 1996 Oxford University Press
Human Molecular Genetics, 1996, Vol. 5, No. 10 1657–1661
Deletion/insertion mutation that causes biotinidase
deficiency may result from the formation of a
quasipalindromic structure
Robert J. Pomponio1, Vasant Narasimhan1, Thomas R. Reynolds2, Gregory A. Buck2,
Lawrence F. Povirk3 and Barry Wolf1,4,*
Departments of 1Human Genetics, 2Microbiology and Immunology, 3Pharmacology and Toxicology, and
4Pediatrics, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA, USA
Received June 3, 1996; Revised and Accepted July 8, 1996
Biotinidase is responsible for recycling the vitamin
biotin from biocytin that is formed after the proteolytic
degradation of the biotin-dependent carboxylases. We
have identified a deletion/insertion mutation within
exon D of the human biotinidase gene in a child with
biotinidase deficiency. The mutation causes a frame
shift and premature termination which are predicted to
result in a truncated protein. We propose that the
mutation occurred during DNA replication by either of
two mechanisms. Both mechanisms involve formation
of a quasipalindromic hairpin loop in the template and
dissociation of DNA polymerase α. This mutation
supports the formation of palindromic structures as a
possible cause of deletions in eukaryotes, and supports
the proposal, derived from in vitro studies, that
polymerase α may preferentially arrest or dissociate at
specific template sequences.
alopecia, skin rash, ketolactic acidosis, and organic aciduria, which
may result in coma (7,8). Treatment with pharmacologic doses of
biotin resolves and reverses most or all of the clinical symptoms
(3). Early diagnosis and treatment of biotinidase deficiency are
important to prevent permanent neurological damage. Many states
and countries perform newborn screening for biotinidase
deficiency (9).
We have characterized the cDNA for normal human serum
biotinidase (BTD) (10), and localized the gene to chromosome
3p25 (11). We have also described a common 7 bp deletion/3 bp
insertion (G98:d7i3) in about half of the symptomatic children with
profound biotinidase deficiency (12). We now describe a child with
profound biotinidase deficiency who is homozygous for a
deletion/insertion mutation within exon D of the biotinidase gene
(13). We propose that the mutation resulted from the formation of
a quasipalindromic stem-loop structure and the dissociation and
reassociation of polymerase α during replication.
INTRODUCTION
The oligonucleotide primers amplified a 300 bp PCR product.
PCR amplification of this region of the BTD DNA did not reveal
any obvious differences in the size of the PCR product of the
patient when compared to that of the individuals with normal
biotinidase activity by non-denaturing polyacrylamide gel
electrophoresis (data not shown).
Single stranded conformational analysis (SSCA) of the PCR
product from 16 normal individuals, as exemplified by patients
93, 209, 113, 114 and 115, exhibited four bands (the upper two
bands migrating as a doublet) (Fig. 1). The SSCA patterns of
patient 20 revealed three bands (the upper two bands as a doublet)
that were different from any of the normal bands. Patient 20’s
mother, 68, and his father, 69, each had bands that corresponded
to those of the normals and to those of their son. These results
suggested that patient 20 was homozygous for a mutation and
each of his parents were heterozygotes.
Sequences were analyzed from normal individuals, the patient
and his parents. The normal individuals all had sequences that
were identical to the normal cDNA sequence (10). In contrast, the
sequence of patient 20 revealed that he is homozygous for a
Biotin, an essential water-soluble B vitamin, is the coenzyme for
four carboxylases in humans (1,2). These carboxylases are involved
in amino acid catabolism, fatty acid synthesis, and gluconeogenesis.
The carboxyl group of biotin is covalently attached through an
amide bond to an ε-amino group of a lysyl-residue of the
apocarboxylases by holocarboxylase synthetase, thereby forming
the holocarboxylases (3). The holocarboxylases are ultimately
degraded to biocytin (biotin-ε-lysine) which are then hydrolyzed by
biotinidase (biotin-amide amidohydrolase, EC 3.5.1.12) to yield
biotin for reutilization (3,4).
