Burden of rare genetic disorders in India: twenty-two years' experience of a tertiary centre.

BACKGROUND: Rare disorders comprise of ~ 7500 different conditions affecting multiple systems. Diagnosis of rare diseases is complex due to dearth of specialized medical professionals, testing labs and limited therapeutic options. There is scarcity of data on the prevalence of rare diseases in different populations. India being home to a large population comprising of 4600 population groups, of which several thousand are endogamous, is likely to have a high burden of rare diseases. The present study provides a retrospective overview of a cohort of patients with rare genetic diseases identified at a tertiary genetic test centre in India. RESULTS: Overall, 3294 patients with 305 rare diseases were identified in the present study cohort. These were categorized into 14 disease groups based on the major organ/ organ system affected. Highest number of rare diseases (D = 149/305, 48.9%) were identified in the neuromuscular and neurodevelopmental (NMND) group followed by inborn errors of metabolism (IEM) (D = 47/305; 15.4%). Majority patients in the present cohort (N = 1992, 61%) were diagnosed under IEM group, of which Gaucher disease constituted maximum cases (N = 224, 11.2%). Under the NMND group, Duchenne muscular dystrophy (N = 291/885, 32.9%), trinucleotide repeat expansion disorders (N = 242/885; 27.3%) and spinal muscular atrophy (N = 141/885, 15.9%) were the most common. Majority cases of ß-thalassemia (N = 120/149, 80.5%) and cystic fibrosis (N = 74/75, 98.7%) under the haematological and pulmonary groups were observed, respectively. Founder variants were identified for Tay-Sachs disease and mucopolysaccharidosis IVA diseases. Recurrent variants for Gaucher disease (GBA:c.1448T > C), ß-thalassemia (HBB:c.92.+5G > C), non-syndromic hearing loss (GJB2:c.71G > A), albinism (TYR:c.832 C > T), congenital adrenal hyperplasia (CYP21A2:c.29-13 C > G) and progressive pseudo rheumatoid dysplasia (CCN6:c.298T > A) were observed in the present study. CONCLUSION: The present retrospective study of rare disease patients diagnosed at a tertiary genetic test centre provides first insight into the distribution of rare genetic diseases across the country. This information will likely aid in drafting future health policies, including newborn screening programs, development of target specific panel for affordable diagnosis of rare diseases and eventually build a platform for devising novel treatment strategies for rare diseases.

Genetic Testing in Pediatric Epilepsy: Tools, Tips, and Navigating the Traps.

With the advent of high-throughput sequencing and computational methods, genetic testing has become an integral part of contemporary clinical practice, particularly in epilepsy. The toolbox for genetic testing has evolved from conventional chromosomal microarray and epilepsy gene panels to state-of-the-art sequencing techniques in the modern genomic era. Beyond its potential for therapeutic benefits through precision medicine, optimizing the choice of antiseizure medications, or exploring nonpharmacological therapeutic modalities, genetic testing carries substantial diagnostic, prognostic, and personal implications. Developmental and epileptic encephalopathies, the coexistence of neurodevelopmental comorbidities, early age of epilepsy onset, unexplained drug-refractory epilepsy, and positive family history have demonstrated the highest likelihood of yielding positive genetic test results. Given the diagnostic efficacy across different testing modalities, reducing costs of next-generation sequencing tests, and genetic diversity of epilepsies, exome sequencing or genome sequencing, where feasible and available, have been recommended as the first-tier test. Comprehensive clinical phenotyping at the outset, corroborative evidence from radiology and electrophysiology-based investigations, reverse phenotyping, and periodic reanalysis are some of the valuable strategies when faced with inconclusive test results. In this narrative review, the authors aim to simplify the approach to genetic testing in epilepsy by guiding on the selection of appropriate testing tools in the indicated clinical scenarios, addressing crucial aspects during pre- and post-test counseling sessions, adeptly navigating the traps posed by uncertain or negative genetic variants, and paving the way forward to the emerging testing modalities beyond DNA sequencing.

Role of next generation sequencing in diagnosis and management of critically ill children with suspected monogenic disorder.

