lenz et al., 1961; Curth
and Warburton 1965; Wettke-Schfifer and Kantner 1983). Moreover, cutaneous patterns
seen in ichthyosis and follicular atrophoderma resemble those of lines of Blascho in both
CDPX2 and CHILD syndrome (Happle et al., 1979; Elias et al., 2008). EBP (CDPX2)
and NSDHL (CHILD), lie on the X chromosome and encode cholesterogenic enzymes,
and so CDPX2 and CHILD syndromes have been frequently confused.
Subject of Study Medical History
The DNA sample used in this study was derived from a 36-year old female
diagnosed with CDPX2 at birth based on radiological analysis. The diagnosis was
confirmed at the age of 2 by Doctor Pierre Maroteaux, a geneticist. The consultand was
delivered by Cesarian because at the time of delivery, after full gestation, her mother
discharged a greenish fluid, thought to represent meconium and fetal distress (Dr.
Carlo Neidhardt, Pediatrician). From birth, as described by her pediatrician, the subject
presented with features characteristic of CDPX2 such as epiphyseal stippling, skeletal
asymmetry in which her right leg is shorter than her left, right hip dysplasia, scoliosis, flat
nasal bridge, alopecia, and congenital cataract on her right eye. She was also described
with hearing loss in her right ear, nystagmus on both eyes and greasy yellowish scales.
The scales were found to reflect ichthyosis, a condition of the skin, with small scabs,
which bled to the touch.
Our subject’s condition progressed throughout her life that she became dependent
on assistive mobility devices. At age 2, our subject began using assistive mobility
equipment such as crutches and a light corset-brace due to her scoliosis. At the age of 5,
she began walking for long distances independently and climbing stairs, despite the
43 imbalance resulting from spasticity in her left leg and the difference in limb lengths.
Unfortunately, due to her imbalance in walking, her scoliosis and lordosis were
increasing. At 6 years old she began to wear a Milwaukee corset-brace, when she wore
until the age of 17, when she went through a successful spinal surgery. At the age of 21
she under went femur lengthening surgery after she began suffering from acute pain in
her right heel and an increase spasticity in her left leg as well as the start of spasticity on
her right leg. These difficulties prevented her from placing her legs in yoga positions and
from being able to raise her right leg as she lay on her side. Problems also became
apparent with her right foot pronating inwardly, experiencing difficulty urinating and
having spontaneous bowel movements. Personal care such as bathing and getting dressed
became difficult as her condition progressed. The inability to bend due to stiffness
resulted from the pain arising from such attempts. Two months after her femurlengthening surgery, the medical team stopped the lengthening process. Soon after, she
showed improvements by managing personal care activities as her fixator (a device
attached directly to a person’s bone which provides stabilization of a body part;
medicadictionary.com) was removed and lead her from using a walker to using crutches.
X-rays from 2009 revealed severe arthrosis in her right hip showing almost complete loss
of joint cleft subchondral sclerosis, and peripheral osteophytes. Moreover, her X-rays
demonstrated subchondral cyst with a shortened femur and femoral neck, a dysplastic hip
and right knee, as well as some mild degeneration at multiple levels of her lumbar facet
joints. At present, she continues to experience pain in her right heel, right hip, right knee,
along her right tight as she describes as muscle and tendon tightening while sensing warm
palpitations on her right leg and is on anti-inflammatory medication. Due to this pain and
44 weakness, she is unable to come to a standing position from the ground. At present, she
can only walk for short distances, cannot climb stairs, depends on crutches, and a Patella
Stabilizers brace for assistive walking.
Purpose of the Study
The objective of this research was to identify previously reported mutation(s) or a
novel mutation in the EBP gene of a female diagnosed with CDPX2. A DNA sample
from the effected individual described and one from each of her parents was sent to our
laboratory to be analyzed for mutations in the four translated exons of the EBP. The
identification of any mutation in these four exons would have assisted her doctor with the
diagnosis, her family, and medical care providers in treating her disability. Genetic
counselors would have been able to assess her condition and assist her as well as her
family with prenatal diagnosis. A correlation between genotype-phenotype would be
further understood by comparison with other individuals with the same mutations.
45 CHAPTER 2: Methods The sample under investigation for this thesis arrived in a vial containing 89.1ng/uL in a 100uL of genomic DNA previously isolated in Italy. The gDNA sample was diluted to a working concentration of 20.0ng/µL necessary for Polymerase Chain Reaction. The gDNA and the diluted sample were stored at -­‐20°C. Amplification of the Exons
The four coding exons (2, 3, 4 and 5) of the EBP gene were amplified by PCR using a Perkin Elmer 2400 ThermalCycler. A total of six PCR primers (Table 1) ~50 nucleotides each in length were designed. Each set of primers (forward and reverse) were selected 50-­‐130 nucleotides upstream or downstream of its corresponding exon. Due to the close proximity of exon 3 and 4, they were amplified together in a single amplicon. The forward primer was selected ~50 nucleotides upstream of exon 3 whereas the reverse primer was selected ~50 nucleotides downstream of exon 4. All six primers were design with 20-­‐25 nucleotides of a high efficient vector, pGEM3(+)Z (Table 1), to maximize sequencing results. The oligoneucleotide primers (XXIDT, Skokie, Illinois) arrived desiccated and were solubilized in deionized water (diH20) to obtain a stock concentration of 100 pmol/uL. To avoid multiple thawing, a working concentration of 5pmol/uL was obtained by pipetting 1.0uL of the stock concentration into 19.0uL of diH20. Both the stock and working primers’ concentrations were frozen either at -­‐70°C or -­‐20°C, respectively. The genomic DNA sequence of the EBP gene is shown in Figure 8. 46 PRIMER
SEQUENCE (5’à3’)
Tm
CD2F CGAATTCGAGCTCGGTACCCGGGGATCCGGTCCATTTACATTTCTCAT
52°C CD2R CAGGTCGACTCTAGAGGATCCCTGCCCAGCAAATCCCAT
52°C CD34F TAATACGACTCACTATAGGGCGAATTCTTATGAAGTCTGTCTTCTT 52°C CD34R TTGCATGCCTGCAGGTCGACTCTAGAGGATCCTTTGTCATTCAGTCTGACTTA 52°C CD5F TAATACGACTCACTATAGGGCGAATTCATTTCAGAATACTTAGCTCT 52°C CD5R TTGCATGCCTGCAGGTCGACTCTAGAGGATCCTCTGGAATAAGTGAATGAT 52°C Table 1. Sequence of the primers used in the PCR protocol for the coding exons of the EBP gene. The
melting point temperatures are included on the side. The lowest melting point temperature between the two
primers (the forward primer and the reverse primer) used to amplify each exon
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
ggtgggaggc
ttctgtttgc
attactataa
acctccccca
ataggaggct
cctggaaccc
acagtgagta
gagccttaaa
tgtttgatgt
gtggttaacc
gctgaggcct
ctttcccata
caaaatatgt
gcctacaatc
aagaccagcc
aggcgtgata
ttgaacccag
agagcgagac
tttacttgtg
attttttttt
acggcccact
gtagctggga
tagagacagg
cccttgcctc
tttaaatata
aatggtgcga
tcagcctcct
cttttagtag
ggtgatccac
cggccaaaaa
actttaaaat
ccagatttta
cttgcagaat
atgggccctc
aataagaagt
agccctgact
acccaggatg
ccctgaacca
actgtgccct
ggtccagttg
cttgctcttt
cgatgtggat
aagggtggcg
gagctcctca
gtccctctgc
ttatttattt
ccagcacttt
tggccaacct
gcatgcacct
gaggtggagg
tctctcaaaa
ttaattattt
tttttttgag
gcagcctcac
ctacaggtgc
gtctccctat
aacctcctaa
tatatatatt
tctcagctca
gagtagctgg
agatgggatt
ccgcctcagc
aatatatatt
ttttcgttaa
ctccttttta
ccaggttttt
tagatcatgg
acagacaact
caaagccctg
ccctcactgt
tctcttccct
gtgtgtcccc
ccaagtatat
ggagctctgg
gacaccacag
ctatctggcg
gactgtgctg
ttgagtcaca
tacttataaa
gggaggccaa
