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From the * Centre for Cellular and Molecular
Biology, Hyderabad, India; the Institute of
Reproductive Medicine, Kolkata, India; and the
Infertility Institute and Research Centre,
Hyderabad, India.
| Correspondence to: Dr K. Thangaraj, Centre for Cellular and Molecular Biology, Uppal Rd, Hyderabad 500 007, India (e-mail: thangs{at}ccmb.res.in). |
| Received for publication March 9, 2009; accepted for publication June 16, 2009. |
Apolipoprotein B (APOB) plays a key role in lipoprotein metabolism and
plasma lipid transport. It has been shown that about two-thirds of male mice
heterozygous for ApoB were infertile. Moreover, a 3-codon deletion
polymorphism (rs11279109) in the signal peptide region of the APOB
gene has been shown to be a risk factor for infertility in Slovenian men, but
its association with infertility in Indian men has not been evaluated to date.
Hence, in the present study, we have genotyped this polymorphism in 545 Indian
men, including 294 infertile and 251 fertile men. Our results show that the
distribution of this deletion polymorphism was consistent with the
Hardy-Weinberg equilibrium in both infertile and fertile men. No statistically
significant difference was observed in the distribution of the APOB
signal peptide deletion polymorphism between infertile and fertile men
(
2 = 0.156, P = .925 for genotypes; 2
= 0.015, P = .903 for alleles). Moreover, no significant difference
was observed when infertile and fertile men were categorized on the basis of
presence (D/D and D/W genotypes) or absence (W/W genotypes) of deletion (odds
ratio, 0.955; 95% confidence interval, 0.644–01.418; P = .820).
Our study concludes that the APOB gene deletion polymorphism is not a
risk factor for the development of infertility in Indian men.
Key words: Spermatogenesis, apolipoprotein B
The B apolipoproteins APOB-100 and APOB-48 are derived from a common structural gene. They play central roles in lipoprotein metabolism and plasma lipid transport (Young, 1990; Kim and Young, 1998). APOB-100 is essential for the assembly of very–low-density lipoprotein in the liver, whereas APOB-48 is required for chylomicron formation in the intestine. Knockout studies on ApoB gene deletion have shown severely compromised fertility in ApoB heterozygous male mice (Huang et al, 1995, 1996), whereas another study found ApoB heterozygous male mice to be fertile (Farese et al, 1995). A 3–amino acid deletion polymorphism in the signal peptide region has been proven to cause inefficient translocation of the APOB protein (Sturley et al, 1994; Benhizia et al, 2001). The presence of the deletion variant resulted in a 66% reduction in endoplasmic transport compared with proteins containing the wild-type signal peptide (Sturley et al, 1994). Recent studies have associated this deletion polymorphism with oligoasthenoteratozoospermia in Slovene white men (Peterlin et al, 2006). Currently, there is no available report on the association of this polymorphism with infertility in Indian men, and studies on mouse models have yielded a controversial interpretation of the role of the ApoB gene in spermatogenesis. Our study evaluated the possibility of association of the APOB gene deletion polymorphism with infertility in groups of infertile and fertile Indian men.
Materials and Methods
Subjects and Controls
Blood samples were collected from men attending the Institute of
Reproductive Medicine (Kolkata, India) and the Infertility Institute and
Research Centre (Hyderabad, India). Informed written consent was obtained from
all participants, and permission to proceed was granted by the Institute
Ethics Committee. A total of 294 infertile men who did not exhibit any known
medical or genetic cause of infertility, such as obstruction, injury,
endocrinologic defect, or karyotypic abnormality, were included in the study.
Men who exhibited deletion or mutation in our previous studies on the analysis
of Y-chromosome microdeletions (Thangaraj
et al, 2003a; and Thangaraj et al, unpublished results),
DAZ copy deletions (Rani et al, unpublished results), and
mitochondrial ND4 gene analysis
(Selvi Rani et al, 2006) were
excluded. A total of 294 infertile men were further identified as 1 of 3
subtypes in accordance with World Health Organization criteria
(World Health Organization,
2000): 137 azoospermatic (Azoo; no sperm in ejaculate), 55
oligoasthenozoospermatic (OA; <20 x 106/mL sperm
concentration and <50% motile sperm), and 102
oligoasthenoteratozoospermatic (OAT; <20 x 106/mL sperm
concentration, <50% motile sperm, and <30% sperm with normal
morphology). A total of 251 ethnically matched men who, in accordance with WHO
criteria (World Health Organization,
2000), exhibited normal semen parameters (25 x
106/mL to 130 x 106/mL sperm concentration,
>50% motile sperm, and >30% with normal morphology) and possessed normal
levels of inhibin B, testosterone, leutinizing hormone, and
follicle-stimulating hormone were included in this study as controls. In
addition, every fertile man in the control group had fathered at least one
child. Paternity was verified using short tandem repeat–based DNA
fingerprinting (AmpFlSTR Identifiler kit; Applied Biosystems, Foster
City, California).