Biotinidase deficiency is an autosomal recessively inherited
trait (3,5). Children with biotinidase deficiency cannot hydrolyze
biocytin and thus cannot recycle biotin. If these children are not
treated with biotin supplementation, they become biotin
deficient, resulting in multiple carboxylase deficiency and the
accumulation of toxic metabolites (6). Untreated individuals with
biotinidase deficiency may exhibit seizures, hypotonia, ataxia,
developmental delay, hearing loss, ophthalmologic abnormalities,
*To whom correspondence should be addressed
RESULTS
1658 Human Molecular Genetics, 1996, Vol. 5, No. 10
reassociated with the first -AGA (bold), which was previously
located within the quasipalindromic structure (Fig. 2B,C).
Replication then continued (Fig. 2D), ultimately producing the
sequence of the mutation shown in Figure 2E.
Mechanism 2
Figure 1. Single stranded conformation analysis of PCR-amplified DNA. ID
numbers refer to the individuals tested. PCR-amplified allelic conformations
are from several normal individuals (open symbol) , and patient 20, an
individual with profound biotinidase deficiency (blackened symbol). The
heterozygous parents are designated by the half-blackened symbols. Patient 10
is a child with profound biotinidase deficiency confirmed to be homozygous for
the G98:d7i3 mutation.
deletion of 15 bp and an insertion of 11 bp (Fig. 2). This
abnormality is predicted to result in a frame shift and premature
termination at a stop codon producing a truncated protein with an
aberrant amino acid sequence. The parents are both heterozygous
for this mutation and the normal sequence.
Proposed mechanisms
We have identified a child with biotinidase deficiency with a
mutation with a net 15 bp deletion and an 11 bp insertion. The
normal double-stranded sequence of the exon involved in the
mutation is shown in Figures 2A and 3A. The net deleted
sequence (dashed underline), the net inserted sequence
(underline) and the repeated flanking sequences (bolded) are also
shown in these figures. We propose that either of the following
two mechanisms caused the mutation.
Mechanism 1
In this mechanism, the deletion resulted from the formation of a
quasipalindromic stem-loop structure in the sequence of the normal
DNA (Fig. 2B). This stem-loop structure is similar to those reported
for other deletions suspected of involving formation of palindromic
and quasipalindromic structures (14–16). The free energy of the
quasipalindromic structure is –4.2 kcal/mol (17–19).
Following the formation of the quasipalindrome, replication
proceeded along the template (3′→5′), bypassing the stem-loop
structure and resulting in the deletion of the 27 nucleotides of the
quasipalindrome (boxed in Fig. 2B). Replication then continued
along the template incorporating the sequence of the insertion
(Fig. 2B).
We predict that the polymerase dissociated at either the second
-GAG or -AGA (bold) of the template (Fig. 2B). In fact, studies
have shown that polymerase α arrest or dissociation sites are
GC-rich regions, with the best site being -GAG (20). The
trinucleotide AGA, although not as good a dissociation site, is
high on the list of sequences that can result in dissociation. We
propose that the polymerase dissociated from the template at this
point. The stem-loop structure then relaxed and the template
slipped forward. The polymerase, with the newly synthesized
strand ending in -TCT (assuming dissociation occurred at -AGA),
In the second mechanism, we propose that transcription
proceeded through the first -CAGAC (bold) of the template
(Fig. 3B). The polymerase then dissociated from the template at
this -GAC, a trinucleotide that is also favorable for dissociation
(20). The -GTCTG (bold) of the newly synthesized strand then
reannealed to the downstream complementary -CAGAC
sequence (bold in Fig. 3B), which is further stabilized by the
formation of the quasipalindromic structure (Fig. 3C) with a free
energy of –4.7 kcal/mol (17–19). Replication continued to the
second -AGA and then as described in mechanism 1 (Fig. 3D).
The stem-loop relaxed, the polymerase and the newly synthesized
strand dissociated, then reassociated, and resulted in the sequence
of the mutation shown in Figure 3E.
DISCUSSION
Deletions, insertions, and complex deletions/insertions occur with
surprising frequency in human genetic disease (14,15,21–25).
