New insights from the genetic work-up in early onset nephrotic syndrome: report from a registry in western India.

BACKGROUND: Eighty-five percent of infants with congenital nephrotic syndrome (CNS) and 66% with infantile NS (INS) are likely to have a monogenic etiology. There exists a significant genetic variability between different regions and ethnic groups. This study aimed to determine the genetic defects in children with CNS and INS by establishing a registry in western India. METHODS: In this cross-sectional study, pediatric nephrologists from 13 private and government institutions shared relevant clinical data and details of the genetic evaluation of children presenting with NS within the first year of life. RESULTS: The median age at presentation was 9 months (range 1-23, IQR 3-13 months), history of consanguinity between parents existed in 14 patients (34%), family history of similar illness in 6 (15%), and extra-renal manifestations in 17 (41%). Twenty-five (61%) were confirmed to have a monogenic etiology. NPHS1 gene was the most implicated (9/25) followed by PLCE1 (5/25). There were 12 variants of uncertain significance (VUS) involving 10 genes (10/25, 40%), and no definite genetic abnormality was found in 4 (25%). A re-analysis of these VUS attempted 2-3 years later facilitated reclassification of 7/12 (58%); increasing the diagnostic yield from 61 to 68.2%. CONCLUSIONS: Consistent with worldwide data, variants in NPHS1 gene were the most common cause of NS in infancy; however, PLCE1 was implicated more frequently in our cohort. NUP93 and COL4A3 were reported in early onset NS for the first time. Reclassification of VUS should be attempted, if feasible, since it may lead to a useful revision of diagnosis.

High prevalence of CFHR deletions in Indian women with pregnancy-associated hemolytic uremic syndrome.

AIM: Pregnancy-associated hemolytic uremic syndrome (P-aHUS) is an important cause of peripartum acute kidney injury. Studies from Europe have described mutations in complement regulator genes, and data in Indian patients is scarce. Hence this study used multiplex ligation-dependent probe amplification (MLPA) to identify variants in complement genes in P-aHUS patients. METHODS: We present 17 patients of P-aHUS who were investigated for complement protein levels and genetic analysis with MLPA for complement genes. Plasma exchange therapy was offered to all patients presenting in acute phase. RESULTS: Mean age 26.74 (3.36) years with 15/17 delivered by caesarean section. Eleven patients received early (within 7 days) plasma exchange, three were dialysis-dependent at 3 months and seven were dialysis-free. Only one of the three patients receiving late (after 7 days) plasma exchange was dialysis-free. MLPA showed that 11 patients had heterozygous deletions of exons 3, 5, 6 of CFHR1 and upstream region of exons 1, 2, 3, 6 and intron 4 of CFHR3 gene while four patients had homozygous deletions at the same loci. Two patients had no MLPA-detectable variations. CONCLUSION: This study reports a high proportion of deletions of exons of CFHR1 & CFHR3 genes in Indian P-aHUS patients detectable by MLPA by copy number variations. This needs confirmation in large multicentre studies. Plasma exchange can be an effective therapy in the non-availability of Eculizumab.

SMN1 gene copy number analysis for spinal muscular atrophy (SMA) in a Turkish cohort by CODE-SEQ technology, an integrated solution for detection of SMN1 and SMN2 copy numbers and the "2+0" genotype.

INTRODUCTION: Spinal muscular atrophy (SMA) is one of the common autosomal recessive disorders with global heterozygous carrier frequency of 1:50. Due to high carrier frequency, significant morbidity associated with the infantile onset disease and prohibitive cost of recently approved therapy, American College of Medical Genetics and Genomics (ACMG) recommends population based screening for SMA carrier status in eligible individuals in the reproductive age group. CODE-SEQ is a novel proprietary next generation sequencing (NGS) based assay, which is capable of detecting homozygous as well as heterozygous SMN1 exon 7 deletions. Along with the copy number estimation, this assay is capable of detecting single nucleotide polymorphisms (SNPs) associated with silent SMA carrier status or "2+0" genotype. METHODS: We have validated a proprietary CODE-SEQ technology in a blinded cohort of 80 clinically well characterized samples from Turkish population for the detection of SMA carriers as well as affected cases. The results were correlated with gold standard MLPA assay. RESULTS: The copy numbers in exon 7 of SMN1 gene matched with MLPA results in all 80 samples giving 100% correlation. The assay accurately detected the presence/ absence of SNPs associated with "2+0" genotype in the reference samples. None of the tested clinical samples had these SNPs. CONCLUSION: The results of this study support the notion that CODE-SEQ will be extremely useful in detecting SMA genotypes in large-scale population-based screening studies.