Genotyping of APOB Deletion
To genotype the 3-codon deletion polymorphism (rs11279109) in the signal
peptide region of the APOB gene, we designed and custom synthesized
(Sigma-Proligo; Proligo Singapore Pty Ltd, Singapore, Singapore) primers as
described previously (Peterlin et al,
2006). The sequences of the primers used were forward primer
5'-FAM-CTCCTCAGCCCCTCCATC-3', and reverse primer
5'-GAGTGCCCTTCTCGGTTG-3', which amplified polymerase chain
reaction (PCR) products of 366/375 bp. PCR for each sample was performed in
0.2-mL, thin-walled tubes using 2.0 ng of DNA, 2 to 5 pm of each primer, 200
µM dinucleotide triphosphates, 10x PCR buffer, 1.5 mM
MgCl2, and 0.5 units of AmpliTaq Gold (Applied Biosystems). Because
the guanine cytocine content of the amplified region is very high (76.8%), 20%
(v/v), 5M Betaine (Sigma-Aldrich, St Louis, Missouri) was added as a PCR
enhancer. The PCR reaction was carried out in a GeneAmp 9700 Thermal Cycler
(Applied Biosystems) under the following conditions: 96°C for 10 minutes,
35 cycles at 96°C for 45 seconds, 66°C for 30 seconds, and 72°C
for 1 minute, and a final extension at 70°C for 7 minutes. Amplified
products were analyzed in an ABI 3700xl DNA Analyzer (Applied
Biosystems) using GeneScan. For GeneScan, 0.5 µL of PCR product was mixed
with 0.2 µL of GeneScan-500 LIZ Size Standard (Applied Biosystems) and 9.3
µL of HiDi Formamide (Applied Biosystems). Raw data were analyzed using
GeneMapper Software, version 4.0 (Applied Biosystems). To reconfirm the
results, we sequenced approximately 80% samples. Sequencing samples were
reamplified using primers without florescent tags (FAM), and the sequencing
reaction was performed as described previously
(Khattri et al, 2009).
Statistical Analysis
Through statistical analysis, the risk of infertility in relation to the
APOB gene signal peptide deletion polymorphism was estimated using
the Statistics Package for Social Sciences, version 16 (SPSS Inc, Chicago,
Illinois). In a 2 test for association, odds ratios at their
95% confidence intervals were calculated, and P < .05 was
considered significant. The online resource facility of the Institute for
Human Genetics (Munich, Germany;
http://ihg2.helmholtz-muenchen.de/ihg/snps.html)
was used to test for deviation of the Hardy-Weinberg equilibrium and
evaluation of different models of association.
Results
PCR amplification of deletion polymorphism rs11279109 in the signal peptide
region of the APOB gene resulted in products of 375 bp (wild type
[W]) or 366 bp (deletion [D]), which were confirmed by sequencing
(Figure 1a) and GeneScan
(Figure 1b). Analysis of the
deletion polymorphism revealed that 24.3% of fertile and 23.47% of infertile
men carried the deletion (D/D and D/W) genotypes
(Table;
Figure 2). This deletion
polymorphism was in Hardy-Weinberg equilibrium in both infertile and fertile
men. The heterozygosity index of this locus in our population was 0.213. No
significant difference was observed when distribution of genotypes or alleles
was compared between fertile and infertile men (2 = 0.156,
P = .925 for genotypes; 2 = 0.015, P = .903
for alleles). Furthermore, no significant difference was observed when any of
the individual patient subgroups (Azoo, OAT, and OA) was compared either with
another subgroup or with fertile men, or when categorizing fertile and
infertile men (and their subgroups) based on the presence (D/D and D/W) or
absence (W/W) of deletion
(Table). The frequency of
individuals carrying the deletion was, however, increased in azoospermic
men.
