Subodh Kumar Singh

Idiopathic cases of male infertility from a region in India show low incidence of Y-chromosome microdeletion

Chromosomal and Y-chromosomal microdeletion analysis has been done in cases of idiopathic infertility with the objective of evaluating the frequency of chromosomal and molecular anomaly as the causal factor of infertility. Barring a few cases of Klinefelter syndrome (XXY or XY/XXY mosaics), no chromosomal anomaly was encountered. Y-microdeletion was analysed by PCR-screening of STSs from different regions of the AZF (AZFa, AZFb, AZFc) on the long arm of the Y, as well as by using DNA probes of the genes RBM, DAZ (Yq), DAZLA (an autosomal homologue of DAZ) and SRY (Yp; sex determining gene). Out of 177 cases examined, 9 (azoospermia -8 and oligoasthenospermia -1) showed partial deletion of AZF. The size of deletion varied among patients but AZFc was either totally or partially removed in all of them. In contrast, no deletion was detected in AZFa. Testis biopsy done on a limited number of cases (50) showed diverse stages of spermatogenic arrest with no specific correlation with the genotype. The frequency of Y-chromosome microdeletion in our samples (∼ 5%) is much lower than the frequency (∼ 10%) reported globally and the two previous reports from India. We contend that the frequency may be affected by population structures in different geographical regions.

MTHFR 677TT Alone and IRF6 820GG Together with MTHFR 677CT, but Not MTHFR A1298C, Are Risks for Nonsyndromic Cleft Lip with or without Cleft Palate in an Indian Population

AIM To determine the association of three SNPs, IRF6 G820A, MTHFR C677T, and MTHFR A1298C, with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in an Indian population. METHOD A total of 323 NSCL/P patients, 116 of their mothers, 108 of their fathers, and 214 normal controls have been examined for the above three SNPs. RESULT Frequency of IRF6 GG was 65% in controls, 78% in cases, 84% in case-fathers, and 80% in case-mothers. MTHFR 677T homozygosity was lower than 1% in controls and unaffected parents, while in the group of probands it was much higher (3.4%; OR 4.30). The frequency of CT genotype was also high in the cases and case-mothers (OR 1.89 and 2.2, respectively). MTHFR A1298C did not reveal a statistically significant deviation in allele and genotype frequencies. CONCLUSION While MTHFR 677T homozygotes show a significant association with NSCL/P, heterozygotes 677CT are minor risk factors. MTHFR A1298C does not show a risk in any combination of alleles. IRF6 820GG too forms a minor risk. However, combined genotypes IRF6 GG/MTHFR 677CT together form greater risk for NSCL/P.

MTHFR A1298C polymorphism and idiopathic male infertility.

BACKGROUND DNA methylation is an important epigenetic feature of DNA that plays a pivotal role in gene expression regulation during spermatogenesis. The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyses the formation of folate intermediates that are vital for DNA synthesis and methylation reactions. C677T and A1298C variants of MTHFR result in reduced plasma folate and increase the susceptibility to various multifactorial disorders. We have already shown that homozygosity for 677 (C ®T) mutation in the MTHFR gene, is a risk factor for idiopathic male infertility in an Indian population. AIM Recently, we showed that homozygosity for the 677(C;T) mutation in the MTHFR gene is a risk factor for idiopathic male infertility and now we aim to assess whether the A1298C mutation in the same gene is an additional risk factor for idiopathic male infertility in an Indian population. SETTING AND DESIGNS In a case-control study 151 idiopathic male infertile patients and 140 healthy fertile control individuals were recruited from the University hospital and infertility clinics in Varanasi city, India. MATERIALS AND METHODS Genotyping for A1298C change of the MTHFR gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Statistical Analysis : Allele frequencies were calculated using Fishers exact test. Odds ratio was calculated as the measure of the association between the MTHFR genotype and idiopathic male infertility. RESULTS The homozygous (C/C) A1298C polymorphism of the MTHFR gene was present at a statistically high significance in idiopathic azoospermic infertile men (OR=3.4494, CI: 1.0092 to 11.7899, P<0.05). CONCLUSION The MTHFR 1298CC genotype is an additional genetic risk factor for idiopathic male infertility in an Indian population.

Lower incidence of nonsyndromic cleft lip with or without cleft palate in females: is homocysteine a factor?

