Variants in DOK7 results in fetal akinesia deformation sequence: A case report and review of literature.

We report the third case of FADS due to biallelic DOK7 variants, which further strengthens the association of DOK7 with this lethal phenotype and lack of genotype phenotype correlation.

Neurodegeneration with brain iron accumulation: a case series highlighting phenotypic and genotypic diversity in 20 Indian families.

Neurodegeneration with brain iron accumulation (NBIA) is an umbrella term encompassing various inherited neurological disorders characterised by abnormal iron accumulation in basal ganglia. We aimed to study the clinical, radiological and molecular spectrum of disorders with NBIA. All molecular-proven cases of NBIA presented in the last 5 years at 2 tertiary care genetic centres were compiled. Demographic details and clinical and neuroimaging findings were collated. We describe 27 individuals from 20 unrelated Indian families with causative variants in 5 NBIA-associated genes. PLA2G6-associated neurodegeneration (PLAN) was the most common, observed in 13 individuals from 9 families. They mainly presented in infancy with neuroregression and hypotonia. A recurrent pathogenic variant in COASY was observed in two neonates with prenatal-onset severe neurodegeneration. Pathogenic bi-allelic variants in PANK2, FA2H and C19ORF12 genes were observed in the rest, and these individuals presented in late childhood and adolescence with gait abnormalities and extrapyramidal symptoms. No intrafamilial and interfamilial variability were observed. Iron deposition on neuroimaging was seen in only 6/17 (35.3%) patients. A total of 22 causative variants across 5 genes were detected including a multiexonic duplication in PLA2G6. The variants c.1799G > A and c.2370 T > G in PLA2G6 were observed in three unrelated families. In silico assessments of 8 amongst 9 novel variants were also performed. We present a comprehensive compilation of the phenotypic and genotypic spectrum of various subtypes of NBIA from the Indian subcontinent. Clinical presentation of NBIAs is varied and not restricted to extrapyramidal symptoms or iron accumulation on neuroimaging.

Retrospective diagnosis by parental testing in the next generation sequencing era and utility of reanalysis of exome data.

OBJECTIVE: Genetic diseases are an important cause of neonatal and childhood mortality. For couples with a history of demise of previous children, screening for carrier status can be done by exome sequencing (ES) of the parents. Our aim was to describe the clinical utility of "targeted parental ES" in such couples and to assess the utility of reanalysis of parental ES data. METHOD: We analyzed previous records, including ES reports, of 52 families with demise of previous offspring with a suspected genetic disorder. We also retrieved and reanalyzed raw data of parental ES in FASTQ format from the testing lab. RESULTS: A potential diagnosis was obtained in 30/52 (57.7%) of couples. We found 38/70 (54.3%) novel variants in this cohort. Shared carrier status for more than one autosomal or X-linked recessive disorder was identified in 18% of couples. Reanalysis of raw data resulted in a reclassification of variants in 15% of cases. CONCLUSION: Targeted parental ES can be helpful for families with demise of previous offspring with a suspected genetic disorder.

Monosomy 1p36: Report of a cohort of 13 Asian Indian patients.

Monosomy 1p36 is one of the common microdeletion syndromes with a recognizable facial phenotype. Failure to thrive, developmental delay, congenital heart disease, and other abnormalities are common in these patients. This is the first study on Asian Indian patients with monosomy 1p36, documenting the phenotypic characteristics of 13 patients, indicating phenotypic similarities in a diverse population and broadening the clinical spectrum.

Meckel Gruber and Joubert Syndrome Diagnosed Prenatally: Allelism between the Two Ciliopathies, Complexities of Mutation Types and Digenic Inheritance.

