Human Reproduction, Vol.24, No.5 pp. 1229– 1236, 2009
Advanced Access publication on January 30, 2009 doi:10.1093/humrep/den500
ORIGINAL ARTICLE Reproductive genetics
Heterogenous spectrum of CFTR gene
mutations in Indian patients with
congenital absence of vas deferens
N. Sharma 1, N. Acharya2, S.K. Singh2, M. Singh 3, U. Sharma 1,
and R. Prasad 1,4
1
Department of Biochemistry, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India 2Department of
Urology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India 3Department of Pediatrics, Advanced
Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India
4
Correspondence address. Tel: þ91 172-2755178; Fax: þ91-172-2744401/2745078; E-mail: [email protected]
background: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause congenital bilateral absence
of vas deferens. Yet, the spectrum and frequency of CFTR mutations in Indian males with congenital absence of vas deferens (CAVD) is
unknown.
methods: We investigated 50 Indian males, diagnosed with unilateral or bilateral absence of vas deferens at the PGIMER, Chandigarh, for
the presence of the most common CFTR gene mutations as well as unknown mutations by single-strand conformation polymorphism followed by sequence analysis.
results: This study led to the identification of 12 CFTR gene mutations on 48% of 100 Indian CAVD chromosomes. CFTR mutations were
identified on both alleles in 11 patients (22%) and on one allele in 26 patients (52%). Novel CFTR mutations identified were L69H, F87I,
G126S, F157C, E543A, Y852F and D1270E. The T5 allele (25%) and F508del (11%) were the most common mutations identified. The
most common intragenic marker haplotype for F508del was 2111 (GATT, TUB9, M470V and T854T). No mutations could be detected
in 13 CAVD patients (26%), including 4 with renal malformations.
conclusions: This study confirms the molecular heterogeneity of CFTR mutations in CAVD. Although the mutation detection rate is
indeed lower in Indian CAVD patients, 74% of the patients tested had at least one CFTR mutation. CAVD alleles with no mutations suggest
that other changes may be located at the non-screened sites that require extensive search by direct sequencing. Furthermore, the novel CFTR
mutations identified require functional studies in a cell-based system.
Key words: congenital absence of vas deferens / cystic fibrosis / CFTR / F508del / single-strand conformational polymorphism
Introduction
Congenital bilateral absence of vas deferens (CBAVD) occurs in 1–2%
of infertile but otherwise healthy men (Holsclaw et al., 1971). It also
accounts for as much as 25% of infertile males with obstructive azoospermia (Patrizio and Salameh, 1998). CBAVD is present in more than
95% of cystic fibrosis (CF) males. Different studies have shown a high
frequency of cystic fibrosis transmembrane conductance regulator
(CFTR) gene mutations in CBAVD patients (Costes et al., 1995;
Dork et al., 1997; Mak et al., 1999; Casals et al., 2000; Dayangac
et al., 2004; Grangeia et al., 2004). In intron 8 of the CFTR gene, the
T5 allele, in contrast with the other two alleles, T7 and T9, leads to
a high proportion of mRNA transcripts lacking exon 9. Consequently,
the T5 variant produces abnormally low levels of CFTR protein. The
T5 variant is the most frequent mutation associated with the CBAVD
phenotype (Chillon et al., 1995).
Low frequencies of CFTR mutations have been detected in patients
with unilateral absence of vas deferens (CUAVD) (Casals et al., 1995;
Mickle et al., 1995). Between 11 and 26% of patients with congenital
absence of vas deferens (CAVD) have renal agenesis in association
with it (Schlegel et al., 1996), and initial negative results in the analysis
of CFTR mutations in these patients suggested that urogenital anomalies
have a different etiology to isolated CAVD (Augarten et al., 1994).
Little is known about the spectrum and frequency of CFTR gene
mutations in India. Our recent study as well as few other investigations
have provided evidence for an extensive allelic heterogeneity in Indian
patients with classic CF (Sharma et al., 2009; Shastri et al., 2008). Congential absence of vas deferens, as a distinct clinical entity with regard
& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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1230
to CFTR mutations, has never been investigated in our population. We
recently investigated the family history of infertility among CF subjects
and characterized mutations in infertile members (Sharma et al.,
2008). We here report the detailed investigation of CFTR mutation
genotypes in 50 Indian CAVD patients. We have identified CFTR
gene alterations that are distinct from other populations.
