Thirteen Indians with camptodactyly-arthropathy-coxa vara-pericarditis syndrome.

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome (MIM# 208250) is a rare monogenic disorder, characterized by early onset of camptodactyly, progressive coxa vara, bilateral arthropathy and constrictive pericarditis. The syndrome is caused by biallelic loss-of-function variants in PRG4. Deficiency of PRG4 results in progressive worsening of joint deformity with age. Thirteen individuals with CACP syndrome from eight consanguineous Indian families were evaluated. We used exome sequencing to elucidate disease-causing variants in all the probands. These variants were further validated and segregated by Sanger sequencing, confirming the diagnosis of CACP syndrome in them. Seven females and six males aged 2-23 years were studied. Camptodactyly (13/13), coxa vara (11/13), short femoral neck (11/13) and arthritis in large joints (12/13) [wrists (11/13), ankle (11/13), elbow (10/13) and knee (10/13)] were observed commonly. Five novel disease-causing variants (c.3636G>T, c.1935del, c.1134dup, c.1699del and c.962T>A) and two previously reported variants (c.1910_1911del and c.2816_2817del) were identified in homozygous state in PRG4. We describe the phenotype and mutations in one of the large cohorts of patients with CACP syndrome, from India.

Biallelic loss of function variants in FUZ result in an orofaciodigital syndrome.

Orofaciodigital syndrome is a distinctive subtype of skeletal ciliopathies. Disease-causing variants in the genes encoding the CPLANE complex result in a wide variety of skeletal dysplasia with disturbed ciliary functions. The phenotypic spectrum includes orofaciodigital syndrome and short rib polydactyly syndrome. FUZ, as a part of the CPLANE complex, is involved in intraflagellar vesicular trafficking within primary cilia. Previously, the variants, c.98_111+9del and c.851G>T in FUZ were identified in two individuals with a skeletal ciliopathy, manifesting digital anomalies (polydactyly, syndactyly), orofacial cleft, short ribs and cardiac defects. Here, we present two novel variants, c.601G>A and c.625_636del in biallelic state, in two additional subjects exhibiting phenotypic overlap with the previously reported cases. Our findings underscore the association between biallelic loss of function variants in FUZ and skeletal ciliopathy akin to orofaciodigital syndrome.

Genetic and phenotypic landscape of pediatric-onset epilepsy in 142 Indian families: Counseling and therapeutic implications.

The application of genomic technologies has led to unraveling of the complex genetic landscape of disorders of epilepsy, gaining insights into their underlying disease mechanisms, aiding precision medicine, and providing informed genetic counseling. We herein present the phenotypic and genotypic insights from 142 Indian families with epilepsy with or without comorbidities. Based on the electroclinical findings, epilepsy syndrome diagnosis could be made in 44% (63/142) of the families adopting the latest proposal for the classification by the ILAE task force (2022). Of these, 95% (60/63) of the families exhibited syndromes with developmental epileptic encephalopathy or progressive neurological deterioration. A definitive molecular diagnosis was achieved in 74 of 142 (52%) families. Infantile-onset epilepsy was noted in 81% of these families (61/74). Fifty-five monogenic, four chromosomal, and one imprinting disorder were identified in 74 families. The genetic variants included 65 (96%) single-nucleotide variants/small insertion-deletions, 1 (2%) copy-number variant, and 1 (2%) triplet-repeat expansion in 53 epilepsy-associated genes causing monogenic disorders. Of these, 35 (52%) variants were novel. Therapeutic implications were noted in 51% of families (38/74) with definitive diagnosis. Forty-one out of 66 families with monogenic disorders exhibited autosomal recessive and inherited autosomal dominant disorders with high risk of recurrence.

Intragenic homozygous duplication in HEPACAM is associated with megalencephalic leukoencephalopathy with subcortical cysts type 2A.

Disease-causing variants in HEPACAM are associated with megalencephalic leukoencephalopathy with subcortical cysts 2A (MLC2A, MIM# 613,925, autosomal recessive), and megalencephalic leukoencephalopathy with subcortical cysts 2B, remitting, with or without impaired intellectual development (MLC2B, MIM# 613,926, autosomal dominant). These disorders are characterised by macrocephaly, seizures, motor delay, cognitive impairment, ataxia, and spasticity. Brain magnetic resonance imaging (MRI) in these individuals shows swollen cerebral hemispheric white matter and subcortical cysts, mainly in the frontal and temporal regions. To date, 45 individuals from 39 families are reported with biallelic and heterozygous variants in HEPACAM, causing MLC2A and MLC2B, respectively. A 9-year-old male presented with developmental delay, gait abnormalities, seizures, macrocephaly, dysarthria, spasticity, and hyperreflexia. MRI revealed subcortical cysts with diffuse cerebral white matter involvement. Whole-exome sequencing (WES) in the proband did not reveal any clinically relevant single nucleotide variants. However, copy number variation analysis from the WES data of the proband revealed a copy number of 4 for exons 3 and 4 of HEPACAM. Validation and segregation were done by quantitative PCR which confirmed the homozygous duplication of these exons in the proband and carrier status in both parents. To the best of our knowledge, this is the first report of an intragenic duplication in HEPACAM causing MLC2A.

c.202_204del in NUP214 causes late onset form of febrile encephalopathy.