Background: Antenatally detected occipital encephalocele and polycystic kidneys are a common presentation of ciliopathies like Joubert syndrome and Meckel Gruber syndrome which have considerable genetic and phenotypic overlap. Case reports: We describe 3 cases of antenatally diagnosed occipital encephalocele and enlarged kidneys with fetal autopsy, histopathology & exome sequencing results. A novel nonsense variant in the CEP290 gene was reported in first case (Meckel syndrome). The second case shows the importance of fetal exome where the parents were carriers for 2 ciliopathy genes (TMEM138 & SDCCAG8). Diagnosis in this case was confirmed by fetal exome sequencing (Joubert syndrome). Multiexon deletion in TMEM67 and KIF14 present in trans was identified in the third case (Meckel syndrome), likely resulting in digenic inheritance. Conclusion: We report 2 cases of Meckel syndrome with a novel variant and multiexon deletion, and 1 case of Joubert syndrome which depicts the limitations of preconceptional carrier screening in ciliopathies due to overlapping phenotypes.

Carrier frequency of SMN1-related spinal muscular atrophy in north Indian population: The need for population based screening program.

Chromosome 5q related Spinal muscular atrophy (SMA) is an autosomal recessive, progressive, neuromuscular disorder most commonly caused by homozygous deletion of exon 7 or exon 7 and 8 of SMN1 gene. Being the leading genetic cause of infant mortality, studies of its prevalence and incidence are necessary. Carrier testing for the common pathogenic variant for SMA is offered to the couples visiting our tertiary care hospital in North India. Subjects were tested for SMA carrier status by Multiplex Ligation-dependent Probe amplification (MLPA) technique for deletion of exons 7 and 8 of SMN1 gene. The retrospective data of individuals tested for SMA carrier status in last 4 years (2016-2019) was evaluated. Six hundred and six individuals without family history of SMA or carrier of SMA who were subjected to MLPA based screening for SMA carrier status were included in the study. The carrier frequency of SMN1 deletion (deletion of exon 7 and/or exon 8) was found to be 1 in 38 (16 out of 606). The catchment area of our medical genetics clinic covering the state of Uttar Pradesh (16.5% of Indian population according to censusindia.gov.in, 2011) and neighboring states, showing SMA carrier frequency of 1:38 in a cohort with no prior positive family history has important significance for policy making.

Genetic heterogeneity of disorders with overgrowth and intellectual disability: Experience from a center in North India.

Overgrowth, defined as height and/or OFC ≥ +2SD, characterizes a subset of patients with syndromic intellectual disability (ID). Many of the disorders with overgrowth and ID (OGID) are rare and the full phenotypic and genotypic spectra have not been unraveled. This study was undertaken to characterize the phenotypic and genotypic profile of patients with OGID. Patients with OGID were ascertained from the cohort of patients who underwent cytogenetic microarray (CMA) and/or exome sequencing (ES) at our center over a period of 6 years. Thirty-one subjects (six females) formed the study group with ages between 3.5 months and 13 years. CMA identified pathogenic deletions in two patients. In another 11 patients, a disease causing variant was detected by ES. The spectrum of disorders encompassed aberrations in genes involved in the two main pathways associated with OGID. These were genes involved in epigenetic regulation like NSD1, NFIX, FOXP1, and those in the PI3K-AKT pathway like PTEN, AKT3, TSC2, PPP2R5D. Five novel pathogenic variants were added by this study. NSD1-related Sotos syndrome was the most common disorder, seen in five patients. A causative variant was identified in 61.5% of patients who underwent only ES compared to the low yield of 11.1% in the CMA group. The molecular etiology could be confirmed in 13 subjects with OGID giving a diagnostic yield of 42%. The major burden was formed by autosomal dominant monogenic disorders. Hence, ES maybe a better first-tier genomic test rather than CMA in OGID.

Resolving fetal hydrops - A rare entity.