Sharma et al.
were subjected to automated DNA sequencing with forward and reverse
primers, using an ABI Prism BigDye Terminator Sequencing Ready Reaction
Kit (Perkin Elmer, USA) and a DNA sequencer ABI Prism (Model 3100,
Perkin Elmer). Nucleotide numbering was based on the A of the ATG translation start codon at position þ133 (GenBank NM-000492.2).
Detection of intragenic markers for haplotype framework
Materials and Methods
Patients and clinical evaluation
A total of 50 consecutive men with a diagnosis of CAVD were studied.
Healthy subjects (n ¼ 50) with no signs and symptoms of disease served
as controls. All samples were representative of the Indian population.
None of the men had been diagnosed with CF (Welsh and Smith, 1995).
In all cases, the initial evaluation included a scrotal examination and a
semen analysis. Overall, CBAVD was found in 40 men, while 10 men had
CUAVD which affected either the right (n ¼ 6) or left (n ¼ 4) side. Complete clinical data concerning infertility were obtained. The diagnosis of
CAVD was based on physical examination, when one or both vasa deferentia were non-palpable in the scrotal portion. Semen analysis included
volume, pH, sperm count and motility, in accordance with WHO guidelines
(World Health Organization, 1999). Concentration of fructose was
measured with commercial kits (FructoScreen, New York, NY, USA).
Hormone assays were done by chemiluminescence. Transrectal ultrasonography was performed for the morphology and size of the seminal vesicles,
prostate and ejaculatory ducts. Abdominal ultrasonography was performed
in order to evaluate the pelvis and the upper urinary tract. Testicular fine
needle aspiration cytology (FNAC) was carried out for studying the patterns
of spermatogenesis. Sweat chloride analysis was performed in all the subjects (Gibson and Cooke, 1959). None of the patient had a family history
of CF, and only patients who were apparently unrelated were included in
this study. Classic lung and gastrointestinal symptoms of CF were not documented in any of the subjects. Of the 50 subjects, 40 individuals had consulted for couple infertility. Female partners (n ¼ 40) of these infertile
subjects were analyzed only for the most common F508del mutation.
CFTR gene analysis
The study was approved by Institutional Ethics Committee in the Post Graduate Institute of Medical Education and Research, Chandigarh. DNA analyses
were performed after written informed consent. Genomic DNA was isolated
from whole blood following the method of Daly et al. (1996).
For CFTR mutation analysis, chromosomes were first screened for the presence of the F508del by ARMS PCR as described by Ferrie et al. (1992). We
next screened R117H, N1303K and R553X each by single ARMS PCR and
621 þ 1G-T, G542X, G551D and W1282X by multiplex ARMS PCR.
CFTR mutations investigated by restriction analysis of PCR products
were R334W (MspI), R347P (NcoI), A455E (AciI), 2789 þ 5 . G-A
(SspI), R1162X (DdeI) and 3849 þ 10 kb . C-T (HphI) as described previously (Dean et al., 1990; Highsmith et al., 1990; Kerem et al., 1990; Gasparini et al., 1991a, b).
The T5 variant in the polymorphic region IVS8-6(T) was analyzed as
described previously (Chillon et al., 1995). Bidirectional sequencing
(Applied Biosystems) of intron 8 of the CFTR gene was carried out to
determine TG repeat number and phase of TG-T repeat combinations.
The presence of mutations in the 27 exons and exon–intron boundaries of
the CFTR gene were assessed by single-strand conformational polymorphism
(SSCP) (Orita et al., 1989). To avoid the overestimation of the pathological
significance of the identified novel sequence variants, we used normal controls in each run to prevent over interpretation of SSCP patterns as abnormal.
The patients’ samples exhibiting shifts relative to normal samples on SSCP
The polymorphic markers, TUB9 (Dork et al., 1992), M470V and T854T
(Kerem et al., 1990), were determined by restriction digests of RFLP-PCR.
The GATT polymorphism (Gasparini et al., 1991a, b) was detected by the
difference in mobility of the PCR product on 12% polyacrylamide gels. Haplotypes were assigned according to the standard nomenclature. The absence
of the restriction site for each enzyme was indicated as allele 1 and the presence of the site as allele 2. For GATT polymorphism, alleles 1 and 2 denoted
for seven and six GATT repeats, respectively (Dork et al., 1992).