ggcgaaaccc
gtaattccag
tcacagtgag
aaaaaaatca
cacttcatta
acaaggtctg
attcctgggc
atgacactaa
gttgcccagg
agtgctagga
tttagacgga
ctgcaacctc
gattacaggc
tcgtcatgtt
ctcccaaagt
ttattttact
aatgtatgca
aattaagaac
gcttctcttt
47 tggggatttg
tcatacttgt
ccactcttcc
gaacagtacc
atctcagggg
tgggctcccc
ccatctgtcc
ccatcttcca
aggagcaggt
gggggaaacc
cttcgcctgg
ctgttaggtc
ataactttta
ggtgggcaga
cgtctctacc
ctactcggga
cccatgccac
ctttatagag
ttgtttaggt
tcacccaggc
tcaagccatc
gcccggctaa
ctggtcttga
taacaggcat
gtcttgctct
cgcctcctgg
acgcaccacc
ggccaggctg
gttgggatta
taggttgagt
ctatttttgt
aatttaaaaa
tagttaggtt
gttttgttgt
ctcccaccca
tcctctgcct
ctcatcttgg
gtaccctggg
cgtgcaggag
cccacagggc
cctggcctac
gaggtggggc
atgaggacaa
gcagccagtg
ttttatttgg
ggctgggcat
tcacttgagg
aaaaatataa
ggctaaggca
tgcactccag
atggagtgga
agaatttgca
taaagtgcag
cttctgcctc
tgttttttta
actcctaggc
gagcaccctg
tgttgcccag
gttcaagcaa
atgctgggct
gtcttgaact
caggcgtgag
gtacaaaccc
tcatgttgag
taacatttat
tcagtcaaaa
gtgtttttca
actcccgttc
ggagactcgc
atgcctctgc
catcctcagt
gaggtgggac
ctgggggtgc
ctgggctccc
cctgggctgt
aggctgggaa
ctcctcggtc
gtgaagtgtg
gctggctcac
tcaggagttc
aaaattagcc
ggagaatcac
cctgggcaac
aaatccaggt
agacttctta
tggtgtgatc
aacctcctga
attttttttg
tcaagcaatc
ttttatttaa
gctggagtgc
ttctcctgcc
aatttttgta
ccaggcctca
ccaccacgcc
tggattctgc
tatgcaaata
tcatatggct
acaaaacttt
1981
2041
2101
2161
2221
2281
2341
2401
2461
2521
2581
2641
2701
2761
2821
2881
2941
3001
3061
3121
3181
3241
3301
3361
3421
3481
3541
3601
3661
3721
3781
3841
3901
3961
4021
4081
4141
4201
4261
4321
4381
4441
4501
4561
4621
4681
4741
4801
4861
4921
4981
5041
5101
5161
5221
5281
5341
gtgttttttg
ttttcttttt
acaatcatag
tcccaagtag
ataaagtttt
ttcaaaaacc
cctaccctct
caaactcctg
cgtgagccac
agcatatcaa
aaagataaca
agtgtcctca
tgtggacatc
tgtacccaag
tggagtagta
gtacagagtg
ccgggcgggg
acttgaggtc
aagtacaaaa
ctgaggcagg
cactgcactc
tgaggaagat
cctttactgg
ttaggagctc
aaaatgattt
gcccttctgc
tttcctaaga
gctacggcat
tcacttttgg
ataaccctct
tctactcctt
acacacaaaa
ggggtggtgc
agggcctccc
aggaaatatt
gggagcacag
attggttcat
gtcctgcttg
attggctgga
agagaaagca
gttctaattg
ggcccactgg
tcttcatgcc
tgaggtcggg
agagctgggg
gcattatact
gctaatttca
tgataaggga
cgcccactaa
gccacgcgcg
gggccgcctt
atgagcccga
agcgtgggag
cctcccccag
gccacccgcc
tgtccagagg
ggattgaccg
atattaaaaa
tttttttgag
ctcactgcag
ctgggactag
tgttttgttt
aaacagtatt
tacaggttac
acctcaggta
cgcactgagc
acaaatataa
catgaatgat
ttcatttttt
aggtaatttc
tggatccaca
cttttgtgct
cctctcagac
tggctcatgc
aggagttcga
attagctggg
agaatctctt
aagcctgggt
tattaatgag
aatgacctat
tttatatatt
ttaaagaaga
ggggcctcgc
acttctttgt
ggcatacact
atgctggatc
taagacccct
gaccccctcc
tcacaccatt
ctgctggggc
cgccttcctt
caataggctt
catggataaa
ggttagttta
gtttgatcag
aaactgaagg
agagagatga
gatgggaggt
tggttaggcg
aattggtcag
agaagagaga
agggggtggt
aagggttact
ccagcggaag
atggggaaat
ccttggcgcg
ggggcggggc
cttcgcttca
actagggatg
gccgctgccg
ctctccccgg
ccccccaccg
acagaagatc
gctttgtgtt
gtttttaatt
acacggtctc
cctcaaactc
agatgcatgc
tcattaaaat
caagatattt
atggtgagac
atccactcac
ccacttttat
aaatatattt
actgttctgc
ccactatgag
tagtcctttc
caggtgtgga
ctcccaccag
ttcctggata
ctgtaatccc
gaccagcctg
catggtggtg
gaacctggga
gacagaatga
gctgagcatc
tcaccttctc
agggagataa
agacaaaaac
ctagagcagt
ccttgctaag
gtccacaagt
ttctaccgtc
ctcagggtgc
atccttgacc
ttcatgcctg
ctagaggggg
cttccttttt
cttcttcact
ggtttggaag
tgattgatta
ggcagggaca
atccccattc
agggttggtg
ctgggatggg
tttgattggc
taggactgag
atgaccgctg
tgcagtgtat
ctacctagaa
aagcgggtac
tgaagccagg
tcccagtctc
tagaggtaca
ccattggctc
tgacagagcg
tcgcgcgcct
ctacgcggcc
cccttttgcg
cgtcttctca
ccgttctagc
48 caggttaagt
actctgttac
ccaggctcga
caccacacct
attttaaaat
agagaaaaga
ggggtttcac
ctcagcctcc
ccttttatta
taattctacc
aatttttgaa
gaagtgacac
ctattgcaag
cagatctgta
caatgcctga
tgtttgccac
agcactttgg
accaacatgg
cagcatctgt
ggtagaggtt
gagcctgtct
ttttcatatg
tggcaatttt
caggtcagag
tatatctggt
cacagaaatg
tgggaccatc
ggtcagagct
tcataggctg
cactgatggg
cccaacacct
tcaatcccca
atgcttgggg
atatacaatt
gctcagaggg
ctggaaacca
gatgatgagc
gattgcatag
tgattggttg
cctatttatc
tctggattct
tttttagaac
aggcctgggt
ctattaggac
agcgtgtgat
aagggcggag
agaccttaat
actcgatgca
caccccaaac
aagagagtgt
gctccgtaag
cgagacccag
tggtgagtgc
agccctcggc
cctgcgcgag
ttgggcagcg
gctgcacgcc
gtgcaaaatc
ccaggctaaa
gcagtccacc
ggctttttgc
agtgaatgcc
tttgctccca
catgttagcc
caaagtgctg
aaatatcttt
cccttgccag
aatcttccac
agtttatttt
tgatgttgca
ggatctgttg
atgtgcctgc
ttcaataaag
gacgcccagg
tgaaacccca
aatcccagct
gcagtgagct
caaaaagaaa
tttaagagcc
tctttccaaa
aatgggatca
cacctttcat
tccttgggca
acctctttct
cagctgggcc
aggcacatct
ggatgaggga
caacacacac
ccccaccaca
aaacagagaa
tgttattgtc
gaataggaat
tgaagagaga
ctgaagatgg
ttggatgtgg
ctctgagtgg
cttaggactg
gattgattgc
tggcatatgg
tctaactagt
tgaggagttc
ctgcttgtaa
gtgaaaggat
gagtccccgt
gattggctac
ctaactcagg
ctattggttg
gcaagagaac
cctaaagaga
cctccacccg
gtgccagcgc
acccccagct
ggactggagg
agacaccggc
tgggatttgc
gtgcagtggc
cacctcagcc
ttctttttaa
tatcatgtgc
ccctctgccc
aggctggtct
agattgcagg
ccaaaattta
tttttaccct
atcaggatat
ttctcttatt
gcaggaactc
atagtctcaa
tgccccacca
aggaagatgg
caggcagatc
tctctactaa
actcaggagg
cagatcgtgc
ataaataaaa
atttgttttt
aaattaattt
ctttgttttt
atgtacctag
aaggatgggt
ctcccacagg
agtcactggg
gtggaccctc
aggccctctc
acacacacac
aggcaggaag
ggggagatcc
aaataaaagt
gagtgaggaa
gcagactgtg
caggatcctg
gggaggcctg
gacacagagg
agggttgtgg
tccatctggg
agattgaggg
tcttctgact
tgagcactga
cagtttcgtt
gcaacgcaac
tttgatcgac
ggatccaaaa
tgaaaatggc
ggagcggcac
ccactagggg
gcccggagcc
gcccctgctc
gagacccttt
accgcacggt
gttctggttc
ctttcaatat
5401
5461
5521
5581
5641
5701
5761
5821
5881
5941
6001
6061
6121
6181
6241
6301
6361
6421
6481
6541
6601
6661
6721
6781
6841
6901
6961
7021
7081
7141
7201
7261
7321
7381
7441
7501
7561
7621
7681
7741
7801
7861
7921
7981
8041
8101
8161
8221
8281
8341
8401
8461
8521
8581
8641
8701
8761
ccgtctcttc
aaacgctggc
agttttcagg
ctgtgcaact
aggactgggt
tcctgtatct
gtgcctccca
taatatggct
tcatgttctt
aggctggagt
gattctcctg
ctaatttttg
ctcctgagct
agccaccgcg
ggtcactgct
acctactagc
agagacagtt
ctgcatcagc
tcttaaaaca
aggctgaggc
aaccccgtct
cccagctact
gtgagccgag
aaaaaaaaaa
attcggtcca
ttctgtactt
ctgcctatac
cacccatact
tggcatatac
ttgtcaggtc
tttgcagtgt
gacctgcttg
gctgtgggat
ttcttccatt
ggcacagtgg
tgaggtcagg
tacaaaaatt
ggcaggcaga
tctctactaa
ctagggaggc
agaccatgcc
ttattatgta
aacttatttt
attgctcact
gtagctggga
gtagagacaa
ccttccacct
tatttaactt
gcagtgaaaa
agttgtttat
aaaggaggat
cagtggttgc
acaaatggta
agaaaaccaa
gagtgcagtg
ctcctgcctc
ttttttttta
cctgcaggtt
accgagggtt
actacgtggg
ggaccacgcc
ttggtggtcg
ccctcgccag
ccagactttg
tctaaagggc
tttcttttct
gcagtggcat
cctcagcctc
tatttttagt
caagtggtcc
cccggccttc
cacttcagcc
tgggactaca
tcgctgtgtt
ctcccaaagc
gaatgtaggc
gggcggatcg
ctactaaaaa
cgggaggctg
gtcgcaccac
aaaaacggaa
tttacatttc
tctatttgtc
acacgcagcc
ggcctcagca
tggctggcct
gtgctgcggt
gtgggttcat
gagaccaagc
gggatttgct
gatttatttt
ctcacgcttg
aatttgagac
agccaggcgc
tcacgaggtc
aaatacaaaa
tgaggcagga
attgcactcc
ttgttattat
tgagacaagg
gcaacctcca
ctatactata
ggtcttgcta
cagcctccca
ttttgttgca
tctgctttct
gtttcttttc
aatagtaata
catgtaactg
atatgctata
agaagccatt
gcatgattat
agcctcccta
acaacttttt
gacggcgttg
gtagttctga
ggagggaata
tggttcctgt
tgagcagttt
actgtgacca
acccccgagg
tggaacaagc
tttttatttt
gaccccggct
ctgagtagct
agagacgggg
actcgtcttg
ttttttagag
ttgaagtcct
ggcctttgtc
gcccaggccg
actggaatta
cgggcgcagt
tgaggtcagg
tacaaaaaat
aggcaggaga
tccactccag
tgtaattaga
tcatgataat
caggtttttc
atcagcccac
cctaagactg
cttctctgtc
tgtcccattg
tcacctggtg
cttcttatct
gggcagggat
aaatttttat
taatcccagc
cagcctggtc
ggtggctcaa
gggagttgaa
attagccggg
gaattgcttg
agcctgggca
tattcttttt
tcttgctctg
actcctgggt
ggtgcacacc
tgttgctcag
aaattctggg
tgtatattca
atccactaag
agtttattta
accaactata
ttcactatta
tatacttcta
ttcttttttg
agctcactgc
gtagctggga
tttttaagag
49 cacgctctcg
ttcgttttct
gctttttgtt
gtctcttacc
gtgacccttg
gggagggctg
gctgagaccg
tttcagcctt
attttgagac
cactgcagcc
gtaactacag
ttttgccatg
gcctcccaaa
gcagagtctc
gggctcaaag
accacacctg
gacttgaatt
cctgccacgg
ggctcacgcc
agatcaagac
tagctgggcg
atcgtttgaa
cctgggtgac
agtgttacaa
aaactatttg
tgttcctttt
aaagacatga
gacaactttg
acaggggtct
gggacttggc
atcgagggct
caactctgtg
cggcttgcat
ttaatctttt
actttgggag
aacatggcaa