Non-immune hydrops fetalis (NIHF) is abnormal accumulation of serous fluid in ≥2 interstitial spaces with no evidence of maternal red cell alloimmunization. Leaving a few treatable conditions, it is generally considered as a sign of poor fetal outcome. Bi-allelic variants in THSD1 have been found to be to be associated with phenotypes ranging from lethal NIHF to persistent edema. Here, we report a family with non-immune hydrops in two successive pregnancies. Whole exome sequencing in second pregnancy identified a homozygous truncating variant in THSD1 (NM_018676:c.892G>T:p.Glu298Ter). Postnatal follow up showed gradual resolution of the accumulated fluid and normal development. This report further strengthens the association of variants in THSD1 with NIHF.

Red cell alloimmunization and associated risk factors in multiply transfused thalassemia patients: A prospective cohort study conducted at a tertiary care center in Northern India.

BACKGROUND: One of the complications of chronic transfusions in thalassemia is the development of red cell alloimmunization. AIMS: The aim of the study was to determine the frequency, specificity of red cell alloantibodies, and factors influencing alloimmunization in multiply transfused thalassemia patients. MATERIALS AND METHODS: The study was carried out prospectively on beta-thalassemia patients over 10 months. Plasma samples were used for antibody screening and identification using the column agglutination technique. Patients' clinical, laboratory, and transfusion details were obtained from hospital information system and patient files. STATISTICAL ANALYSIS: Continuous variables were reported as median and quartile, whereas categorical variables were provided as numbers and proportions. P < 0.05 was considered statistically significant. RESULTS: Out of 255 patients, 17 (6.6%) patients developed alloantibodies. Alloimmunized patients had significantly higher median ages at their first transfusions (1 year vs. 0.5 years; P = 0.042) than nonalloimmunized patients. Alloimmunized patients had significantly higher conjugated bilirubin (P = 0.016) and serum ferritin (P = 0.007). The majority of alloantibodies had specificity toward K antigen, followed by E, C, D, JKa, and JKb antigens. Alloimmunized patients received more units per year than nonalloimmunized patients (median, 30 vs. 24 units; P < 0.001). The average transfusion interval time between two successive transfusions showed a significant difference (P < 0.001). CONCLUSIONS: The prevalence of alloimmunization in thalassemia patients in North India is relatively low. Since most of the alloantibodies belong to Rh and Kell blood group system, extended phenotype-matched blood for Rh and Kell will be helpful in further preventing or decreasing the development of alloantibodies in multiply transfused thalassemia patients.

Integrated Management Strategies for Epidermolysis Bullosa: Current Insights.

Epidermolysis bullosa (EB) is a group of rare genodermatoses that is characterized by skin fragility resulting from minor trauma. There are four major subtypes, namely, EB simplex, junctional EB, dystrophic EB and Kindler EB, depending upon the localization of defective protein and resulting plane of blister formation. The phenotype is heterogeneous in terms of severity and majority of them present at birth or neonatal period. Currently, the treatment is mainly supportive and requires multidisciplinary care. The complex molecular pathology creates difficulty in discovering a unified curative treatment approach. But with arduous efforts, significant progress has been made in the development of treatment strategies in the last decade. The management strategies range from targeting the underlying causative factor to symptom-relieving approaches, and include gene, mRNA, protein, cell and combination therapies. In this review, we enumerate the promising approaches that are currently under various stages of investigation to provide effective treatment for patients with EB.

Management of pregnant female with Haemophilia-A: A case report.

Haemophilia-A is a rare, X-linked recessive inherited disease affects males and females are carrier results in prolonged bleeding after minor injuries, procedures or surgery. Spontaneous or recurrent bleeding may occur in deep muscles, joints but intracranial haemorrhage can be dangerous. Women with a family history of bleeding disorder, personal history of bleeding (menorrhagia, mucous membrane, postoperative bleeding and PPH) or a prolonged activated partial thromboplastin time (aPTT) should be screened for haemophilia by measuring coagulation factor VIII level during hospital visits for these reason or antenatal care (ANC). Female heterozygote carriers may have variable presentation of bleeding due to reduced levels of clotting FVIII and may experience excessive bleeding due to gynaecologic issues, during spontaneous abortion, medical termination of pregnancy or invasive prenatal diagnostic techniques in first trimester of pregnancy, antepartum and postpartum haemorrhage in later part of gestation and after delivery.

