Spectrum of Rare and Novel Indel Mutations Responsible for ß Thalassemia in Eastern India.

There is limited data available regarding the clinical utility of routine molecular diagnosis of ß Thalassaemia in addition to HPLC-based screening in low resource settings. The current study highlights the caveats of an HPLC-based screening compared to the inclusion of genetic confirmation as a second-tier test and its implications in terms of genotype-phenotype correlation. A prospective, institution-based, observational study was conducted at the Department of Paediatric Medicine, including 103 children aged up to 12 years. Five common mutations for ß Thalassemia and the HbE mutation in the HBB gene were tested by a two-tiered approach using multiplex ARMS PCR and PCR RFLP methods respectively. Sanger sequencing of all three exons of the HBB gene was performed in all negative cases. Sequencing revealed many rare pathogenic mutations like c.316-106 C > G (dbSNP: 34,690,599); Hb Kairouan (c.92G > C); c.33 C > A (dbSNP rs35799536); c.47G > A (dbSNP rs63750783); c.51delC (HbVar ID 799); c.[93-2 A > C] and c.118 C > T (HbVar ID 845). We detected a novel Pathogenic M_000518.5(HBB):c.164_168delinsGGCATCA (p.Val55fs) mutation in a heterozygous state which was reported in the ClinVar database with accession ID VCV000590977.2. We also encountered several cases of silent carrier on HPLC and de novo occurrence of mutation. We conclude that the multiplex touchdown ARMS PCR methodology employed in the present study provides a low-cost solution for molecular diagnostics of Β Thalassaemia. The problem of silent carriers in HPLC is significant enough to rethink if we need supplemental genetic testing in the couple when one of the partners is a carrier. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01098-w.

Ectodysplasin pathogenic variants affecting the furin-cleavage site and unusual clinical features define X-linked hypohidrotic ectodermal dysplasia in India.

Hypohidrotic ectodermal dysplasia (HED) is a rare genetic disorder caused by mutational inactivation of a developmental pathway responsible for generation of tissues of ectodermal origin. The X-linked form accounts for the majority of HED cases and is caused by Ectodysplasin (EDA) pathogenic variants. We performed a combined analysis of 29 X-linked hypohidrotic ectodermal dysplasia (XLHED) families (including 12 from our previous studies). In addition to the classical triad of symptoms including loss (or reduction) of ectodermal structures, such as hair, teeth, and sweat glands, we detected additional HED-related clinical features including facial dysmorphism and hyperpigmentation in several patients. Interestingly, global developmental delay was identified as an unusual clinical symptom in many patients. More importantly, we identified 22 causal pathogenic variants that included 15 missense, four small in-dels, and one nonsense, splice site, and large deletion each. Interestingly, we detected 12 unique (India-specific) pathogenic variants. Of the 29 XLHED families analyzed, 11 (38%) harbored pathogenic variant localized to the furin cleavage site. A comparison with HGMD revealed significant differences in the frequency of missense pathogenic variants; involvement of specific exons and/or protein domains and transition/transversion ratios. A significantly higher proportion of missense pathogenic variants (33%) localized to the EDA furin cleavage when compared to HGMD (7%), of which p.R155C, p.R156C, and p.R156H were detected in three families each. Therefore, the first comprehensive analysis of XLHED from India has revealed several unique features including unusual clinical symptoms and high frequency of furin cleavage site pathogenic variants.

Charcot-Marie-Tooth disease type 4J with spastic quadriplegia, epilepsy and global developmental delay: a tale of three siblings.

Charcot-Marie-Tooth (CMT) disease is mainly a disease of peripheral nervous system and patients typically present with features of demyelinating neuropathy or axonal neuropathy or both. Rarely patients present with features of central nervous system involvement. Parkinsonism, aphemia and familial epilepsy syndrome have previously come up as case reports in association with CMT type 4 J.We hereby describe a family with 3 siblings affected with CMT4J with homozygous FIG4 mutation who presented with global developmental delay, epilepsy and spastic quadriparesis.

Schuurs-Hoeijmakers syndrome in a patient from India.