gcctgtaatc
gaccagccag
tgtggtggca
aacctgggag
acagagtgag
gttttttgcc
ttgcccaggc
tcaagcgatc
tttatgcctg
gctgttcttt
atgacaggtg
cgtgggtcaa
tttccccaaa
cacacgtaag
tgtaagatac
ttatcattat
atcttttctc
agacggggtc
aaccttgaac
ttataggcat
atggcgtctt
cggggaggct
ttcctctgtc
gaacggttta
aaaccgtgac
aaggaagaca
ggatcaggtg
ggactgtttc
tctgtggcct
agagtctcgc
tctgcctccc
gcgcacgcca
ttggccgggc
gtgctgggat
agactggagg
cgatcctcct
actgattttc
cctggcctca
tgcctggcct
tgtaatccca
catcctggct
tggtggcagg
cccggaaggc
agagcaagac
tcctgtctgt
aatttgattt
tttttttttt
ctaccaacgc
tacctaatga
tagtcgtgac
ggcgactgtc
ggttcgttct
agtcctgatt
gtttacctat
aaaaaattta
gccgaggcag
aaccccatct
ccagcacttt
gccaagatga
cacgcctgta
gcggaggttg
actttgtctc
cttctctaaa
tggagtgcag
gtcttgcctc
gctaatattt
aactcctggg
cgcaccaccg
aaaaccattt
cgctgttagg
tctcaatttg
ttagaacagt
ttgcactttt
tttttctttc
tcgctctgtc
tcctgggcaa
gtgccacccc
gctttgttgc
ctggctttcc
ccccagttgc
agacgctgac
cccggagcac
ccaggcctgg
ttgttcccat
taccacatca
tgtgtttcag
tctgttgccc
aggttcaagc
ccacacctgg
tggtcttgaa
tacaggcgtg
gcagtgatgt
gcctcagcct
ttattttttt
ggtgctgctc
ttagtcatgg
gcactttggg
aacacggtga
cacctgtagt
agaggttgca
tccgtctgaa
taactggtaa
tattatctca
ttttaacttc
gggccccttg
ccgccccacc
cacatggctg
cctgtgctgg
ctactacgaa
tctttcatat
ccacctattc
ttataggccg
gtggatcact
ctactaaaaa
gggaggccga
tgaaacccca
atcccagcta
cagtgagcca
aaaaaaaaaa
actggtagat
tggtatgatg
agcctcccca
tcactttttt
ctcaagcaat
tgcccagcct
aaagagattt
cactgttgat
ctcaattgta
gtttggcaaa
ccctttttac
tttccttaca
acccaggcta
tcaagcaatc
accaagttat
ccaggctgtt
8821
8881
8941
9001
9061
9121
9181
9241
9301
9361
9421
9481
9541
9601
9661
9721
9781
9841
9901
9961
10021
10081
10141
10201
10261
10321
10381
10441
10501
10561
10621
10681
10741
10801
10861
10921
10981
11041
11101
11161
11221
11281
11341
11401
11461
11521
11581
11641
11701
11761
11821
11881
11941
12001
12061
12121
12181
ctccaactcc
agcatgagcc
agagctttca
ttccctcaat
cagcactttg
ggccaacata
aggtgcctgt
ggcggaggtt
caaggctccg
gcccggctgg
attatggttt
cttattggcc
atattggatg
cctgttgtcc
ggctcaagac
aatttttgta
tcctttatgc
gccatcgtgc
ctgttcacct
gcttcctttt
aggctttaca
ccatttcacg
gacccccagc
ttcttttctt
gcctctgtca
gggctaacct
ggtgacaact
ctgtgggtgg
gtctctgtgg
gatccacaga
taagtcagac
ctgcactctc
tctcctgtga
atcagggctc
ggctctgtca
cccagactca
ccaccgtgcc
ggctggtctc
gattacaggt
tttctttagt
ttgaggattt
cagctctctg
tcagaatact
ttggcgaaag
cagatctatg
gagctgggcc
gtgctgcctg
ctggatgcca
cgaacactgg
tttggaggga
ggaataaagg
gccaggaggt
acactgagag
caccatcatt
cacaaacccc
ctggccccac
cttaaaccac
tggcttcaag
accgcaccca
aagcatggac
cttctgccat
tgaggcgaag
gtgaaacccc
aatcccagct
gtggtgagct
tctcaaaaaa
tcagatgttt
taatttgctt
atttagaaat
atctttttct
aggctgagca
atcctcccac
tgttttatag
aagccatcat
ccggcggtct
acattctctc
ataccacaca
catattacct
gatgaggaag
tctcacaagt
cagggaaaga
atggagactg
gtaggaagag
tcacagtgtg
tgatcgcctt
gtaaggaaag
cacagatgta
tgaatgacaa
aggtggggag
aggttacatg
tgagatccct
cccaggccag
agcgatcttc
tggctaactt
aagctcctga
gtgagccact
tctagcgata
tgaacttcct
gtgtctggaa
tagctctgag
tgtccccttc
gggatgtgct
accctctcta
gagtccttgt
aggccacaaa
ccgaggagga
caaagctaat
ggctgtgtga
ctatgatggt
atccttaatt
cacttattcc
tatcactgac
agtcccctgg
cgaagttcat
tgatctgccc
gctgcttttt
attgtacagt
tataaacaag
gcgggcagat
gtctctacta
actcaggagg
gagattgcgc
aaaaaaaaaa
cttaaatttt
ttccttagtg
cctctttgtg
ttctttcttt
ctgtggtgct
tgcagcctgg
agttgggggt
cccacctcag
ttttctttct
tctctcattg
cacttcttga
tgttttagag
cagacgcatg
gtgtgttcct
gtatgccaag
gcattggttt
cacaccgata
catggaaacc
tctccgccag
ggcactagag
tccctgtggg
accccctgag
ggttcatttt
aggtggccca
tcatgtaaga
agtgctatgg
ctgcctcagt
ttgtattttt
gctcaaacag
gtgcccggcc
tgagttatct
taaatctgaa
tctcactttt
accttgaatg
ctcactgggg
ctacttcctg
cttctggttt
gcttgatgct
agccaagagc
gctctctgcc
tgatctgtca
aggcactgct
gacgattttt
actaacccca
agacccccat
cccccgccag
tcttaactcg
tttctcacag
50 acctcagcct
ttaatgaggt
tgcttgactt
gccgggcaga
cacctgaggt
aaagtctaaa
ctgaggcagg
cactgcacac
aaaaaaaaca
agtcattcca
actaatggaa
aagaacctat
tttttttttt
atcatagctc
gactgtaggc
ctcactatat
cctcccaaag
gattctctgc
gctacttcac
gtaccaatca
aaataaacca
ggaaggttat
ttcactgcct
ggagacagcc
tcggggggtg
ccagtgtccc
atcacagctt
catcccctcc
gggcactggg
tgggatctct
gctctggaaa
tcttcctcct
gggtggcaag
ttctgtcata
tgtgatctcg
ctcctgagta
agtagagatg
tcctcctggc
tcagtcatct
ttagtcccag
ggtgtgagct
ctgatttctc
atgacttgga
cttctccttc
acagagcacc
tactttgtct
gtgaagcacc
aagaagaact
tgccagaaga
cactcaggct
gggagccatt
aaaatcagga
gagatgcgct
cagcatctaa
accccataac
agctgccata
ttctggagtc
cccaaagtgc
cactcctcat
gtgtagtatt
gtggctcatg
caggagtttg
aattagctgg
agaatctctg
tccagcctga
ttacaggcat
gtagatgtga
gtttgaacat
tcaagtcttc
tttttgagac
actgcagcct
acgtgccacc
tgttcagact
tgttgggatt
tcactcttgt
tcactccatc
cagccacact
ctagaatcca
gaagtctgtc
ttattcttca
gatacatcct
gtgagttggg
ctcatgcttt
gcctgtgggg
gcttcattct
cactagaggg
caacggtgcc
ggtcatgccc
cctccttctc
aagcccctgg
ttttttttaa
gctcactgca
gctgggacta
gggtttcgcc
ttggcctccc
tgtgttatta
aatcctaagc
ctcctgagtt
agattttcag
aagtgctttg
ccctcctgcc
gcgacggatt
tcatgaatgc
tcactcatgc
gaggagtggt
gtctagtcct
catgggcagg
agaacacaga
aataaaagat
atgactgacc
agacctccca
tcactacaca
acagaatgcc
caagatcaag
taggattaca
gatttcataa
gatgggcatt
cctgtaatcc
agaccagtct
gcgtggtggc
ggacctggga
gcgataagag
gagccattgt
gtgatatctc
ctttcatata
tgcctatttt
agggtcttac
caacctcctg
acgtccagct
ggtcttgaac
acaggcatga
attcactcat
atctacacct
gttttgtgtc
aacagttacc
ttcttgcagg
tatctctctc
gtaagtgttt
gagcactaat
ctcctgcagg
accactcagc
acagcttgtg
gttgatgggg
cttccagggg
ttctctgagc
catcacaaag
aacctccagg
aacagggtct
acctccacct
caggcatgtg
atgttgccca
gaagtactgg
aattgttagc
tgtcttggat
ctgaaattct
ctctgtgatt
gaatagagaa
acccacaggc
ccagcacgga
cctgtggctg
ccagagcacg
ggaccaggct
gctcccacag
cacaagaagg
tacaagagaa
cttgactcta
ccacagatcc
cctctgacct
gaccctatca
acagactagg
gtgtcagcag
12241
12301
12361
12421
12481
12541
12601
12661
12721
12781
12841
12901
12961
13021
13081
13141
13201
13261
13321
13381
13441
13501
13561
13621
13681
13741
13801
13861
13921
gtttgacttc
cacatggtct
cttttttttt
tatgatcatg
ctcctaagta
tatagcaatg
tcctcccacc
tttgttttgt
atggcacgat
cagcctcccg
tttagtagag
tgatctaccc
gcaatatttt
aaactcctag
gtgaactacc
caacatggtg
gccacggtgg
gtgagagcct
gctcatgcct
cagcctggcc
ggtggcacat
ctgggaagtt
acagagtgag
ccactctatg
gtcacattct
tcagtctata
accttcaccc
gctctgtggc
tcactatgtt
tcctgaggcc
ctatgaacta
tttttttttt
gttcactgca
gctgggacta
gactctcact
tcaaccttcc
tttttttttt
cttggctcac
ggtaggtggg
acggggtttc
gcctctgcct
ttcaattttt
gctcaagtgg
atgcctagcc
aaaccccatc
gaggatcatc
gtcttaaaaa
ataatcccag
aacatggtaa
gcctgtaatc
gaggtggcag
actctgtccc
agtcccattt
gtggtattgg
acattctgct
ccatcccagc
ccaggctgga
ggcctggctg
cctgtccgta
gcgtccctgt
gagacagggc
gcctctacct
caggtgtgca
atgttgccca
aagtaactgg
tgagacggag
cacaacctct
atcacaggca
tccatgttgg
cccaaagtgc
tgtagagatg
tcctcccacc
caaatttcct
tctacaaaaa
tgagctaggg
aaaaaaggag
gctgacacaa
aactccatct
ccagctactt
tgagccaaca
aaaaaaaaaa
taacttaacc
gggttagggc
ctctctggcc
agcccaaaac
gtgcaacggc
g
gcttgcaggt
ccctggtgtc
ctcgctctgt
ccctggctcc
ctaccacaca
ggctggtctc
gacaacaggc
tttcactctt
gcctcctggg
tgtgccacca
tcaagctggt
caggattaca
gggtctcact
tcagcctccc
tttatgagga
aaattttgaa
aggtgtaggc
gtggggggtg
gtggatcacc
ctactaaaaa
gggaggctga
tcatggcact
gtggggggca
acccttttaa
tttaacatat
tcccagaatt
tctcttttgt
acgatctcag
ggccaccttc
tctctgtgtg
gtcaggctgg
agcgatcctc
tggctatatt
aaacacctgg
acatgccatt
gttgcccagg
ttaaagcaat
tgctcagcta
cgcgaactcc
ggtgtgaacc
atgttgccca
aagtagctga
caccatttga
aattagctgg
tgcagtgagc
ggcaccagtc
tgaggtcagg
tacaaaaatt
ggcaggagaa
actgcactcc
ccatttgtat
agtccctatc
gaatttgggg
catgtccttg
ttttttggag
gtcactgcaa
tcagtgtcct
tccaaatttc
agtgcagtgg
ctgcctcagc
tatttttttt
gctcaagtga
atacctaatt
ctggagtgca
tctcctgcct
attttgtatt
cgacctcagg
accatgtccg
agctgatctc
gactacaggt
ccagcctggg
gcatcatgag
catgatcacg
aggcacagtg
agtttgagac
agccatgcat
tcacttgaac
agtctgggca
tggattaggc
tccaaatata
aggacacagt
catgcaaaat
acagggtctt
ccacagggtt
Figure 8. Genomic sequence of the emopamil-binding protein gene. Source :OMIM 300205, Locus:
NC_007452, Accession: NG_007452 . Sequences in green = exon, in blue = PCR and sequencing forward
primers, in red = PCR and sequencing reverse primers. Exons 3 and 4 were amplified as a single amplicon.
