SETD2 controls m6A modification of transcriptome and regulates the molecular oncogenesis of glioma.

SETD2 is known for its epigenetic regulatory function and a frequently mutated gene in multiple cancers. Recently, it has been inferred that SETD2 regulates m6A mRNA methylation (epitranscriptome) via H3K36me3. The m6A RNA methylation is vital for tumor maintenance, self-renewal, and tumorigenesis. RNA modifications are executed by writers, readers, and erasers. m6A modifiers work along with the molecular cues, H3K36me3, laid down by SETD2. A positive correlation observed between SETD2 and RNA modifiers signifies their direct role in epitranscriptomics. Hence, understanding the epitranscriptomics will provide a new facet for molecular oncogenesis. Glioma is a common, malignant grade IV tumor with limited therapeutic alternatives and a poor prognosis. Yet, its function in glioma is not fully defined. In the present study, thorough investigations were done in the m6A RNA methylation regulators expression, the molecular pathways leading to tumor progression, and their respective outcomes in SETD2-mediated RNA methylation. In vitro analysis reveals that SETD2 knockdown positively affected the oncogenic properties of the glioma cell line and a global reduction in m6A levels in the transcriptome. The reduction of m6A in the transcriptome can be attributed to the decreased expression of METTL3 and METTL14. Therefore, we conclude that SETD2-mediated m6A modifications are crucial for glioma oncogenesis.

Deciphering the RNA-binding protein interaction with the mRNAs encoded from human chromosome 15q11.2 BP1-BP2 microdeletion region.

Microdeletion of the 15q11.2 BP1-BP2 region, also known as Burnside-Butler susceptibility region, is associated with phenotypes like delayed developmental language abilities along with motor skill disabilities, combined with behavioral and emotional problems. The 15q11.2 microdeletion region harbors four evolutionarily conserved and non-imprinted protein-coding genes: NIPA1, NIPA2, CYFIP1, and TUBGCP5. This microdeletion is a rare copy number variation frequently associated with several pathogenic conditions in humans. The aim of this study is to investigate the RNA-binding proteins binding with the four genes present in 15q11.2 BP1-BP2 microdeletion region. The results of this study will help to better understand the molecular intricacies of the Burnside-Butler Syndrome and also the possible involvement of these interactions in the disease aetiology. Our results of enhanced crosslinking and immunoprecipitation data analysis indicate that most of the RBPs interacting with the 15q11.2 region are involved in the post-transcriptional regulation of the concerned genes. The RBPs binding to this region are found from the in silico analysis, and the interaction of RBPs like FASTKD2 and EFTUD2 with exon-intron junction sequence of CYFIP1 and TUBGCP5 has also been validated by combined EMSA and western blotting experiment. The exon-intron junction binding nature of these proteins suggests their potential involvement in splicing process. This study may help to understand the intricate relationship of RBPs with mRNAs within this region, along with their functional significance in normal development, and lack thereof, in neurodevelopmental disorders. This understanding will help in the formulation of better therapeutic approaches.

RNA N6-methyladenosine modification in regulating cancer stem cells and tumor immune microenvironment and its implication for cancer therapy.

Therapy resistance is a well-known phenomenon in cancer treatment. It can be intrinsic or acquired, accountable for frequent tumor relapse and death worldwide. The interplay between cancer cells and their neighboring environment can activate complex signaling mechanisms influencing epigenetic changes and maintain cancer cell survival leading to the malignant phenotype. Cancer stem cells (CSCs) are tumor-initiating cells (TICs) and constitute the primary source of drug resistance and tumor recurrence. Studies have shown that cancer cells exhibit dysregulated RNA N6-methyladenosine (m6A) "writers," "erasers," and "readers" levels after acquiring drug resistance. The present review provides novel insight into the role of m6A modifiers involved in CSC generation, cancer cell proliferation, and therapy resistance. m6A RNA modifications in the cross-talk between CSC and the tumor immune microenvironment (TIME) have also been highlighted. Further, we have discussed the therapeutic potential of targeting m6A machinery for cancer diagnosis and the development of new therapies for cancer treatment.

