Sher Ali

Molecular dissection of the human Y-chromosome.

Human Y chromosome, earlier thought to be gene deficient, has attracted a great deal of attention owing to its supremacy in male sex determination and unique haplotype status in the genome. Studies on Y chromosome have shown the presence of different types of satellite DNA and several genes implicated with a variety of physical and physiological functions. The interaction of these repetitive DNA with genes in normal individuals and in patients with Y-chromosome-related genetic anomalies is still an unresolved issue and is actively being pursued. The fast changing scenario of the human genome project is likely to effect our overall understanding of the Y chromosome and Y-linked genetic anomalies in a big way. We provide a brief overview of the organization of Y chromosome with respect to several important loci encompassing both the arms and their likely involvement/modulation in genetic anomalies. The experimental approaches discussed here are envisaged to be of clinical relevance for the molecular diagnosis of the Y-linked disorders.

Detection of Salmonella typhi by polymerase chain reaction: Implications in diagnosis of typhoid fever

The present study was conducted to detect Salmonella typhi by polymerase chain reaction (PCR) in a clinical setting. A group of 40 clinically suspected cases of typhoid fever, lasting for about 3-11 days, with or without chills and rigors and hepatosplenomegaly were selected. Of these, 20 were culture positive and the remaining 20 were found to be negative by conventional blood culture technique. Primary PCR was followed by nested PCR using two sets of primers corresponding to flagellar gene of S. typhi strain. Two bands of about 458 and 343 bp were detected in 20 blood culture positive cases and 12 of the 20 culture negative ones. In the simulated group of samples, no amplification was detected. Our results suggest that PCR-based diagnosis is particularly useful for all clinically suspected cases of typhoid fever. The sensitivity of PCR and its potential use in routine diagnosis and epidemiological studies of typhoid fever can be exploited to complement studies by including bone marrow culture, faeces and bile samples.

AZFc somatic microdeletions and copy number polymorphism of the DAZ genes in human males exposed to natural background radiation

Ionizing radiations are known to induce tumors, chromosomal lesions and minisatellite length variations, yet no correlation has been demonstrated between radiation exposure and indels or copy number polymorphism (CNP) of the genes. We studied the impact of natural background radiation (NBR) on the human Y chromosome owing to its haploid status and clonal inheritance. We analyzed the AZFc region using the DNA from blood and semen of 100 males living near the coastal peninsula in Kerala (India), exposed to NBR along with other 50 normal fertile males. STS mapping of AZFc region showed random microdeletions without conclusive gr/gr or b1/b3 phenotypes. Using a highly specific novel Taqman assay based on sY587 sequence, we detected four copies of the DAZ genes in normal males and 4–16 in those exposed to NBR. Amongst NBR exposed males with multiples copies of the DAZ genes, 75% showed varying FISH signals for DAZ genes with cosmid 18E8 whereas 30% showed mosaicism in terms of presence/absence of the signals in 6–8% cells and unexpected number of signals in 9–12% interphase nuclei. Startlingly, all germline samples studied were found to be free from AZFc microdeletions and CNP of the DAZ genes. Since the DAZ genes are heavily implicated with the germ cell development, the cells with DAZ deletion/duplication are unlikely to survive. Alternatively, an innate mechanism may be operative to protect the germline from the effects of NBR.

Startling mosaicism of the Y-chromosome and tandem duplication of the SRY and DAZ genes in patients with Turner Syndrome

Presence of the human Y-chromosome in females with Turner Syndrome (TS) enhances the risk of development of gonadoblastoma besides causing several other phenotypic abnormalities. In the present study, we have analyzed the Y chromosome in 15 clinically diagnosed Turner Syndrome (TS) patients and detected high level of mosaicisms ranging from 45,XO:46,XY = 100:0% in 4; 45,XO:46,XY:46XX = 4:94:2 in 8; and 45,XO:46,XY:46XX = 50:30:20 cells in 3 TS patients, unlike previous reports showing 5–8% cells with Y- material. Also, no ring, marker or di-centric Y was observed in any of the cases. Of the two TS patients having intact Y chromosome in >85% cells, one was exceptionally tall. Both the patients were positive for SRY, DAZ, CDY1, DBY, UTY and AZFa, b and c specific STSs. Real Time PCR and FISH demonstrated tandem duplication/multiplication of the SRY and DAZ genes. At sequence level, the SRY was normal in 8 TS patients while the remaining 7 showed either absence of this gene or known and novel mutations within and outside of the HMG box. SNV/SFV analysis showed normal four copies of the DAZ genes in these 8 patients. All the TS patients showed aplastic uterus with no ovaries and no symptom of gonadoblastoma. Present study demonstrates new types of polymorphisms indicating that no two TS patients have identical genotype-phenotype. Thus, a comprehensive analysis of more number of samples is warranted to uncover consensus on the loci affected, to be able to use them as potential diagnostic markers.