Nucleoporins (NUPs) are a group of transporter proteins that maintain homeostasis of nucleocytoplasmic transport of proteins and ribonucleic acids under physiological conditions. Biallelic pathogenic variants in NUP214 are known to cause susceptibility to acute infection-induced encephalopathy-9 (IIAE9, MIM#618426), which is characterized by severe and early-onset febrile encephalopathy causing neuroregression, developmental delay, microcephaly, epilepsy, ataxia, brain atrophy, and early death. NUP214-related IIAE9 has been reported in eight individuals from four distinct families till date. We identified a novel in-frame deletion, c.202_204del p.(Leu68del), in NUP214 by exome sequencing in a 20-year-old male with episodic ataxia, seizures, and encephalopathy, precipitated by febrile illness. Neuroimaging revealed progressive cerebellar atrophy. In silico predictions show a change in the protein conformation that may alter the downstream protein interactions with the NUP214 N-terminal region, probably impacting the mRNA export. We report this novel deletion in NUP214 as a cause for a late onset and less severe form of IIAE9.

A novel homozygous variant in PMVK is associated with enhanced IL1ß secretion and a hyper-IgD syndrome-like phenotype.

The evolutionarily conserved mevalonate pathway plays an important role in the synthesis of cholesterol and isoprenoid compounds. Mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) enzymes regulate key rate-limiting steps in this pathway by sequentially phosphorylating mevalonic acid to yield downstream metabolites that regulate protein prenylation and cell signaling. Biallelic pathogenic variants in MVK cause a spectrum of rare autoinflammatory disorders that encompass milder forms of hyper-IgD syndrome (HIDS) at one end and the more severe mevalonic aciduria on the other. In contrast, pathogenic variants reported in PMVK are heterozygous and associated with porokeratosis, a skin disorder with no systemic manifestations. Recently, biallelic variants in PMVK were reported as a cause for an autoinflammatory disorder for the first time in two unrelated patients. In this study, we describe a child with recurrent arthritis and a HIDS-like phenotype harboring a novel homozygous variant c.398 C>T (p.Ala133Val) in PMVK. Mononuclear cells isolated from the patient showed significantly elevated production of interleukin 1ß, a key cytokine that shapes the inflammatory response in HIDS. Protein modeling studies suggested potential defects in PMVK enzyme activity. These results posit a further expanding of the genotypic spectrum of autoinflammatory disease to include biallelic PMVK variants.

LPIN2 -related Majeed syndrome: report of two Indian patients with novel variants in LPIN2 and review of literature.

LPIN2 -related Majeed syndrome (MIM# 609628) is a rare non-inflammasome autoinflammatory disease, caused due to biallelic variants in LPIN2 (MIM* 605519). To date, only 31 individuals from 18 families have been reported with this rare condition. Exome sequencing was done in two affected individuals from two unrelated families. Additionally, phenotypic, and genotypic information from the literature was reviewed. Two novel homozygous missense variants, c.2207G>A p. (Arg736His) and c.1157C>G p. (Ser386Ter) in LPIN2 , were identified in family 1 and family 2 respectively. Chronic recurrent osteomyelitis involving the lower extremities was the most common clinical presentation. LPIN2 -related Majeed syndrome should be considered as a differential diagnosis in an individual with clinical or radiological evidence of recurrent sterile osteomyelitis and chronic anaemia.

De novo variants underlying monogenic syndromes with intellectual disability in a neurodevelopmental cohort from India.