http://www.ncbi.nlm.nih.gov/nucleotide/169881268?report=genbank&log$=nucltop&blast_rank=1
&RID=PEPM517J01S For all PCR reactions, the following mix solution was prepared in a 0.2mL PCR tube; 71.5 uL of diH20, 1.0uL of 25.0 mM dNTPs, 20.0uL of 5x Phusion Buffer, 12.0uL of 5.0 pmoles forward primer, 2.0uL of 5.0pmoles reverse primer (XXIDT, Skokie, Illinois), 1.0uL of 50ng/uL gDNA, and 1.0uL of Phusion Polymerase (New England BioLabs) for a 100.0uL reaction with the addition of 30.0uL of Mineral oil to prevent evaporation. A negative control was prepared for each PCR reaction in a similar mixture as described above, however 1.0uL of diH20 was substituted for the 51 1.0uL of 50.0ng/uL gDNA. Immediately after the addition of the Mineral oil, all reactions were subjected to PCR using a thermocycler (GeneAmp PCR System 97000, Applied Bossytems, Foster City, CA). The thermocycler was programed to run 35 amplification cycles. The first 10 cycles consisted of a denaturizing temperature of 97 oC for 15 seconds, an annealing temperature of 54 oC for 15 seconds, and an extension temperature of 72 oC for 90 seconds. Immediately following the 10th cycle, 25 cycles were programed at a denaturizing temperature of 97 oC for 15 seconds and an extension temperature of 72 oC for 90 seconds. PCR samples were kept at an indefinite temperature of 4 oC before being subjected to gel electrophoresis purification. Purification of PCR Amplicons
To purify the desired PCR fragments, electrophoration was performed for all three PCR reactions. Since the size of the PCR amplicons ranged from 640-­‐866 bases, a 7.5% polyacrylamide gel was poured using standard techniques (see Appendix for protocol). The first well was loaded with 1.0uL of 0.5ug/uL Ready to Use GeneRuler 1 Kb Plus DNA Ladder (fermentas). The second, fifth and seventh wells were left empty in between samples. The third well was loaded with a mixture of 10.0uL of the control sample and 2.0uL of 6x Dye. The fourth well was loaded with the first PCR amplicon containing Exon 2 (640 bases) and 2.0uL of 6x Dye. The sixth well was loaded with a mixture of 10.0uL of the second PCR amplicon containing Exon 3 and Exon 4 (660 bases) and 2.0uL of 6x Dye. The eight well was loaded with a mixture of 10.0uL of the third PCR amplicon containing Exon 5 (860 bases) and 2.0uL of 6x Dye. A 750mL of running buffer, 1x TAE (Tris Acetate EDTA), 52 was prepared from a 1:50 dilution of a stock concentration of 50x TAE (Appendix). The gels were emersed in the 1x TAE buffer and electrophoresed for about 1.5 hours at 90 volts. The gel was stained in a 400mL water bath comprising of 200uL of 10mg/ml ethidium bromide and stored at room temperature in the dark for five minutes followed by a 2 minute submersion in deionized H2O (di H2O). The identification of the correct band was determined by the visualization of the gel under the exposure of UV using a UV transiluminator (Alpha Innotech Cell Biosciences Fluorchem HD2, San Leandro, CA Figure 4). Upon confirmation of the correct size bands, each sample band was excised using a razor blade. To ensure each sample band was properly excised, each sample band was excised at a time while the the gel remained under UV exposure. The bands were collected in their respective 1.5mL Eppendorf tubes containing 350uL of 100mM NaCl / 1mM EDTA (elution buffer) and left overnight at room temperature to allowed diffusion of the DNA out of the gel fragment. For each sample, its DNA fragment was subjected to Ethanol Precipitation in order to obtain a suitable concentration. The clear supernatant was transferred from the 1.5mL Eppendorf tube containing the excised gel into a new 1.5mL Eppendorf tube while avoiding the uptake of any gel debris. 1mL of 100% cold EtOH was added proceeded by the addition of 1uL of 20ug/uL molecular biology grade glycogen (Roche, Indianapolis, IN). After being subjected to centrifugation for 20 minutes at 14,000rpm (Eppendorf Centrifuge 5417C), the clear supernatant was discarded without disturbing the pellet. Two washing steps of 1mL of 70% EtOH with a centrifugation of 14,000rpm for 5 minutes were performed successively 53 while removing much of the EtOH as possible and not to disturb the pellet. To ensure all residual volume of EtOH were removed, all three tubes were air dried for 30 minutes. Each DNA pellet was resuspended in 20uL of diH2O. The concentration for all three samples were obtained through the NanoDrop 1,000 (ThermoScientific NanoDrop 2000C Spectrophotometer) in which it measures the optical density at 260nm and 280nm, where nucleic acids and proteins are absorbed maximally, respectively Insertion of Amplicon into pGEM3Z(+)
To achieve a highly readable chromatograph, each amplicon ranging from 640-­‐866 bases was independently inserted to pGEM3Z following Gibson method protocol. pGEM3Z(+) was chosen due to being a highly efficient vector with the lacZ alpha peptide utilize for the selection of recombinants by the screening of blue and white colonies (DNA persist versus protein). From pGEM3Z’s multiple cloning site, EcoR1 and BamH1 were selected for the vector’s 40uL (100ng/uL) one-­‐hour digestion at 37 oC. The digested vector was EtOH precipitated following same protocol as for the DNA extraction from a gel and resuspended in 6uL of diH2O. Following digestion, Gibson method was performed for each of the EtOH precipitate by transferring three microliters of the EtOH precipitate sample and two microliters of digested pGEM3Z into a new 0.2mL PCR tube already containing 15.0uL of polymerase ligase and exonuclease. All LIC samples were subjected to a one-­‐hour incubation period of 50oC to achieve pGEM3Z-­‐CDPX2Exon2, pGEM3Z-­‐
CDPX2Exon3,4, and pGEM3Z-­‐CDPX2Exon5 construct.
Transformation of Constructs
54 Transformation of home made competent XL1 blue cells with each construct was performed to obtain higher concentrations of the three constructs. Four 1.5mL falcon tubes were labeled, each corresponding to one of the constructs while the fourth falcon tube corresponded to a control plasmid, pUC19 (Life Technologies-­‐
Invitrogen Corporation, Grand Island, NY). Two tubes each containing 200uL of XL1 blue competent cells were immediately thawed and 100 microliters were transferred into each of the four tubes having previously been placed on ice. one microliter of each construct (50pg/uL) was transferred to its respective labeled tube as one microliter of pUC19 was transferred to the fourth tube labeled pUC19. All four tubes with their contents were kept on ice for 30 minutes. The tubes were subjected to heat shock by subsequently having their conical region submerged into a 42oC water bath for a period of 45 seconds. Immediately following heat shock, the tubes were placed on ice for 2 minutes. Each tube was then added 900uL of home made S.O.C. medium (Appendix) and placed on a shaker at 37 oC for one hour. A series of three LB agar plates (12 plates in total) for each tube were prepared by adding the antibiotic ampicillin marker once the LB Agar was comfortable to the touch (~50 oC). About 30mL of LB Agar was poured onto each petri dish and were left to acclimatize and solidify for 30minutes. Four of the eight plates, following sterile techniques were spread evenly with 30uL of X-­‐Gal and 30uL of IPTG. Once all 12 plates were dried, all four falcon tubes were removed from the 37 oC shaker. To observe colony forming efficiencies, 10.0uL of sample was transferred to the first plate of three series, 100.0uL of sample was transferred to the second plate of the three series and the rest of the solution was poured onto the third plate of the three 55 series. Each sample was evenly spread on the surface of the LB Agar following sterile techniques. All 12 plates were placed at 37 oC in an incubator for 16.0hours (VWR VWR Scientific Incorporated). Purification of Plasmids
For each transformant studied, 5.0mL of Luria Broth were inoculated with a single isolated colony, and left shaking at 240rpm at 37 oC grown overnight. Using Promega’s Wizard Plus SVMiniPreps DNA Purification System (Appendix), several constructs were successfully obtained at a desirable concentration for sequencing using the NanoDrop 1000(>50ng/uL). For the purified plasmid samples with lower than 50ng/uL, EtOH precipitation, as previously described, was performed to obtain 500ng at a volume of 10uL. Conformation of pGEM3Z-CDPX2-Exons constructs
To confirm each construct contain its proper insert, a 1% Agarose gel was prepared for all constructs. The constructs were electrophoresed in a single gel. These constructs ranged in size from 3383-­‐3609 bases. Into lane one was loaded 1.0uL of 0.5ng/uL Ready to use Gene Ruler 1kb plus DNA Ladder (Fermentas) Lanes two, four and six were left emptied. Lanes three, five, and seven were loaded with 10uL of 20ng/uL pGEM3Z-­‐CDPX2-­‐Exon 2, pGEM3Z-­‐CDPX2-­‐Exon3&4, pGEM3Z-­‐
CDPX2-­‐Exon5, respectively. Upon confirmation of base length for each construct, constructs were prepared at a concentration of 50ng/uL at a 10uL volume using 0.5mL PCR tubes. The 5.0pmol/uL primers for each exon were diluted to 1.0pmol/uL at 10uL total 56 volume using 0.5mL PCR tubes. Both the three constructs and primers were sent for sequencing (Retrogen inc, San Diego, CA) A.
The data obtained, both in a computer-interpreted nucleotide sequence and
a chromatogram, were analyzed through 4Peaks (4Peaks.en.softonic.com/mac. Version
1.7.1) and later through T-Coffee (T-Coffee Multiple Sequence Alignment Server;
tcoffee.vital-it.ch/). A Multiple Sequence Alignment (MSA) for a nucleotide and later a
protein alignment were performed using T-Coffee. The wild-type EBP (sterol isomerase
gene (exons 2-5 sequences) (GenBank, accession number NG_007452) was used for the
analysis of the MSAs .
57 CHAPTER 3: Results
Molecular Construction and Confirmation of three pGEM3Z(+)-EBP Exon
Constructs
Each of the coding exons of the EBP gene were successfully inserted into the
highly expressived pGEM3Z(+) vector using the Gibson method thus creating three
distinct constructs (Figure 8A-D). Since pGEM3Z(+) contains the LacZ and the
Ampicillin resistant gene, each construct was tested for its transformation efficiency
using basic recombinant DNA techniques. Blue vs white colonies were screened in the
presence of ampicillin (Data Not Shown).
9A
9B
9B
6901
6961
7021
7081
7141
7201
7261
7321
cggtcca
ttctgtactt
ctgcctatac
cacccatact
tggcatatac
ttgtcaggtc
tttgcagtgt
gacctgcttg
gctgtgggat
tttacatttc
tctatttgtc
acacgcagcc
ggcctcagca
tggctggcct
gtgctgcggt
gtgggttcat
gagaccaagc
gggatttgct
9D
9C
tcatgataat
caggtttttc
atcagcccac
cctaagactg
cttctctgtc
tgtcccattg
tcacctggtg
cttcttatct
gggcagggat
aaactatttg
tgttcctttt
aaagacatga
gacaactttg
acaggggtct
gggacttggc
atcgagggct
caactctgtg
c
58 aatttgattt
tttttttttt
ctaccaacgc
tacctaatga
tagtcgtgac
ggcgactgtc
ggttcgttct
agtcctgatt
tattatctca
ttttaacttc
gggccccttg
ccgccccacc
cacatggctg
cctgtgctgg
ctactacgaa
tctttcatat
9C
9D
10141
10201
10261
10321
10381
10441
10501
10561
10621
gacccccagc
ttcttttctt
gcctctgtca
gggctaacct
ggtgacaact
ctgtgggtgg
gtctctgtgg
gatccacaga
taagtcagac
tctcacaagt
cagggaaaga
atggagactg
gtaggaagag
tcacagtgtg
tgatcgcctt
gtaaggaaag
cacagatgta
tgaatgacaa
gtgtgttcct
gtatgccaag
gcattggttt
cacaccgata
catggaaacc
tctccgccag
ggcactagag
tccctgtggg
a
11341
11401
11461
11521
11581
11641
11701
11761
11821
11881
11941
12001
tcagaatact
ttggcgaaag
cagatctatg
gagctgggcc
gtgctgcctg
ctggatgcca
cgaacactgg
tttggaggga
ggaataaagg
gccaggaggt
acactgagag
caccatcatt
tagctctgag
tgtccccttc
gggatgtgct
accctctcta
gagtccttgt
aggccacaaa
ccgaggagga
caaagctaat
ggctgtgtga
ctatgatggt
atccttaatt
cacttattcc
accttgaatg
ctcactgggg
ctacttcctg
cttctggttt
gcttgatgct
agccaagagc
gctctctgcc
tgatctgtca
aggcactgct
gacgattttt
actaacccca
aga
ttat
ttcactgcct
ggagacagcc
tcggggggtg
ccagtgtccc
atcacagctt
catcccctcc
gggcactggg
tgggatctct
gaagtctgtc
ttattcttca
gatacatcct
gtgagttggg
ctcatgcttt
gcctgtgggg
gcttcattct
cactagaggg
caacggtgcc
ttcttgcagg
tatctctctc
gtaagtgttt
gagcactaat
ctcctgcagg
accactcagc
acagcttgtg
gttgatgggg
cttccagggg
atgacttgga
cttctccttc
acagagcacc
tactttgtct
gtgaagcacc
aagaagaact
tgccagaaga
cactcaggct
gggagccatt
aaaatcagga
gagatgcgct
aagtgctttg
ccctcctgcc
gcgacggatt
tcatgaatgc
tcactcatgc
gaggagtggt
gtctagtcct
catgggcagg
agaacacaga
aataaaagat
atgactgacc
att
gaatagagaa
acccacaggc
ccagcacgga
cctgtggctg
ccagagcacg
ggaccaggct
gctcccacag
cacaagaagg
tacaagagaa
cttgactcta
ccacagatcc
Figure 9A-D: pGEM3Z(+) EBP Exon Constructs
A. pGEM3Z(+) vector indicating its Ampicillin resistant gene (Ampr), beta-galactosidase gene
(LacZ), and its Multiple Cloning Site (MSC) along with BamHI and EcoRI digestive sites.