Genes, Genetics, and Environment in Type 2 Diabetes: Implication in Personalized Medicine

Type 2 diabetes (T2D) is a multifactorial anomaly involving 57 genes located on 16 different chromosomes and 136 single nucleotide polymorphisms (SNPs). Ten genes are located on chromosome 1, followed by seven genes on chromosome 11 and six genes on chromosomes 3. Remaining chromosomes harbor two to five genes. Significantly, chromosomes 13, 14, 16, 18, 21, 22, X, and Y do not have any associated diabetogenic gene. Genetic components have their own pathways encompassing insulin secretion, resistance, signaling, and β-cell dysfunction. Environmental factors include epigenetic changes, nutrition, intrauterine surroundings, and obesity. In addition, ethnicity plays a role in conferring susceptibility to T2D. This scenario poses a challenge toward the development of biomarker for quick disease diagnosis or for generating a consensus to delineate different categories of T2D patients. We believe, before prescribing a generic drug, detailed genotypic information with the background of ethnicity and environmental factors may be taken into consideration. This nonconventional approach is envisaged to be more robust in the context of personalized medicine and perhaps would cause lot less burden on the patient ensuring better management of T2D.

Unique Signatures of Natural Background Radiation on Human Y Chromosomes from Kerala, India

Background The most frequently observed major consequences of ionizing radiation are chromosomal lesions and cancers, although the entire genome may be affected. Owing to its haploid status and absence of recombination, the human Y chromosome is an ideal candidate to be assessed for possible genetic alterations induced by ionizing radiation. We studied the human Y chromosome in 390 males from the South Indian state of Kerala, where the level of natural background radiation (NBR) is ten-fold higher than the worldwide average, and that from 790 unexposed males as control. Results We observed random microdeletions in the Azoospermia factor (AZF) a, b and c regions in >90%, and tandem duplication and copy number polymorphism (CNP) of 11 different Y-linked genes in about 80% of males exposed to NBR. The autosomal homologues of Y-linked CDY genes largely remained unaffected. Multiple polymorphic copies of the Y-linked genes showing single Y-specific signals suggested their tandem duplication. Some exposed males showed unilocus duplication of DAZ genes resulting in six copies. Notably, in the AZFa region, approximately 25% of exposed males showed deletion of the DBY gene, whereas flanking genes USP9Y and UTY remained unaffected. All these alterations were detected in blood samples but not in the germline (sperm) samples. Conclusions Exposure to high levels of NBR correlated with several interstitial polymorphisms of the human Y chromosome. CNPs and enhanced transcription of the SRY gene after duplication are envisaged to compensate for the loss of Y chromosome in some cells. The aforesaid changes, confined to peripheral blood lymphocytes, suggest a possible innate mechanism protecting the germline DNA from the NBR. Genome analysis of a larger population focusing on greater numbers of genes may provide new insights into the mechanisms and risks of the resultant genetic damages. The present work demonstrates unique signatures of NBR on human Y chromosomes from Kerala, India.

Analysis of Conserved Microsatellite Sequences Suggests Closer Relationship between Water Buffalo Bubalus bubalis and Sheep Ovis aries

The distribution and evolutionary pattern of the conserved microsatellite repeat sequences (CA)n, (TGG)6, and (GGAT)4 were studied to determine the divergence time and phylogenetic position of the water buffalo, Bubalus bubalis. The mean allelic frequencies of these repeat loci showed a high level of heterozygosity among the euartiodactyls (buffalo, cattle, sheep, and goat). Genetic distances calculated from the allelic frequencies of these microsatellites were used to position Bubalus bubalis in the phylogenetic tree. The tree topology revealed a closer proximity of the Bubalus bubalis to the Ovis aries (sheep) genome than to other domestic species. The estimated time of divergence of the water buffalo genome relative to cattle, goat, sheep, pig, rabbit, and horse was found to be 21, 0.5, 0.7, 94, 20.3, and 408 million years (Myr), respectively. Although water buffaloes share morphological and biochemical similarities with cattle, our study using the microsatellite sequences places the bubaline species in an entirely new phylogenetic position. Our results also suggest that with respect to these repeat loci, the water buffalo genome shares a common ancestry with sheep and goat after the divergence of subfamily Bovinae (Bos taurus) from the family Bovidae.

