Focused Exome Sequencing Gives a High Diagnostic Yield in the Indian Subcontinent.

The genetically isolated yet heterogeneous and highly consanguineous Indian population has shown a higher prevalence of rare genetic disorders. However, there is a significant socioeconomic burden for genetic testing to be accessible to the general population. In the current study, we analyzed next-generation sequencing data generated through focused exome sequencing from individuals with different phenotypic manifestations referred for genetic testing to achieve a molecular diagnosis. Pathogenic or likely pathogenic variants are reported in 280 of 833 cases with a diagnostic yield of 33.6%. Homozygous sequence and copy number variants were found as positive diagnostic findings in 131 cases (15.7%) because of the high consanguinity in the Indian population. No relevant findings related to reported phenotype were identified in 6.2% of the cases. Patients referred for testing due to metabolic disorder and neuromuscular disorder had higher diagnostic yields. Carrier testing of asymptomatic individuals with a family history of the disease, through focused exome sequencing, achieved positive diagnosis in 54 of 118 cases tested. Copy number variants were also found in trans with single-nucleotide variants and mitochondrial variants in a few of the cases. The diagnostic yield and the findings from this study signify that a focused exome test is a good lower-cost alternative for whole-exome and whole-genome sequencing and as a first-tier approach to genetic testing.

Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy in Patients Clinically Suspected of FSHD Using Optical Genome Mapping.

Background and Objectives: Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common muscular dystrophy in the general population and is characterized by progressive and often asymmetric muscle weakness of the face, upper extremities, arms, lower leg, and hip girdle. In FSHD type 1, contraction of the number of D4Z4 repeats to 1-10 on the chromosome 4-permissive allele (4qA) results in abnormal epigenetic derepression of the DUX4 gene in skeletal muscle. In FSHD type 2, epigenetic derepression of the DUX4 gene on the permissive allele (4qA) with normal-sized D4Z4 repeats (mostly 8-20) is caused by heterozygous pathogenic variants in chromatin modifier genes such as SMCHD1, DNMT3B, or LRIF1. We present validation of the optical genome mapping (OGM) platform for accurate mapping of the D4Z4 repeat size, followed by diagnostic testing of 547 cases with a suspected clinical diagnosis of FSHD and next-generation sequencing (NGS) of the SMCHD1 gene to identify cases with FSHD2. Methods: OGM with Bionano Genomics Saphyr and EnFocus FSHD analysis software was used to identify FSHD haplotypes and D4Z4 repeat number and compared with the gold standard of Southern blot-based diagnosis. A custom Agilent SureSelect enrichment kit was used to enrich SMCHD1, followed by NGS on an Illumina system with 100-bp paired-end reads. Copy number variants were assessed using NxClinical software. Results: We performed OGM for the diagnosis of FSHD in 547 patients suspected of FSHD between December 2019 and December 2022, including 301 male (55%) and 246 female patients (45%). Overall, 308 of the referred patients were positive for D4Z4 contraction on a permissive haplotype, resulting in a diagnosis of FSHD1. A total of 252 of 547 patients were referred for concurrent testing for FSHD1 and FSHD2. This resulted in the identification of FSHD2 in 9/252 (3.6%) patients. In our FSHD2 cohort, the 4qA allele size ranged from 8 to 18 repeats. Among FSHD1-positive cases, 2 patients had biallelic contraction and 4 patients had homozygous contraction and showed early onset of clinical features. Nine of the 308 patients (3%) positive for 4qA contraction had mosaic 4q alleles with contraction on at least one 4qA allele. The overall diagnostic yield in our cohort was 58%. Discussion: A combination of OGM to identify the FSHD haplotype and D4Z4 repeat number and NGS to identify sequence and copy number variants in the SMCHD1 gene is a practical and cost-effective option with increased precision for accurate diagnosis of FSHD types 1 and 2.

Utility and performance of bacterial artificial chromosomes-on-beads assays in chromosome analysis of clinical prenatal samples, products of conception and blood samples.

