Sakthivel Murugan

Retinoblastoma in India

AbstractObjectives: This study was conducted with two objectives. The first was to estimate the frequency of loss of heterozygosity (LOH) of the RB1 gene as a mechanism in disease causation in tumors of patients from India. The second objective was to employ RB1 molecular deletion and microsatellite-based linkage analysis as laboratory tools, while counseling families with a history of retinoblastoma (RB). Methods: DNA was extracted from peripheral blood and tumors of 54 RB patients and their relatives. Eight fluorescent microsatellite markers, both intragenic and flanking the RB1 gene, were used. After PCR amplification, samples were run on an ABI PRISM® 310 genetic analyzer for LOH, deletion detection, and haplotype generation. Results: LOH was found in conjunction with tumor formation in 72.9% of RB patients (39/54 patients; p = 0.001; 95% CI 0.6028, 0.8417); however, we could not associate various other clinical parameters of RB patients with the presence or absence of RB1 LOH. Seven germline deletions (13% of RB patients) were identified, and the maternal allele was more frequently lost (p = 0.01). A disease co-segregating haplotype was detected in two hereditary autosomal dominant cases. Conclusion: LOH of the RB1 gene could play an important role in tumor formation. Large deletions involving RB1 were observed, and a disease co-segregating haplotype was used for indirect genetic testing. This is the first report from India where molecular testing has been applied for RB families in conjunction with genetic counseling. In tertiary ophthalmic practice in India, there is an emerging trend towards the application of genetical knowledge in clinical practice.

A splice site mutation in HERC1 leads to syndromic intellectual disability with macrocephaly and facial dysmorphism: Further delineation of the phenotypic spectrum.

We report on a sib pair of Indian origin presenting with intellectual disability, dysmorphism, and macrocephaly. Exome sequencing revealed a homozygous splice site HERC1 mutation in both probands. Functional analysis revealed use of an alternate splice site resulting in formation of a downstream stop codon and nonsense mediated decay. In the light of recent reports of HERC1 mutations in two families with a similar phenotypic presentation, this report reiterates the pathogenic nature and clinical consequences of HERC1 disruption.

Comprehensive genomic analysis identifies pathogenic variants in maturity-onset diabetes of the young (MODY) patients in South India

BackgroundMaturity-onset diabetes of the young (MODY) is an early-onset, autosomal dominant form of non-insulin dependent diabetes. Genetic diagnosis of MODY can transform patient management. Earlier data on the genetic predisposition to MODY have come primarily from familial studies in populations of European origin.MethodsIn this study, we carried out a comprehensive genomic analysis of 289 individuals from India that included 152 clinically diagnosed MODY cases to identify variants in known MODY genes. Further, we have analyzed exome data to identify putative MODY relevant variants in genes previously not implicated in MODY. Functional validation of MODY relevant variants was also performed.ResultsWe found MODY 3 (HNF1A; 7.2%) to be most frequently mutated followed by MODY 12 (ABCC8; 3.3%). They together account for ~ 11% of the cases. In addition to known MODY genes, we report the identification of variants in RFX6, WFS1, AKT2, NKX6–1 that may contribute to development of MODY. Functional assessment of the NKX6–1 variants showed that they are functionally impaired.ConclusionsOur findings showed HNF1A and ABCC8 to be the most frequently mutated MODY genes in south India. Further we provide evidence for additional MODY relevant genes, such as NKX6–1, and these require further validation.

Next-Generation Sequencing Reveals Novel Mutations in X-linked Intellectual Disability