Analysis of the evolutionarily conserved repeat motifs in the genome of the highly endangered central Indian swamp deer Cervus duvauceli branderi.

We have analyzed the genome of central Indian swamp deer Cervus duvauceli branderi, an inhabitant of the Kanha National Park, a wildlife conservatory in Central India, with a view to provide a genetic basis for their extinction. Evolutionarily conserved repeat sequence motifs (GATA)3.75, TA(GATA)4, (GACA)3.75, (TGG)6 and a set of mouse beta-actin primers were used to uncover the sequence variation within and between related species by employing techniques of hybridization and AP-PCR amplification. The oligo probe carrying the GACA and TGG repeat motifs was found to be positive with Cervus genome, whereas (GATA)3.75, TA(GATA)4 and beta-actin probes did not cross-hybridize with the same. AP-PCR amplification with (GACA)3.75, unlike the (TGG)6 primer, generated distinct bands in the range of 0. 37-2.10kb amongst different genomes including Cervus. A comparative genome analysis of other species using the AP-PCR approach with (GACA)3.75 primer revealed the phylogenetic status of Cervus duvauceli branderi. From the analysis of a very limited number of Cervus DNA samples, we observed a high level of genetic homogeneity that may be a prime reason for the extinction of this species. This study has implications in the context of conservation of this endangered Cervus duvauceli branderi species.

Organization and differential expression of the GACA/GATA tagged somatic and spermatozoal transcriptomes in Buffalo Bubalus bubalis

BackgroundSimple sequence repeats (SSRs) of GACA/GATA have been implicated with differentiation of sex-chromosomes and speciation. However, the organization of these repeats within genomes and transcriptomes, even in the best characterized organisms including human, remains unclear. The main objective of this study was to explore the buffalo transcriptome for its association with GACA/GATA repeats, and study the structural organization and differential expression of the GACA/GATA repeat tagged transcripts. Moreover, the distribution of GACA and GATA repeats in the prokaryotic and eukaryotic genomes was studied to highlight their significance in genome evolution.ResultsWe explored several genomes and transcriptomes, and observed total absence of these repeats in the prokaryotes, with their gradual accumulation in higher eukaryotes. Further, employing novel microsatellite associated sequence amplification (MASA) approach using varying length oligos based on GACA and GATA repeats; we identified and characterized 44 types of known and novel mRNA transcripts tagged with these repeats from different somatic tissues, gonads and spermatozoa of water buffalo Bubalus bubalis. GACA was found to be associated with higher number of transcripts compared to that with GATA. Exclusive presence of several GACA-tagged transcripts in a tissue or spermatozoa, and absence of the GATA-tagged ones in lung/heart highlights their tissue-specific significance. Of all the GACA/GATA tagged transcripts, ~30% demonstrated inter-tissue and/or tissue-spermatozoal sequence polymorphisms. Significantly, ~60% of the GACA-tagged and all the GATA-tagged transcripts showed highest or unique expression in the testis and/or spermatozoa. Moreover, ~75% GACA-tagged and all the GATA-tagged transcripts were found to be conserved across the species.ConclusionPresent study is a pioneer attempt exploring GACA/GATA tagged transcriptome in any mammalian species highlighting their tissue, stage and species-specific expression profiles. Comparative analysis suggests the gradual accumulation of these repeats in the higher eukaryotes, and establishes the GACA richness of the buffalo transcriptome. This is envisaged to establish the roles of integral simple sequence repeats and tagged transcripts in gene expression or regulation.

Insect sex chromosomes

The two X chromosomes in tetraploid spermatogonial cells from Gryllotalpa fossor respond differentially to the production of chromatid aberrations by 3H-uridine (3H-U). As in diploid female somatic cells, only the euchromatic arm of one X shows such aberrations. The equivalent arm of the other X and the constitutive arms of both Xs are not affected. This differential response of the homologous arms of the two Xs appears to be due to a facultative heterochromatinization of one of them. It is suggested that an imprinting process, which has been assumed to occur during fertilization in other cases of X-inactivation, may not be necessary for the differential regulation of two X chromosomes in this case.