|
|
|
Discussion
Sperm maturation involves a broad range of processes that occur during sperm migration through the seminiferous tubules and the epididymis, including the remodeling of sperm membrane components (De Vriese and Christophe, 2003). The plasma membrane of sperm plays a very active role in its fertilization capacity and in spermatozoon-oocyte cross-talk. Its biochemical constitution is one of the main fields of interest in the study of sperm physiology and pathology (Lenzi et al, 1996). Because APOB is one of the apolipoproteins that play an important role in lipoprotein metabolism, there is a potential role for APOB in male infertility, because this polymorphism alters the structure of the signal peptide of the gene central to lipid metabolism (Visvikis et al, 1990). Moreover, some other genes affecting lipid metabolism have been associated with infertility in knockout mice, such as the apolipoprotein E receptor-2 (Apoer2; Andersen et al, 2003), acid sphingomyelase (Asm; Butler et al, 2002), and adenosine triphosphate–binding cassette transporter 1 (Abca1; Selva et al, 2004) genes. A prior study (Peterlin et al, 2006) has found significantly increased proportions of D/D and D/W genotypes among OAT men in the Slovene white population, but we did not find any significant difference in the deletion frequency (D/D and D/W genotypes) between fertile and infertile men or subgroups of infertile men (Azoo, OA, and OAT). Rather, the frequency of deletion was slightly decreased in OAT (20.59%) and OA (20.20%) men (Table). Although the authors of the Slovenian study reported the association of deletion within OAT, it was marginally significant (P = .014; at 95% significance level, when Bonferroni correction applied: P < .05/2 = .025). The difference between these 2 studies could very well be explained by the ethnic difference of the study populations. The most notable difference is the frequency with which D/D and D/W deletion genotypes occur: 52.8% of fertile men carried a deletion in the population studied by Peterlin et al (2006), whereas only 24.4% carried a deletion in our study. This difference was not surprising to us, knowing the unique ethnic origin of Indian populations. Earlier, we contrasted the results of the studies performed on Indian populations compared with other populations; for example, CAG repeat expansion in the androgen receptor was reported to be associated with male infertility (Patrizio et al, 2001), but our study did not find an association (Thangaraj et al, 2002). Similarly, the A386G polymorphism in the DAZL gene was not associated with male infertility in Indian males (Thangaraj et al, 2006), but it was found to be associated with severe spermatogenic failure in the Taiwanese population (Teng et al, 2002). Similar patterns were observed in other genes involved in sex determination (Rajender et al, 2006), alcoholism (Bhaskar et al, 2007; Rao et al, 2007), mitochondrial disorders (Vanniarajan et al, 2006), and cardiomyopathies (Dhandapany et al, 2009).
This is the first study on Indian men and second study worldwide addressing the association of the APOB gene signal peptide deletion polymorphism with male infertility. Men with 1 of 3 different phenotypes of infertility (Azoo, OA, and OAT) were included in our study, but no association with the APOB signal peptide deletion polymorphism was detected in any of them. Our results suggest that the polymorphism is not associated with infertility in Indian men, although it has been shown to be associated with OAT in Slovene white men (Peterlin et al, 2006). Knockout mouse studies on the APOB gene also produced confusing results: two-thirds of male ApoB heterozygous mice were found infertile, despite normal genitourinary system function and mating behavior (Huang et al, 1995). Sperm from these heterozygous knockout males failed to fertilize eggs, either in vivo or in vitro. These sperm were able to fertilize eggs once the zona pellucida was removed, but they displayed persistent abnormal binding to the egg after fertilization. In vitro fertilization–related and other experiments revealed that reduced sperm count, motility, and survival time also contributed to the infertility phenotype. Furthermore, when the genomic sequence encoding human APOB was introduced into genetically engineered animals, normal fertility was restored (Huang et al, 1996). Contrary to the above studies, infertility was not reported in the study by Farese et al (1995) on APOB heterozygous male mice. Peterlin et al (2006) and our study have produced contrasting findings on the APOB gene signal peptide deletion polymorphism. Accordingly, we expect similar trends in other populations. Therefore, it is important to perform replicate studies on patients from diverse ethnic origins before either designating or excluding this polymorphism as a risk for male infertility.
Acknowledgments
We thank Dr Alkes Price, Assistant Professor, Harvard School of Public Health (Boston, Massachusetts) for going through the manuscript and editing it for English. This work is supported from the grant from the Indian Council of Medical Research and the Council of Scientific and Industrial Research, government of India, New Delhi, India.
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