Results
Overall mutations identified
A total of 12 mutations were identified on 48 of 100 alleles, as listed in
Table I. Two mutations were found to be very common: first, the
IVS8-T5 allele was observed on 25 chromosomes; thus confirming the
association of this splice site variant with CAVD in Indian patients. Secondly, the F508del mutation was detected in 11 chromosomes. In the
CAVD patients with normal renal development, the initial screening identified one extra, R117H mutation in three chromosomes. SSCP analysis
performed in patients with only one or no mutation revealed nine
further mutations on one allele each including seven new sequence alterations: L69H, F87I, G126S, F157C, E543A, Y852F and D1270E (Table I).
Other identified mutations R117H, 3120 þ 1 . G-A and P1021S have
been described previously in studies of patients with CAVD. None of
the other screened mutations were identified in our population.
Pathological predictions and multiple
sequence alignments of novel substitution
mutations
The output prediction scores (http://blocks.fhrc.org/sift/SIFT.html) for
L69H, E543A and D1270E were less than the 0.05 (threshold for pathological mutations) (Table II). Pathological predictions confirmed by
another computer algorithm (http://genetics.bwh.harvard.edu/pph)
revealed L69H, E543 and D1270E as deleterious mutations and other
four mutations, F87I, G126S, F157C and Y852F, as benign sequence
alterations (Table II). Human splicing finder matrices (http://www.
umd.be/HSF/HSF.html) predicted no difference in splicing between
the novel mutants and their respective wild-type reference sequences
(Table II). Multiple sequence alignment using ClustalW (http://align.
genome.jp/) confirmed that the wild-type form of all the novel sequence
variants, except F87I, was conserved across human, rhesus monkey,
bovine, sheep, horse, dog, pig, rabbit and mouse (Fig. 1).
Genotype and haplotype framework
of Indian CAVD patients
Two allelic mutations were identified in 11 (22%) cases of which 5
were homozygous for IVS8-T5 (Table III). Monoallelic mutations
were found in 26 (52%) cases. We failed to identify mutations in
either allele in 13 (26%) cases. Since the disease penetrance of T5 is
1231
CFTR mutations in Indian CAVD patients
Table I CFTR mutations identified and characterized in the Indian patients with CAVD
Mutation
Nucleotide change
Consequence
Exon/intron
No. of alleles
.............................................................................................................................................................................................
T5
Reduction of oligo T tract to 5T at 1342-6
Aberrant splicing
Intron 8
25
F508del
Deletion of 3 bp (CTT or TTT) between 1652 and 1655
Deletion of phenylalanine at 508
Exon 10
11
L69Ha
T to A at 338
Leucine to histidine at 69
Exon 3
1
F87I
T to A at 391
Phenylalanine to isoleucine
Exon 3
1
R117H
G to A at 482
Arginine to histidine at 117
Exon 4
3
G126Sa
G to A at 508
Glycine to serine at 126
Exon 4
1
F157Ca
a
T to G at 602
Phenylalanine to cystine at 157
Exon 4
1
a
E543A
A to C at 1760
Glutamate to alanine at 543
Exon 11
1
Y852Fa
A to T at 2687
Tyrosine to phenylalanine at 852
Exon 14a
1
3120 þ 1 G-A
G to A 3120 þ 1
Aberrant splicing
Intron 16
1
P1021S
C to T at 3193
Proline to serine at 1021
Exon 17a
1
T to A at 3942
Aspartate to glutamate at 1270
Exon 20
1
a
D1270E
Total chromosomes: 100; known mutations: 48%; unknown mutations: 52%.
a
Indicates novel substitution mutations.
Table II Pathological prediction and splicing defect of novel substitution mutations identified in Indian CAVD patients
Wild-type
Mutant
Position
Score
Pathological consequence
Splicing defect
.............................................................................................................................................................................................
