Katta Mohan Girisha

Autoinflammatory disorders (AIDs) are a clinically heterogeneous group of inborn errors of immunity primarily caused by dysregulation in the innate immune system. Clinical diagnosis is often challenging due to clinical heterogeneity and the overlapping phenotypes with other inborn errors of immunity and monogenic conditions that mimic AIDs. Worldwide, and especially in India, there is a lack of systematic large cohort studies employing genomic testing early in the course of evaluation for individuals with autoinflammatory disease. Herein, we describe our experience using exome sequencing as a first‐tier investigation in evaluating 202 individuals from 196 unrelated families with possible monogenic AIDs. Consanguinity was noted in 19% of the families. Recurrent fever was the most common manifestation (90%), frequently associated with arthritis (54%), skin lesions (38%), hepatosplenomegaly (23%), oral ulcers (21%) and lymphadenopathy (21%). Exome sequencing could identify a molecular diagnosis in 50 of 196 (25%) families of whom 30 (60%) had variants in genes associated with monogenic AIDs, 10 (20%) had other distinct inborn errors of immunity and 10 (20%) had monogenic conditions with phenotypic overlap with AIDs. We identified 21 variants in genes associated with monogenic AIDs, of which 20 (95%) were single nucleotide variants and one (5%) was a copy number variant. Notably, seven (33%) of the 21 variants were novel. This study highlights the diagnostic challenges posed by phenotypic overlap in this group of disorders and demonstrates the utility of exome sequencing in enabling a timely diagnosis, including conditions that mimic AIDs thereby facilitating targeted therapy and genetic counseling.

Background BHLHE22 encodes a basic helix-loop-helix transcription factor expressed exclusively in the retina and central nervous system and functions as an important regulator of neuronal differentiation. However, BHLHE22 has not yet been associated with a Mendelian neurodevelopmental or neurological disorder. Methods 15 individuals from 13 unrelated families carrying BHLHE22 variants identified by exome sequencing were collected through an international collaboration. Results De novo missense variants located in the highly conserved helix-loop-helix domain of the protein were found in six individuals, and one recurrent homozygous frameshift variant, NP_689627.1:p.Gly74AlafsTer18, was found in nine individuals. Frequent clinical features include absent or limited speech (10/13), delayed or impaired motor abilities (11/13), intellectual disability (ID; 9/12), partial or complete agenesis of the corpus callosum (12/15), involuntary movements and/or stereotypies (11/13) and abnormal muscle tone (13/13), depending on data availability. Two individuals developed spastic paraplegia, without ID or callosal anomalies. One individual had moderate developmental delay and ID but without callosal anomalies. Conclusion Collectively, our data establish BHLHE22 as a previously unrecognized neurodevelopmental disease gene. Disruption of BHLHE22, through either dominant or recessive variants, results in a distinct syndrome characterised by abnormalities in brain development, cognition, tone and movement.

Systematic analysis of copy number variants (CNVs) in large datasets is challenging, and there are limited studies of homozygous copy number losses in rare disease exomes. Here, we leveraged the genomic uniqueness and relative under-representation of the Indian population in the current public genomic databases and identified 42,386 possible homozygous losses (median 20 per individual) in a heterogeneous cohort of 2021 individuals with suspected Mendelian disorders, who had undergone exome sequencing using 12 different capture kits in a resource-limited setting. Employing a genomic position loss-count-based approach, we filtered 1224 rare homozygous loss calls in 718 individuals (median 1 per individual) for further analysis, thus significantly reducing the analysis burden. Clinical correlation and validation of these rare calls enabled 10 new diagnoses in 240 unsolved individuals. This led to a two-fold increase in diagnosis owing to homozygous deletions. Further analysis of the data and identification of additional affected individuals through collaboration led to identification of biallelic FILIP1 and FAM177A1 variants as causes of a syndromic arthrogryposis and a neuromuscular disorder respectively. Both conditions were recently reported as ultra-rare recessive disorders, thus validating our approach. We also show that biallelic loss-of-function TFCP2L1 variants cause chronic kidney disease and VPS36 variants cause a severe recessive neurodevelopmental disorder characterised by microcephaly, motor delay, agenesis of the corpus callosum, cerebellar atrophy, seizures, hypotonia, spasticity and early death. Overall, these results demonstrate a scalable approach to screen homozygous losses for improving diagnostic yield and discovering disease-genes in large exome cohorts.

Hennekam lymphangiectasia‐lymphedema syndrome (HKLLS) is an autosomal recessive disorder, caused by biallelic variants in CCBE1 , FAT4 , and ADAMTS3 genes. We herein report a 15‐month‐old male with peripheral lymphedema, facial dysmorphism, camptodactyly and generalized hypotonia. Solo exome sequencing revealed a homozygous splice site variant, c.12479+3A>G in intron 14 of FAT4 (NM_001291303.3). Reverse transcriptase‐PCR (RT‐PCR) was performed using cDNA isolated from patient‐derived fibroblasts, which revealed aberrant splicing. This study provides a report of an additional family with a novel biallelic splice site variant in FAT4 which disrupts the normal splicing of the FAT4 mRNA, leading to aberrant splicing causing a milder form of HKLLS2.