Variable neurological phenotypes of homocystinuria caused by biallelic methylenetetrahydrofolate reductase variants.

Inherited methylenetetrahydrofolate reductase (MTHFR) deficiency is associated with a wide spectrum of disorders including homocystinuria. This study aims to describe the neurological phenotypes and molecular profiles of patients with homocystinuria caused by biallelic variants in MTHFR. We report six subjects with MTHFR deficiency who presented with variable neurological phenotypes which could be viewed as a continuous spectrum. Fatal infantile encephalopathy was observed in one family, whereas another patient presented at 27 years with acute leukoencephalopathy and recovered within 3 months. Intermediate forms presenting as complicated hereditary spastic paraparesis of variable severity were observed in four subjects. Clinical and molecular information of the 207 cases reported in literature were also retrieved and analyzed. We categorized all subjects into three categories - severe, intermediate and mild forms according to the clinical presentation. In addition, a total of 286 disease-causing variations reported to date were analyzed. These included seven disease-causing variants reported in this study of which one is novel. Some genotype-phenotype correlation could be seen which corroborated with previous observations. However, inter- and intrafamilial variability was also noted. Treatment with betaine, B12 and folic acid was started in four subjects with variable outcomes.

Deciphering the molecular landscape of microcephaly in 87 Indian families by exome sequencing.

Microcephaly is a frequent feature of neurodevelopmental disorders (NDDs). Our study presents the heterogeneous spectrum of genetic disorders in patients with microcephaly either in isolated form or in association with other neurological and extra-neural abnormalities. We present data of 91 patients from 87 unrelated families referred to our clinic during 2016-2020 and provide a comprehensive clinical and genetic landscape in the studied cohort. Molecular diagnosis using exome sequencing was made in 45 families giving a yield of 51.7%. In 9 additional families probable causative variants were detected. We identified disease causing variations in 49 genes that are involved in different functional pathways Among these, 36 had an autosomal recessive pattern, 8 had an autosomal dominant pattern (all inherited de novo), and 5 had an X-linked pattern. In 41 probands where sequence variations in autosomal recessive genes were identified 31 were homozygotes (including 16 from non-consanguineous families). The study added 28 novel pathogenic/likely pathogenic variations. The study also calls attention to phenotypic variability and expansion in spectrum as well as uncovers genes where microcephaly is not reported previously or is a rare finding. We here report phenotypes associated with the genes for ultra-rare NDDs with microcephaly namely ATRIP, MINPP1, PNPLA8, AIMP2, ANKLE2, NCAPD2 and TRIT1.

Molecular analysis of severe hemophilia B in Indian families: Identification of mutational hotspot and novel variants.

INTRODUCTION: Hemophilia B is associated with molecular heterogeneity, with more than 1200 unique variants in the F9 gene. We hereby describe the mutational spectrum of severe hemophilia B patients presenting in a tertiary-care center in India. METHOD: DNA was extracted from peripheral blood samples of 35 diagnosed severe hemophilia B patients belonging to 32 families, and were subjected to Sanger sequencing. Determination of the effect of novel variants on the protein structure and correlation between genotype and phenotype was attempted using in-silico tools. RESULTS: Twenty-seven different mutations were detected in 30 probands, including 20 known and 7 novel variants. Also, we found one suspected case of whole gene deletion. The serine peptidase domain harbored most of the variants (48.1%). Inhibitory antibodies were found in two patients. CONCLUSIONS: This study provides a comprehensive mutational spectrum and mutation screening strategy by Sanger sequencing of F9 gene in severe hemophilia B patients, in a resource-constraint setting.

Genotype-phenotype spectrum of 130 unrelated Indian families with Mucopolysaccharidosis type II.