Various mechanisms have been proposed to explain how these
mutations arose. In general, they involve either slipped mispairing
through the formation of various secondary structural intermediates
or strand-switching or base misincorporation (21). Some deletions
have been hypothesized to have resulted from the formation of
palindromic or quasipalindromic cruciform (hairpin or stem-loop)
structures (14,15,26). In the case of deletion/insertion mutations,
the origin of the insertion is usually unknown and the mechanism
of its formation is unexplained.
Although the quasipalindromic structure has a negative free
energy in both mechanisms, it is not usually expected to form a
stable structure. This is supported by our findings that this mutation
has only been observed in one of 58 patients with profound
biotinidase deficiency studied (Pomponio, Norrgard and Wolf,
unpublished data). It is, however, important that the hypothesized
conformation existed for only a short time and at one moment in
time. The nucleotide loop at the 3′-base of the stem-loop structure
is mildly strained and would not be expected to result in a stable
conformation. If the structure did produce a stable conformation,
such as occur in tRNAs, then the chances of mutation would be
greatly increased and would likely have occurred often.
Because there are repeated nucleotide sequences at the 5′-end
(GTCTG) and at the 3′-end (TCT) of the deleted sequence and
surrounding the downstream sequence that is copied in the
insertion, the actual size of the deletion and/or insertion is
impossible to determine. The proposed mechanisms allow us to
predict the origin of the bases in the mutation.
Formation of the secondary structure in the lagging strand is
more likely than in the leading strand, because the lagging strand
template becomes transiently single-stranded at the replication
fork (27). As suggested by Bissler et al. (15), the cruciform
structure may be stabilized when the RNA segment at the
Okazaki fragment is replaced by the polymerase. The locations of
the replication origins in the BTD gene, and thus the identity of
leading and lagging strands in this region are not known.
We cannot definitively exclude other possibilities, such as a
double crossover or gene conversion, as mechanisms that resulted
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Figure 2. Proposed mechanism 1 for the formation of the deletion/insertion mutation. (A) Normal double-stranded sequence of exon D of the BTD gene that is involved
in the mutation. The net deleted sequence (dashed underline), the net inserted sequence (underline), and the repeated flanking sequences (bold) are indicated. (B) The
quasipalindromic stem-loop structure is formed. Replication proceeded along the template until DNA polymerase α (DNA POL α; represented above the newly
replicated strand), paused and dissociated at the second -AGA (bold). (C) The hairpin loop relaxed and the template slipped forward allowing the polymerase to reanneal
to the first -AGA (bold). (D) Replication then continued along the template. (E) The resulting 5′→3′ sequence of the strand with the mutation of patient 20. The repeated
sequences are double underlined.
in the mutation described here. However, the above mechanisms
precisely encompass available experimental data supporting
palindromic formation and predict the site of polymerase α
dissociation. Undoubtedly, other mutations that occur in
eukaryotes and cause diseases in humans will be found to occur
through similar mechanisms.
MATERIALS AND METHODS
Subjects
The child (designated patient 20) is an 18 month old Hispanic
male. At 1 month of age he exhibited shaking of the extremities,
fluttering eyelids, foaming at the mouth, and staring. He
subsequently had multiple similar intermittent episodes which
were controlled with phenobarbital. Serum bicarbonate
concentrations were 15 and 16 mg/dl. An EEG was abnormal with
high amplitude sharp and slow wave activity bilaterally beginning
in the right central region. There was also intermittent asynchrony
of activity between the hemispheres. Magnetic resonance
imaging of the brain was normal. Newborn screening for
biotinidase deficiency indicated that the child was affected.
Multiple attempts to locate the child were unsuccessful. At 2
months of age, he was admitted to the hospital for seizures and
biotinidase deficiency testing was performed. His serum
biotinidase activity is 0.17 nmol/min per ml (mean normal activity
is 7.1 nmol/min per ml; range is 4.4–12) (5), confirming that he had
profound biotinidase deficiency (less than 10 % of mean normal
serum biotinidase activity) (28). The child’s serum contained no
cross-reacting material to antibodies prepared against normal
serum biotinidase purified to homogeneity (28). There was also no
biotinyl-transferase activity detected in his serum (29). He was
subsequently treated with biotin and his seizures stopped within
hours. With continued oral biotin supplementation, he has had no
more seizure activity and is developmentally normal.