Schuurs-Hoeijmakers syndrome (SHMS), or Autosomal Dominant Mental Retardation Syndrome type 17 (MRD17) is a rare form of intellectual disability with distinct facial features. A recurrent de novo heterozygous c.607C>T, p.Arg203Trp mutation in the PACS1 gene accounts for all reported cases except for one patient with a de novo heterozygous c.608G>A, p.Arg203Trp mutation. Ethnic background is known to affect the clinical manifestation of dysmorphic syndromes. Here we describe the first Indian patient with Schuurs-Hoeijmakers syndrome (SHMS) with a de novo heterozygous NM_018026.3 (PACS1):c.607C>T (p.Arg203Trp) variant. He is the only child with SHMS with a cleft lip. Thus our report expands the phenotypic spectrum of SHMS and establishes its occurrence across populations.

Genetic analysis of consanguineous families presenting with congenital ocular defects.

Anophthalmia and microphthalmia (A/M) are a group of rare developmental disorders that affect the size of the ocular globe. A/M may present as the sole clinical feature, but are also frequently found in a variety of syndromes. A/M is genetically heterogeneous and can be caused by chromosomal aberrations, copy number variations and single gene mutations. To date, A/M has been caused by mutations in at least 20 genes that show different modes of inheritance. In this study, we enrolled eight consanguineous families with A/M, including seven from Pakistan and one from India. Sanger and exome sequencing of DNA samples from these families identified three novel mutations including two mutations in the Aldehyde Dehydrogenase 1 Family Member A3 (ALDH1A3) gene, [c.1310_1311delAT; p.(Tyr437Trpfs*44) and c.964G > A; p.(Val322Met)] and a single missense mutation in Forkhead Box E3 (FOXE3) gene, [c.289A > G p.(Ile97Val)]. Additionally two previously reported mutations were identified in FOXE3 and in Visual System Homeobox 2 (VSX2). This is the first comprehensive study on families with A/M from the Indian subcontinent which provides further evidence for the involvement of known genes with novel and recurrent mutations.

Pharmacogenomics-assisted schizophrenia management: A hybrid type 2 effectiveness-implementation study protocol to compare the clinical utility, cost-effectiveness, and barriers.

OBJECTIVES: The response to antipsychotic therapy is highly variable. Pharmacogenomic (PGx) factors play a major role in deciding the effectiveness and safety of antipsychotic drugs. A hybrid type 2 effectiveness-implementation research will be conducted to evaluate the clinical utility (safety and efficacy), cost-effectiveness, and facilitators and barriers in implementing PGx-assisted management compared to standard of care in patients with schizophrenia attending a tertiary care hospital in eastern India. METHODS: In part 1, a randomized controlled trial will be conducted. Adult patients with schizophrenia will be randomized (2: 1) to receive PGx-assisted treatment (drug and regimen selection depending on the results of single-nucleotide polymorphisms in genes DRD2, HTR1A, HTR2C, ABCB1, CYP2D6, CYP3A5, and CYP1A2) or the standard of care. Serum drug levels will be measured. The patients will be followed up for 12 weeks. The primary endpoint is the difference in the Udvalg for Kliniske Undersøgelser Side-Effect Rating Scale score between the two arms. In part 2, the cost-effectiveness of PGx-assisted treatment will be evaluated. In part 3, the facilitators and barriers to implementing PGx-assisted treatment for schizophrenia will be explored using a qualitative design. EXPECTED OUTCOME: The study findings will help in understanding whether PGx-assisted management has a clinical utility, whether it is cost-effective, and what are the facilitators and barriers to implementing it in the management of schizophrenia. TRIAL REGISTRATION: The study has been registered with the Clinical Trials Registry-India (CTRI/2023/08/056210).

A Rapid and Scalable Multiplex PCR-Based Next-Generation Amplicon Sequencing Method for Familial Hypercholesterolemia Genetic Screening.