Circular RNA: A novel and potential regulator in pathophysiology of schizophrenia.

Circular RNAs (CircRNAs) are a sub-class of non-coding RNA, which are covalently closed at the ends through a non-canonical process called, backsplicing from the precursor linear RNAs. These molecules are involved in several biological phenomena including regulation of gene expression, synaptic plasticity, and cognition. Several studies have shown that circRNA are present abundantly inside the mammalian brain and they are believed to be associated with the development of neurons and neuronal functions. It is also evident that alterations in intracellular and extracellular levels of circRNAs are linked with various neurological and neuropsychiatric disorders including schizophrenia (SZ). Detailed studies of circRNAs are required to decode the molecular mechanism behind the onset of SZ and the related biological activities during disease progression. This can help unravel their role in this neurobehavioral disorder and develop effective therapeutics against the disease. The present review mainly focuses on the expression and activities of the circRNAs in the post-mortem brain, peripheral blood, and exosomes. It also gives an insight into the role of circRNA interaction with RNA binding proteins (RBPs) and nucleotide modification and their therapeutic potential in the context of schizophrenia.

Identification of Novel Genomic Variations in Susceptibility to Nonsyndromic Cleft Lip and Palate Patients.

BACKGROUND: Nonsyndromic cleft lip with or without palate (NSCL/P) is a multifactorial and common birth malformation caused by genetic and environmental factors, as well as by teratogens. Genome-wide association studies found genetic variations with modulatory effects of NSCL/P formation in Chinese and Iranian populations. We aimed to identify the susceptibility of single-nucleotide polymorphisms (SNPs) to nonsyndromic cleft lip with or without palate in the Indian population. MATERIAL AND METHODS: The present study was conducted on NSCL/P cases and controls. Genomic DNA was extracted from peripheral blood and Axiom- Precision Medicine Research Array (PMRA) was performed. The Axiom-PMRA covers 902,527 markers and several thousand novel risk variants. Quality control-passed samples were included for candidate genetic variation identification, gene functional enrichment, and pathway and network analysis. RESULTS: The genome-wide association study identified fourteen novel candidate gene SNPs that showed the most significant association with the risk of NSCL/P, and eight were predicted to have regulatory sequences. CONCLUSION: The GWAS study showed novel candidate genetic variations in NSCL/P formations. These findings contribute to the understanding of genetic predisposition to nonsyndromic cleft lip with or without palate.

Brain exosomes as minuscule information hub for Autism Spectrum Disorder.

INTRODUCTION: Autism spectrum disorder (ASD) is a neurodevelopmental disorder initiating in the first three years of life. Early initiation of management therapies can significantly improve the health and quality of life of ASD subjects. Thus, indicating the need for suitable biomarkers for the early identification of ASD. Various biological domains were investigated in the quest for reliable biomarkers. However, most biomarkers are in the preliminary stage, and clinical validation is yet to be defined. Exosome based research gained momentum in various Central Nervous System disorders for biomarker identification. However, the utility and prospect of exosomes in ASD is still underexplored. AREAS COVERED: In the present review, we summarized the biomarker discovery current status and the future of brain-specific exosomes in understanding pathophysiology and its potential as a biomarker. The studies reviewed herein were identified via systematic search (dated: June 2021) of PubMed using variations related to autism (ASD OR autism OR Autism spectrum disorder) AND exosomes AND/OR biomarkers. EXPERT OPINION: As exosomess are highly relevant in brain disorders like ASD, direct access to brain tissue for molecular assessment is ethically impossible. Thus investigating the brain-derived exosomes would undoubtedly answer many unsolved aspects of the pathogenesis and provide reliable biomarkers.

Molecular spectrum, family screening and genetic counselling of Spinocerebellar Ataxia (SCA) cases in an Indian scenario.