AIM: Chromosome analysis of prenatal samples and products of conception (POC) has conventionally been done by karyotyping (KT). Shortcomings of KT like high turnaround time and culture failure led to technology innovations, such as the bacterial artificial chromosomes (BAC)s-on-Beads (BoBs)-based tests, Prenatal BoBs (prenatal samples) and KaryoLite BoBs (POC samples). In the present study, we validated and evaluated the utility of each test on prenatal, POC and blood samples. METHODS: Study A (n = 305; 259 prenatal + 46 blood/POC) and Study B (n = 176; 146 POC/chorionic vill + 30 blood/amniotic fluid) samples were analyzed using Prenatal and KaryoLite BoBs kits, respectively. KT, array-based Comparative Genomic Hybridization (arrayCGH) and fluorescence in situ hybridization (FISH) were used for comparison of results. Ability of KaryoLite BoBs to identify ring chromosomes was tested. RESULTS: Prenatal BoBs had zero test failure rate and results of all samples were concordant with KT results. Totally four microdeletions were identified by Prenatal BoBs but not by KT. In Study B, all but two POC samples (one triploid and one tetraploid) were concordant with KT and arrayCGH. Partial chromosomal imbalance detection rate was ~64% and KaryoLite BoBs indicated the presence of a ring chromosome in all four cases. The failure rate of KaryoLite BoBs was 3%. CONCLUSION: We conclude that Prenatal BoBs (common aneuploidies and nine microdeletions) together with KT constitutes more comprehensive prenatal testing compared to FISH and KT. KaryoLite BoBs for aneuploidies of all chromosomes is highly successful in POC analysis and the ability to indicate presence of ring chromosomes improves its clinical sensitivity. Both tests are robust and could also be used for different specimens.

Relationship between Plasma Albumin Concentration and Plasma Volume in 5 Inbred Rat Strains.

Using the Evans Blue procedure, we previously found strain-related differences in plasma volumes in 5 inbred rat strains. Because albumin binds strongly with Evans blue, this protein is important in the Evans blue method of plasma volume determination. Therefore, we speculated that interstrain differences in plasma albumin concentration (PAC) could distort calculated plasma volumes. To address this concern, we used ELISA techniques to measure PAC in these inbred rat strains. In study A, the blood volume was measured by using Evans blue dye, and albumin was measured at the start of hemorrhage. In study B, blood volume was not measured, and albumin was measured twice, near the start and end of hemorrhage (approximately 14 min apart). Neither study revealed any interstrain differences in PAC, which decreased after hemorrhage in all 5 strains. No correlation was found between PAC and plasma volume, survival time, blood lactate, or blood base excess. Percentage changes in PAC during hemorrhage were greater in salt-sensitive compared with Lewis rats. Moreover, these percentage changes were associated with survival time in Fawn hooded hypertensive rats. Our data show that the plasma volumes we measured previously were not misrepresented due to variations in PAC.

Arterial blood gases, electrolytes, and metabolic indices associated with hemorrhagic shock: inter- and intrainbred rat strain variation.

We have previously shown interstrain variation (indicating a genetic basis), and intrastrain variation in survival time after hemorrhage (STaH) among inbred rat strains. To assist in understanding physiological mechanisms associated with STaH, we analyzed various arterial blood measures (ABM; pH, Paco2, oxygen content, sodium, potassium, glucose, bicarbonate, base excess, total CO2, and ionized calcium) in inbred rats. Rats from five inbred strains (n = 8-10/strain) were catheterized and, ≈ 24 h later, subjected to a conscious, controlled, 47% hemorrhage. ABM were measured at the start (initial) and end (final) of hemorrhage. Inter- and intrainbred strain variations of ABM were quantified and compared, and correlations of ABM with STaH were determined. All final ABM values and some initial ABM values were different among strains. Most ABM changed (Δ) during hemorrhage, and these changes differed among strains (P <0.03). Some strain-dependent correlations (r ≥ 0.7; P ≤ 0.05) existed between ΔABM and STaH (e.g., BN/Mcwi, ΔK(+), r = -0.84). Dark Agouti rats (longest STaH) had the smallest ΔPaco2, ΔHCO3(-), and Δbase excess, and the highest final glucose. High coefficients of variation (CVs, >10%), strain-specific CVs, and low intraclass correlation coefficients (rI < 0.5) defined the large intrastrain ABM variation that exceeded interstrain variation for most ABM. These results suggest that some ABM (K(+), Paco2, glucose, oxygen content) could predict subsequent STaH in an inbred rat strain-dependent manner. We speculate that whereas genetic differences may be responsible for interstrain variation, individual-specific epigenetic processes (e.g., DNA methylation) may be partly responsible for both inter- and intrastrain ABM variation.

Impact of occupational health hazards on serum markers of bone formation in spray painters of Chennai region in Tamil Nadu.

CONTEXT: The association between spray paint exposure and bone remodeling received little attention despite the high usage of spray paints in automobile industries, steel furniture workshops etc. AIM: The present study was aimed at investigating the level of serum markers of bone formation in spray painters. The spray painting subjects were selected from automobile body repair workshops in Chennai region of TamilNadu which constitutes 30% of India's automobile industry. SETTING AND DESIGN: All the study subjects, exposed to spray paint were working in a workshop without standard spraying room and did not wore any aerosol removing respirator. The controls were selected from random population irrespective of occupation. Data relevant to the socioeconomic features and personal history was collected using a questionnaire. The current study included 50 spray painters and 25 control subjects of same age group. MATERIALS AND METHODS: We examined the level of serum calcium, serum phosphorus, serum differentiation markers of bone such as alkaline phosphatase (bone specific) and serum osteocalcin in which these levels were found to be high in serum of spray painters. CONCLUSION: The current study concludes dysregulation in bone remodeling of spray painters exposed to chronic solvents and paint pigments.