BACKGROUND: Familial hypercholesterolemia (FH) is a frequently underdiagnosed genetic disorder characterized by elevated low-density lipoprotein (LDL) levels. Genetic testing of LDLR, APOB, and PCSK9 genes can identify variants in up to 80% of clinically diagnosed patients. However, limitations in time, scalability, and cost have hindered effective next-generation sequencing of these genes. Additionally, pharmacogenomic variants are associated with statin-induced adverse effects in FH patients. To address these challenges, we developed a multiplex primer-based amplicon sequencing approach for FH genetic testing. METHODS: Multiplex primers were designed for the exons of the LDLR, APOB, and PCSK9 genes, as well as for pharmacogenomic variants rs4149056 (SLCO1B1:c.521T > A), rs2306283 (SLCO1B1:c.388A > G), and rs2231142 (ABCG2:c.421C > A). Analytical validation using samples with known pathogenic variants and clinical validation with 12 FH-suspected probands were conducted. Library preparation was based on a bead-based tagmentation method, and sequencing was conducted on the NovaSeq 6000 platform. RESULTS: Our approach ensured no amplicon dropouts, with over 100× coverage on each amplicon. Known variants in 2 samples were successfully detected. Further, we identified one heterozygous LDLR (p.Glu228Ter) variant and 2 homozygous cases of LDLR (p.Lys294Ter) and LDLR (p.Ser177Leu) variants in patients. Pharmacogenomic analysis revealed that overall 3 patients may require reduced statin doses. Our approach offered reduced library preparation time (approximately 3 h), greater scalability, and lower costs (under $50) for FH genetic testing. CONCLUSIONS: Our method effectively sequences LDLR, APOB, and PCSK9 genes including pharmacogenomic variants that will guide appropriate screening and statin dosing, thus increasing both efficiency and affordability.

Genetic factors affecting susceptibility to alcoholic liver disease in an Indian population.

INTRODUCTION. Indians are more likely to develop alcoholic cirrhosis compared to Caucasians, though the cause remains obscure. North Indians tend to consume more alcohol than other parts of the country. Genetic factors are likely to play a major role in these observations. This study investigated whether 10 different polymorphisms were associated with alcohol dependence and/or cirrhosis in North Indians. These were in ADH2*2 (rs1229984), ADH3*2 (rs698), CYP2E1*1D, CYP2E1*5 (rs3813867 and rs2031920), TNF-α(rs1800629), TNF-α (rs361525), IL-1ß (rs3087258), CD-14 (rs2569190), IL-10 (rs1800872) and PNPLA3 (rs738409). MATERIAL AND METHODS. Hundred healthy controls and 120 chronic alcoholics (60 alcoholic noncirrhotics and 60 alcoholic cirrhotics) attending various departments of PGIMER, Chandigarh were genotyped using PCR-RFLP methods. RESULTS. Alcoholic cirrhotics compared to healthy individuals demonstrated a statistically significant increase in PNPLA3 (10109G) allele (p = 0.037, OR = 2.12, 95% CI 1.29-3.4). Rest of the associations were not significant after correction for multiple testing. CONCLUSION. PNPLA3 10109G predisposed North Indian subjects to alcoholic cirrhosis.

Developing a Blueprint for Theoretical Assessment of Biochemistry of Phase I MBBS Students.

Here, we have systematically developed a blueprint for the biochemistry theory assessment of phase I MBBS students in India which we have been using for both formative and summative assessments for the past 2 academic years. The blueprint has ensured the content validity and construct reliability and fairness of the assessment.

Comparative Evaluation of Serum Lithium Estimation Using Plain Glass Vial and Serum Clot Activator Vacutainer by Reflectance Photometry.

Introduction The collection of blood samples in different vacutainers can affect the result of serum lithium estimation due to the presence of distinct additives in the blood collection vacutainer for enhancing the clot formation process. Due to the low therapeutic index and threat of toxicity of lithium, it is imperative to correctly report the test result. Thus, it has become a challenge for the laboratory physician to estimate lithium in any clinical laboratory setup. Materials and Methods Sample of 100 patients were collected and paired into clot activator vacutainers and plain glass vials. After centrifugation, samples from the paired collection tubes were processed immediately for serum lithium estimation by VITROS 4600 analyzer working on the principle of reflectance photometry. Both the paired tubes were stored at 2 to 8°C and were further analyzed, at 24 and 48 hours, respectively, from the time of their collection. The statistical analysis was done in IBM SPSS software version 23. Results There was a statistically significant differences between the mean of lithium values when processed within 1st hour of collection, obtained from clot activator vacutainers in comparison to glass vials. However, within tube comparison, there was no statistical difference in the lithium values estimated at 1st hour, 24 hours, and 48 hours of collection. Conclusion In this study, lithium values measured by clot-activated vacutainers are found to be lower as compared with values measured through glass vials.