Systematic analysis of copy number variants (CNVs) in large datasets is challenging and there are limited studies of homozygous copy number losses in rare disease exome datasets. Here we leveraged the genomic uniqueness and relative under-representation of the Indian population in the current public genomic databases and identified 42,386 possible homozygous losses (median count 20 per individual, range 0 – 55; median size 2.95 kb, range 99 bp – 4.76 Mb) in a heterogeneous cohort of 2,021 individuals with suspected Mendelian disorders, who had undergone exome sequencing using 12 different capture kits in a resource-limited setting. Employing a genomic position loss-count based approach, we filtered 1,224 rare homozygous loss calls in 718 individuals (median count 1 per individual, range 0 – 22; median size 3.49 kb, range 121 bp – 4.76 Mb) for further analysis, thus significantly reducing the analysis burden. Clinical correlation and validation of these rare calls enabled 10 new diagnoses in 240 unsolved individuals with at least one filtered rare homozygous loss call. This, led to nearly two-fold increase in diagnosis owing to homozygous deletions in our cohort. Further analysis of the data and identification of additional affected individuals through collaboration led to identification of biallelic FILIP1 and FAM177A1 variants as causes of a syndromic arthrogryposis and a neuromuscular disorder respectively. Both these conditions have been recently proven as ultra-rare recessive disorders, thus validating our approach. We also show that biallelic loss-of-function TFCP2L1 variants cause chronic kidney disease and VPS36 variants cause a severe recessive neurodevelopmental disorder characterised by microcephaly, motor delay, agenesis of the corpus callosum, cerebellar atrophy, seizures, hypotonia, spasticity and early death. Overall, these results demonstrate a scalable approach to screen homozygous losses for improving diagnostic yield and discovering disease-genes in large exome cohorts.

Osteogenesis imperfecta type VI is a rare genetic disorder caused by biallelic disease‐causing variants in SERPINF1. The phenotype is characterized by severe osteopenia, recurrent fractures, and moderate to severe skeletal deformities. We report an 11‐year‐old individual who presented with multiple fractures of the long bones of the upper and lower extremities, severe osteopenia, and skeletal deformities. Whole exome sequencing revealed compound heterozygous variants c.[‐37C>A];[829_831del] in SERPINF1 . To determine the functional consequence of the variants, quantitative real‐time PCR and immunoblotting analyses in patient‐derived fibroblasts were performed, which showed reduced expression of SERPINF1 transcript and protein levels.

Mucopolysaccharidosis type 10 (MPS10) is a recently discovered lysosomal storage disorder caused by biallelic loss of function variants in ARSK . To date, 10 ARSK‐related MPS10 patients from six families have been reported, with a median age at presentation of 9.5 years. The affected individuals usually present with progressive hip joint abnormalities. Coarse facial features, genu valgum, and joint abnormalities are variable. None demonstrated hepatosplenomegaly, neurological deficits, or cognitive impairment. Radiographically, platyspondyly, inferior tapering of the ilea with acetabular hypoplasia, irregularity of the central part of the femoral head, and metaphyseal striation of the long bones characterize this condition. Biochemical analyses reveal variable dermatan sulfate (DS) excretion in urine, with some patients showing increased DS, while others had normal urinary glycosaminoglycans (GAGs), suggesting that a normal GAG profile does not exclude this condition. This report provides a comprehensive review of existing knowledge on ARSK‐related mucopolysaccharidosis.

Background Inherited ARPC1B deficiency (ARPC1BD) is a rare autosomal recessive inborn error of immunity that manifests with allergic, infective, and autoimmune/inflammatory features. Only about three dozen patients with ARPC1BD have been reported in the literature thus far. Methods We describe 14 patients (ten families) with ARPC1BD from Nepal. Many of these patients have unique/rare clinical features that expand the phenotypic spectrum of ARPC1BD. The study included data on clinical-epidemiological features, immuno-hematological parameters, treatment, and outcome. Homozygosity mapping and haplotype analysis were used to evaluate the founder effect. Results We noted allergic/autoimmune and infective manifestations in all of our patients. Notable clinical features noted in our study include rheumatoid factor positive (RF+) chronic arthritis (mimicking RF+ juvenile idiopathic arthritis), significantly elevated anti-tissue transglutaminase antibody titers, generalized skin hyperpigmentation, distal phalangeal enlargement, frontal bone hypertrophy, hypertrophic skin scars, and hyperkeratosis. All cases harbored the founder splice-site variant c.64+2T>A in the ARPC1B gene. Long-term outcomes in our patients appeared worse than reported previously. Comparative phenotypic analysis showed significantly greater proportions of otitis, gastroenteritis, inflammatory bowel disease-like manifestations, and elevated IgA in patients with c.64+2T>A variant as compared to other variants. Homozygosity mapping (in eight probands) revealed that the gene/variant is contained within the only overlapping region of homozygosity (>1 Mb) across all autosomal chromosomes. Besides, the included probands share a similar haplotype around the said variant. Conclusions c.64+2T>A is a founder variant in Nepalese patients with ARPC1BD. This variant is associated with a severe clinical phenotype and diverse clinical complications. Graphical Abstract

UNC50 encodes a transmembrane protein that plays a crucial role in L‐acetylcholine receptor trafficking and thus cholinergic transmission at the neuromuscular junction. Previously, a biallelic loss‐of‐function variant in UNC50 was reported in an individual with lethal arthrogryposis multiplex congenita. We herein describe affected individuals from two unrelated families with arthrogryposis multiplex congenita in one family and a severe early‐onset neuromuscular dysfunction in the other, both within the spectrum of congenital myasthenia syndrome. A biallelic variant, c.644‐13_644‐9del, p.? in intron 5 of UNC50 (NM_014044.7) was identified in both families. Transcript analysis in the peripheral blood cDNA of the heterozygous carrier parents of family 1 revealed that the c.644‐13_644‐9del variant leads to aberrant splicing. With these findings, we validated the association of disease‐causing variants in UNC50 with congenital myasthenia syndrome.