Spectrum of prenatally detected central nervous system malformations: Neural tube defects continue to be the leading foetal malformation.

BACKGROUND & OBJECTIVES: Prenatal diagnosis of malformations is an important method of prevention and control of congenital anomalies with poor prognosis. Central nervous system (CNS) malformations amongst these are the most common. The information about the prevalence and spectrum of prenatally detected malformations is crucial for genetic counselling and policymaking for population-based preventive programmes. The objective of this study was to study the spectrum of prenatally detected CNS malformations and their association with chromosomal abnormalities and autopsy findings. METHODS: This retrospective study was conducted in a tertiary care hospital in north India from January 2007 to December 2013. The details of cases with prenatally detected CNS malformations were collected and were related with the foetal chromosomal analysis and autopsy findings. RESULTS: Amongst 6044 prenatal ultrasonographic examinations performed; 768 (12.7%) had structural malformations and 243 (31.6%) had CNS malformations. Neural tube defects (NTDs) accounted for 52.3 per cent of CNS malformations and 16.5 per cent of all malformations. The other major groups of prenatally detected CNS malformations were ventriculomegaly and midline anomalies. Chromosomal abnormalities were detected in 8.2 per cent of the 73 cases studied. Foetal autopsy findings were available for 48 foetuses. Foetal autopsy identified additional findings in eight foetuses and the aetiological diagnosis changed in two of them (4.2%). INTERPRETATION & CONCLUSIONS: Amongst prenatally detected malformations, CNS malformations were common. NTD, which largely is a preventable anomaly, continued to be the most common group. Moreover, 60 per cent of malformations were diagnosed after 20 weeks, posing legal issues. Chromosomal analysis and foetal autopsy are essential for genetic counselling based on aetiological diagnosis.

Clinical and mutation profile of multicentric osteolysis nodulosis and arthropathy.

​Multicentric osteolysis nodulosis and arthropathy (MONA) is an infrequently described autosomal recessive skeletal dysplasia characterized by progressive osteolysis and arthropathy. Inactivating mutations in MMP2, encoding matrix metalloproteinase-2, are known to cause this disorder. Fifteen families with mutations in MMP2 have been reported in literature. In this study we screened thirteen individuals from eleven families for MMP2 mutations and identified eight mutations (five novel and three known variants). We characterize the clinical, radiographic and molecular findings in all individuals with molecularly proven MONA from the present cohort and previous reports, and provide a comprehensive review of the MMP2 related disorders.

Recurrent benign copy number variants & issues in interpretation of variants of unknown significance identified by cytogenetic microarray in Indian patients with intellectual disability.

BACKGROUND & OBJECTIVES: Cytogenetic microarray (CMA) is now recommended as a first-tier clinical diagnostic test in cases with idiopathic intellectual disability and/or developmental delay (ID/DD). Along with clinically relevant variants, CMA platforms also identify variants of unknown significance (VUS). This study was done to look for utility and various issues in interpretation of copy number variants (CNVs) in Indian patients with ID/DD. METHODS: The CMA was performed in 86 Indian patients with idiopathic ID/DD with or without dysmorphic features. CNV was reported if copy number gain was >400 kb in size and copy number loss was > 200 kb in size. RESULTS: Pathogenic CNVs were found in 18 of 86 (20.9%) patients. One large (14 Mb size) de novo heterozygous copy number gain was found in one patient. VUS (total 31) were present in 17 of 86 (19.7%) patients. Five novel recurrent benign CNVs were also present in our patients. INTERPRETATION & CONCLUSIONS: Our findings highlight the difficulties in interpretation of CNVs identified by CMA. More Indian data on VUS and recurrent benign CNVs will be helpful in the interpretation of CMA in patients with ID/DD.

Hemiconvulsion-hemiplegia-epilepsy syndrome with 1q44 microdeletion: causal or chance association.