Myocilin mutations among POAG patients from two populations of Tamil Nadu, South India, a comparative analysis.

PURPOSE: Primary open angle glaucoma (POAG) is the most common type of glaucoma. Among the POAG genes identified so far, myocilin (MYOC) is the most frequently mutated gene in POAG patients worldwide. The MYOC Gln48His mutation is unique among Indian POAG patients. This mutation has not been observed in some populations within India and in other populations worldwide. The objectives of this work were to characterize and compare the mutation spectrum among POAG patients from two places of South India and identify the occurrence and prevalence of Gln48His mutation in our study populations. METHODS: One hundred-one (101) POAG patients from Chennai, South India were recruited for the study. Earlier, 100 patients from the southernmost part of India, Kanyakumari district, were screened. MYOC was screened by polymerase chain reaction based single stand conformation polymorphism (PCR-SSCP) methodology. DNA sequencing of deviant samples was performed. Secondary structures of the proteins with amino acid sequence variations were predicted. RESULTS: The mutation frequency of MYOC among POAG patients in Chennai was 2%. Three types of mutations were observed. The MYOC Gln48His mutation was observed among 2 POAG patients from Chennai. However, absence of this mutation among patients from Kanyakumari suggests possible involvement of demographic factors in disease causation via this mutation. Two heterozygous sequence variants, Thr353Ile and Asn480Lys, in the same exon (exon III) of MYOC were observed in one POAG patient who had a severe disease phenotype. This is the first such report of a compound heterozygote individual with two mutations in the same exon of MYOC. CONCLUSIONS: The presence of mutations at a rate similar to other studies suggests the causative role of MYOC among POAG patients from Chennai. Screening of more patients and families from all parts of India is required to identify the actual frequency of the Gln48His mutation and thus highlight its importance. The compound heterozygote with a severe disease phenotype reiterates the importance of MYOC in certain POAG patients.

Genetic influences on survival time after severe hemorrhage in inbred rat strains.

To find a genetic basis for differential ability to survive severe hemorrhage, we previously showed eightfold differences in survival times among inbred rat strains. We assumed that rat strains had similar normalized blood volumes (NBV; ml/100 g body wt). As NBV might vary among strains and constitute one genetic variable affecting survival time to hemorrhage, in experiment 1 of the current studies we first measured total blood volumes and calculated NBV in specific inbred rat strains (Brown Norway/Medical College of Wisconsin, BN; Dark Agouti, DA; Fawn Hooded Hypertensive, FHH; Lewis, LEW; and Dahl Salt-Sensitive, SS) previously found to be divergent in survival time. NBV differed by 20% (P < 0.01; BN > SS > FHH = LEW = DA) and had a heritability (h(2)) of 0.56. Hence, differential survival times in our previously published study might reflect strain-dependent differences in NBV. Then studies were conducted wherein rats were catheterized and, ∼24 h later, 47% of their blood volume was removed; these rats were observed for a maximum of 4 h. In experiment 2, blood volumes were measured the day prior to hemorrhage. Percent survival and survival time did not differ among strains. To obviate possible confounding effects of blood volume determination, in experiment 3 the average NBV for each strain was used to determine hemorrhage volumes. Percent survival (P < 0.01) and survival times (P < 0.001) were different with DA demonstrating the best (62.5%, 190 ± 29 min) and BN the worst (0%, 52 ± 5 min) survival responses. These data indicate that both blood volume and survival time after hemorrhage in rats are heritable quantitative traits, and continue to suggest that genetic assessment of these phenotypes might lead to novel therapeutics to improve survival to hemorrhage.

Life or death? A physiogenomic approach to understand individual variation in responses to hemorrhagic shock.

Severe hemorrhage due to trauma is a major cause of death throughout the world. It has often been observed that some victims are able to withstand hemorrhage better than others. For decades investigators have attempted to identify physiological mechanisms that distinguish survivors from nonsurvivors for the purpose of providing more informed therapies. As an alternative approach to address this issue, we have initiated a research program to identify genes and genetic mechanisms that contribute to this phenotype of survival time after controlled hemorrhage. From physiogenomic studies using inbred rat strains, we have demonstrated that this phenotype is a heritable quantitative trait, and is therefore a complex trait regulated by multiple genes. Our work continues to identify quantitative trait loci as well as potential epigenetic mechanisms that might influence survival time after severe hemorrhage. Our ultimate goal is to improve survival to traumatic hemorrhage and attendant shock via regulation of genetic mechanisms and to provide knowledge that will lead to genetically-informed personalized treatments.