MPS II is an X linked recessive lysosomal storage disorder with multi-system involvement and marked molecular heterogeneity. In this study, we explored the clinical and molecular spectrum of 144 Indian patients with MPS II from 130 unrelated families. Clinical information was collected on a predesigned clinical proforma. Sanger method was employed to sequence all the exons and exon/intron boundaries of the IDS gene. In cases where causative variation was not detected by Sanger sequencing, MLPA and RFLP were performed to identify large deletions/duplications and complex rearrangements. Cytogenetic microarray was done in one patient to see the breakpoints and extent of deletion. In one patient with no detectable likely pathogenic or pathogenic variation, whole-genome sequencing was also performed. Novel variants were systematically assessed by in silico prediction software and protein modelling. The pathogenicity of variants was established based on ACMG criteria. An attempt was also made to establish a genotype-phenotype correlation. Positive family history was present in 31% (41/130) of patients. Developmental delay and intellectual disability were the main reasons for referral. Macrocephaly, coarse facies and dysostosis were present in almost all patients. Hepatosplenomegaly, joint contractures and short stature were the characteristic features, seen in 87% (101/116), 67.8% (74/109) and 41.4% (41/99) patients respectively. Attenuated phenotype was seen in 32.6% (47/144) patients, while severe phenotype was seen in 63% (91/144) patients. The detection rate for likely pathogenic or pathogenic variants in our cohort is 95.5% (107/112) by Sanger sequencing, MLPA and RFLP. We also found two variants of unknown significance, one each by Sanger sequencing and WGS. Total of 71 variants were identified by Sanger sequencing and 29 of these variants were found to be novel. Amongst the novel variants, there was a considerable proportion (51%) of frameshift variants (15/29). Almost half of the causative variants were located in exon 3,8 and 9. A significant genotype-phenotype correlation was also noted for both known and novel variants. This information about the genotype spectrum and phenotype will be helpful for diagnostic and prognostic purposes.

Novel pathogenic variants in an Indian cohort with epidermolysis bullosa: Expanding the genotypic spectrum.

BACKGROUND: Epidermolysis bullosa (EB) is a genodermatosis characterized by skin fragility and blisters with variable severity. Patients with Dystrophic EB (DEB) or Junctional EB (JEB) mainly present to clinic due to greater functional impairment. Pathogenic sequence variations in COL7A1 are implicated in DEB. OBJECTIVE: We have tried to decipher the molecular spectrum and genotype phenotype correlation of 21 Indian patients with EB. METHODS: Next generation sequencing (NGS) was performed to determine the pathogenic variants. Sanger sequencing was also done for validation of the variants in eleven individuals. RESULTS: Pathogenic variants were detected in 20 individuals (diagnostic yield of 95%). Majority of them (90%) had sequence variation in COL7A1 while two had pathogenic variants in ITGB4 and KRT14 respectively. Out of the 18 patients confirmed to have DEB, 3 had Dominant DEB (DDEB) whereas 15 patients had Recessive DEB (RDEB). Amongst 23 sequence variations identified, 12 were found to be novel (3 were missense, 5 were premature termination codon variants while 4 were splice-site changes). CONCLUSION: Genotype phenotype correlation was noted with milder manifestations in those with dominant inheritance types. Exact molecular diagnosis can be ascertained by NGS in majority of cases.

Phenotypic and genotypic spectrum of CTSK variants in a cohort of twenty-five Indian patients with pycnodysostosis.