B. pGEM3Z (+) EBP Exon 2 construct.
C. pGEM3Z (+) EBP Exons 3 and 4 construct.
D. pGEM3Z (+) EBP Exon 5 construct.
*In blue are the forward primers, in green are the exons, in red are the reverse primers.
To further investigate the white colonies containing their respective construct,
several isolated white colonies from each plate were purified and nucleotide
concentrations were meassured (Figure 9A-D.). The nucleotide concentration of each
construct was determined by UV-irradiation of DNA with intercalated ethidium bromide.
Among the pGEM3Z(+) Exon 2 constructs, the highest nucleotide concentration was
found to be 81.5ng/uL with an 260/280 ratio of 1.90, indicating relative purity. Among
the pGEM3Z(+) Exon 3,4 constructs, the highest nucleotide concentration was found to
be 159.5ng/uL with an 260/280 ratio of 1.88, indicating relative purity. Among the
pGEM3Z(+) Exon 5 constructs, the highest nucleotide concentration was found to be
90.2ng/uL with an 260/280 ratio of 1.88, indicating relative purity.
59 10A
10B
60 10C
10D
Figure 10A-D: Concentrations of each purified construct
A. pGEM3Z (+) - Exon 2 Constructs
B. pGEM3Z (+) - Exon 3 and 4 Constructs
C. pGEM3Z (+) - Exon 5 Constructs and Exon 3 and 4 Constructs
D. pGEM3Z (+) - Exon 5 Constructs
61 Sequence Analysis of: pGEM3Z(+) Exon 2, pGEM3Z(+) Exon 3&4, pGEM3Z(+)
Exon 5
Samples representing each of the purified constructs were prepared and diluted to
50ng/uL and sent to Retrogen inc., San Digego, CA for sequencing. Electropherograms
were analyzed for possible mutations using Mek&Tosj.com 2006 freeware program
4Peaks version 1.7.2 (Figure 10A-D).
11A
11B
62 11C
11D
Figure 11A-D: Representative Sequences for all samples from each construct subjected to sequencing
A. Exon 2 sequences for the pGEM3Z (+)-Exon2 Construct
B. Exon 3 sequences for the pGEM3Z (+)-Exon34 Construct
C. Exon 4 sequences for the pGEM3Z (+)-Exon34 Construct
D. Exon 5 sequences for the pGEM3Z (+)-Exon5 Construct
63 Amino Acid sequences from our samples which matched to Uniprot’s EBP (Tables 3-5),
were analyzed using EMBL-EBI’s Clustal Omega - Multiple Sequence Alignment online
software (Figure 11-14). Sample CDPX2 2-3_M13F only resulted in evidently
“missequencing”. Nevertheless no mutation was detected.
Sequence Analysis of Exon 2: Missequencing of Sample 23M and 24M
Of the eight Exon2 Samples sequenced, seven were analyzed as shown
previously. Of these seven samples, two samples were further scrutinized for the
possibility of novel mutations (Figure 15-16). Sample Ex23M amino acid sequence had
three changes whereas sample Ex24 amino acid sequence was predicted to have one
change.
Exon 2
Sample
Amino Acid Sequence
UniProt TTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAAVVPLGTWR
2-1M
2-2M
RLSLCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQL
IRLTIGRIRARYPGIRSIYISHDNKLFEFDFIISFCTFYLSRFFCSFFFFFFFNFLPIHTQ
PSAHKDMTTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAAV
VPLGTWRRLSLCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQLCES*FLSYAVGWDLLG
RDPLESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS
*LTLIALRSLPAFQSGNLSCQLH**IGQRAGRGGLRIGRSSASSLTDSLRSVVRLRRAVSA
HSKAVI-VIHRIRG-RRK-TCEQKGQQK--K--KCIRL-IGRIRARYPGIRSIYISHDNKLFEFDFIISFCTFYLSRFFCSFFFFFFFNFLPIHT
QPSAHKDMTTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAA
VVPLGTWRRLSLCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQLCES*FLSYAVGWDLL
GRDPLESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**V
S*LTLIALRSLPAFQSGNLSCQLH**IGQRAGRGGLRIGRSSASSLTDSLRSVVRLRRAVS
AHSKAG----STEI-G-T-GK--CEQK--
2-3M
CIRL-IGRIRARYPGIRSIYISHDNKLFEFDFIISFCTFYLSRFFCSFFFFFFFNFLPIHT
QPSAHKDMTTNAGPLHPYWPQHLKLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAA
VVPLGTWRRLSLCWFAVCGFIHLVIEGWFVLYYKNLLGDQAFLSQLCES*FLSYAVGWDLL
GRDPLESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**V
S*LTLIALRSLPAFQSGNLSCQLH**I-PTRG--AVCVLGALPLPRSLTRC-RSFGCGRY-LPQG----I------A-----------*-----VF---------
2-4M
CIRL-IGRIRARYPGIRSIYISHDNKLFEFDFIISFCTFYLSRFFCSFFFFFFFNFLPIHT
QPSAHKDMTTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAA
VVPLGTWRRLSLCWFAVCGFIHLVIEGWFVLYYENLLGDQAFLSQLCES*FLSYAVGWDLL
GRDPLESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**V
S*LTLIALRSLPAFQSGNLSCQLH**IGQRAGRGGFAYWALFRFLAH*LAALGRSAASGISSLKGGNTVIH-I-G*R-K-N--AKGSK---RKK-A-AGVF-*---P-
Table 2: Amino Acid Sequences for four exon 2 samples. AA in Magenta. Intronic region in
black. UniProt: http://www.uniprot.org/blast/?about=Q15125%5B2-230%5D
64 Exon 2
Sample
UniProt
2-1A
2-4A
2-5A
Amino Acid Sequence
TTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAAVVPLGTWRR
LSLCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQL
YTTHYRANSSSVPGDPVHLHFS***TI*I*FYYLILYFLFVQVFLFLFFFFFNFLPIHTQPS
AHKDMTTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAAVVPL
GTWRRLSLCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQLCES*FLSYAVGWDLLGRDPL
ESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS*LTLI
ALRSLPAFQSGNLSCQLH**IGQRAGRG-LRIGRSSASSLTDSLRSVVRLRRAVSAHSKVI--STESGDNAG-NM*A--AK--NVKGRVA-VF--SAPL-SITKIDA-V-------------P---------------------------------G--------TT-YRANSSSVPGDPVHLHFS***TI*I*FYYLILYFLFVQVFLFLFFFFFNFLPIHTQP
SAHKDMTTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAAVVP
LGTWRRLSLCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQLCES*FLSYAVGWDLLGRDP
LESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS*LTL
IALRSLPAFQSGNLSCQLH**IGQRAGRGGLRIGRSSASSLTDSLRSVVRLRRAVSAHSKAV
I--IHRIRG-RRKEHVSKRPA-GQEP*KGP-CWRF-*A--P*RAS-NR-SV---------K----------------------------------TT-YRANSSSVPGDPVHLHFS***TI*I*FYYLILYFLFVQVFLFLFFFFFNFLPIHTQP
SAHKDMTTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAAVVP
LGTWRRLSLCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQLCES*FLSYAVGWDLLGRDP
LESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS*LTL
IALRSLPAFQSGNLSCQLH**IGQRAGRGGLRIGRSSASSLTDSLRSVVRLRRAVSAHKAVIRLS-ESGDNAGKE-CEQ--AKGQE-*KG-VAGVF-----P----KIDAQV----KP--------P---------------------------------------
12
Figure 12: Multiple Sequence Alignment of all Exon 2 Samples
Samples run in March designated with the letter ‘M’
Samples run in April designated with the letter ‘A’
Ex2 sample is from UniProt;
* = no change;
: = change
65 Exon 3&4
Sample
Amino Acid Sequence
UniProt
L*SLSSCRDPQLSQVCVPFTAFILHISLFFSSGKEYAKGDSRYIL*VFASVNGDWHWF
SGGGELGSTNGLTCRKSTPIPVSPHAFSCRGDNFTVCMETITACLWGPLSLWVVIAFL
RQHPLRFILQLVVSVGKERALEGHWALEGLMGDPQTQMYPCGWDLSTVPFQG*VRLND
K
L*SLSSCRDPQLSQVCVPFTAFILHISLFFSSGKEYAKGDSRYIL*VFASVNGDWHWF
SGGGELGSTNGLTCRKSTPIPVSPHAFSCRGDNFTVCMETITACLWGPLSLWVVIAFL
RQHPLRFILQLVVSVGKERALEGHWALEGLMGDPQTQMYPCGWDLSTVPFQG*VRLND
K
VYD-L*GEFL*SLSSCRDPQLSQVCVPFTAFILHISLFFSSGKEYAKGDSRYIL*VF
ASVNGDWHWFSGGGELGSTNGLTCRKSTPIPVSPHAFSCRGDNFTVCMETITACLWGP
LSLWVVIAFLRQHPLRFILQLVVSVGKERALEGHWALEGLMGDPQTQMYPCGWDLSTV
PFQG*VRLNDKGSSRVDLQACKLEYSIVSPK*LGVIMVIAVSCVKLLSAHNSTQHTSR
KHKV*SLGCLMSELTHINCVALTARFPVGKPVVPAALMNRPTRGERRFAYWALFRFLA
H*LAALGRSAAASGISSLKGGNTVIHRIRG*RRKEHVSKRPAKGQEP*KG-VA-VHRLRPPD-HH-KSTLKV-R--
34-2M
34-9M
34-10M
34-10M
34-11A
34-13A
VYD-L*GEFL*SLSSCRDPQLSQVCVPFTAFILHISLFFSSGKEYAKGDSRYIL*VF
ASVNGDWHWFSGGGELGSTNGLTCRKSTPIPVSPHAFSCRGDNFTVCMETITACLWGP
LSLWVVIAFLRQHPLRFILQLVVSVGKERALEGHWALEGLMGDPQTQMYPCGWDLSTV
PFQG*VRLNDKGSSESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAG
SIKCKAWGA**VS*LTLIALRSLPAFQSGNLSCQLH**IGQRAGRGGLRIGRSSASSL
TDSLRSVVRLRRAVSAHSKAVIRLSTESGDNAGKNM*AKGQQK-R-RK--RLL-FFHSAPL--I-KNRRSR
VYD-L*GEFL*SLSSCRDPQLSQVCVPFTAFILHISLFFSSGKEYAKGDSRYIL*VF
ASVNGDWHWFSGGGELGSTNGLTCRKSTPIPVSPHAFSCRGDNFTVCMETITACLWGP
LSLWVVIAFLRQHPLRFILQLVVSVGKERALEGHWALEGLMGDPQTQMYPCGWDLSTV
PFQG*VRLNDKGSSRVDLQACKLEYSIVSPK*LGVIMVIAVSCVKLLSAHNSTQHTSR
KHKV*SLGCLMSELTHINCVALTARFPVGKPVVPAALMNRPTRGERRFAYWALFRFLA
H*LAALGRSAAASGISSLKGGN--IHRI-G-R-KNM*A--Q-PG-*K-ALL-FSI-S--DVYD-L*GEFL*SLSSCRDPQLSQVCVPFTAFILHISLFFSSGKEYAKGDSRYIL*VF
ASVNGDWHWFSGGGELGSTNGLTCRKSTPIPVSPHAFSCRGDNFTVCMETITACLWGP
LSLWVVIAFLRQHPLRFILQLVVSVGKERALEGHWALEGLMGDPQTQMYPCGWDLSTV
PFQG*VRLNDKGSSRVDLQACKLEYSIVSPK*LGVIMVIAVSCVKLLSAHNSTQHTSR
KHKV*SLGCLMSELTHINCVALTARFPVGKPVVPAALMNRPTRGERRFAYWALFRFLA
H*LAALGRSAAASGISSLKGGNTVIHRI-D-AGKNM*AKG-Q--G-VKRP--WFP*A-P-TSI-KSTLK--------Y-----P-----------------------------L-GE-L*SLSSCRDPQ---VCVPFTAFILHISLFFSSGKEYAKGDSRYIL*VFASVN
GDWHWFSGGGELGSTNGLTCRKSTPIPVSPHAFSCRGDNFTVCMETITACLWGPLSLW
VVIAFLRQHPLRFILQLVVSVGKERALEGHWALEGLMGDPQTQMYPCGWDLSTVPFQG
*VRLNDKGSSRVDLQACKLEYSIVSPK*LGVIMVIAVSCVKLLSAHNSTQHTSRKHKV
-SLGCLMSELTHI*LRCAHCPLSS-ETCRASCINESNARGEAVCVLGALPLPRSLTRCARSFGCGER-QLTQRR*Y-Y-QNQ-*R-RTCEQRA-ARNRKRPRC-V-H--APL-SI--I-RS---W--P--N-------------------T----------------G---------------
Table 3: Amino Acid (AA) sequences for exon 3 (red) and 4 (Magenta) samples.