1660 Human Molecular Genetics, 1996, Vol. 5, No. 10
Figure 3. Proposed mechanism 2 for the formation of the deletion/insertion mutation. (A) Normal double-stranded sequence of exon D of the BTD gene that is involved
in the mutation. The net deleted sequence (dashed underline), the net inserted sequence (underline), and the repeated flanking sequences (bold) are indicated. (B)
Replication proceeded to the first -CAGAC (bold), where DNA polymerase α (DNA POL α) arrested, dissociated and reannealed to the downstream CAGAC (bold).
(C) Reannealing is concomitantly stabilized by the formation of the quasipalindromic stem-loop structure. Replication continued along the template until the second
-AGA (bold). The polymerase again arrested and dissociated. (D) The hairpin loop relaxed, the template slipped forward and is stabilized by pairing with the first
-CTCTG in the template. The polymerase then reassociated to the first -AGA (bold). Replication then continued along the template as described in mechanism 1 (Fig.
2). (E) The resulting 5′→3′sequence of the strand with the mutation of patient 20. The repeated sequences are double underlined.
The biotinidase activity of the father (designated 69) is
2.29 nmol/min per ml serum and the activity of the mother
(designated 68) is 2.47 nmol/min per ml serum. The family
history reveals that he is the only child of parents whose families
were both from the same town or region in Puerto Rico. The
mother was adopted at 3 years of age. The parents are unaware of
consanguinity.
Materials
Whole blood was obtained from the child with biotinidase
deficiency, his parents, and 33 individuals with normal serum
biotinidase activity. Lymphoblast cultures were established as
described previously (12). Genomic DNA was isolated from
lymphoblast cultures and whole blood samples using the Gentra
(Minneapolis, MN) Pure Gene DNA isolation kit according to the
manufacturer’s recommendations. The concentration of each
DNA sample was calculated from the optical density at 260 nm
and diluted to a concentration of 0.2 µg/µl.
Amplification of genomic BTD DNA
One µg of gDNA from each subject was amplified in a 50 µl
polymerase chain reaction (PCR) (30) using the previously
described amplification parameters (12) and oligonucleotide
primers 277.S (5′-AACGGACCCATCCCATAGT-3′) and 267.A
(5′-GTAAGTGCCATGTACTGT-3′) based on the cDNA sequence
of human biotinidase (10). The annealing temperature used for this
oligonucleotide pair was 58C.
Screening for mutations using single strand
conformation analysis (SSCA)
SSCA was performed on PCR-amplified products from the
patient, his parents, and individuals with normal biotinidase
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activity for this region of exon D of the genomic biotinidase gene
using previously published methods (12,31,32).
Sequencing analysis
Templates for sequence analysis of patient and normal gDNA
were prepared by non-radioactive PCR amplification of this
region of exon D [nucleotides 1059–1359 based on the published
cDNA sequence (10)] on using primers 277.S and 267.A using
the method described previously (12). Briefly, six separate 50 µl
PCR reactions and a negative control lacking DNA template were
prepared for each individual. Sequencing reactions were
performed using at least 200 ng of purified PCR product, 20 ng
of primer (either 277.S and 267.A), and fluorescently labeled Taq
DyeDeoxy terminator reaction mix (Applied Biosystems,
Foster City, CA) according to the manufacturer’s instructions.
Reactions were performed in a Perkin Elmer 9600 thermocycler
and DNA sequence was determined using a 373A Automated
DNA Sequencer (Applied Biosystems). Both strands were
sequenced on at least three separate occasions for verification.
Sequence editing was performed using Sequencher software,
version 3.0 (Gene Codes Corporation, Ann Arbor, MI).
Sequences of interest were screened for potential secondary
structure using the STEMLOOP function of the University of
Wisconsin/GCG program.
ACKNOWLEDGEMENTS
This work was supported by NIH grant HD48258 to B.W. We
thank Karen Norrgard, Kristin Fleischhauer, Jeanne Hymes, and
Greg Meyers for technical assistance and advice, and Dr Marilyn
Cowger and Cheryl Clow, RN, at the Children’s Hospital at
Albany Medical Center for providing samples and medical
history from this patient and his family.
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