Next generation sequencing based diagnosis has emerged as a promising tool for evaluating critically ill neonates and children. However, there is limited data on its utility in developing countries. We assessed its diagnostic rate and clinical impact on management of pediatric patients with a suspected genetic disorder requiring critical care. The study was conducted at a single tertiary hospital in Northern India. We analyzed 70 children with an illness requiring intensive care and obtained a precise molecular diagnosis in 32 of 70 probands (45.3%) using diverse sequencing techniques such as clinical exome, whole exome, and whole genome. A significant change in clinical outcome was observed in 13 of 32 (40.6%) diagnosed probands with a change in medication in 11 subjects and redirection to palliative care in two subjects. Additional benefits included specific dietary management (three cases), avoidance of a major procedure (one case) and better reproductive counseling. Dramatic therapeutic responses were observed in three cases with SCN1A, SCN2A and KCNQ2-related epileptic encephalopathy. A delayed turn-around for sequencing results was perceived as a major limiting factor in the study, as rapid and ultra-rapid sequencing was not available. Achieving a precise molecular diagnosis has great utility in managing critically ill patients with suspected genetic disorders in developing countries.

The molecular landscape of oculocutaneous albinism in India and its therapeutic implications.

Oculocutaneous albinism is an inherited disorder of melanin biosynthesis, characterized by absent or reduced pigmentation of the skin, hair, and eyes. Molecular alterations of genes that cause non-syndromic albinism in Asian Indians are poorly characterized. This information would be useful for developing therapies for this disorder. We analyzed 164 persons with non-syndromic albinism, belonging to unrelated families from all parts of India, for molecular changes in the causative genes. Subjects with white hair, white skin, and red iris had their tyrosinase gene sequenced and were also tested by MLPA for deletions/duplications. Subjects with negative results or with darker skin, golden/brown or darker hair had sequencing of TYR, P, TYRP1, SLC45A2 and GPR143 genes. Pathogenic variants in TYR (OCA1) were observed in 139 (84.7%) patients, in the P gene (OCA2) in 20 (12.2%), in TYRP1 (OCA3) in two (1.2%), in SLC45A2 (OCA 4) in one (0.61%), and in GPR143 (X-linked ocular albinism) in two (1.2%) patients. Of 278 alleles with variants in TYR, 179 (64.3%) alleles had (p.R278*) alteration, suggesting the possibility of therapy with a stop codon readthrough molecule. We report 20 patients with 13 disease associated variants in the P gene and 18 novel pathogenic variants in TYR, P, TYRP1, SLC45A2 and GPR143 genes. The data are compared with those reported from India, Pakistan and rest of the world. The therapeutic options in albinism are briefly described, opening this field for future therapies.

A Rare Case of Mosaic 3pter and 5pter Deletion-Duplication with Autism Spectrum Disorder and Dyskinesia.

Introduction: There is evidence that neurodevelopmental disorders are associated with chromosomal abnormalities. Current genetic testing can clinch an exact diagnosis in 20-25% of such cases. Case Description. A 3 years and 11 months old boy with global developmental delay had repetitive behaviors and hyperkinetic movements. He was stunted and underweight. He had ataxia, limb dyskinesia, triangular face, microcephaly, upward slanting palpebral fissure, hypertelorism, retrognathia, posteriorly rotated ears, long philtrum, thin lips, broad nasal tip, polydactyly, tappering fingers, and decreased tone in the upper and lower limbs with normal deep tendon reflexes. Magnetic resonance imaging of the brain, ultrasound of the abdomen, and ophthalmological evaluation were normal. Brain evoked response auditory revealed bilateral moderate hearing loss. He fulfilled the Diagnostic Statistical Manual 5 criteria for autism. In the Vineland Social Maturity Scale, his score indicated a severe delay in social functioning. His genetic evaluation included karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray analysis (CMA). The karyotype report from high-resolution lymphocyte cultures was mos 46, XY, der(3)t(3; 5)(p26; p15.3)[50]/46, XY,der(5) t(3;5) (p26;p15.3)[50].ish. His karyotype report showed a very rare and abnormal mosaic pattern with two cell lines (50% each). Cell-line#1: 3pter deletion with 5pter duplication (3pter-/5pter+) and cell-line#2: 3pter duplication with 5pter deletion (3pter+/5pter-) derived from a de novo reciprocal translocation t(3; 5)(p26; p15.3) which was confirmed by FISH. The chromosomal microarray analysis report was normal. The two cell lines (50% each) seem to have balanced out at the whole genome level. Occupational, sensory integration, and behavior modification therapy were initiated for his autistic features, and anticholinergic trihexiphenidyl was prescribed for hyperkinetic movements. Conclusion: This case highlights a rare genetic finding and the need for timely genetic testing in a child with dysmorphism and autism with movement disorder to enable appropriate management and genetic counselling.

Clinical and Genetic Profile of Children With Short Stature Presenting to a Genetic Clinic in Northern India.

