COASY related pontocerebellar hypoplasia type 12: A common Indian mutation with expansion of the phenotypic spectrum.

Pontocerebellar hypoplasia (PCH) type 12 is a rare, perinatal lethal neurodegenerative genetic disorder caused by biallelic mutations in the COASY gene. Herein, we describe the clinical and neuroradiological profile of nine affected fetuses/neonates from five families identified with a common COASY: c.1486-3C>G biallelic variant. Four of the five families were identified after data reanalysis of unresolved, severe PCH like phenotype and the fifth family through collaboration. The common antenatal phenotype was cerebellar hypoplasia. Microcephaly, arthrogryposis, and intrauterine growth restriction were the shared postnatal findings. The neurological manifestations included seizures, poor sucking, and spasticity. Novel findings of corpus callosum agenesis, simplified gyral pattern, normal sized pons, optic neuropathy, and a small thorax are reported in this series. The allele frequency of the COASY: c.1486-3C>G variant was 0.62% in the available Asian Indian database. We describe this as a possible common Indian origin variant. To the best of our knowledge, this is the largest PCH12 series reported.

Prenatal phenotypic spectrum of full trisomy 18 in an Indian cohort.

Trisomy 18 or Edward syndrome is a chromosomal disorder due to the presence of an extra chromosome 18. We describe the phenotype of five fetuses at different gestational ages, each highlighting a different aspect of trisomy 18. The clinical spectrum included increased nuchal translucency, fetal hydrops, congenital malformations of the central nervous system, congenital heart disease, radial ray defects, and characteristic facial gestalt. We made a comparison of prenatal ultrasonography and the autopsy findings. The fetal autopsy defined the craniofacial and digit anomalies better compared with sonography. The facial features of tall forehead, hypoplastic nares, microstomia, micrognathia, low set abnormal ears along with clenched hands, and short hallux are typical for trisomy 18 and help in planning the targeted cytogenetic or molecular tests. The diagnosis was established by either fluorescence in situ hybridization or quantitative fluorescent polymerase chain reaction or chromosomal microarray in the patients. This communication emphasizes the importance of detailed assessment for craniofacial and limb anomalies on prenatal ultrasonography which can prompt an early evaluation for trisomy 18.

Functional characterization of novel variants in SMPD1 in Indian patients with acid sphingomyelinase deficiency.

Pathogenic variations in SMPD1 lead to acid sphingomyelinase deficiency (ASMD), that is, Niemann-Pick disease (NPD) type A and B (NPA, NPB), which is a recessive lysosomal storage disease. The knowledge of variant spectrum in Indian patients is crucial for early and accurate NPD diagnosis and genetic counseling of families. In this study, we recruited 40 unrelated pediatric patients manifesting symptoms of ASMD and subnormal ASM enzyme activity. Variations in SMPD1 were studied using Sanger sequencing for all exons, followed by interpretation of variants based on American College of Medical Genetics and Genomics & Association for Molecular Pathology (ACMG/AMP) criteria. We identified 18 previously unreported variants and 21 known variants, including missense, nonsense, deletions, duplications, and splice site variations with disease-causing potential. Eight missense variants were functionally characterized using in silico molecular dynamic simulation and in vitro transient transfection in HEK293T cells, followed by ASM enzyme assay, immunoblot, and immunofluorescence studies. All the variants showed reduced ASM activity in transfected cells confirming their disease-causing potential. The study provides data for efficient prenatal diagnosis and genetic counseling of families with NPD type A and B.

Levels of Lyso GL-1 in Gaucher and Lyso GL-3 in Fabry patients from India: Diagnostic aids for these lysosomal storage disorders.

BACKGROUND & AIMS: Lysosomal storage disorders (LSDs) remain a significant cause of morbidity in the Indian population and treatment is largely out of reach for most patients. Although data on enzymatic and molecular diagnosis of Gaucher disease (GD) and Fabry disease (FD) in Indian patients are available, the present study intended to establish the pathogenic levels of Lyso GL-1 and Lyso GL-3 in patients of GD and FD respectively as diagnostic aids. MATERIALS AND METHODS: From 2017 to 2019, ninety confirmed Gaucher cases (by enzymatic and molecular analysis) were tested for chitotriosidase (fluorometrically) and Lyso GL-1 (LC-MS/MS) and ten confirmed Fabry cases were analyzed for Lyso GL-3 (LC-MS/MS). RESULTS: Lyso GL-1 (median: 685.5 ng/mL, cut-off: 14) and Lyso GL-3 (median: 75.6 ng/mL, cut-off: 3.5) were found to be elevated in all enzymatically deficient patients of GD and FD respectively, however, no specific trend was observed between the levels of these biomarkers and the pathogenic variant(s) present in the patients of these disorders. CONCLUSIONS: This is the first report on Lyso GL-1 and Lyso GL-3 levels in Indian patients of GD and FD respectively. These results will be useful for early diagnosis to improve management of these LSDs.

Clinical and genetic spectrum of 104 Indian families with central nervous system white matter abnormalities.