Spinocerebellar Ataxia (SCA) is a heterogeneous adult-onset disorder with an autosomal dominant inheritance pattern mainly caused by triplet repeat expansions. Clinical diagnosis of SCA is based on phenotypic features followed by confirmation through molecular diagnosis. To identify status of repeat range in Indian SCA cases and provide extended family screening, we enrolled 70 clinical SCA suspects. For molecular diagnosis, multiplex PCR (M-PCR) was used for common Indian SCA subtypes 1, 2, 3, 6, 7, 10, 12 and 17. TP-PCR was further used in SCA2, 7 and 10 to identify larger expansions. Eighteen out of 70 SCA suspects (25%) were found to be positive for various SCA subtypes- (5 SCA1 (28%), 6 SAC2 (34%), 2 SCA3 (12%), 3 SCA7 (16%) and one each for SCA6 (1%) and SCA17 (1%) subtypes). Genetic counselling and extended family screening were offered to all positive cases and yielded additional nine cases. We have established M-PCR and TP-PCR to detect the CAG repeat expansion in SCA suspects. This method can confirm SCA subtypes in a reliable, rapid and cost-effective way. Genetic characterization of SCA-related genes has great clinical relevance, as it could provide additional information and guidance to clinicians and family members regarding prognosis.

Defining the role of FMR1 gene in unexplained recurrent spontaneous abortion.

INTRODUCTION: Recurrent spontaneous abortion is a multifactorial disorder and till date, various factors have been attributed in its pathogenesis. Still, approximately 50% of RSA cases remain unexplained. Premutation (PM) expanded allele of fragile-X mental retardation 1 (FMR1) gene is known to contribute to ovarian dysfunction in 20% of the cases. Recently, the link between expanded FMR1 allele and recurrent miscarriages has been reported. METHOD: In the present prospective case-control study, we have investigated the status of CGG repeat size at 5'UTR of the FMR1 gene in women with unexplained RSA in comparison to age-matched healthy control women (n = 100 each). The genomic DNA from these samples was subjected to molecular analysis for characterization of CGG repeat size and composition at FMR1 gene RESULTS: As compared to the control women, the RSA women cohort had a higher frequency of carriers with alleles in gray zone (GZ) and expanded PM range, i.e., 2% (2/100) versus 5% (5/100), respectively. Also, the RSA cohort had a significantly higher number of normal alleles with ≥ 35 CGG repeats (24 out of 200 alleles) as compared to control cohort (8 out of 200 alleles). The number of larger FMR1 alleles with pure CGG repeat tract was found to be significantly higher (P = 0.0063) in the RSA cohort (15 out of 200 alleles) as compared to that in control cohort (3 out of 200 alleles). CONCLUSION: Henceforth, the CGG expanded uninterrupted FMR1 allele might be associated with recurrent abortions and may help to explain many of these unexplained cases.

Fragile X molecular investigation and genetic counseling of intellectual disability/developmental delay patients in an Indian scenario.

Background: Fragile X Syndrome (FXS), the most common cause of inherited intellectual disability (ID), is caused by a CGG repeat expansion (full mutation (FM), >200 CGG) at the Fragile X Mental Retardation 1 (FMR1) gene. Early identification of FXS has prognostic significance for affected individuals due to early initiation of interventions. Genetic counseling and family screening can aid parents and at-risk asymptomatic carriers (premutation (PM), 55-200 CGG) in taking proper reproductive decisions. Methodology: The present study utilizes Triplet Primed-Polymerase Chain Reaction (TP-PCR) methodology for detecting the repeat expansion at FMR1 gene in 233 Indian intellectual disability/developmental delay (ID/DD) patients. Results: We have identified 18/233 (7.7%) FXS positive cases. Early diagnosis was made in 66.7% cases (<10 years). Extended family screening in 14 affected individuals identified 9 additional FM cases (7 males and 2 females) and 23 carrier PM females, which otherwise could have been missed. Four prenatal diagnoses were also performed, leading to the identification of 1 PM and 1 FM carrier fetus. Conclusion: A high frequency (7.7%) of FXS among Indian ID/DD subjects obtained in this study depicted the need for more professional recommendations concerning prompt referral for genetic testing, and increased exposure to information about FXS to pediatricians.