OBJECTIVE: To define the spectrum of genetic disorders in patients with short stature visiting the genetic out-patient department in a tertiary care hospital. METHODS: A chart review was done for 455 individuals (10 months-16 yrs) with short stature, who were evaluated at the genetic clinic from 1 January, 2017 upto 31 October, 2018. 226 patients who needed detailed evaluation, the spectrum of genetic diagnosis is presented. RESULTS: Proportionate short stature was identified in 63% individuals (n=142) of which 93 (65%) were recognizable syndromes such as Turner syndrome, and William syndrome, and RASopathies. In clinically undefined syndromes (39, 27%), a diagnosis could be made by karyotype (n=3/10), chromosomal microarray (6/12) and exome sequencing (1/6). In the 84 children in the disproportionate short stature group (37%), lysosomal storage disorders (LSDs) (45%, n=38) were identified by enzyme analysis in 86.8% and skeletal dysplasias (44%, n=37) identified by skeletal survey in 89% cases. CONCLUSIONS: In undefined syndromic short stature, chromosomal microarray may be the first investigation of choice if phenotyping is not suggestive of a specific genetic syndrome. Exome sequencing can be useful in identifying newer genes among idiopathic and familial short stature cohorts.

Hypervitaminosis D and Acute Interstitial Nephritis: Tale of Injections.

A 33-year-old man came with nausea, vomiting and abdominal pain due to hypercalcaemia and renal dysfunction following two doses of intramuscular vitamin D injections. Levels of vitamin D were repeatedly above 300 ng/ml over a period of 10 months. Whole-body PET CT scan revealed a thin-walled collection in the right gluteal region. The patient refused a surgical intervention for the same. After 7 months of follow-up, the abscess ruptured spontaneously and was then surgically debrided. At this point, a history of pentazocine addiction was uncovered. One month later, vitamin D levels began to fall along with improvement in serum calcium and creatinine. This case unravels a diagnostic odyssey which ended with a simple surgical debridement. We aim to highlight that vitamin D supplementation in 'megadoses' in the presence of active infection can have an exaggerated response and may take months to resolve.

Osteopathia Striata with Cranial Sclerosis: A Face-to-Radiograph-to-Gene Diagnosis.

Osteopathia striata with cranial sclerosis is an X-linked dominant bone dysplasia with osteosclerosis. It should be suspected in girls with macrocephaly, intellectual disability with unique facial dysmorphic features. We described the clinical and radiological profile of a patient with this rare disorder. A novel heterozygous variant was identified in the AMER1 gene which leads to premature truncation of the AMER1 protein. Facial gestalt recognition using artificial intelligence and radiographic features were used to narrow the differential diagnosis.

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.

Growth and neurodevelopmental disorder with arthrogryposis, microcephaly and structural brain anomalies caused by Bi-allelic partial deletion of SMPD4 gene.

Neutral sphingomyelinases have an important role in generation of ceramide and phosphorylcholine from sphingomyelins which then act as secondary messengers in various signaling pathways of the cellular machinery. They function ubiquitously with a predominant role in the central nervous system. Neutral sphingomyelinase type 3, encoded by SMPD4 gene has recently been reported to cause a severe autosomal recessive neurodevelopmental disorder with congenital arthrogryposis and microcephaly. We report a 22-month-old girl having characteristic features of neurodevelopmental delay, prenatal onset growth failure, arthrogryposis, microcephaly and brain anomalies including severe hypomyelination, simplified gyral pattern and hypoplasia of corpus callosum and brain stem. In addition, she was noted to have nystagmus and visual impairment secondary to macular dystrophy and retinal pigment epithelial stippling at posterior pole. Copy number variant analysis from trio whole exome sequencing (ES) enabled identification of a homozygous 11 kb deletion encompassing exons 18-20 of SMPD 4 gene, confirming the diagnosis of SMPD4-related disorder in her.

LMNB1 Duplication-Mediated Autosomal Dominant Adult-Onset Leukodystrophy in an Indian Family.

Autosomal dominant leukodystrophy is an adult onset neurodegenerative disorder presenting with progressive symptoms of ataxia and autonomic dysfunction in fourth or fifth decade in life. It has clinical similarity with multiple sclerosis, but shows characteristic magnetic resonance imaging findings of diffuse bilaterally symmetrical leukodystrophy which can distinguish this disorder. It is a rare disorder with no known treatment till date, and has never been described from the Indian subcontinent. We present an Indian family with autosomal dominant adult-onset demyelinating leukodystrophy with multiple members affected over four generations, and demonstrate a cheap and accurate molecular method of real-time polymerase chain reaction to detect the LMNB1 gene duplication, which is the genetic basis of this devastating disorder.

Clinical and genetic spectrum of 104 Indian families with central nervous system white matter abnormalities.