In India, as in other parts of the world, nonsyndromic cleft lip with or without cleft palate (NSCL±P) is a highly prevalent birth defect, its incidence in males being twice that in females. A case–control association study has been carried out with respect to homocysteine level and MTHFR C677T, A1298C and SLC19A1 (RFC1) G80A genotypes from an eastern Indian cohort to investigate whether Hcy and other Hcy-pathway genes also contribute to the risk level. While MTHFR 677T and SLC19A1 80G are individually and cumulatively risk factors, SLC19A1 80A appears to be protective against MTHFR 677T risk allele. Elevated Hcy associates with NSCL±P both in case mothers and cases. Significantly, this difference shows a gender bias: the level of elevation of Hcy in female cases is distinctly higher than in males, and more case females are hyperhomocyteinemic than the case males. It implies that compared with the males, higher level of Hcy is needed for NSCL±P to manifest in the females. We consider this as one of the possible factors why the incidence of this disorder in females is much lower than in males.

High levels of genetic variation in Indian field and house mice

Genetic variation in the Indian pygmy field miceMus booduga and theMus terricolor complex and in the house mouseMus musculus tytleri was analysed electro phoretically at 20 enzymatic and nonenzymatic protein loci. The results show an unusually high genetic variation in the field mice in terms of per cent polymorphism (P = 75 to 90 at 0-95 level), observed heterozygosity (Ho = 0.215[ ± 0.213] to 0.314 [±0.236]) and average number of alleles(A = 2.0[± 0.858] to 2.38 [±0.868]). Very high values of P,Ho andA were also observed for the house mouse. Genetic distance (D) determined by using Nei’s (1978) formula in theM. terricolor complex ranged from the lowest,D = 0.082, betweenM. terricolor I and II to the highest,D = 0.155, betweenM. terricolor II and III. Genetic distance betweenM. booduga and theM. terricolor complex was 0-259 and that between the house mouseM. m. tytleri and theM. booduga-terricolor lineage was 0.285. TheseD values corroborate that the pygmy field and house mice are closely allied.

Coding Region of IRF6 Gene May Not Be Causal for Van Der Woude Syndrome in Cases From India

Objective: Evaluation of the IRF6 gene in Van der Woude syndrome cases from an Indian population. Subjects: Nine affected and four unaffected individuals from seven families with Van der Woude syndrome as well as five normal controls (with no history of Van der Woude or any other congenital malformation and belonging to the same geographical area as the families with Van der Woude syndrome). Method: Direct sequencing of all coding regions and exon-intron boundaries of the IRF6 gene. Results: Five novel variants: IVS1+3900 A>G, 191 T>C, IVS4+775 C>T, IVS8+218 C>T, 1511 T>A (Ser 416 Arg) and two known variants: IVS6+27 C>G, 1083 G>A (V274I) were detected. Except for one, all were in noncoding regions either in 3′UTR or in introns. There was only one mutation in the coding region, detected in a normal control. Conclusion: The present report indicates that point mutations in the coding region of the IRF6 gene may not be a major cause of Van der Woude syndrome in Indian populations.

GLI3 mutations in syndromic and non-syndromic polydactyly in two Indian families.

The GLI3 protein is a zinc finger transcription factor, expressed early in development. The GLI3 gene exhibits allelic heterogeneity as mutations in this gene are associated with several developmental syndromic and non‐syndromic polydactyly. The present study reports two cases: first, a familial case of Greig Cephalopolysyndactyly Syndrome (GCPS); the second is a sporadic case with both postaxial polydactyly (PAP) type A and B. Resequencing of GLI3 gene reveals a previously reported nonsense truncation mutation g.42007251G > A (p.R792X; rs121917714) in the GCPS family and a novel single nucleotide insertion g.42004239_42004240insA (p.E1478X) in the sporadic case of postaxial polydactyly (PAP). Both nonsense truncation mutations; p.R792X (GCPS) and p.E1478X (PAP) introduce a premature stop codon leading to loss of C‐terminal domains.