Hemiconvulsion-hemiplegia-epilepsy (HHE) syndrome is a rare syndrome characterized by childhood onset partial motor convulsions, hemiplegia, and epilepsy in sequence. Exact pathogenesis is not clear. Here we are describing a 3-year-old girl with HHE syndrome with cytogenetic microarray (CMA) showing deletion of 1.8 Mb in 1q44 region. Along with HHE syndrome, the patient also had global developmental delay, subtle facial dysmorphism, and preaxial polydactyly. Clinical phenotype of 1q44 microdeletion syndrome is quite variable. Main clinical features are microcephaly, seizures, and abnormality of corpus callosum. We compared the patient's phenotype with other patients in 10 previously published papers of 1q44 microdeletion syndrome. HNRNPU and FAM36A are two important genes in the deleted region. HNRNPU gene mediate long range control of SHH gene which is likely explanation of preaxial polydactyly in the present patient. HHE may be a chance co-occurrence.

GALNS mutations in Indian patients with mucopolysaccharidosis IVA.

Mucopolysaccharidosis IV A (Morquio syndrome A, MPS IVA) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS). The mutation spectrum in this condition is yet to be determined in Indians. We aimed to analyze the mutations in the GALNS gene in Asian Indians with MPS IVA. All the exons and the adjacent intronic regions of the gene were amplified and sequenced in sixty-eight unrelated Indian families. We identified 136 mutant alleles comprising of 40 different mutations. We report twenty-two novel mutations that comprise of seventeen missense (p.Asn32Thr, p.Leu36Arg, p.Pro52Leu, p.Pro77Ser, p.Cys79Arg, p.His142Pro, p.Tyr191Asp, p.Asn204Thr, p.Gly188Ser, p.Phe216Ser, p.Trp230Cys, p.Ala291Ser, p.Gly317Arg, p.His329Pro, p.Arg386Ser, p.Glu450Gly, p.Cys501Ser), three splice-site variants (c.120+1G>C, c.1003-3C>G, c.1139+1G>A), one nonsense mutation (p.Gln414*) and one frameshift mutation (p.Pro420Leufs*440). Eighteen mutations have been reported earlier. Among these p.Ser287Leu (8.82%), p.Phe216Ser (7.35%), p.Asn32Thr (6.61%) and p.Ala291Ser (5.88%) were the most frequent mutations in Indian patients but were rare in the mutational profiles reported in other populations. These results indicate that the Indian patients may have a distinct mutation spectrum compared to those of other populations. Mutant alleles in exon 1, 7 and 8 accounted for 44.8% of the mutations, and sequencing of these exons initially may be a cost-effective approach in Asian Indian patients. This is the largest study on molecular analysis of patients with MPS IVA reported in the literature, and the first report from India.

Angelman syndrome and prenatally diagnosed Prader-Willi syndrome in first cousins.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by loss of function of imprinted genes in the 15q11-13 critical region. Reports of PWS and AS in close relatives within the same family are rare. We report on the diagnosis of a familial unbalanced 10;15 translocation causing AS in a child that led to the prenatal diagnosis of an unbalanced 10;15 translocation with resultant deletion of the Prader-Willi critical region in her maternal uncle's offspring.

Late termination of pregnancy for fetal abnormalities: The perspective of Indian lay persons and medical practitioners.

OBJECTIVE: The objective of this article was to ascertain the opinion of lay persons and medical practitioners in India regarding late termination of pregnancies (LTOP) for fetal abnormalities. METHODS: One hundred and fifty lay persons and 120 medical practitioners were given separate questionnaires and asked their opinions regarding LTOP for prenatally detected fetal abnormalities of varying severity. The views regarding legalisation of LTOP and the acceptability of feticide by the lay persons were also ascertained. RESULTS: More than two-thirds of the lay persons and majority (85.8%) of clinicians felt that LTOP should be allowed for fetal conditions with poor prognosis. At least 70% of lay persons felt that LTOP should be legalised for severe fetal abnormalities. For potentially treatable conditions, continuation of pregnancy in late gestation was the preferred option. For lethal malformations like anencephaly and disorders requiring lifelong treatment like meningomylocele and thalassemia major, majority of clinicians (86.7%, 69.2% and 55.8%, respectively) and lay persons (65%, 51% and 25%, respectively) had the opinion that termination of pregnancy can be offered at any gestational age. CONCLUSION: Both the lay persons as well as the medical fraternity in India feel the need to look into revision of legalisation of LTOP particularly for fetal conditions with poor outcome.

Effect of dentin biomodification techniques on the stability of the bonded interface.

