Inborn Errors of Ketogenesis: Novel Variants, Clinical Presentation, and Follow-Up in a Series of Four Patients.

Inborn errors of ketogenesis are rare disorders that result in acute and fulminant decompensation during lipolytic stress, particularly in infants and children. These include mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGCS) deficiency and HMG-CoA lyase (HMGCL) deficiency. In this series, we describe the clinical, biochemical, and molecular profiles of four patients along with dietary interventions and their outcomes on a long-term follow-up. Two patients each of HMGCS and HMGCL deficiency were evaluated with clinical history, biochemical investigations, including tandem mass spectrometry (TMS) and urine gas chromatography-mass spectrometry (GCMS). Molecular analysis was performed by whole-exome sequencing, as well as exon array validated by long-range polymerase chain reaction. All individuals were diagnosed with acute metabolic decompensation in the early infancy period except one with HMGCL deficiency who had the first presentation at 5 years of age. Central nervous system manifestations, severe metabolic acidosis, hyperammonemia, hypoglycemia with a normal lactate, and absence of urinary ketones were observed in all the affected individuals. The disorder was life-threatening in three individuals and one succumbed to the illness. TMS was nonspecific and urine GCMS revealed dicarboxylic aciduria in HMGCS deficiency. Both the patients with HMGCL deficiency demonstrated elevated 3 hydroxyisovaleryl carnitine levels in TMS and metabolites of leucine degradation in urine GCMS. We identified five novel variants that included a large deletion involving exon 2 in HMGCL gene. There was no evidence of long-term neurological sequelae in the living individuals. Diet with moderation of fat intake was followed in two individuals with HMGCS deficiency. Low leucine and protein diet with moderation of fat intake was followed in the individual with HMGCL deficiency. All affected individuals are thriving well with no further major metabolic decompensation.

Complex Hereditary Spastic Paraparesis Caused by de novo p.Arg480Ser in FAR1.

FAR1 (MIM *616107) is required for the reduction of fatty acyl CoAs to fatty alcohols which is important for plasmalogen biosynthesis. Recently, heterozygous de novo variants in FAR1 have been associated with cataracts, spastic paraparesis, and speech delay (MIM# 619338). Three different heterozygous de novo variants, all located in the same codon, causing substitution of arginine at position 480 into cysteine, histidine, or leucine, were reported in patients in the latter disorder.Here, authors have identified a novel substitution in the same Arg480 position into serine. The authors also provide in silico docking analysis of the mutant protein.

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.

Proband only exome sequencing in 403 Indian children with neurodevelopmental disorders: Diagnostic yield, utility and challenges in a resource-limited setting.

Whole exome sequencing is recommended as the first tier test for neurodevelopmental disorders (NDDs) with trio being an ideal option for the detection of de novo variants. Cost constraints have led to adoption of sequential testing i.e. proband-only whole exome followed by targeted testing of parents. The reported diagnostic yield for proband exome approach ranges between 31 and 53%. Typically, these study designs have aptly incorporated targeted parental segregation before concluding a genetic diagnosis to be confirmed. The reported estimates however do not accurately reflect the yield of proband only standalone whole -exome, a question commonly posed to the referring clinician in self pay medical systems like India. To assess the utility of standalone proband exome (without follow up targeted parental testing), we retrospectively evaluated 403 cases of neurodevelopmental disorders referred for proband-only whole exome sequencing at Neuberg Centre for Genomic Medicine (NCGM), Ahmedabad during the period of January 2019 and December 2021. A diagnosis was considered confirmed only upon the detection of Pathogenic/Likely Pathogenic variants in concordance with patient's phenotype as well as established inheritance pattern. Targeted parental/familial segregation analysis was recommended as a follow up test where applicable. The diagnostic yield of the proband-only standalone whole exome was 31.5%. Only 20 families submitted samples for follow up targeted testing, and a genetic diagnosis was confirmed in twelve cases increasing the yield to 34.5%. To understand factors leading to poor uptake of sequential parental testing, we focused on cases where an ultra-rare variant was detected in hitherto described de novo dominant neurodevelopmental disorder. A total of 40 novel variants in genes associated with de novo autosomal dominant disorders could not be reclassified as parental segregation was denied. Semi-structured telephonic interviews were conducted upon informed consent to comprehend reasons for denial. Major factors influencing decision making included lack of definitive cure in the detected disorders; especially when couples not planning further conception and financial constraints to fund further targeted testing. Our study thus depicts the utility and challenges of proband-only exome approach and highlights the need for larger studies to understand factors influencing decision making in sequential testing.