A novel GLI3c.750delC truncation mutation in a multiplex Greig cephalopolysyndactyly syndrome family with an unusual phenotypic combination in a patient

Greig cephalopolysyndactyly (GCPS) syndrome is an autosomal dominant disorder with high penetrance in majority of cases, characterized by a triad of polysyndactyly, macrocephaly and hypertelorism. GCPS is known to be caused by mutations in the transcription factor GLI3 gene (7p13) which results in functional haploinsufficiency of this gene. The present study reports a large multiplex family having 12 members affected with GCPS in 3 generations and several unaffected members showing autosomal dominant pattern of inheritance with complete penetrance. Interestingly an affected member of the family had unusual features including thumb which is although biphalangeal (confirmed with X-ray) but morphologically looks like finger and a unilateral tiny bony outgrown (externally indistinguishable) on the distal phalanx of the first toe of the left foot. This member also presented with mild ichthyosis. Although it is also possible that one or more of these features are coincidentally present in this member and might not be part of GCPS. Resequencing of the GLI3 gene detected a novel frame-shift mutation c.750delC in heterozygous state transmitting in the family and co-segregating with the disorder suggesting it to be the causal for the GCPS phenotype in the family. In silico analysis suggests that this mutation creates a truncated GLI3 protein resulting in its haploinsufficiency leading to GCPS syndrome. Furthermore, genotype-phenotype correlation is supported by the mutation as it lies in the amino terminal domain of the protein.

TGFβ3, MSX1, and MMP3 as Candidates for NSCL±P in an Indian Population

Objective: To evaluate the association of transforming growth factor β3 (TGFβ3), muscle segment homeobox 1 (MSX1), Metalloproteinases 3 (MMP3), and MMP9 genes as candidates for nonsyndromic cleft lip and/or palate in an Indian population. Design: Case–control association study, mutational screening, and functional evaluation of obtained mutations. Setting: Mutational screening of the developmental genes, TGFβ3 and MSX1, along with functional evaluation and association of promoter region SNPs—one each in MMP3 and MMP9. Patients, Participants: Two hundred forty five NSCL±P cases from G. S. Memorial Plastic Surgery Hospital and Trauma Center, Varanasi and 201 healthy controls without a family history of congenital malformations from nearby schools, primary health centers, and the university hospital. Main Outcome Measure(s): Sequencing, SSCP, and PCR-RFLP were used for candidate gene screening. MatInspector and electrophoretic mobility shift assay (EMSA) were used to check the differential transcription factor binding of the variants at promoter region. Luciferase assay was used to test the transcriptional potential of the variant, and evaluation of the alternative splice site was carried out using exon-trapping experiment. Results: Metalloproteinases3 −1171 5A/6A was associated with NSCL±P, whereas MMP9 −1562 C/T did not show association. A rare variant in the promoter region of TGFβ3 (rs117462711) creates a differential binding site, confirmed by EMSA. Luciferase assay showed 3.7-fold increased expression level in mutant construct. A synonymous change in MSX1 (rs34165410) showed association with NSCL±P, which may create an alternative splice site or lead to low codon usage. Exon-trapping experiment failed to confirm alternative splicing, indicating low codon usage frequency of the mutant affecting the gene function. Conclusions: TGFβ3, MSX1, and MMP3 are candidates for NSCL±P.

A novel non-coding RNA within an intron of CDH2 and association of its SNP with non-syndromic cleft lip and palate

BACKGROUND Genome-wide linkage analysis and whole genome sequencing in a Van der Woude syndrome (VWS) family revealed that the SNP, rs539075, within intron 2 of the cadherin 2 gene (CDH2) co-segregated with the disease phenotype. RESULTS A study with nonsyndromic cleft lip with or without cleft palate (NSCL ± P) cases (N = 292) and controls (N = 287) established association of this SNP with NSCL ± P as a risk factor. RT-PCR based expression analysis of the SNP-harbouring region of intron 2 of CDH2 in the clefted lip and/or palate tissues of 16 patients revealed that the mutant allele expressed in all those individuals having it (hetero-/homozygous), whereas the wild type allele expressed in <50% of the samples in which it was present. The intronic transcript was also present in the prospective lip and palate region of 13.5 dpc mouse embryo, detected by RNA in situ hybridization and RT-PCR. CONCLUSIONS These results including the in silico, characterization of the ~200 nt-intronic transcript showed that conformationally it fits best with noncoding small RNA, possibly a precursor of miRNA. Its function in the orofacial organogenesis remains to be elucidated which will enable us to define the role of this mutant ncRNA in the clefting of lip and palate.