The contribution of de novo variants as a cause of intellectual disability (ID) is well established in several cohorts reported from the developed world. However, the genetic landscape as well as the appropriate testing strategies for identification of de novo variants of these disorders remain largely unknown in low-and middle-income countries like India. In this study, we delineate the clinical and genotypic spectrum of 54 families (55 individuals) with syndromic ID harboring rare de novo variants. We also emphasize on the effectiveness of singleton exome sequencing as a valuable tool for diagnosing these disorders in resource limited settings. Overall, 46 distinct disorders were identified encompassing 46 genes with 51 single-nucleotide variants and/or indels and two copy-number variants. Pathogenic variants were identified in CREBBP, TSC2, KMT2D, MECP2, IDS, NIPBL, NSD1, RIT1, SOX10, BRWD3, FOXG1, BCL11A, KDM6B, KDM5C, SETD5, QRICH1, DCX, SMARCD1, ASXL1, ASXL3, AKT3, FBN2, TCF12, WASF1, BRAF, SMARCA4, SMARCA2, TUBG1, KMT2A, CTNNB1, DLG4, MEIS2, GATAD2B, FBXW7, ANKRD11, ARID1B, DYNC1H1, HIVEP2, NEXMIF, ZBTB18, SETD1B, DYRK1A, SRCAP, CASK, L1CAM, and KRAS. Twenty-four of these monogenic disorders have not been previously reported in the Indian population. Notably, 39 out of 53 (74%) disease-causing variants are novel. These variants were identified in the genes mainly encoding transcriptional and chromatin regulators, serine threonine kinases, lysosomal enzymes, molecular motors, synaptic proteins, neuronal migration machinery, adhesion molecules, structural proteins and signaling molecules.

Further validation of craniosynostosis as a part of phenotypic spectrum of BCL11B-related BAFopathy.

Heterozygous disease-causing variants in BCL11B are the basis of a rare neurodevelopmental syndrome with craniofacial and immunological involvement. Isolated craniosynostosis, without systemic or immunological findings, has been reported in one of the 17 individuals reported with this disorder till date. We report three additional individuals harboring de novo heterozygous frameshift variants, all lying in the exon 4 of BCL11B. All three individuals presented with the common findings of this disorder i.e. developmental delay, recurrent infections with immunologic abnormalities and facial dysmorphism. Notably, craniosynostosis of variable degree was seen in all three individuals. We, thus add to the evolving genotypes and phenotypes of BCL11B-related BAFopathy and also review the clinical, genomic spectrum along with the underlying disease mechanisms of this disorder.

Further evidence of biallelic variants in KCNK18 as a cause of intellectual disability and epilepsy with febrile seizure plus.

INTRODUCTION: KCNK18 , a potassium channel subfamily K member 18 (MIM*613655), encodes for TWIK-related spinal cord K+ channel (TRESK) and is important for maintaining neuronal excitability. Monoallelic variants in KCNK18 are known to cause autosomal dominant migraine, with or without aura, susceptibility to, 13 (MIM#613656). Recently, biallelic missense variants in KCNK18 have been reported in three individuals from a non-consanguineous family with intellectual disability, developmental delay, autism spectrum disorder (ASD), and seizure. METHODS: Singleton exome sequencing was performed for the proband after detailed clinical evaluation to identify the disease-causing variants in concordance with the phenotype. RESULTS: We herein report an individual with intellectual disability, developmental delay, ASD, and epilepsy with febrile seizure plus with a novel homozygous stopgain variant, c.499C>T p.(Arg167Ter) in KCNK18 . CONCLUSION: This report further validates KCNK18 as a cause of autosomal recessive intellectual disability, epilepsy, and ASD.

Intracranial nanomedicine-gel with deep brain-penetration for glioblastoma therapy.

Glioblastoma Multiforme (GBM) is one of the challenging tumors to treat as it recurs, almost 100%, even after surgery, radiation, and chemotherapy. In many cases, recurrence happens within 2-3cm depth of the resected tumor margin, indicating the inefficacy of current anti-glioma drugs to penetrate deep into the brain tissue. Here, we report an injectable nanoparticle-gel system, capable of providing deep brain penetration of drug up to 4 cm, releasing in a sustained manner up to >15 days. The system consists of ∼222 nm sized PLGA nanoparticles (NP-222) loaded with an anti-glioma drug, Carmustine (BCNU), and coated with a thick layer of polyethylene glycol (PEG). Upon release of the drug from PLGA core, it will interact with the outer PEG-layer leading to the formation of PEG-BCNU nanocomplexes of size ∼33 nm (BCNU-NC-33), which could penetrate >4 cm deep into the brain tissue compared to the free drug (< 5 mm). In vitro drug release showed sustained release of drug for 15 days by BCNU-NP gel, and enhanced cytotoxicity by BCNU-NC-33 drug-nanocomplexes in glioma cell lines. Ex vivo goat-brain phantom studies showed drug diffusion up to 4 cm in tissue and in vivo brain-diffusion studies showed almost complete coverage within the rat brain (∼1.2 cm), with ∼55% drug retained in the tissue by day-15, compared to only ∼5% for free BCNU. Rat orthotopic glioma studies showed excellent anti-tumor efficacy by BCNU-NP gel compared to free drug, indicating the potential of the gel-system for anti-glioma therapy. In effect, we demonstrate a unique method of sustained release of drug in the brain using larger PLGA nanoparticles acting as a reservoir while deep-penetration of the released drug was achieved by in situ formation of drug-nanocomplexes of size <50 nm which is less than the native pore size of brain tissue (> 100 nm). This method will have a major impact on a challenging field of brain drug delivery.