Genetic disorders with predominant central nervous system white matter abnormalities (CNS WMAs), also called leukodystrophies, are heterogeneous entities. We ascertained 117 individuals with CNS WMAs from 104 unrelated families. Targeted genetic testing was carried out in 16 families and 13 of them received a diagnosis. Chromosomal microarray (CMA) was performed for three families and one received a diagnosis. Mendeliome sequencing was used for testing 11 families and all received a diagnosis. Whole exome sequencing (WES) was performed in 80 families and was diagnostic in 52 (65%). Singleton WES was diagnostic for 50/75 (66.67%) families. Overall, genetic diagnoses were obtained in 77 families (74.03%). Twenty-two of 47 distinct disorders observed in this cohort have not been reported in Indian individuals previously. Notably, disorders of nuclear mitochondrial pathology were most frequent (9 disorders in 20 families). Thirty-seven of 75 (49.33%) disease-causing variants are novel. To sum up, the present cohort describes the phenotypic and genotypic spectrum of genetic disorders with CNS WMAs in our population. It demonstrates WES, especially singleton WES, as an efficient tool in the diagnosis of these heterogeneous entities. It also highlights possible founder events and recurrent disease-causing variants in our population and their implications on the testing strategy.

Prenatal Diagnosis by Chromosome Microarray Analysis, An Indian Experience.

BACKGROUND: Karyotyping has been the gold standard for prenatal chromosome analysis. The resolution should be higher by chromosome microarray analysis (CMA). The challenge lies in recognizing benign and pathogenic or clinically significant copy number variations (pCNV) and variations of unknown significance (VOUS). The aim was to evaluate the diagnostic yield and clinical utility of CMA, to stratify the CMA results in various prenatal referral groups and to accumulate Indian data of pCNVs and VOUS for further interpretation to assist defined genetic counseling. METHODS: Karyotyping and CMA were performed on consecutive referrals of 370 prenatal samples of amniotic fluid (n = 274) and chorionic villi (n = 96) from Indian pregnant women with high maternal age (n = 23), biochemical screen positive (n = 61), previous child abnormal (n = 59), abnormal fetal ultrasound (n = 205) and heterozygous parents (n = 22). RESULTS AND CONCLUSION: The overall diagnostic yield of abnormal results was 5.40% by karyotyping and 9.18% by CMA. The highest percentage of pCNVs were found in the group with abnormal fetal ultrasound (5.40%) as compared to other groups, such as women with high maternal age (0.81%), biochemical screen positive (0.54%), previous abnormal offspring (0.81%) or heterozygous parents group (1.62%). Therefore, all women with abnormal fetal ultrasound must undergo CMA test for genotype-phenotype correlation. CMA detects known and rare deletion/duplication syndromes and characterizes marker chromosomes. Accumulation of CNV data will form an Indian Repository and also help to resolve the uncertainty of VOUS. Pretest and posttest genetic counseling is essential to convey benefits and limitations of CMA and help the patients to take informed decisions.

The fatal fetal tumor: a geneticist's perspective.

Epignathus is an extremely rare oral teratoma which leads to high mortality in the early neonatal period. Various theories have been put forward for the genesis of such a tumor, though none is completely convincing. A genetic basis is not well established for the tumor. Microdeletions/duplications, as well as single gene disorders, have been known to cause epignathus, all with additional malformations. Evidence of single gene involvement in an isolated epignathus is lacking. We present a case of a 19-week-fetus with oro-pharyngeal teratoma detected on the level II ultrasound. The couple was counseled regarding the grave prognosis of the fetal condition following which they opted for termination of pregnancy and fetal autopsy. The autopsy revealed fetus-like body attached to the tumor. Genetic testing including a whole genome microarray did not reveal any significant variant. An explanation for the fetus-like body maybe a common origin of the teratoma and the additional fetus-like bodies due to an erroneous process of early embryonic development. Another possibility is of an acardiacus acranius twin masquerading as a fetus-like body. Thus, we conclude that in the absence of an associated malformation, an epignathus is unlikely to have a genetic etiology. This study highlights the importance of performing a fetal autopsy as a part of deep phenotyping to ascertain the etiology, as it identified additional fetal-like body which was not detected on the antenatal ultrasound.

Biallelic Pathogenic GFRA1 Variants Cause Autosomal Recessive Bilateral Renal Agenesis.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are one of the most common malformations identified in the fetal stage. Bilateral renal agenesis (BRA) represents the most severe and fatal form of CAKUT. Only three genes have been confirmed to have a causal role in humans (ITGA8, GREB1L, and FGF20). METHODS: Genome sequencing within a diagnostic setting and combined data repository analysis identified a novel gene. RESULTS: Two patients presented with BRA, detected during the prenatal period, without additional recognizable malformations. They had parental consanguinity and similarly affected, deceased siblings, suggesting autosomal recessive inheritance. Evaluation of homozygous regions in patient 1 identified a novel, nonsense variant in GFRA1 (NM_001348097.1:c.676C>T, p.[Arg226*]). We identified 184 patients in our repository with renal agenesis and analyzed their exome/genome data. Of these 184 samples, 36 were from patients who presented with isolated renal agenesis. Two of them had loss-of-function variants in GFRA1. The second patient was homozygous for a frameshift variant (NM_001348097.1:c.1294delA, p.[Thr432Profs*13]). The GFRA1 gene encodes a receptor on the Wolffian duct that regulates ureteric bud outgrowth in the development of a functional renal system, and has a putative role in the pathogenesis of Hirschsprung disease. CONCLUSIONS: These findings strongly support the causal role of GFRA1-inactivating variants for an autosomal recessive, nonsyndromic form of BRA. This knowledge will enable early genetic diagnosis and better genetic counseling for families with BRA.