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.

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.

COASY related pontocerebellar hypoplasia type 12: A common Indian mutation with expansion of the phenotypic spectrum.

Pontocerebellar hypoplasia (PCH) type 12 is a rare, perinatal lethal neurodegenerative genetic disorder caused by biallelic mutations in the COASY gene. Herein, we describe the clinical and neuroradiological profile of nine affected fetuses/neonates from five families identified with a common COASY: c.1486-3C>G biallelic variant. Four of the five families were identified after data reanalysis of unresolved, severe PCH like phenotype and the fifth family through collaboration. The common antenatal phenotype was cerebellar hypoplasia. Microcephaly, arthrogryposis, and intrauterine growth restriction were the shared postnatal findings. The neurological manifestations included seizures, poor sucking, and spasticity. Novel findings of corpus callosum agenesis, simplified gyral pattern, normal sized pons, optic neuropathy, and a small thorax are reported in this series. The allele frequency of the COASY: c.1486-3C>G variant was 0.62% in the available Asian Indian database. We describe this as a possible common Indian origin variant. To the best of our knowledge, this is the largest PCH12 series reported.

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.

Indian Undiagnosed Diseases Program (I-UDP) - The Unmet Need.

Genomics is an integral part of many pediatric diseases spanning all sub-specialities. While many genetic disorders are diagnosed with the currently available genomic tests, there still are many patients who do not receive a definitive diagnosis. The Indian Undiagnosed Diseases Program is a multicenter effort to address these challenges and unmet needs of rare disease patients where current available genetic tests have failed to make a diagnosis. It embodies the principles of collaborative effort across multispecialty disciplines, and uses detailed phenotype. Diagnostic methods are tailored to patient specifics and the large genomic data is interrogated with precise, in-house bioinformatics pipelines using patient-specific phenotype to build the diagnostic algorithm. The inception of this research initiative in India is a step towards creating awareness and appreciation of the needs for our undiagnosed cohorts to enable appropriate management in this era of precision medicine.

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.

Autosomal recessive spinocerebellar ataxia-20 due to a novel SNX14 variant in an Indian girl.

Autosomal recessive spinocerebellar ataxia-20 is a rare disorder having distinctive coarse facies in addition to intellectual disability and cerebellar ataxia, with less than 35 cases reported worldwide. It is caused by biallelic variants in the SNX14 gene and is classified under the group of autophagy disorders. We report a 9-year-old girl who presented with classic clinical features of autosomal recessive spinocerebellar ataxia-20 and cerebellar atrophy on magnetic resonance imaging of brain. Trio exome sequencing with Sanger confirmation revealed a novel splice site variant, c.140 + 3A > T in the SNX14 gene. The variant pathogenicity established by mRNA expression study showed a significant reduction in the expression levels of SNX14 gene in proband and her parents on comparison to the control. The electron microscopy of the skin fibroblasts of proband depicted numerous cytoplasmic vacuoles with variable degrees of dense staining material. In addition, we have briefly reviewed and compared the phenotypic features of published cases of autosomal recessive spinocerebellar ataxia-20 in the literature. Coarse facies, intellectual disability with severe speech delay, hypotonia, and cerebellar atrophy were universal findings in the published cases. This is the second reported case from the Indian subcontinent.

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.