Robust diagnostics for many human genetic disorders are much needed in the pursuit of global personalized medicine. Next-generation sequencing now offers new promise for biomarker and diagnostic discovery, in developed as well as resource-limited countries. In this broader global health context, X-linked intellectual disability (XLID) is an inherited genetic disorder that is associated with a range of phenotypes impacting societies in both developed and developing countries. Although intellectual disability arises due to diverse causes, a substantial proportion is caused by genomic alterations. Studies have identified causal XLID genomic alterations in more than 100 protein-coding genes located on the X-chromosome. However, the causes for a substantial number of intellectual disability and associated phenotypes still remain unknown. Identification of causative genes and novel mutations will help in early diagnosis as well as genetic counseling of families. Advent of next-generation sequencing methods has accelerated the discovery of new genes involved in mental health disorders. In this study, we analyzed the exomes of three families from India with nonsyndromic XLID comprising seven affected individuals. The affected individuals had varying degrees of intellectual disability, microcephaly, and delayed motor and language milestones. We identified potential causal variants in three XLID genes, including PAK3 (V294M), CASK (complex structural variant), and MECP2 (P354T). Our findings reported in this study extend the spectrum of mutations and phenotypes associated with XLID, and calls for further studies of intellectual disability and mental health disorders with use of next-generation sequencing technologies.

First Report of Kufor‐Rakeb Syndrome (PARK 9) from India, and a Novel Nonsense Mutation in ATP13A2 Gene

To the Editor: Kufor-Rakeb syndrome (KRS; PARK 9) is a rare autosomalrecessive form of juvenile-onset Parkinson’s disease (PD) caused by ATP13A2 gene mutations. The classical description of KRS is that of rapidly progressive symptoms in the form of parkinsonism, spasticity, supranuclear upgaze paresis, facial-faucialfinger minimyoclonus, visual hallucinations, oculogyric dystonic spasms, and dementia, usually noted between 12 and 16 years of age, resulting in early severe motor handicap. World-wide prevalence of KRS is unknown, with only case reports/series being published. We report on the first case of KRS from India, with previously unreported nonsense mutation in exon 22 of ATP13A2 gene (chr1: 17316187; G>A). An 18-year-old adolescent male born of consanguineous parentage presented with history of reduced interactions, withdrawn behavior, which was noted at the age of 16. Over a period of time, he started to have flexed posturing of the upper limb with small and illegible handwriting along with keeping his mouth open constantly. During the last 6 to 8 months, he started to notice mild tremors of the right upper limb with dragging of the right foot while walking. He was initially evaluated with a diagnosis of depression/behavior disorder by various doctors. Subsequently, he was considered to have PD and was started on levodopa (50 mg, twice a day) with which he developed dyskinetic movements of the tongue and foot, at which time he was referred to us for further evaluation. On evaluation, he was noted to have masked facies, rigidity, bradykinesia (right > left), and slow saccades (horizontally and vertically). Range of eye movements, power, sensory examination, reflexes, and coordination were normal (see Video 1). There was no family history of parkinsonism. The only nonmotor symptom noted was of anxiety. All his blood and metabolic workup for early-onset parkinsonism, including brain MRI, was normal (Supporting Fig. 1). He was started on dopamine agonist (pramipexole), with which significant clinical improvement has been noticed and is currently attending his college, albeit mild difficulty of writing still persists (slowness). Genomic DNA from the proband was used to perform exome sequencing using the Agilent SureSelectXT capture kit (Agilent Technologies, Santa Clara, CA). The sample was sequenced with mean coverage of 80 to 1009 on an Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA). Sequenced data were aligned to the human reference genome (GRCh37/hg19) using the BWA program. Subsequent to this, the GATK-Lite pipeline was used for realignment, base recalibration, variant calling, and variant filtering. Filtered variants were annotated using the VariMAT pipeline (internal data analysis pipeline of MedGenome), and clinically relevant mutations were identified from published literature and various databases, including ClinVar, OMIM, SwissVar, HGMD, GWAS, ExAC, 1000-Genome, dbSNP, EVS. Only nonsynonymous and splice-site variants detected in the targeted genes were used for clinical interpretation. Whole-exome sequencing, performed to identify the causative gene variation, did not detect any literature-reported variations for the clinical symptoms. A homozygous nonsense variation in exon 22 of ATP13A2 gene (chr1: 17316187; G>A), which results in a stop codon and premature truncation of the protein at codon 826 (ENST00000341676:c.2476C>T, ENSP00000341115: p.Q826Ter), was detected. This variant was not found in different common variation databases, such as 1000Genome, ExAC, dbSNP, and EVS. The variant was also not detected in 100 normal controls that were screened by us. The Exome Aggregation Consortium does not report this variant in its database. After this, analysis of both the parents showed the same mutation in the heterozygous state, indicating that they are the carriers for this likely pathogenic mutation (Fig. 1). Since the initial description of KRS, case reports/series have been published from various parts of the world, including Pakistan and Afghanistan. To date, no documented cases have been published from India. Initial description of KRS was a rapidly progressive disorder with early development of significant motor disabilities. However, newer publications have indicated variable phenotypes, including that of intact cognition, absence of myoclonus/tremors, and slower clinical progression. Most of these patients have good L-dopa response. In comparison to published literature, our case had a clear autosomal-recessive inheritance, with