Intronic region in black UniProt: http://www.uniprot.org/blast/?about=Q15125%5B2-230%5D
66 13
Figure 13: Multiple Sequence Alignment of all Exon 3 and Exon 4 Samples
Samples run in the month of March are designated with the letter ‘M’.
Samples run in the month of April are designated with the letter ‘A’.
Ex34 sample is from UniProt; * = no change
67 Exon 5
Sample
Amino Acid Sequence
UniProt QIYGDVLYFLTEHRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDA
VKHLTHAQSTLDAKATKAKSKKN
5-5M VYDL*GEFISEYLALRP*MTWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFLTE
5-6M
5-7M
5-8M
5-10M
HRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSKK
N*GVVDQARTLAEEELSACQKSLVLLPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTAGS
H*NTDTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPEDPLE
STCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGCLMSELTHI
NCVALTARFPVGKPVVPAALMN--NARGEAGLRIGRS--SSLTDSLRS--RL--ASAHSK--L*GEFISEYLALRP*MWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFLTEHRDG
FQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSKKN*GV
VDQARTLAEEELSACQKSLVLLPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTAGSH*NT
DTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPEDPLESTCR
HASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS*LTLIALR
SLPAFQSGNLSCQLH**I-PTRGERRFAYWALF--RSLTRCARS-GC-S--QLTQ---DL*GEFISEYLALRP*MMTWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFLTEH
RDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSKKN
*GVVDQARTLAEEELSACQKSLVLLPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTAGSH
*NTDTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPEDPLES
TCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWG**VS*LTLIALRSLPAFQSGNLSCQLH**I--RAGEAG---WAL--LPSLTRCARSFGC-E--SSLK-GVYDL*GEFISEYLALRP*MMTWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFLT
EHRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSK
KN*GVVDQARTLAEEELSACQKSLVLLPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTAG
SH*NTDTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPEDPL
ESTCRHASLSIL*CHLNSLA*SWS*LFPV*N-YPLTIPHNIRAGSIKCKAWG**VS*LTLIALRSLPAFQSGNLSCQLH**IGPTRG-RR-AYWALFRFPRSLTRCAR-FG-R--SA--K--VYDL*GEFISEYLALRP*MMTWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFLT
EHRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSK
KN*GVVDQARTLAEEELSACQKSLVLLPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTAG
SH*NTDTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPEDPL
ESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS*LT
LIALRSLPAFQSGNLSCQLH**I-PTRG-RR-CVLGALRF-AH*LAAL-R-AAA---SSK--
Table 4: Amino Acid sequences for five exon 5 samples. AA in Magenta. Intronic sequence in
black. UniProt: http://www.uniprot.org/blast/?about=Q15125%5B2-230%5D
68 Exon 5
Sample
Amino Acid Sequence
UniProt QIYGDVLYFLTEHRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDA
VKHLTHAQSTLDAKATKAKSKKN
5-12A
5-13A
5-20A
5-22A
VDL*GEFISEYLALRP*MMTWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFLTE
HRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSKK
N*GVVDQARTLAEEELSACQKSLVLPPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTAGS
H*NTDTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPEDPLE
STCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKWGA**VS*LTLIALRSLPAFQSGNLSCQLH**I--RG----AYWALFR-S-TDS----------QL--------P-N------------KG---VYDL*GEFISEYLALRP*MMTWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFLT
EHRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSK
KN*GVVDQARTLAEEELSACQKSLVLLPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTAG
SH*NTDTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPEDPL
ESTCRHASLSIL*CHLNSL*SWS*LFPV*NCYPLTIPHNIRAGSIKCKGLGCLMSELTHINCVALTRFPVGKPVVPAAL-SA--G-------G------H*-A---------------R--------G------------------K-----VYDSL*GEFISEYLALRP*MMTWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFL
TEHRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKS
KKN*GVVDQARTLAEEELSACQKSLVLLPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTA
GSH*NTDTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPEDP
LESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS*L
TLIALRSLPAFQSGNLSCQLH**I-PRAG-GGLRIGRSSA--L-DSL---------QL---N-----G-------Q------K--------------------VYDL*GEFISEYLALRP*MMTWKVLWNRELAKVSPSSLGLLLPLLPPTGQIYGDVLYFLT
EHRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSK
KN*GVVDQARTLAEEELSACQKSLVLLPQFGGTKLIDLSHSGSWAGTRRGIKGLCEGTAG
SH*NTDTREARRSMMVTIFKIRK*KILTLTLRDP*LLTPEMRYD*PHRSHHHSLIPDPLESTCRHASLSIL*CHLNSLA*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS
*LTLIALRSLPAFQSGNLSCQLH**------G-L-IGR-SLP-SLT---------R-----
Table 4: Amino Acid sequences for four exon 5 samples. AA in Magenta. Intronic sequence in
black. UniProt: http://www.uniprot.org/blast/?about=Q15125%5B2-230%5D
69 14
Figure 14: Multiple Sequence Alignment of all Exon 5 Samples
Samples run in the month of March are designated with the letter ‘M’.
Samples run in the month of April are designated with the letter ‘A’.
Ex5 sample is from UniProt; * = no change
70 15
NNNNNNNNNNNNNTGTATACGACTCNCTATAGGGCGAATTCGAGCTCGGTACCCGGGGA
TCCGGTCCATTTACATTTCTCATGATAATAAACTATTTGAATTTGATTTTATTATCTCA
TTCTGTACTTTCTATTTGTCCAGGTTTTTCTGTTCCTTTTTTTTTTTTTTTTTTTTTAA
CTTCCTGCCTATACACACGCAGCCATCAGCCCACAAAGACATGACTACCAACGCGGGCC
CCTTGCACCCATACTGGCCTCAGCACCTAAAACTGGACAACTTTGTACCTAATGACCGC
CCCACCTGGCATATACTGGCTGGCCTCTTCTCTGTCACAGGGGTCTTAGTCGTGACCAC
ATGGCTGTTGTCAGGTCGTGCTGCGGTTGTCCCATTGGGGACTTGGCGGCGACTGTCCC
TGTGCTGGTTTGCAGTGTGTGGGTTCATTCACCTGGTGATCGAGGGCTGGTTCGTTCTC
TACTACAAAAACCTGCTTGGAGACCAAGCCTTCTTATCTCAACTCTGTGAGTCCTGATT
TCTTTCATATGCTGTGGGATGGGATTTGCTGGGCAGGGATCCTCTAGAGTCGACCTGCA
GGCATGCAAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCA
TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGG
AAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGT
TGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC
NGGCCAACGCGCGGGGANNANGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTC
ACTGACTCGCTGCNCTCGGTCGTTCGGCTGCGGCGANCGGTATCANCTCCCTCAAGGCG
GNANACNNGTNATCCNCNNANTCNGGGNNNNNGCAGNAANNNNNNNANNNNNNNNCAAN
GNCANNANNTAANNNNCNCNTNGCTGNGTTTTCNTAGNNNCNNCCCNCNNACNNNNATC
NNN
Figure 15: Sample CDPX2-3_M13F Sequence
Sequence of the CDPX2-3_M13F containing exon 2.
In green is exon 2. In red are the changes (A50G; G262A, G265A)
71 H.
E.
I.
G.
Figure 16: Possible Mutation of CDPX2-3_M13F
A change seen in both the Nucleotide sequence and Amino Acid of CDPX2-3_M13F sample showing an Arginine to Lysine (R17K); G50A. (A)
Electropherogram of sample CDPX2-3M13F; blue arrows point to the expected sequence, and the red arrow points to the discrepancy; amino acid sequence
shown on the far right in which the R17K change is highlighted in gray. (B) ClustalOmega MSA
16
J.
F.
72 O.
N.
P.
M.
L.
K.
73 Figure 17: Possible Mutation of CDPX2-3_M13F and CDPX2-4_M13F
A change seen in both the Nucleotide sequence and Amino Acid of CDPX2-3_M13F sample and CDPX2-4_M13 F showing Glutamic acid to
Lysine (E88K; G262A) for sample CDPX2-3 and Aspartic acid to Asparagine (D89N; G265A) for both samples . (A) Electropherogram of samples;
blue arrow point to the expected sequence, and the red arrows point the discrepancies; amino acid sequence shown on the far right in which changes
are highlighted in gray. (B) ClustalOmega MSA
17
CHAPTER 4: Conclusion
CDPX2 Review
Conradi-Hunermann-Happle syndrome (Chondrodysplasia punctata 2: CDPX2) is an Xlinked disorder effecting mostly females, with a prevalence of 1:40,000 in newborns.