BACKGROUND: Pycnodysostosis is an autosomal recessive skeletal dysplasia with easily recognizable clinical features and marked molecular heterogeneity. In this study, we explored the clinical and molecular spectrum of 25 Indian patients with pycnodysostosis from 20 families. METHODS: Clinical information was collected on a predesigned clinical proforma. Sanger method was employed to sequence all the exons and exon/intron boundaries of the CTSK gene. Novel variants were systematically assessed by prediction softwares and protein modelling. The pathogenicity of variant was established based on ACMG-AMP criteria. An attempt was also made to establish a genotype-phenotype correlation and devise a diagnostic scoring system based on clinical and radiological findings. RESULTS: Consanguinity and positive family history were present in 65% (13/20) and 45% (9/20) of the families respectively. Short stature and fractures were the predominant presenting complaints and was evident in 96% (24/25) and 32% (8/25) of affected individuals respectively. Gestalt facial phenotype and acro-osteolysis were present in 76% (19/25) and 82.6% (19/23) of the individuals respectively. Hepatosplenomegaly was present in 15% (3/20) of the individuals with one of them having severe anaemia. Causative sequence variations were identified in all of them. A total of 19 variants were identified from 20 families amongst which 10 were novel. Homozygous variants were identified in 90% (18/20) families. Amongst the novel variants, there was a considerable proportion (40%) of frameshift variants (4/10). No significant genotype-phenotype correlation was noted. Scoring based on clinical and radiological findings led to the proposal that a minimum of 2 scores in each category is required in addition to high bone density to diagnose pycnodysostosis with certainty. CONCLUSION: This study delineated the genotypic and phenotypic characterisation of Indian patients with pycnodysostosis with identification of 10 novel variants. We also attempted to develop a clinically useful diagnostic scoring system which requires further validation.

Diversity-oriented synthesis of benzimidazole, benzoxazole, benzothiazole and quinazolin-4(3H)-one libraries via potassium persulfate-CuSO4-mediated oxidative coupling reactions of aldehydes in aqueous micelles.

Libraries of 2-substituted-benzimidazoles, benzoxazoles, benzothiazoles as well as quinazolin-4(3H)-ones were synthesized via potassium persulfate-CuSO(4)-mediated oxidative coupling of aldehydes with o-phenylenediamines, o-aminophenols, o-aminothiophenols, and anthranilamide, respectively, in aqueous micelles. The strategy opens a way for rapid generation of libraries of small heterocycles for biological screening. The reagent is commercially available, cheap, and highly chemoselective. The yields were superior in aqueous micelles to those in organic solvents. Short reaction times, large-scale synthesis, excellent chemoselectivity, excellent yields, as well as environmental friendliness are the main advantages of this diversity-oriented synthesis.

Craniovertebral junction instability in Larsen syndrome: An institutional series and review of literature.

OBJECTIVE: Larsen syndrome (LS) is characterized by osteo-chondrodysplasia, multiple joint dislocations, and craniofacial abnormalities. Symptomatic myelopathy is attributed to C1-C2 instability and sub-axial cervical kyphosis. In this article, we have analyzed the surgical outcome after posterior fixation in LS with craniovertebral junction instability. METHODS: Ten symptomatic pediatric patients, operated between 2011 and 2019, were included, and the clinical outcome was assessed by Nurick grade, neurological improvement, and complications. The requirement of anti-spasticity drugs, the degree of bony fusion, and restriction of neck movement were also noted. At last follow-up, patient satisfaction score (PSS) and back to school status were studied. We also reviewed the literature and categorized two types of presentation of reported LS patients and discussed the pattern of disease progression among both. RESULTS: Ten patients, age range 1.5-16 years, underwent 12 surgeries (6 C1-C2 fixation, 4 long-segment posterior cervical fixation, and 2 trans-oral decompressions as the second stage); the mean follow-up was 23 (range, 6-86 months). All the ten patients in our study had the characteristic "dish-" like face and nine patients had acral anomalies. The median Nurick grade improved from preoperative (median = 4) to follow-up (median = 3). The requirement of anti-spasticity drugs decreased in seven patients and the neck-pain improved in nine patients. The median satisfaction at follow-up was good (median PSS = 2); five patients were going back to school. CONCLUSION: Craniovertebral junction instability in LS is rare and surgically challenging. Early posterior fixation showed a promising outcome with a halt in the disease progression.

Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A.

BACKGROUND: Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture. RESULTS: Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression. CONCLUSION: At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.