Identification of HDM2 as a regulator of VEGF expression in cancer cells.

Overexpression of vascular endothelial growth factor (VEGF) and the extent of neoangiogenesis are closely correlated with tumor development and cancer metastases. To assess whether VEGF mediated angiogenesis is regulated by HDM2, we treated the GI-101A and HL-60 cells with HDM2 gene specific antisense phosphorothioate oligodeoxynucleotide (AS5). The antisense treatment resulted in a significant reduction of both basal as well as phorbol 12,13-dibutyrate (PDB) and Diethylstilbestrol (DES) induced VEGF mRNA and protein expressions. Furthermore, when the Human Umbilical Vein Endothelial Cells (HUVECs) were exposed to medium obtained from AS5 transfected GI-101A and HL-60 cells, the angiogenesis was significantly reduced compared to the controls in the in vitro angiogenesis assay. On the contrary, the medium obtained from PDB treated cells that expressed HDM2 and VEGF at a higher level showed an increase in the tube formation by HUVEC. Thus, our present study suggests that modulation of HDM2 expression could play an important role in tumor angiogenesis and the metastatic process via transcriptional regulation of VEGF.

Detection of HDM2 and VEGF co-expression in cancer cell lines: novel effect of HDM2 antisense treatment on VEGF expression.

The human homologue of murine double minute 2 (HDM2) oncogene is amplified in approximately 7% of all human cancers. Overexpression of HDM2 protein impairs cell cycle control and confers growth advantage to cancer cells. In several cancers the progression of tumor growth and formation of distant metastases are found to be dependent on tumor angiogenesis, a process that is regulated by vascular endothelial growth factor (VEGF). In this study, we have investigated the co-expression of HDM2 and VEGF in various types of human cancer cell lines and have shown that the co-expression is not cell-type-specific. Furthermore, when different types of cell lines were treated with a HDM2 gene specific antisense phosphorothioate oligodeoxynucleotide (HDMAS5), the expression of VEGF mRNA as well as the levels of VEGF protein was found to be decreased. Interestingly, the higher basal levels of VEGF mRNA and the protein observed in HDM2 transfected LNCaP-MST cells were effectively suppressed by HDMAS5 treatment. On the contrary, the mutant oligodeoxynucleotide containing 4 mismatched bases (M4) did not alter the expression of either HDM2 or VEGF in any of the cell lines tested. In conclusion, our findings are the first time evidence showing that HDM2 and VEGF are co-expressed in various cancer cell lines that have aggressive growth and high metastatic abilities. Furthermore, the decrease in VEGF expression observed at the transcriptional as well as translational levels, subsequent to HDMAS5 treatment of p53 null cells, strongly suggests that HDM2 has a regulatory role on VEGF expression in a p53 independent manner.

Myocilin mutations among primary open angle glaucoma patients of Kanyakumari district, South India.

PURPOSE: Glaucoma can be defined as optic neuropathy leading to irreversible blindness if not treated in time. Primary open angle glaucoma (POAG) is the most common form of glaucoma. The myocilin (MYOC) gene has been found to mutate in both sporadic and familial cases of POAG worldwide. About 90% of these mutations have been seen to cluster at exon III of the gene. There are documented reports of mutations in the MYOC gene among POAG patients from different parts of India. The southernmost tip of the Indian subcontinent (Kanyakumari district) has remained isolated from all these studies. The aim of this study was to indicate or rule out the disease causative role of the MYOC gene mutations in these patients by screening the MYOC gene for mutations among POAG patients of the Kanyakumari district. METHODS: One hundred POAG patients from the Kanyakumari District of South India were recruited for the study. The MYOC gene was screened using the PCR-SSCP methodology followed by DNA sequencing. The sequences were analyzed using BLAST. Secondary structures of the amino acid sequences with a variation were predicted. RESULTS: Two probable disease-causing variations (mutations), Ser331Thr and Pro370Leu, were each observed in one patient apiece. Two polymorphisms, (Tyr347Tyr and Thr325Thr) were also observed in the patients. Ser331Thr is a novel conservative change while Pro370Leu is a widely reported mutation with an associated severe disease phenotype. CONCLUSIONS: The presence of the mutations in the patients suggests the causative role of the MYOC gene among POAG patients in the Kanyakumari district of India. The mutation frequency of 2% corresponds well with the other reports from India and other countries. However, the mutation rate reported from a population in the eastern part of India was much higher. Screening of patients from different parts of India is essential to estimate the overall mutation frequency. More functional studies on the MYOC gene are required to elucidate the pathophysiology of POAG.