Exploration of clinical and genetic findings in Ataxia-Telangiectasia (AT) patients from the Indian subcontinent.

BACKGROUND: Ataxia-Telangiectasia (AT) is a rare autosomal recessive neurodegenerative disorder. It is caused by mutations in the Ataxia-Telangiectasia mutated (ATM) gene, which codes for protein ATM serine/threonine kinase. OBJECTIVE: We aim to describe the clinical and radiological findings in children and adolescents of 20 molecularly confirmed cases of AT. We aim to correlate these findings with the genotype identified among them. METHODS: This retrospective study included 20 patients diagnosed clinically and genetically with AT over 10 years. The clinical, radiological and laboratory data were extracted from the hospital's electronic medical records. Molecular testing was done using next generation sequencing and Sanger sequencing. In silico predictions were performed for the variants identified by applying Cryp-Skip, Splice site prediction by Neural Network, Mutation Taster and Hope prediction tool. RESULTS: Consanguinity was documented in nearly half of the patients. Telangiectasia was absent in 10%. Microcephaly was seen in 40% cases. The incidence of malignancy in our study population was low. Molecular testing done in the 18 families (20 patients) identified 23 variants of which ten were novel. Biallelic homozygous variants were noted in 13 families and compound heterozygous in 5 families. Out of the 13 families who were homozygous, 8 families (61.5%) (9 patients) have history of consanguinity. In silico prediction of novel missense variants, NM_000051.4 (ATM_v201): c.2702T > C showed disruption of the α-helix of ATM protein and NM_000051.4 (ATM_v201): c.6679C > G is expected to disturb the rigidity of protein structure in the FAT domain. The four novel splice site variants and two intronic variants result in exon skipping as predicted by Cryp-Skip. CONCLUSIONS: AT should be confirmed by molecular testing in young-onset cerebellar ataxia, even without telangiectasia. Awareness of this rare disease will facilitate study of larger cohorts from Indian population to characterize variants and determine its prevalence in this population.

A curious case of hyperbilirubinemia.

A 60-year-old patient suffering from easy fatigability was noted to have high total but normal conjugated bilirubin level though serum colour was pale. On further investigation the patient was found to be suffering from multiple myeloma and gamma globulin was possibly interfering with total bilirubin estimation.

Correlation of ATP7B gene mutations with clinical phenotype and radiological features in Indian Wilson disease patients.

INTRODUCTION: Wilson disease (WD) is characterized by a wide variety of clinical manifestations. Our study aimed to correlate genotype with clinical and radiological features in Indian WD patients. METHODS: We conducted a descriptive observational study in a tertiary care neurology referral center of eastern India over a period of 2 years. Demographic data collection, clinical examination and relevant investigations were done for all WD patients meeting the inclusion criteria. Based on previous reports of mutation hotspots for WD in Eastern India, we performed PCR-Sanger sequencing of selected exons of ATP7B gene. To understand the role of each of these covariates on the occurrence of common mutation, we applied a logistic regression as well as random forest in a supervised learning framework. RESULTS: Fifty-two WD patients were included in the study. c.813C > A (p.C271X) was the commonest identified mutation. The statistical methods applied to our data-set reveal the most important features for predicting common mutation or its absence. We also found that the state-of-the-art classification algorithms are good at predicting the absence of common mutation (with true positive rates being 0.7647 and 0.8823 for logistic classifier and random forest, respectively), but predicting the occurrence remains a harder modeling challenge. CONCLUSIONS: WD patients in eastern India have significant genotypic and phenotypic diversity. Statistical methods for binary classification show some early promise of detecting common mutations and suggest important covariates, but further studies with larger samples and screening of remaining exons are warranted for understanding the full genetic landscape of Wilson disease.

Host genomics of COVID-19: Evidence point towards Alpha 1 antitrypsin deficiency as a putative risk factor for higher mortality rate.