L
H
69
0.00
Possibly damaging
No
F
G
I
87
0.16
Benign
No
S
126
0.29
Benign
No
F
C
157
0.22
Benign
No
E
A
543
0.02
Probably damaging
No
Y
F
852
0.16
Benign
No
D
E
1270
0.01
Probably damaging
No
SIFT (http://blocks.fhrc.org/sift/SIFT.html) was used to generate the output scores of novel sequence variants, pathological predictions were confirmed by polyphen (http://genetics.
bwh.harvard.edu/pph) and potential effect on splicing by human splicing finder (http://www.umd.be/HSF/HSF.html). Threshold for pathological mutations was 0.05.
affected by its adjacent polymorphic TG repeats, it was found that T5
on one or both alleles (n ¼ 25) was associated with either (TG)11
(n ¼ 4), (TG)12 (n ¼ 14) or (TG)13 (n ¼ 7). F508del was mostly
found in patients carrying the (TG)10T9 haplotype. Rare or undescribed haplotypes (TG)13T9 (n ¼ 1) or (TG)13T7 (n ¼ 1) were
also identified in Indian CAVD chromosomes. Haplotype studies
including (GATT)n, TUB9, M470V and T854T were not found relevant
for identified CFTR mutations, except for F508del. The most common
intragenic marker haplotype for F508del was 2111 (72.27%), with the
order of polymorphism being (GATT)n, TUB9, M470V and T854T
(Table III).
T5 and T9 alleles were significantly more frequent in the chromosomes of the patients with CBAVD (26 and 31%, respectively) and
CUAD (20 and 45%, respectively) compared with control subjects
(5 and 5%, respectively) (Table IV). Further, (TG)12 was frequent in
both CBAVD (46%) and CUAD (45%). However, (TG)11 was predominant in control subjects (68%). (TG)10 and (TG)13 were rare (1%)
in control subjects in contrast to CBAVD and CUAD patients where
its frequency was more than 10% (Table IV).
Clinical phenotype
Of 50 patients with CAVD, 40 had CBAVD and 10 had CUAVD
(Table V). Unilateral renal agenesis was found in one CBAVD and
three CUAVD subjects. Both CBAVD and CUAVD patients with the
absence of kidney presented no CFTR mutations. Unilateral absence
of seminal vesicles (n ¼ 5) typically on the same side of the absent
vas deferens predominated in CUAVD, whereas bilateral dysplasia
(n ¼ 2) was found only in CBAVD (Table V). There were no associations between the mutations detected and biochemical variables
studied (data not shown). However, median sweat chloride was
higher in CBAVD compared with CUAVD subjects. Further, semen
volume, pH, fructose and citrate contents were significantly lower
(P , 0.05) in CBAVD compared with CUAVD subjects (Table V).
None of the patients showed pulmonary or gastrointestinal symptoms
of CF, but repeated respiratory infections or bronchitis and raised
sweat chloride were documented in a CBAVD patient with
F508del/G126S genotype. A compound heterozygous, R117H/F87I,
genotype was identified in a CBAVD subject with normal sweat
chloride and no CF-like symptoms.
1232
Sharma et al.
Figure 1 Multiple alignments of CFTR amino acid sequences from different species (human, rhesus monkey, bovine, sheep, pig and mouse) and
seven novel substitution mutations (L69H, F87I, G126S, F157C, E543A, Y852A and D1270E) identified in Indian CAVD patients. Amino acids in
box indicate alignment across different species. Full-length CFTR sequences of different species were obtained from UCSC genome browser
(http://genome.ucsc.edu/).
Discussion
In Indian infertile men with CAVD, the initial screening for the most
common CFTR mutations led to the identification of 39% alleles.
Further, SSCP analysis detected 9% of the CAVD alleles with at
least one CFTR mutation detected (Table I). The present report is
of one of the lowest frequencies of CFTR mutations in comparison
with most other countries (71 –82%), such as the USA (Oates and
Amos, 1994), Germany (Dork et al., 1997), France (Claustres et al.,
2000), Greece (Kanavakis et al., 1998) and Spain (Casals et al.,
2000). SSCP is simple and easy to set up, but its sensitivity depends
on PCR fragment size. More than 90% of the sequence variations
can be detected for most fragments shorter than 200 nucleotides,
but about 80% for 300 –350 nucleotides PCR fragments (Hayashi
and Yandell, 1993). In the present study, the primers generated
PCR products mostly above 250 base pairs which may have contributed to the decreased number of mutations detected. Sensitivity is
also dependent on electrophoretic conditions such as temperature,
ionic strength, composition of the gel and glycerol concentration. Previously, we standardized all parameters for each CFTR exon to optimize the sensitivity of SSCP (Sharma et al., 2009). Failure to detect
mutations in about 50% of CAVD alleles could be indeed due to
the presence of other defects in introns or the promoter region of
CFTR, but also to the presence of large rearrangements such as
exon deletions, insertions or duplications, which have already been
found in CBAVD patients (Hantash et al., 2006; Ratbi et al., 2007;
Taulan et al., 2007).