Prevalence and Genetic Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency in Anemic Subjects from Uttar Pradesh, India.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. It affects approximately 400 million people worldwide. The purpose of this study was to detect the prevalence of G6PD deficiency and G6PD gene mutations in the hospital-based settings in patients referred for suspected G6PD deficiency. A qualitative fluorescent spot test and dichlorophenol-indolphenol (DCIP) test were performed. G6PD-deficient, positive samples were further processed for mutation analysis by Sanger sequencing. Out of 1,069 cases, 95 (8.8%) were detected as G6PD deficient (by DCIP test) and were sent for molecular analysis. The G6PD Mediterranean mutation (563C > T) is the most common variant among G6PD-deficient individuals followed by the Coimbra (592C→T) and Orissa (131C→G) variants. We concluded that all symptomatic patients (anemic or jaundiced) should be investigated for G6PD deficiency. Our findings will inform our population screening approach and help provide better management for G6PD-deficient patients.

Identification of microdeletion and microduplication syndromes by chromosomal microarray in patients with intellectual disability with dysmorphism.

BACKGROUND: A retrospective analysis using chromosomal microarray in syndromic patients with intellectual disability from genetic clinics of a tertiary healthcare center in India was conducted. AIM: To identify the spectrum of chromosomal abnormalities detected on microarray analysis. SETTINGS AND DESIGN: Cases were identified among those with intellectual disability with dysmorphism attending genetic clinics of a tertiary care center. PATIENTS AND METHODS: All patients attending genetic clinics over a 3-year period were analyzed. Clinical profile and baseline investigations were noted on a predesigned proforma. Among the 65 studied cases, there were 12 cases suggested to be having Prader-Willi syndrome (PWS), 27 cases with DiGeorge/velocardiofacial syndrome (DGS), and 1 case with Williams-Beuren syndrome (WBS). These were detected by fluorescent in situ hybridization (FISH) analysis with specific probes and were excluded from the final analysis. Chromosomal microarray analysis (CMA; single-nucleotide polymorphism-based array-comparative genomic hybridization) was performed as per the clinical indication in selected patients with dysmorphism, microcephaly, mental retardation, and/or multiple malformations. These patients had a negative result on FISH analysis. RESULTS: In suspected patients with PWS, FISH and methylation testing confirmed six cases to be really PWS. FISH also detected five cases of DGS and one case of WBS. These were excluded from the final analysis. Among the 18 cases tested by CMA, in 13 patients, abnormalities with potential clinical significance were identified. Genetic counseling was done in all these cases. Prenatal diagnosis was done in one family. CONCLUSION: In cases with dysmorphism with or without mental retardation or cardiac defect, advanced studies such as CMA can lead to a definitive diagnosis. Genetic counseling is mandatory in all these cases and a prenatal diagnosis is also feasible in selected families.

Current molecular insight to reveal the dynamics of CAG repeating units in spinocerebellar ataxia.

Spinocerebellar ataxia (SCA) is a heterogeneous genetic disorder with overlapping clinical phenotypes arising from the degeneration of purkinje cells and other regions of the brain. There are approximately 36 different subtypes of SCA, but SCA 1, 2, 3, 6 and 7 are most prevalent in the Indian population. Many findings suggested that cerebellar Purkinje cells region may be a uniquely vulnerable neuronal cell type, and more susceptible to a wider variety of genetic or cellular problems than other neuron types. In this review we emphasized mainly five common subtypes of SCA (1, 2, 3, 6 and 7) their pathophysiology, therapeutics, drugs studies and the technical challenges in the field of molecular genetic diagnosis.