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.

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.

Homozygous Missense Variation in PNPLA8 Causes Prenatal-Onset Severe Neurodegeneration.

The patatin-like protein family plays an important role in various biological functions including lipid homeostasis, cellular growth, and signaling. Conserved across species, the patatin domain is shared by all 9 members of the PNPLA family without redundancy in the coding sequences. The defective function of PNPLA2, PNPLA6, and PNPLA9 are known to cause mitochondrial-related neurodegeneration. Recently, PNPLA8 has been associated with mitochondrial myopathy and poor weight gain with lactic acidosis in 3 unrelated families. Using whole-exome sequencing, we identified a homozygous novel missense variation c.1874A>G in the patatin domain of PNPLA8. The patient had prenatal-onset severe and progressive neurodegeneration with mortality in infancy.

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.

Twins with PEX7 related intellectual disability and cataract: Highlighting phenotypes of peroxisome biogenesis disorder 9B.

Peroxisome biogenesis disorders (PBDs) are a group of autosomal recessive disorders caused due to impaired peroxisome assembly affecting the formation of functional peroxisomes. PBDs are caused by a mutation in PEX gene family resulting in disease manifestation with extreme variability ranging from the onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults. Disease causing variations in PEX7 is known to cause severe rhizomelic chondrodysplasia punctata type 1 and PBD 9B, an allelic disorder resulting in a milder phenotype, often indistinguishable from that of classic Refsum disease. This case report highlights the variability of PEX7 related phenotypes and suggests that other than RCDP1 and late onset phenotype similar to Refsum disease, some cases present with cataract and neurodevelopmetal abnormalities during childhood without chondrodysplasia or rhizomelia. This report also underlines the importance of considering PBD 9B in children presenting with neurodevelopmental abnormalities especially if they have congenital cataract.

A data set of variants derived from 1455 clinical and research exomes is efficient in variant prioritization for early-onset monogenic disorders in Indians.

Given the genomic uniqueness, a local data set is most desired for Indians, who are underrepresented in existing public databases. We hypothesize patients with rare monogenic disorders and their family members can provide a reliable source of common variants in the population. Exome sequencing (ES) data from families with rare Mendelian disorders was aggregated from five centers in India. The dataset was refined by excluding related individuals and removing the disease-causing variants (refined cohort). The efficiency of these data sets was assessed in a new set of 50 exomes against gnomAD and GenomeAsia. Our original cohort comprised 1455 individuals from 1203 families. The refined cohort had 836 unrelated individuals that retained 1,251,064 variants with 181,125 population-specific and 489,618 common variants. The allele frequencies from our cohort helped to define 97,609 rare variants in gnomAD and 44,520 rare variants in GenomeAsia as common variants in our population. Our variant dataset provided an additional 1.7% and 0.1% efficiency for prioritizing heterozygous and homozygous variants respectively for rare monogenic disorders. We observed additional 19 genes/human knockouts. We list carrier frequency for 142 recessive disorders. This is a large and useful resource of exonic variants for Indians. Despite limitations, datasets from patients are efficient tools for variant prioritization in a resource-limited setting.

Novel FOXP1 pathogenic variants in two Indian subjects with syndromic intellectual disability.

We describe two unrelated Indian boys with Mental retardation with language impairment with or without autistic features (OMIM#613670). Novel pathogenic variants c. 593_599 delins AGAAG and c.1556T>C in FOXP1 were identified in Patients 1 and 2, respectively by exome sequencing. The patients shared the cardinal features of significant language impairment, prominent forehead, downslanted palpebral fissures, frontal upsweep of hair, and behavioral abnormalities. Camptodactyly (with pterygia in Patient 2) was an additional feature noted in our study. The phenotype was consistent with previous reports of patients with monogenic defects in FOXP1. The facial features overlap with Sotos syndrome. However, presence of frontal upsweep of hair is a good pointer toward FOXP1 related syndromic intellectual disability.