CDPX2, both a Chondrodysplasia punctata syndrome and a cholesterol biosynthesis
disorder, is due to a faulty protein Empamil Binding Protein (EBP), known as Sterol
D8,D7 Isomerase. Sterol D8, D7 Isomerase is a 230 amino acid transmembrane protein
originally described only in the Endoplasmic Reticulumn (ER); it has now been shown to
be located in the Nuclear membrane as well as in the Lipid Droplets (LDs) (Hanner et al.,
1995; Dussossoy et al., 1999; Caldas et al., 1993). It is possible that the protein maybe
located in several different subcellular compartments because of the various functions of
this protein. Since Sterol D8,D7 Isomerase acts as the penultimate catalytic enzyme
converting lanosterol to cholesterol, any mutation along the 1.0kb length of the EBP gene
proves to be detrimental for the synthesis of cholesterol and steroid hormones. As of
today, only four exonic regions and several of the intronic regions of the Sterol D8,D7
Isomerase’s have been found with mutations. These mutations include
misssense/nonsense mutations (63%), deletions (23%), insertions (0.07%) splice cite
mutations (0.04%) and small insertions or deletions (indels) (the inclusion of insertions
and deletions resulting in a net change of nucleotides ;0.01%) (the Human Mutation Gene
Database). Finding a novel mutation or identifying a change with a known mutation in
these boundaries would help explain an effected person’s condition.
74 The aim of this study was to analyze the DNA of a female presenting phenotypic
characteristics of CDPX2 since birth. To enhance sequencing output, each of the four
transcribable exons (exons 2-5) were independently cloned into a high-expressing vector
(pGEM3Z(+)). However, exon three and exon four along with the intronic region in
between them, were cloned as a single fragment due to the close proximity of the two
exons.
Summary of Results
I successfully cloned a total of 27 pGME3Z(+) CDPX2 constructs (7 pGEM3Z(+)
Exon 2: 10 pGEM3Z(+) Exon 34 (Exons 3 and 4); 10 pGEM3Z(+) Exon 5 (data not
shown). These successes were highlighted through nucleotide analysis showing each
construct free of impurities such as ethanol and proteins as seen by the A260:A280 raito
in the NanoDrop results in figures 2A-2D. The criteria that I used for accessing sample
purity depended upon the presence of three spectrophotometric features in the NanoDrop.
The first, and most important, was a sharp peak between the 240 and 280nm peaks, with
260nm being the optimal point of absorbance for nucleic acids. The second feature is
seen as a sharp decline followed by a steady increase between 220nm and 230nm, which
reflects a low level of various impurities, such as in ethanol and salts. The 230nm
absorbance reflects a trough in the graph. The third feature of the graphical representation
of the absorbance spectrum of a sample is a continuous declination starting at 260nm,
which is indicative of low protein contaminants. The results show the absence of proteins
in the sample of study. This analysis was important for the success of this study, because
the presence of any contaminate can interfere with nucleotide sequencing. As shown in
75 three of my constructs’ nanodrop results, their relative purities were evident as 81.5ng/uL
with an 260/280 ratio of 1.90, 159.5ng/uL with an 260/280 ratio of 1.88, and 90.2ng/uL
with an 260/280 ratio of 1.88, respectively. Constructs for Exon 2 contained the lowest
concentration yields (11ng/uL to 24.9ng/uL) and the highest 260/280 ratios (1.92 to 2.07)
when compared to the other constructs. In addition to all of my constructs respective
purities based on nucleotide concentration measurements and 260/280 ratio, all three
constructs had a sharp decline in absorbance between 220nm and 230nm followed by an
inclination leading to 260nm.
Having all 27 constructs free from impurities, six to nine samples with high
nucleotide concentration (>50ng/uL) and an adequate 260/280 ratio (1.8-1.9) for each
construct were selected for sequence analysis. Of all the samples sequenced, samples
23M and 24M returned with three discrepancies. The first discrepancy was found to be at
amino acid position 17 (R to K); nucleotide position 50 (G>A; nucleotide residue AGA
to AAA). This first change was only detected in sample Ex23M. The second discrepancy
was found at amino acid position 88 (E to K); nucleotide position 262 (G>A; nucleotide
residue GAA to AAA). This 88 E to K (G>A; 262) change was also seen only in sample
Ex23M. The third discrepancy was found at amino acid position 89 (D to N); nucleotide
position 265 (G>A; nucleotide residue GAC to AAC). This last discrepancy was found in
both sample Ex23M and sample Ex24M. None of these three discrepancies (R17K,
E88K, D89N) have been reported among the 65 mutations described thus far in literature
(Human Gene Mutation Database). Perhaps these discrepancies in the sequence of these
clones is most likely reflective of the subject’s heterozygosity in which they were silent
and would not result in amino acid substitution.
76 To further address the unlikelihood of novel mutations, it is important to note that two of
the seven pGEM3Z(+) Exon 2 constructs showed changes. Also, sequencing was
performed on the forward strand and not the reverse strand. I did not have the reverse
strands to compare complimentary nucleotide residues, but the findings may be better
explained as sequencing misreads. I constructed 10 pGEM3Z(+) Exon34 clones for the
sequence analysis of exon 3 and exon 4. In all of these 10 constructs, there were no
changes in the predicted amino acid sequence, because no changes were observed in the
nucleotide sequences. There were also no changes observed in the nucleotide sequences
of the 10 pGEM3Z(+) Exon 5 constructs. The observation that there was no difference
between multiple clones from the blood lymphocytes of an individual with apparent
clinical signs of CDPX2, raises the possibility that this subject is affected with a subtype
of CDP.
Possible Phenocopy of EBP
To attempt to answer how an individual may show clinical features of CDPX2
and yet DNA analysis did not present any of the known mutations in EBP, one must ask
several questions. Several of these questions arise from current knowledge of CDP, the
Cholesterol biosynthetic pathway, and EBP studies. For instance, what is/are the actual
defective protein(s) involving CDPX2 and its similarity with CHILD syndrome, and how
do these disorders’ proteins overlap phenotypic features, EBP and NSDHL respectively.
Also, what are the overall localizations of these two proteins and is there a functionality
disparity between their locations. As demonstrated by Caldas and Ohashi 2003, do lipid
droplets (LDs) regulatory just NSDHL or do they also regulate EBP or any other
77 cholesterol biosynthetic protein. Recent studies have demonstrated how the cells’
environmental impact the functionality of proteins, so how are cholesterol biosynthetic
proteins affected by the presence of teratogens? It has also been shown that abnormal
levels of their intermediates/sterol precursors affect functionality of proteins. A set of
question to rise would be if intermediates obstruct more than one protein and if so, do
two distinctive disorders have one or two phenotypic characteristics because of such
anomaly. There are 30 known enzymatic steps in the sterol biosynethic pathway and it
may be probable that defective enzymes from this pathway may lead to similar yet
distinctive clinical trademarks. Clinical trademarks to consider of such degree of
similarity are skin abnormalities as seen in CDPX2 and CHILD syndrome and cranial
facial deformities as seen in both CDP and Cholesterol biosynthetic disorders. Enzymes
within the Cholesterol biosynthetic pathway with somewhat similar clinical
manifestations include EBP, NSDHL, 7DHCR, PEX7, GNPAT/DHAPAT, and AGPS.
Changes in the genetic code for these proteins gives rise to four distinctive disorders
with cranialfacial and long bones malformations. These disorders include CDPX2,
CHILD syndrome, SLO, and the three subtypes of RCDP. There maybe an additional
cholesterol biosynthesis defect syndrome, described by the subject of this study.
Peroxisomes, Endoplasmic Reticulum, defective enzymes, and overlapping symptoms of CDPX2, CHILD and SLO
When considering the development of closely related disease, it may be important
to carefully consider organelles involved in the entire biochemical pathway other than the
organelle known to house the protein in question. An example of a commonly neglected
subcellular compartment in etiology is that of the peroxisomes. The enzymes defective in
RCDP3 and RCDP2 are essential for proper peroxisome activity. AGPS, the enzyme
78 found to be defective in RCDP3, is involved in Ether lipid metabolism whereas
GNPAT/DHAPAT, the enzyme found to be defective in RCDP2, is involved in
Glycerophospholipid metabolism. Also, PEX7 is involved in peroxisomal activity by
acting as a transporter protein, which has been found to be defective in RCDP1
individuals. As we already know EBP, NSDHL, and 7DHCR are localized in the
Endoplasmic Reticulum (ER) and are involved with steroid biosynthesis. Defects in EBP,
AGPS or GNPAT/DHAPAT result in CDP disorders such as CDPX2, RCDP3, or
RCDP2, respectively. Defects in NSDHL, EBP, and 7DHCR result in Cholesterol
biosynthesis disorders such as CHILD syndrome, CDPX2, and SLOS. A defective
organelle resulting from a malfunction in one of these enzymes may suggest that some
disorders may have overlapping symptoms.
Several overlapping phenotypic characteristics have been found in various
disorders including those involving cholesterol biosynthesis and peroxisomal
biosynthesis. One such characteristic is stippling of the epiphysis, which has been
observed in the X-linked recessive, the autosomal dominant, and recessive forms of CDP
(Herman, 2003). All CDP subtypes involve either defective enzymes in the cholesterol
biosynthesis pathway or peroxisomes. Other disorders not involving these two
metabolism pathways include lysosomal storage disorders, abnormalities in vitamin K
metabolism, trisomy 13, 21, and 18 (Poznanski et al., 1994). A second overlapping
phenotypic characteristic is ichtyosioform dermatoses, which has been observed in the
dominant and recessive forms for both the autosomal and the X-linked CDP subtypes
(Cameo et al., 2000). In regards to the similarity between CHILD syndrome and CDPX2,
there have been cases in which biochemical analysis fail to make a distinction between
79 the disorders. A few individuals with CHILD syndrome have been found to have EBP
mutations in CDPX2, as well as the similar accumulation of sterols.(Grange et al., 2000).
There has not been a correlation observed between levels of sterol accumulation and the
severity of the disorder. One large study found EBP mutations in all females of 7
unrelated families, yet failed to identify an EBP mutation in the grandmother of an
affected female, who had some symptoms of the disorders. (Has et al., 2000). She had
dwarfness and sectorial cataracts. Individuals in another study, three unrelated females
with CDPX2 whos mothers lacked a known responsible mutation from their peripheral
blood, failed to present a correlation between genotype and phenotype (Herman et al.,
2002). In later study involving CDPX2 and biochemical/mutational analysis, it has been
declared that biochemical alterations in serum do not reflect nor predict the clinical
manifestations (Maurer et al., 2008). An even more recent case study showed the
presence of a CDPX2-causing mutation both in a female child and in the affected skin of
the mother, yet not in the blood lymphocytes of either parent (Morice-Picard et al., 2011).
Morice described this phenomenon as the results of gonadal mosaicism and the first
description of such a mosaic pattern in CDPX2. For most individuals affected with
CDPX2, bilateral and asymmetrical abnormalities are present whereas most individuals
affected with CHILD syndrome present unilateral and symmetrical abnormalities. Given
that both disorders give rise to CDP manifestations and are involved in the same
metabolic pathway, one may speculate the involvement of regulatory sites throughout cell
compartments. Such regulatory sites may control proper enzymes’ activities where
intermediates levels may be maintained within the cell or compartments
80 Lipid Droplets and possible role for Cholesterol Biosynthesis
There has been difficulty in detecting any correlation between genotype and
phenotype in disorders of cholesterol metabolism, probably in part because some of the
disorders are X-linked, and expression is affected by X-inactivation. The defective
protein in CDPX2 is the Sterol D7,D8 Isomerase (EBP) which is located in the E.R., in
the N.M and possibly in LDs. In a complex metabolic pathway as is the case for
cholesterol biosynthesis, perhaps location is the result of regulatory mechanisms yet to be
understood. Furthermore, their proteins were originally localized strictly to the ER, but
recent studies have demonstrated that these two proteins are located in distinctive
organelles. Both CHILD syndrome and CDPX2 result from defects in cholesterol
biosynthesis but the EBP protein has been found to be in both the E.R. and N.M. whereas
the protein NSDHL has been found to be located in the ER and LDs (Hanner et al., 1995;
Dussossoy et al.1999; Caldas et al., 2003; Ohashi et al., 2003). Transition of the NSDHL
protein from the LDs to the ER was performed in CHO cells grown in medium depleted
in oleic acid. (Ohashi et al., 2003). In contrast, the addition of Oleic acid to the growth
medium of CHO cells reversed the redistribution by inhibiting the localization of NSDHL
to the E.R. Not only was NSDHL redistributed from the E.R. to the LDs by elevating
Oleic acid, but the conversion of lanosterol to cholesterol was reduced. Moreover, it was
found that the A105V, a CHILD syndrome mutation, resulted in the localization of
NSDHL on LDs. The incorporation of another CHILD syndrome mutation (G205S) was
observed as displacing NSHDL from LDs into the cytoplasm. Perhaps LDs served as a
regulatory site for maintaining proper levels of cholesterol as NSDHL has been
demonstrated to be active on the ER, yet inactive on the surface of LDs. Given that
81 NSDHL is the first cholesterol biosynthesis enzyme to be attributed to LDs, the need for
further localization studies on the remaining 29 enzymes within cholesterol biosynthesis
is crucial.