Corona Virus Disease 2019 (COVID-19) has emerged as a pandemic leading to unprecedented disruption of global health and economy. Transmembrane protease serine 2 (TMPRSS2) has been found to be critical in priming the viral spike protein and the host ACE2 receptor before the virus enters into the host cell. Recent studies have experimentally demonstrated that Alpha 1 antitrypsin (encoded by SERPINA1 gene) is an inhibitor of TMPRSS2 and provided support to the already approved therapy as a candidate for COVID-19. Interestingly Alpha 1 antitrypsin deficiency is common among Europeans. Here we have provided in silico evidence that Alpha 1 antitrypsin can interact with TMPRSS2 and both of them are co-expressed in the human liver and lung. We then analyzed the gnomAD dataset to show that Europeans and Latinos have a substantially higher carrier frequency of Alpha 1 Antitrypsin Deficiency (~12%) compared to other large ethnicities. Therefore, we hypothesize that Alpha 1 antitrypsin deficiency might be a risk factor for severe infection with SARS-CoV-2. We propose Alpha 1 antitrypsin status as a potential prognostic predictor of COVID-19 outcome.

Zinc and COVID-19: Basis of Current Clinical Trials.

Coronavirus disease-2019 (COVID-19) pandemic continues to threaten patients, societies, and economic and healthcare systems around the world. Like many other diseases, the host immune system determines the progress of COVID-19 and fatality. Modulation of inflammatory response and cytokine production using immunonutrition is a novel concept that has been applied to other diseases as well. Zinc, one of the anti-inflammatory and antioxidant micronutrient found in food with well-established role in immunity, is currently being used in some clinical trials against COVID-19. This review integrates the contemporary studies of role of zinc in antiviral immunity along with discussing its potential role against COVID-19, and ongoing COVID-19 clinical trials using zinc.

Evaluation of e-OSPE as compared to traditional OSPE: A pilot study.

Objectively Structured Clinical/Practical Examination (OSCE/OSPE) has been the backbone of the assessment system of graduate medical education for over three decades. We have developed an electronic Objectively Structured Practical Examination (e-OSPE) in Medical Biochemistry using the freely available Google forms to mitigate the academic disruption posed by COVID-19 pandemic in our resource-poor setting. Ten e-OSPE stations created, interlinked, and time-restricted. Fifty undergraduate students appeared for the e-OSPE examination on a prefixed date and time. Learner feedback was collected immediately after the completion of the examination. Facilitator feedback was also collected. Students' mean scores in e-OSPE and traditional OSPE were 78.15% and 74.56%, respectively. Their difference was not statistically significant (paired t-test two-tailed p-value 0.0979). Thus, the results of e-OSPE are reliable as compared to traditional OSPE. Bland Altman Plot revealed 92% of students had scores that were in the agreeable limit of both traditional OSPE and e-OSPE. Both the learners and facilitators were in consensus that the online format of e-OSPE is a good alternative for assessment (0.67 and 0.82); their experience was good (0.72 and 0.92) and conduction was well organized (0.73 and 0.86). Several suggestions were also received to make e-OSPE even more effective. In conclusion, this pilot study showed e-OSPE can be an effective alternative to traditional OSPE when "in-person" evaluation is not possible such as in the current era of COVID-19 even in resource-limited settings.

The transition from objectively structured practical examination (OSPE) to electronic OSPE in the era of COVID-19.

The coronavirus (COVID-19) pandemic is forcing the medical educators to innovate and embrace online education and assessment platform. One of the most significant challenges we are facing is the formative assessment of practical skills in the undergraduate medical biochemistry education. We have designed the electronic objectively structured practical examination to facilitate the formative assessment.

"Eye of tiger sign" mimic in an adolescent boy with mitochondrial membrane protein associated neurodegeneration (MPAN).

Neurodegeneration with brain iron accumulation (NBIA) refers to an inherited heterogeneous group of disorders pathologically characterized by focal brain iron deposition. Clinical phenotype, imaging findings and genotype are variable among the different types of this disorder. In this case report, we describe the imaging finding of an adolescent boy with mitochondrial membrane protein associated neurodegeneration (MPAN), a subentity of NBIA. Magnetic resonance imaging of brain revealed hypointensity of globi pallidi with medial medullary lamina appearing as a hyperintense streak in T2 weighted images. Mild cerebellar atrophy in T2 weighted images and blooming of substantia nigra and globi pallidi in susceptibility weighted images were also observed. Imaging findings in patients with MPAN mimics the eye of tiger appearance in patients with pantothenate kinase associated neurodegeneration. Classical phenotype and eye of tiger sign mimic in imaging of patients with NBIA should raise the suspect for MPAN. Genetic studies helps in the confirmation of diagnosis of this neurodegenerative disorder.