Young mothers and higher incidence of maternal meiosis-I non- disjunction: Interplay of environmental exposure and genetic alterations during halt phase in trisomy 21.

Trisomy 21 is a genetic condition caused when chromosomes fail to separate during meiosis. We have studied conventional karyotype and QF-PCR using STR markers with high polymorphism and heterogeneity and the results were analyzed, to determine the paternal and meiotic origin of trisomy 21. This study was conducted using a detailed questionnaire to include: paternal, maternal, clinical and family history for various confounding factors such as age and regional environmental exposures where the parents resided. Out of 120 samples 95% (N = 114) were of maternal origin, including 92% (N = 105) of meiosis 1 errors and 8% (N = 9) meiosis 2 errors. Paternal origin accounted for 5% (N = 6) and were all due to meiosis-I errors. The higher incidence of maternal meiosis-I observed in the present study suggests that human trisomy 21 non-disjunction is a result of multiple factors contributing to the origin of the genetic condition.

CRELD1 gene variants and atrioventricular septal defects in Down syndrome.

Congenital heart defects (CHD) are seen in around 40% of the Down syndrome patients. Atrioventricular Septal Defect (AVSD) or endocardial cushion defect is commonest form of CHD in these children. CRELD1 gene is implicated in causation of sporadic AVSD. In the present study, we evaluated the association and significance of CRELD1 variants with AVSD in Down syndrome (DS) patients. Sequencing was done in blood samples from 3 groups: group I (DS with AVSD), group II (DS without AVSD) and group III (non-syndromic AVSD cases). Twenty two variants in CRELD1 gene were identified, comprising of sixteen novel and six previously reported variants. However, on the basis of sequence, as well as structure analysis, the variant c.973G>A(p.Glu325Lys) variant was identified only in DS having AVSD group which was predicted to have significant effects on calcium binding of putative CRELD1 protein. Since CRELD1 gene acts as a regulator of calcineurin/NFATc1 signaling which is crucial for the regulation of cardiac development by dephosphorylation of the transcription factor, NFAT(nuclear factor of activated T cells),in cytoplasm, the variation in cb-EGF-like calcium binding domain in CRELD1 protein is likely to have pathogenic consequences. Thus, we conclude that the CRELD1 gene is likely to have a major role in causation of AVSD phenotype in selected DS patients.

Segmental Duplication QF-PCR: A Simple and Alternative Method of Rapid Aneuploidy Testing for Developing Country Like India.

BACKGROUND: Aneuploidy screening is becoming an integral part of routine prenatal screening in developing countries like India, and the need for more cheaper and rapid aneuploidy testing methods are required to relive the anxiety and financial burden among the high-risk couples. Segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR) emerged as an alternative aneuploidy diagnostic method. METHODS: This study was conducted to optimize and access the utility of SD-QF-PCR in routine prenatal diagnosis to complement existing short tandem repeats (STR) based QF-PCR. About 50 control samples, 50 Down's syndrome samples, and one each trisomy 18 and Klinefelter samples were studied to optimize the assay. Later, 100 amniotic fluid samples were also studied. RESULTS AND CONCLUSION: The assay was able to successfully identify normal and aneuploidy samples with 100% sensitivity and specificity. The results of amniotic fluid analysis by SD-QF-PCR were in agreement with results of STR-QF-PCR. Observed results qualify SD-QF-PCR as a preliminary aneuploidy diagnosis method.

Molecular screening of intellectually disabled patients and those with premature ovarian failure for CGG repeat expansion at FMR1 locus: Implication of combined triplet repeat primed polymerase chain reaction and methylation-specific polymerase chain reaction analysis.