SREBP Cleavage-Activating Protein
A possible regulatory mechanism for proteins found in the E.R. is through the
SREBP cleavage-activating protein (SCAP). Proteins regulated by SCAP contain a
sterol-sensing domain (SSD), which allows for the binding of protein-sterols (Kuwabara
and Labouesse 2002; Weber et al., 2004). Proteins that are transcriptionally regulated
through SCAP maybe retained in the E.R. or maybe excorted into the Golgi apparatus.
(Brown and Goldstein 1999; Brown et al.m 2002; Horton et al., 2002; Goldstein et al.,
2006). Some of these SSD proteins have been shown to play a role in sterol-dependent
proteolysis in which SCAP escorted SREBP from the ER to the Golgi in cells depleted of
sterols and thus having SREBP target genes activated. (Goldstein et al., 2006; Korade et
al., 2009). Moreover, these SREBP target genes include those for fatty acid, triglyceride
and cholesterol biosynthesis which are targeted by two of the three SREBP isoforms
known to be produced by mammalian cells (Horton et al., 2003; Goldstein et al., 2006).
Given that SREBP-1c and SREBP-2 are involved through SCAP in lipid biosynthesis, it
will be most effective to investigate their regulatory mechanism(s). In particular,, the
way in which regulatory mechanisms are impacted through the presence of anomalies in
the cholesterol biosynthesis pathway. The anomalies to be scrutinized are the
accumulation of sterol intermediates, defective peroxisomes, and translocalization of
enzymes such as NSDHL.
82 Importance of Peroxisomes in Pre-Squalene Pathway
Most of the cholesterol biosynthesis’ enzymatic steps are known to occur in the
ER, however peroxisomes have been known to initiate cholesterol biosynthesis.
Compromised peroxisomes leads to several cholesterol biosynthesis disorders and other
disorders not related to such pathways. For example, RCDP is comprised of three distinct
subtypes and all three result from compromised peroxisomes. Each of the subtypes of
RCDP have distinctive malfunctioning proteins, which are not involved in the synthesis
of cholesterol. However, these three proteins are crucial for proper activity of
peroxisomes since they serve as transport proteins for essential entry of enzymes. Such
essential enzymes contain peroxisomal targeting signals, which are recognizable by
peroxisomal transport proteins. Three of the pre-squalene enzymes, MK, PvK, and
Mevalonate diphosphate decarboxylase, have such targeting signals. However, these
three pre-squalene enzymes have yet to be localized in peroxisomes although they were
originally thought to be there in human cells (Kovacs et al., 2007). Nevertheless,
accumulation of sterol intermediates has been shown to present abnormalities in genetic
disorders. Any perturbation disrupting proper peroxisomal activity or enzymatic activity
results in CDP. This is the reason why abnormal levels of sterol precursors and
cholesterol should be investigated in cohort.
Maternal Autoimmune Diseases and CDP
There have been correlative studies in the past two decades on connective CDP
and maternal autoimmune diseases. Three diseases that have been associated with CDP
have been MCTD, scleroderma and SLE (Umranikar et al., 2006; Chitayat et al., 2008).
The first CDP case presented with maternal SLE was in 1993 and in the next decade
83 several other cases have been presented. Most of the mothers were diagnosed with SLE
after delivery with just a few before or during pregnancy. Of the cases in Chitayat’s
investigation, all mothers had been exposed to at least one hormone/medication including
prednisone, verapamil, lansoprazole, metoclopramide hydrochloride, amlodipine
besylate, fexofenadine hydrochloride, acetylsalicylic acid (ASA), acetazolamide,
hydroxychloroquine sulfate, ibuprofen, azathiprine, or methyldopa. So the underlying
question would be if CDP infants resulted because of any of these prescribed
hormones/medications to mothers. Besides the intake of these hormones/medications,
several of these mothers either were diagnosed or had a family history of at least one of
the following diseases; MCTD, scleroderma, Rheumatoid arthritis, raynaud phenomenon,
dermatomyositis, arthritis, Tourette syndrome and dysphagia. To those mothers
diagnosed with MCTD, prednisone was prescribed and taken during the first trimester
and was either discontinued or monitored throughout pregnancy. Prednisone was also
prescribed to treat symptoms of MCTD including pericarditis, pancytopenia, myalgia,
and fevers. Along with these diseases, few of the mothers suffered from mitral valve
prolapse, skin thickening and tightening of both hands or face. Half of these cases
resulted in delivery by Cesarean. Several studies have further associated CDP with
maternal exposure to medications, teratogens and to viruses such as cytomegalovirus
(Poznanski et al., 1994; Hertzber et al., 1999; Umranikar et al., 2006; Chitayat et al.,
2008). All of these CDP cases were characterized by epiphyseal stippling, asymmetric
limb shortening, ectopic cartilaginous calcifications, vertebral segmentation anomalies,
facial features such as midface hypoplasia and hypoplastic nasal septum, as well as
cataracts and skin abnormalities. Maternal autoimmune diseases have elucidated the
84 correlation between genotype and phenotype by addressing the effects of environmental
changes in the womb. Monitoring the prevalence of the mother’s autoimmune diseases,
the infant’s sterol profiles and mutational analysis of genes encoding proteins found in
peroxisomes, ER or any other organelle has proven to be vital for determining a
genotype-phenotype correlation for CDP cases.
Medical History of Subject: First and Third trimester
In the current study, the subject’s mother faced a peculiar situation, which was
described as a threatened abortion. In the first trimester of pregnancy, the mother was
prescribed high-doses of a hormone to maintain the pregnancy. Unfortunately, neither the
mother nor her medical records reveal the name of the hormone. Also, the duration of
exposure to the prescribed hormone was not reported. However, the mother does recall
felling normal movements and being able to hear the heart beats of the subject at the third
month of pregnancy. These movements were decreased and recorded at the seventh
month of pregnancy. At the ninth month, the mother was hospitalized and her obstetrician
observed a green amniotic fluid, which resulted in a Caesarean delivery. At two days of
age , the subject expelled meconium orally which resulted in the improvement of her
health. This maybe because exposure of meconium is correlated with a reduction in illicit
or prescribed drugs. At present, the subject suffers from many complications including
severe arthritis in her right hip and her mother has experienced general arthritis.
Prednisone, may have been prescribed for the treatment of arthritis. Perhaps the contents
of the meconium should have been analyzed for the for the presence of steroids or other
compounds, as it is now analyzed for maternal substance abuse during pregnancy.
85 Significance of the Study
In the study, I presented the DNA analysis of a 36 year old female diagnosed with
CDPX2 at birth. In the DNA analysis, no novel mutations were found nor any of the 64
known mutations presented in the literature. Given that no biochemical analysis has been
performed, the phenotypic manifestations in the subject are the only evidence of CDP.
Furthermore, both of the parents show no abnormalities illustrating the presence of CDP.
It may be postulated that the mother might have had shown an autoimmune response
while under the prescribed hormone during pregnancy thus resulting in CDP in the
subject. To best test this suggestion, the mother should have been or should be tested for
SLE or any of the autoimmune diseases correlated with CDP. In any case, biochemical
analysis for the presence of 8(9)- cholestenol and 8-dehydrocholesterol should be
conducted on the serum of the subject in order to determin if EBP is impaired. Further
studies should investigate tissue samples obtained from skin lesions as well as from
normal skin from the subject. In addition to skin samples, plasma samples should also be
investigated to correlate mosaic-phenotype findings as in the case of somatic mosaicism
vs gonadal mosaicism. Along with these investigations, the localization of proteins
should be considered. For example, EBP, NSDHL, PEX7, AGPS, and GNPAT/DHAPAT
along with the regulatory mechanisms involving LDs and SREBP/SCAP. One should
also investigate the effects on these proteins by the administration of hormones such as
prednisone and compounds use for the treatment of maternal autoimmune response
during pregnancy. It is also possible that CDP may arise in response to environmental
insults to the developing embryo. Compounds such as sterol intermediates and teratogens
86 may give rise to CDP. They have also directly or indirectly been shown to affect the
cholesterol biosynthesis pathway.
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125 Appendix A: 7.5% Polyacrylamide Gel
7.5% Polyacrylamide Gel
In a beaker or erlenmyer flask add the following:
1. 5.950mL of deionized water
2. 1.4mL of 40% acrylamide (Fisher Bioreagents)
3. 0.150mL of 50X TAE
Under the fume hood add the following:
4. 10uL of TEMED
5. 60uL of 10% APS (Ammonium persulfate); 1-14 days fresh
126 Appendix B: 50X TAE
50X TAE (Tris Acetate) (500mL):
1. 121grams of Tris base
2. 300 mililiters of deionized water
3. 28.5 mililiters of Glacial acetic acid
4. 50 mililiters of 0.5 Molar EDTA (pH 8.0)
5. Bring up volume to 500 mililiters
127 Appendix C: S.O.C Medium
SOC Medium (100mL)
1. 0.5% Yeast Extract, 0.5 grams
2. 2% Tryptone, 2.0 grams
3. 10mM NaCl, 0.05 grams or 1.0mL of 1M
4. 1M KCl, 0.25mL
5. 1M MgCl2, 1mL
6. 1M MgSO4, 1mL
7. Sterilize through autoclave
8. 20mM glucose, 2mL
9. Sterilize through 0.2micro filter
128 Promega’s Wizard Plus SV MiniPreps DNA Purification
Production of Cleared Lysate
1. Pellet 1-10mL overnight culture for 1 minute at 3,000 rpm
2. Resuspend pellet with 250uL Cell Resuspension Solution
3. Add 250uL Cell Lysis Solution to each sample; invert 4 times to mix
4. Add 10uL Alkaline Protease Solution; invert 4 times to mix. Incubate for 5
minutes at room temperature
5. Add 350uL Neutralization Solution; invert 4 times to mix
6. Centrifuge at 5,000 rpm for 10 minutes at room temperature
Binding of Plasmid DNA
7. Insert Spin Column into Collection tube
8. Decant cleared lysate into Spin Column
9. Centrifuge at 5,000 rpm for 1 minute at room temperature. Discard flowthrough
and reinsert Column into Collection tube
Washing
10. Add 750uL Wash Solution (ethanol added). Centrifuge at 5,000 rpm for 1 minute.
Discard flowthrough and reinsert column into Collection tube
11. Repeat step 10 with 250uL Wash Solution
12. Centrifuge at 5,000 rpm for 2 minutes at room temperature
Elution
13. Transfer Spin Column to a sterile 1.5mL microcentrifuge tube.
Add 100uL of Nuclease-Free Water to the Spin Column. Centriguge at 14,000 rpm
for 1 minute at room temperature. Discard column and store DNA at -20C 129