A neoepitope derived from a novel human germline APC gene mutation in familial adenomatous polyposis shows selective immunogenicity

Familial adenomatous polyposis (FAP) is an inherited condition arising from genetic defects in the Adenomatous polyposis coli (APC) gene. Carriers with mutations in the APC gene develop polyps in the colon and rectum which if not managed, transition into colon cancer. In this study, we identified a novel germline mutation in the APC gene in members of an FAP-affected (Familial adenomatous polyposis) family. This unique heterozygous variant (c.735_736insT; p.Ser246PhefsTer6) was identified in ten out of twenty six family members, ranging in age from 6 to 60 years. Polyps were detected in six of the ten individuals (35–60 years) carrying this mutation. The remaining four members (6–23 years) remain polyp free. A significant fraction of FAP affected individuals eventually develop colon cancer and therapeutic interventions to prevent cancer progression remain elusive. To address this issue, we sought to determine if peptides derived from the novel APC mutation could induce a cytotoxic T cell response, thereby qualifying them as vaccine candidates. Peptides harboring the variant amino acids were first interrogated in silico for their immunogenicity using a proprietary neoepitope prioritization pipeline, OncoPeptVAC. A single 9-mer peptide was predicted to be immunogenic. Remarkably, CD8+ T cells isolated from either an FAP+/ APCmut individual, or from a FAP-/ APCmut individual, failed to respond to the peptide, whereas those from either an unaffected family member (FAP-/ APCwt) or from healthy unrelated donors, showed a robust response, suggesting that CD8+ T cells from individuals carrying this germline APC mutation have been tolerized to the mutation. Furthermore, experimental testing of six additional reported APC gene mutation-derived peptides revealed one of the six to be immunogenic. While not all APC mutant peptides are inmmunogenic, a few qualify as vaccine candidates offering novel treatment opportunities to patients with somatic APC gene mutations to delay/treat colorectal cancer.

A novel splice variant in EMC1 is associated with cerebellar atrophy, visual impairment, psychomotor retardation with epilepsy

Several genes have been implicated in a highly variable presentation of developmental delay with psychomotor retardation. Mutations in EMC1 gene have recently been reported. Herein, we describe a proband born of a consanguineous marriage, who presented with early infantile onset epilepsy, scaphocephaly, developmental delay, central hypotonia, muscle wasting, and severe cerebellar and brainstem atrophy.

Molecular alterations associated with chronic exposure to cigarette smoke and chewing tobacco in normal oral keratinocytes.

ABSTRACT Tobacco usage is a known risk factor associated with development of oral cancer. It is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, molecular alterations induced by tobacco are poorly understood. We therefore sought to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes (OKF6/TERT1). OKF6/TERT1 cells acquired oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed whole exome sequencing (WES) and quantitative proteomics to investigate the molecular alterations in oral keratinocytes chronically exposed to smoke/ chewing tobacco. Exome sequencing revealed distinct mutational spectrum and copy number alterations in smoke/ chewing tobacco treated cells. We also observed differences in proteomic alterations. Proteins downstream of MAPK1 and EGFR were dysregulated in smoke and chewing tobacco exposed cells, respectively. This study can serve as a reference for fundamental damages on oral cells as a consequence of exposure to different forms of tobacco.