Intriguingly, among the seven novel substitution mutations identified, L69H, E543A and D1270E were predicted to be damaging,
whereas F87I, G126S, F157C and Y852F were possibly neutral
(http://blocks.fhcrc.org/sift/SIFT.html and http://genetics.bwh.
harvard.edu/pph/). It is noteworthy that L69H has been previously
identified on one allele of the Indian classic CF patient (Sharma
et al., 2009). The consequences of exonic alterations are not necessarily restricted to the protein level that is the subject of the SIFT and
polyphen programmes used. Some variants may already be operational at the transcript level. But, the evaluation of potential effect
on splicing (http://www.umd.be/HSF/HSF.html) predicted that
none of these novel substitution mutations have an effect on splicing.
Missense mutations of conserved sequences have been reported to
have pathological significance in CBAVD patients (Grangeia et al.,
2008). Since F87I mutation is not conserved across various species
(http://align.genome.jp/), it implies that it is a rare neutral sequence
variation rather than a disease-causing mutation. Other novel
mutations (G126S, F157C and Y852F) have been identified as possibly
neutral, but are aligned across different species. Furthermore, these
mutations have not been identified in the Indian classic CF patients
(Sharma et al., 2009; Shastri et al., 2008), and not finding a specific
variant in the 100 normal chromosomes tested indicates that the
variant has a frequency of ,5% (Collins and Schwartz, 2002).
Nevertheless, further functional studies are required for their pathological significance.
In CBAVD patients, a high frequency of compound heterozygosity
with severe/mild or mild/mild mutations has been reported
1233
CFTR mutations in Indian CAVD patients
Table III CFTR genotypes in Indian patients with CAVD
Mutation genotype
IVS8-(TG)mTn
GATTn
TUB9
M470V
T854T
No. of patients (%)
.............................................................................................................................................................................................
Two mutations detected
22%
IVS8-T5/IVS8-T5
5 (10)
(TG)12T5/(TG)12T5
2/2
2/2
1/1
1/1, 1/2
(TG)12T5/(TG)13T5
1/1
2/1
2/1 (1), 2/2 (1)
1/1 (1), 1/2 (1)
2 (4)
(TG)11T5/(TG12)T5
1/2
1/1
2/2
2/2
1 (2)
IVS8-T5/F508del
(TG)13T5/(TG10)T9
2/2
1/1
1/1
1/1
1 (2)
IVS8-T5/R117H
(TG)12T5/(TG)12T7
1/1
2/2
2/2
1/1
1 (2)
IVS8-T5/Y852F
(TG)12T5/(TG)12T7
1/1
1/2
2/1‘
1/2
1 (2)
IVS8-T5/D1270E
(TG)12T5/(TG)12T9
1/1
1/1
2/2
2/2
1 (2)
F508del/G126S
(TG)10T7/(TG)11T7
2/2
1/1
1/1
1/1
1 (2)
R117H/F87I
(TG)12T7/(TG)12T7
1/1
2/1
2/2
1/2
1 (2)
One mutation detected
2 (4)
52%
F508del/?
9 (18)
(TG)10T9/(TG)12T7
2/2
1/1
1/1
1/1 (3), 1/2 (1)
4 (8)
2/2
1/1
1/1
2/2
1 (2)
2/2(1), 2/1(1)
1/1
1/1
1/1 (1), 1/2 (1)
2 (4)
(TG)10T7/(TG11)T9
2/2
1/1
1/1
2/2
1 (2)
(TG)10T9/(TG)13T7
2/2
1/2
1/1
2/2
1 (2)
L69H/?
(TG)11T7/(TG13)T9
1/1
1/2
1
2
1 (2)
F157C/?