Genetic disorders with predominant central nervous system white matter abnormalities (CNS WMAs), also called leukodystrophies, are heterogeneous entities. We ascertained 117 individuals with CNS WMAs from 104 unrelated families. Targeted genetic testing was carried out in 16 families and 13 of them received a diagnosis. Chromosomal microarray (CMA) was performed for three families and one received a diagnosis. Mendeliome sequencing was used for testing 11 families and all received a diagnosis. Whole exome sequencing (WES) was performed in 80 families and was diagnostic in 52 (65%). Singleton WES was diagnostic for 50/75 (66.67%) families. Overall, genetic diagnoses were obtained in 77 families (74.03%). Twenty-two of 47 distinct disorders observed in this cohort have not been reported in Indian individuals previously. Notably, disorders of nuclear mitochondrial pathology were most frequent (9 disorders in 20 families). Thirty-seven of 75 (49.33%) disease-causing variants are novel. To sum up, the present cohort describes the phenotypic and genotypic spectrum of genetic disorders with CNS WMAs in our population. It demonstrates WES, especially singleton WES, as an efficient tool in the diagnosis of these heterogeneous entities. It also highlights possible founder events and recurrent disease-causing variants in our population and their implications on the testing strategy.

Functional characterization of novel variants in SMPD1 in Indian patients with acid sphingomyelinase deficiency.

Pathogenic variations in SMPD1 lead to acid sphingomyelinase deficiency (ASMD), that is, Niemann-Pick disease (NPD) type A and B (NPA, NPB), which is a recessive lysosomal storage disease. The knowledge of variant spectrum in Indian patients is crucial for early and accurate NPD diagnosis and genetic counseling of families. In this study, we recruited 40 unrelated pediatric patients manifesting symptoms of ASMD and subnormal ASM enzyme activity. Variations in SMPD1 were studied using Sanger sequencing for all exons, followed by interpretation of variants based on American College of Medical Genetics and Genomics & Association for Molecular Pathology (ACMG/AMP) criteria. We identified 18 previously unreported variants and 21 known variants, including missense, nonsense, deletions, duplications, and splice site variations with disease-causing potential. Eight missense variants were functionally characterized using in silico molecular dynamic simulation and in vitro transient transfection in HEK293T cells, followed by ASM enzyme assay, immunoblot, and immunofluorescence studies. All the variants showed reduced ASM activity in transfected cells confirming their disease-causing potential. The study provides data for efficient prenatal diagnosis and genetic counseling of families with NPD type A and B.

Prenatal Diagnosis by Chromosome Microarray Analysis, An Indian Experience.

BACKGROUND: Karyotyping has been the gold standard for prenatal chromosome analysis. The resolution should be higher by chromosome microarray analysis (CMA). The challenge lies in recognizing benign and pathogenic or clinically significant copy number variations (pCNV) and variations of unknown significance (VOUS). The aim was to evaluate the diagnostic yield and clinical utility of CMA, to stratify the CMA results in various prenatal referral groups and to accumulate Indian data of pCNVs and VOUS for further interpretation to assist defined genetic counseling. METHODS: Karyotyping and CMA were performed on consecutive referrals of 370 prenatal samples of amniotic fluid (n = 274) and chorionic villi (n = 96) from Indian pregnant women with high maternal age (n = 23), biochemical screen positive (n = 61), previous child abnormal (n = 59), abnormal fetal ultrasound (n = 205) and heterozygous parents (n = 22). RESULTS AND CONCLUSION: The overall diagnostic yield of abnormal results was 5.40% by karyotyping and 9.18% by CMA. The highest percentage of pCNVs were found in the group with abnormal fetal ultrasound (5.40%) as compared to other groups, such as women with high maternal age (0.81%), biochemical screen positive (0.54%), previous abnormal offspring (0.81%) or heterozygous parents group (1.62%). Therefore, all women with abnormal fetal ultrasound must undergo CMA test for genotype-phenotype correlation. CMA detects known and rare deletion/duplication syndromes and characterizes marker chromosomes. Accumulation of CNV data will form an Indian Repository and also help to resolve the uncertainty of VOUS. Pretest and posttest genetic counseling is essential to convey benefits and limitations of CMA and help the patients to take informed decisions.

Challenges in Chronic Genetic Disorders: Lessons From the COVID-19 Pandemic.

To examine the impact of the COVID-19 pandemic, we interviewed 26 patients with lysosomal storage disorders receiving enzyme replacement therapy. 20 (77 %) had significant interruption in their treatment, with an average of 8 (range 2-28) missed doses. Alternate methods of delivering uninterrupted care including home therapy were used. Vulnerable patients with chronic genetic disorders require organization for their multidisciplinary needs of care.

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.