AIM: The aim of this study is to evaluate the effect of different bonding techniques ethanol wet bonding and dimethyl sulfoxide (DMSO) wet bonding and a novel collagen cross-linker Quercetin application on the durability of resin-dentin bond and observe the bonded interface under the scanning electron microscope (SEM). MATERIALS AND METHODS: For shear bond strength testing, flat coronal dentin surfaces were prepared on 110 extracted human molars. Teeth were randomly divided into five experimental groups according to different surface pretreatments techniques. Group A was control group without any surface pretreatment. In Group B, ethanol wet bonding pretreatment was done before the application of adhesive. In Group C, DMSO wet bonding was done before the application of adhesive and in Groups D and E, Quercetin along with ethanol and Quercetin along with DMSO pretreatment, respectively, were done before adhesive application. Composite restorations were placed in all the samples. Twenty samples from each group were subjected to immediate and delayed (9 months) shear bond strength evaluation. In addition, two samples per group were subjected to the scanning electron microscopic analysis for the observation of resin-dentin interface. STATISTICAL ANALYSIS: Data collected were subjected to the statistical analysis using the one-way analysis of variance and post hoc Tukey's test at a significance level of P < 0.05. RESULTS: Dentin pretreatment with all the techniques resulted in significantly higher resin-dentin bond strength after 9 months storage with DMSO group showing the highest bond strength values. CONCLUSION: It can be concluded that these biomodification techniques can improve the durability of the resin-dentin bond.

A novel homozygous frameshift variant in the ABCC2-gene in Dubin-Johnson syndrome may predispose to chronic liver disease.

Dubin-Johnson syndrome (DJS), an autosomal recessive disorder presenting with conjugated hyperbilirubinemia, is not associated with progression to chronic liver disease (CLD). Next-generation sequencing, application of bioinformatics pipeline, and segregation analysis were performed on 8 members of a consanguineous family with DJS and CLD. A novel variant, c.4406_4407delTA (p.Leu1469fs), in the ABCC2-gene in a homozygous state was found to be associated with DJS and CLD in proband and afflicted family members. DJS may not be a benign entity and novel genetic variants may be associated with progressive liver disease.

Novel Mutations in the DGKE Gene in Two Indian Patients with Early-onset Atypical Haemolytic Uraemic Syndrome.

Atypical haemolytic uremic syndrome (aHUS) is a clinically and genetically heterogeneous condition caused by a complex interplay between genomic susceptibility factors and environmental influences. Pathogenic variants in the DGKE gene are recently identified in cases with infantile-onset autosomal recessive aHUS. The presence of low serum C3 levels, however, has rarely been described in cases of DGKE-associated aHUS. Molecular genetic testing was performed by a commercial next-generation sequencing (NGS) panel as well and by an in-house developed targeted NGS for DGKE gene. Copy number variations (CNVs) were computed from NGS data by calculating a normalised copy number ratio of aligned number of reads at targeted genomic regions against multiple reference regions of the same sample and multiple controls. We report here two such novel clinically relevant variants (c.727_730delTTGT and c.251_259delGCGCCTTC) in the DGKE gene, in two families of infantile aHUS with low serum C3 levels.

Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congenital adrenal hyperplasia from India.

PURPOSE: Accurate diagnosis is required for management of Congenital adrenal hyperplasia (CAH). The conventional method for detection of mutations in the CYP21A2 gene is targeted capillary sequencing which is labor intensive and has limited multiplexing capability. Next generation sequencing (NGS) provides data with high sequence coverage and depth. Our objective was to develop an accurate NGS-based assay to characterize the mutation spectrum in CYP21A2 gene in Indian patients suspected to have 21-OH CAH. METHODS: Cases with 21-OH CAH from 12 endocrine units across India were studied. DNA was extracted from proband's and parent's(subset) blood. Locus-specific long-range PCR and gel electrophoresis of amplicons was followed by NGS where no visible 30 kb homozygous/whole gene deletion was observed. Orthogonal confirmation was performed by capillary sequencing (ABI 3500) and Multiplex Ligation-dependent Probe Amplification (MLPA, MRC-Holland). PCR products were purified and individual libraries were pooled and sequenced (Illumina). RESULTS: Of the 310 CAH cases, biallelic mutations (pathogenic/ likely pathogenic variants involving both CYP21A2 gene copies) were detected in 256 (82.6%), heterozygous mutations in 13 (4.2 %), and none in 41 (13.2%). Most common mutation was c.293-13A/C>G (29.03%), followed by 30 kb deletion (18.24%). Thirty samples tested orthogonally (by capillary sequencing or MLPA) showed 100% concordance with NGS assay. Nine novel variants were identified. CONCLUSIONS: We have developed and validated a comprehensive NGS-based assay for detection of variants in CYP21A2 gene in patients with 21-OH CAH. We describe CYP21A2 mutation spectrum and novel variants in a large cohort of Indian patients with CAH.