(TG)11T7/(TG)11T9
1/1
1
2
2
1 (2)
E543A/?
(TG)12T7/(TG)12T7
1/1
2
1
2
1 (2)
R117H/?
(TG)11T7/(TG)12T7
1/1
2
1
2
1 (2)
P1021/?
(TG)12T7/(TG)10T9
1/2
2
1
2
1 (2)
3120 þ 1G-A/?
(TG)10T7/(TG)11T7
2/2
1
2
1
IVS8-T5/?
1 (2)
11 (22)
(TG)12T5/(TG)12T7
(TG)13T5/(TG)11T9
(TG)11T5/(TG)12T7
1/1
1/1 (1), 1/2 (1)
2/2
1/2 (1), 2/2 (1)
2 (4)
1/1
2/2
2/2
1/1
2 (4)
1/1 (1), 1/2 (1)
1/2
1/1
2/2
2 (4)
1/1
1/1 (1), 1/2 (1)
2/2
2/2
2 (4)
2/2
2/2
1/1
2/2
1 (2)
1/1
2/2
2/2
1/2
1 (2)
2/2
2/2
2/2
1/2
No mutation detected
1 (2)
26%
The IVS8-(TG)mTn haplotypes were named by the number of upstream TG dinucleotides followed by the number of consecutive thymidines (Groman et al., 2004). The TUB9, M470V and
T854T were designated as 1 for the absence and 2 for the presence of restriction site (Dork et al., 1992). Alleles 1 and 2 denoted for seven and six GATT repeats, respectively. ? denotes
unknown mutation.
(Anguiano et al., 1992). In the Indian CBAVD population, F508del
mutation was 11%, an allelic frequency similar (12 –14%) to Greece
(Kanavakis et al., 1998) and Portugal (Grangeia et al., 2004), but
lower than that found (21– 33%) in Canada (Jarvi et al., 1998), USA
(Oates and Amos, 1994), Germany (Dork et al., 1997), France
(Claustres et al., 2000) and Spain (Casals et al., 2000). In the Indian
CBAVD population, 74% of the patients had at least one CFTR or
T5 mutation; 22% had two mutations and 52% had only one mutation,
whereas 26% had no mutation detected (Table III).
In CBAVD patients, one of the most frequent genotypes found is
the combination of the T5 allele with other CFTR exonic mutations
(Chillon et al., 1995). This suggests that the vas deferens is the most
susceptible tissues to the effect of the changes in CFTR activity
(Anguiano et al., 1992). In Indian CAVD patients, there were 40%
of cases with T5 allele (Table III), having an allele frequency of 25%.
A (TG)12T5 or (TG)13T5 CFTR gene found in compound heterozygosity with a CF-causing mutation, or possibly even in homozygosity,
in general results in CAVD (Castellani et al., 2008). From a genetic
diagnostic standpoint, CFTR mutations have been divided into
(i) CF-causing disorder; (ii) CFTR-related disorder; (iii) no clinical consequence and (iv) unproven clinical relevance (Dequeker et al., 2009).
In the present study, the absence of vas deferens as CFTR-related disorder was confirmed in cases with one CFTR mutation and T5 allele
associated with (TG)12 or (TG)13 (Table III). However, it was not
1234
Sharma et al.
confirmed in cases with 0 –1 CFTR mutation or T5. Since family studies
have not been performed, it cannot be precisely determined at this
point whether patients carrying two CFTR mutations are true compound heterozygotes, especially in cases where novel mutations
could be in cis with the T5 (Table III).
The presence of mutation on more than one haplotype background
in a particular geographical region indicates a longer history of the
mutation that has allowed recombination events (Raskin et al.,
1997). The linkage disequilibrium data between CF and the dimorphic
extragenic B haplotypes (1/2, XV2c-KM19) in different European
populations are compatible with a relatively more recent appearance
of the mutation in Northern Europe, whereas in Southern Europe a
longer history of the same mutation would have allowed time for
recombination with other haplotypes (Claustres et al., 1996). In corroboration, we observed 2111 as the most common intragenic
marker haplotype for F508del, the order of four intragenic
Table IV Frequencies (and percentages) of the poly T
alleles and TG repeats in intron 8 of CFTR in Indian
CAVD subjects
Alleles
CBAVD
(n 5 40)
CUAVD
(n 5 10)
Control subjects
(n 5 50)
........................................................................................