BACKGROUND: Fragile X syndrome (FXS) is also a leading cause of intellectual disability along with Down's syndrome. It is caused by the expansion of CGG triplet repeat at 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. Since the prevalence rate is quite high in the general population, molecular diagnosis is important to establish the cause and the prenatal diagnosis. At present, there are a number of methods available with their own merits and demerits. AIM AND METHODS: Molecular screening of intellectually disabled patients and those with premature ovarian failure with combined triplet repeat primed polymerase chain reaction (TP-PCR) and methylation-specific polymerase chain reaction (MS-PCR) for establishing the diagnosis of FXS. RESULTS: The specificity of the method has been validated with archived previously genotyped samples, facilitating the application of this method in the screening procedure. The combined TP-PCR and MS-PCR approach identified six (10%) of the intellectually disabled cases as full mutation positive, one (4%) of the premature ovarian failure cases as premutation positive, and one (out of two) of the prenatal samples as premutation positive. CONCLUSION: The present study concludes that a combined usage of TP-PCR and MS-PCR will be a useful alternative approach to diagnose patients suffering from fragile X syndrome.

Friedreich Ataxia: From the Eye of a Molecular Biologist.

Friedreich ataxia (FRDA) is caused by the expansion of a GAA triplet repeat in the first intron of the FXN gene. This disease was named after Nicholaus Friedreich, Germany, who depicted the essential finding. Among ataxias, FRDA is the most common hereditary ataxia. It has the autosomal recessive pattern of inheritance. The expansion of the GAA triplet repeat hinders the transcription, thereby reducing the level of the FXN transcript and consequently reducing the level of frataxin, a 210-amino acid protein. The disease pathogenesis is fundamentally due to a lack of frataxin, which is claimed to play a role in iron-sulfur cluster synthesis. Oxidative stress builds up as a result of Fe accumulation in the mitochondria, causing degeneration of the cells, which primarily occurs in the neurons and later in the cardiac tissues, and to some extent in the pancreas. The therapeutic interventions are at infancy; however, current treatments are targeted toward the reduction of iron overload and its effects.

"Down syndrome: an insight of the disease".

Down syndrome (DS) is one of the commonest disorders with huge medical and social cost. DS is associated with number of phenotypes including congenital heart defects, leukemia, Alzeihmer's disease, Hirschsprung disease etc. DS individuals are affected by these phenotypes to a variable extent thus understanding the cause of this variation is a key challenge. In the present review article, we emphasize an overview of DS, DS-associated phenotypes diagnosis and management of the disease. The genes or miRNA involved in Down syndrome associated Alzheimer's disease, congenital heart defects (AVSD), leukemia including AMKL and ALL, hypertension and Hirschprung disease are discussed in this article. Moreover, we have also reviewed various prenatal diagnostic method from karyotyping to rapid molecular methods -  MLPA, FISH, QF-PCR, PSQ, NGS and noninvasive prenatal diagnosis in detail.

Performance of QF-PCR in targeted prenatal aneuploidy diagnosis: Indian scenario.

OBJECTIVE: Among the rapid aneuploidy detection methods, QF-PCR has now become an alternative tool for prenatal aneuploidy diagnosis concomitant with karyotyping. This method has been validated in many of the western clinics but in India no study was conducted to assess its utility as standalone procedure. The study was designed to answer the question whether QF-PCR can be implemented as a standalone diagnostic method for rapid aneuploidy diagnosis in our present clinical setup? MATERIALS AND METHODS: Study was conducted during March 2012 to August 2014 consisting of 270 prenatal samples that underwent for aneuploidy diagnosis. In addition to karyotyping, QF-PCR was also performed on these samples and the results were compared. RESULTS: Of 270 samples screened, 262 samples showed euploid genome (125 normal male and 137 normal female). Eight samples were consistent with aneuploidy--four trisomy 21 male sample, 2 trisomy 21 female sample, 1 trisomy 18 samples and 1 Klinefelter sample. The specificity, sensitivity, positive prediction value and negative prediction values were 100% while false positive rate and false negative rate were 0%. CONCLUSION: Outcome of this study strongly suggests that QF-PCR can be used as standalone procedure for targeted rapid aneuploidy diagnosis.