Atypical HUS Associated with CFH/CFHR-1 Hybrid Gene.

Atypical hemolytic uremic syndrome is a rare form of thrombotic microangiopathy caused by complement pathogenic variants. We describe a case of a 33-year-old woman who presented as rapidly progressing renal failure requiring dialysis and had anemia, microhematuria, low C3, normal C4 levels, and normal platelet count. Renal biopsy revealed arteriolar thrombotic microangiopathy and acute tubular injury. Patient was treated with plasma exchange and hemodialysis as required. This resulted in partial recovery at 1 month. Genetic workup by multiplex ligation-dependent probe amplification revealed a 1.5 times higher signal intensity on downstream region of CFH gene and 50% reduced intensity of exon 6 of CFHR1 gene, suggesting a gene conversion event, similar to those previously reported from Spain and Portugal.

Detection of a novel mutation in the rpoB gene in a multidrug resistant Mycobacterium tuberculosis isolate using whole genome next generation sequencing.

BACKGROUND: Mycobacterium tuberculosis (Mtb) drug resistance is a global concern. Moreover, multiple drug resistant (MDR), extensively drug resistant (XDR), and totally drug resistant (TDR) Mtb cases are on the rise in developing countries like India. Most of these cases are identified only 3-6 months after initiation of treatment owing to incomplete/failed clinical response and incomplete information from phenotypic drug resistance assays and/or targeted Mtb mutation analysis. Here, we report the development of an in-house whole genome sequencing (WGS) assay and bioinformatics pipeline that helped resolve the phenotype-genotype discrepancy in a clinical isolate. METHODOLOGY AND RESULTS: A sample from a suspected drug resistant Mtb case tested by line probe assay (LPA) showed the absence of both the mutant and wild type alleles for an rpoB gene mutation site. An in-house next generation sequencing (NGS) assay was used for WGS of this isolate. Bioinformatics analysis revealed that the isolate harboured a novel insertional mutation in the 81-bp hotspot region of the rpoB gene and a S315T mutation in the katG gene, which could explain resistance to rifampicin and isoniazid, respectively. These results correlated with the clinical diagnosis, LPA, solid culture drug susceptibility testing, and pyrosequencing carried out on the sample. The WGS data also provided information regarding the isolate's lineage and indicated an absence of known mutations conferring resistance to other antitubercular drugs. CONCLUSION: WGS is a highly sensitive, specific, and unbiased approach for identification of all possible drug resistance-conferring mutations, which can help clinicians make more informed treatment-related decisions.

A Targeted Next Generation Sequencing Panel for Non-syndromic Early Onset Severe Obesity and Identification of Novel Likely -Pathogenic Variants in the MC4R and LEP Genes.

OBJECTIVES: To screen for variants in the MC4R and LEP genes in 46 patients with clinical suspicion of non-syndromic early onset severe obesity (NEOSO). METHODS: Children with early onset obesity satisfying WHO criteria of obesity were studied. The MC4R and LEP genes were sequenced using a PCR amplicon based NGS on Illumina MiSeq next generation sequencer using an in-house developed protocol. RESULTS: Of the 46 children tested, four were found to have novel pathogenic/likely-pathogenic variants (one in the MC4R gene and three in the LEP gene). In three out of the 4 families, the presence of the variants was confirmed using standard bidirectional capillary sequencing in the probands. CONCLUSIONS: Four children with novel likely pathogenic variants in the MC4R and LEP genes are reported. Genetic analysis is crucial in children with early onset obesity and should be considered.

A Novel Phosphoinositide-3-kinase Adapter Protein 1 Gene Missense Mutation in Familial Cirrhosis.

Familial cirrhosis is a condition that is associated with the presence of liver disease with genetic linkage among multiple family members in a generation or in multiple generations. With cirrhosis, most of these disease pathogeneses are related to a defect of an enzyme/transport protein leading to a deranged metabolic pathway with variable prevalence. Many studies and high-quality metanalyses have shed light on genetic linkage associated with nonalcoholic fatty liver disease and steatohepatitis such as the PNPLA3, MBOAT7, and TM6SF2 variants. In this report, we shed light on a novel missense mutation associated with cirrhosis in a family of brothers associated with phosphoinositide-3-kinase adapter protein 1 gene through high-output whole exosome gene sequencing methodology.