T5
21 (26.25)
4 (20)
5 (5)
T7
34 (42.5)
7 (35)
90 (90)
T9
25 (31.25)
9 (45)
5 (5)
(TG)10
14 (17.5)
2 (10)
1 (1)
(TG)11
19 (23.75)
7 (35)
68 (68)
(TG)12
37 (46.25)
9 (45)
30 (30)
(TG)13
10 (12.5)
2 (10)
1 (1)
polymorphic sites being GATT, TUB9, M470V and T854T (six
GATT repeats, nucleotide G at TUB9 and M470V, and nucleotide T
at T854T). Thus, we predict a more recent appearance of delta
F508 in India. Haplotype studies were not found relevant for other
identified CFTR mutations. Further studies demand investigation into
the selection of these mutations in our population. In the present
series, one couple showed F508del mutation on either allele (data
not shown). It confirms previous reports (Kapoor et al., 2006;
Sharma et al., 2008, 2009) that carrier frequency is expected to be
high among Indians. Proper counseling was given to the couple of
having a 25% chance of giving birth to a full blown CF child, if entering
assisted reproduction techniques. Genetic counseling is especially difficult in CAVD subjects having a severe allele (F508del) on one loci
and the other allele being uncharacterized or having a sequence
variant with unknown pathological significance. There was no correlation between the CFTR mutation and development of either unilateral or bilateral vas deferens (Table V). It leads us to assume that it
is not just the CFTR mutations per se but there are certain modifiers
genes or environment that collectively determines the clinical phenotype. Although, we and other investigators (Anguiano et al., 1992;
Augarten et al., 1994) did not find any CFTR mutations in CAVD subjects with renal agenesis, Casals et al. (2000) reported that about
one-third of the patients with CAVD and renal agenesis had CFTR
mutations. We attribute this conflicting results to the reduced
number of samples in our and previous studies compared with the
latter report. It is suggested that studying genome-wide data substantiated with complete analysis of CFTR can provide convincing correlation and better counseling of the patients.
Taken together, we have characterized the type and frequency of
CFTR mutations and of IVS8 poly (T) variants in Indian CAVD males
with the absence of CF clinical symptoms. Identification of 7 novel
mutations out of 12 reflects the molecular heterogeneity of CFTR
mutations in CAVD and emphasizes the importance of extensive
Table V Clinical variables in Indian patients with CAVD
Clinical variable
CBAVD (n 5 40)
CUAVD (n 5 10)
.............................................................................................................................................................................................
Median age (years)
31.9
28.1
Median sweat chloride (mEq/l)
67.1 + 9.1
58.1 + 9.2
Semen analysis
Volume
0.7 + 0.2
1.5 + 0.2
pH
6.4. + 0.04
7.0 + 0.1
Sperm concentration (106)
0
0.8 + 0.6
Fructose (mmol/ejaculate)
4.8 + 1.1
18.7 + 4.9
Citrate (mmol/ejaculate)
58.3 + 12.1
88.5 + 11.3
Normal spermatogenesis
Normal spermatogenesis
Testosterone (ng/dl)
432.7 + 266.2
464.2 + 71.1
FSH (mIU/ml)
4.07 + 2.02
4.1 + 1.9
LH (mIU/ml)
5.19 + 2.32
5.01 + 2.03
Absence of seminal vesicle
2
5
Renal agenesis
1
3
FNAC
Hormone assays
CBAVD, congenital bilateral absence of vas deferens; CUAVD, congenital unilateral absence of vas deferens.
CFTR mutations in Indian CAVD patients
CFTR analysis in Indian population. The study also demands further
investigation into the functional aspects of novel variants in the
cell-based system to predict their pathological significance.
Acknowledgements
The authors are thankful to Indian Council of Medical Education and
Research, New Delhi for awarding senior research fellowship (vide
letter no. 45/3/2006-Hum/BMS).
Funding
This is a part of research project funded by Department of Science
and Technology, New Delhi, India (DST No.SR/SO/HS-43/2002).
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Submitted on September 12, 2008; resubmitted on December 16, 2008; accepted
on December 28, 2008