Robinow Syndrome and Brachydactyly: An Interplay of High-Throughput Sequencing and Deep Phenotyping in a Kindred.

We report a family with a spectrum of short stature, craniofacial dysmorphism, and digital anomalies in a father and 2 daughters, with the youngest (proband) displaying a severe phenotype. Clinically, autosomal dominant Robinow syndrome (ADRS) was diagnosed. Whole-exome sequencing identified a heterozygous pathogenic BMP2 variant in the father and his daughters. The phenotype of short stature, facial dysmorphism, and skeletal anomalies with or without cardiac anomalies related to BMP2 haploinsufficiency has some facial and digital resemblance to ADRS. Although this variant segregated in the affected members, it failed to explain the severe phenotype of the proband. A reanalysis of the girl's raw data confirmed 2 disorders: a de novo likely pathogenic DVL1 variant implicated in ADRS and the familial BMP2 variant. A close interplay of high-throughput sequencing and deep phenotyping unraveled the complexities of the blended phenotype in the proband.

Extending the Phenotype and Identification of a Novel Candidate Gene for Immunodeficiency in 5q11 Microdeletion Syndrome.

Array CGH has led to the delineation of innumerable microdeletion syndromes. We present a patient with a 7-Mb deletion at 5q11.2 with previously unreported features, such as immunodeficiency, asymmetry of hands and feet, joint laxity, and agenesis of corpus callosum. The clinical features of this patient are compared with 13 patients reported previously. A common critical region (CCR) of 1.4 Mb (54-55.4 Mb) is defined in all cases including the present one. Of the 14 genes present in CCR, IL6ST is proposed to be the candidate gene for immunodeficiency observed in some of these patients. IL6ST encodes gp130, a signal transduction protein for various interleukins and cytokines. It is involved in the generation of both T and B lymphocytes as well as the production of acute-phase reactants. Microdeletion 5q11.2 should be considered as a recognisable syndrome based on the common phenotype and the novel features described.

Identification of mutations, genotype-phenotype correlation and prenatal diagnosis of maple syrup urine disease in Indian patients.

Maple syrup urine disease (MSUD) is caused by mutations in genes BCKDHA, BCKDHB, DBT encoding E1α, E1ß, and E2 subunits of enzyme complex, branched-chain alpha-ketoacid dehydrogenase (BCKDH). BCKDH participates in catabolism of branched-chain amino acids (BCAAs) - leucine, isoleucine and valine in the energy production pathway. Deficiency or defect in the enzyme complex causes accumulation of BCAAs and keto-acids leading to toxicity. Twenty-four patients with MSUD were enrolled in the study for molecular characterization and genotype-phenotype correlation. Molecular studies were carried out by sequencing of the 3 genes by Sanger method. Bioinformatics tools were employed to classify novel variations into pathogenic or benign. The predicted effects of novel changes on protein structure were elucidated by 3D modeling. Mutations were detected in 22 of 24 patients (11, 7 and 4 in BCKDHB, BCKDHA and DBT genes, respectively). Twenty mutations including 11 novel mutations were identified. Protein modeling in novel mutations showed alteration of structure and function of these subunits. Mutations, c.1065 delT (BCKDHB gene) and c.939G > C (DBT gene) were noted to be recurrent, identified in 6 of 22 alleles and 5 of 8 alleles, respectively. Two-third patients were of neonatal classical phenotype (16 of 24). BCKDHB gene mutations were present in 10 of these 16 patients. Prenatal diagnoses were performed in 4 families. Consanguinity was noted in 37.5% families. Although no obvious genotype-phenotype correlation could be found in our study, most cases with mutation in BCKDHB gene presented in neonatal period. Large number of novel mutations underlines the heterogeneity and distinctness of gene pool from India.

Down syndrome in India--diagnosis, screening, and prenatal diagnosis.

Down syndrome (DS) is the most common genetic cause of mental retardation. Clinical manifestations are variable, and children have psychomotor impairment, multiple malformations, and medical conditions. Confirmation of the diagnosis is by karyotype analysis. The cytogenetic abnormality can be classified into pure trisomy 21, translocation, or mosaicism. Risk of recurrence depends on the primary cytogenetic abnormality in the proband. Prenatal screening is by biochemical and ultrasound markers in the first and second trimester. Definitive prenatal diagnosis is by analysis of fetal chromosomes in fetal chorionic villi, amniocytes, or cord blood. A noninvasive test for trisomy 21 in maternal blood has been developed by massively parallel shotgun sequencing. Therapeutic studies in Ts65Dn mice suggest an exciting prospect of improvement of learning ability and memory deficits.