Further delineation of KIF21B-related neurodevelopmental disorders.

Kinesin Family Member 21B (KIF21B) encoded by KIF21B (MIM*608322), belongs to the Kinesin superfamily proteins, which play a key role in microtubule organisation in neuronal dendrites and axons. Recently, heterozygous variants in KIF21B were implicated as the cause of intellectual disability and brain malformations in four unrelated individuals. We report a 9-year-old male with delayed speech, hyperactivity, poor social interaction, and autistic features. A parent-offspring trio exome sequencing identified a novel de novo rare heterozygous variant, NM_001252102.2: c.1513A>C, p.(Ser505Arg) in exon 11 of KIF21B. In vivo functional analysis using in utero electroporation in mouse embryonic cortex revealed that the expression of Ser505Arg KIF21B protein in the cerebral cortex impaired the radial migration of projection neurons, thus confirming the pathogenicity of the variant. Our report further validates pathogenic variants in KIF21B as a cause of neurodevelopmental disorder.

Bi-allelic variants in CHKA cause a neurodevelopmental disorder with epilepsy and microcephaly.

The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.

Further evidence of affected females with a heterozygous variant in FGF13 causing X-linked developmental and epileptic encephalopathy 90.

Developmental and epileptic encephalopathies (DEE) are a genetically heterogeneous group of disorders characterised by early onset epilepsy, epileptiform activity on electroencephalogram and associated developmental delay or neuroregression. With the advent of high throughput sequencing, novel gene-disease associations have been described for DEEs. Voltage activated sodium channels (Nav) regulate neuronal excitability. Fibroblast growth factor homologous factors (FHFs) are proteins, which bind to the C terminal cytoplasmic tails of alpha subunits of Nav channels and influence their function and surface expression. Gain of function hemizygous or heterozygous variants in FGF13 (also known as FHF2) were recently identified as the cause for X-linked developmental and epileptic encephalopathy 90 (DEE90; MIM# 301058) in seven individuals from five families, which included one female. We report an additional female, providing further evidence for a novel de novo heterozygous missense variant in FGF13, NM_004114.5: c.14T > G p.(Ile5Ser) causing X-linked DEE90. In addition, we review the genotype and phenotype of affected individuals with DEE90.

KCTD7-related progressive myoclonic epilepsy: report of three Indian families and review of literature.

Epilepsy, progressive myoclonic 3, with or without intracellular inclusions (MIM# 611726) is a rare autosomal recessive condition associated with pathogenic variants in KCTD7, which encodes the BR-C,ttk and bab/pox virus and zinc finger domain-containing KCTD7 protein. We report four individuals from three Indian families presenting with an initial period of normal development, progressive myoclonic seizures followed by neuroregression and an abnormal electroencephalogram. We identified two novel missense variants, c.458G>C p.(Arg153Pro) and c.205C>G p.(Leu69Val) and one known disease-causing variant, c.280C>T p.(Arg94Trp) in KCTD7 by exome sequencing. We review the literature of 67 individuals with variants in KCTD7. Our study expands the molecular spectrum of KCTD7-related progressive myoclonic epilepsy.

Multilocus disease-causing genomic variations for Mendelian disorders: role of systematic phenotyping and implications on genetic counselling.

Multilocus disease-causing genomic variations (MGVs) and multiple genetic diagnoses (MGDs) are increasingly being recognised in individuals and families with Mendelian disorders. This can be mainly attributed to the widespread use of genomic tests for the evaluation of these disorders. We conducted a retrospective study of families evaluated over the last 6 years at our centre to identify families with MGVs and MGDs. MGVs were observed in fourteen families. We observed five different consequences: (i) individuals with MGVs presenting as blended phenotypes (ii) individuals with MGVs presenting with distinct phenotypes (iii) individuals with MGVs with age-dependent penetrance (iv) individuals with MGVs with one phenotype obscured by another more predominant phenotype (v) two distinct phenotypes in different individuals in families with MGVs. Consanguinity was present in eight (8/14, 57.1%) of them. Thirteen families had two Mendelian disorders and one had three Mendelian disorders. The risk of recurrence of one or more conditions in these families ranged from 25% to 75%. Our findings underline the importance of the role of a clinical geneticist in systematic phenotyping, challenges in genetic counselling and risk estimation in families with MGVs and MGDs, especially in highly inbred populations.

Report of rapid diagnosis and precise management of MMADHC-related intracellular cobalamin defect.

Disorders of intracellular cobalamin metabolism are a group of metabolic disorders that lead to varied clinical presentation from intrauterine life to adulthood. We report a male infant with developmental regression, macrocytic anaemia and hyperpigmentation. Exome sequencing identified a homozygous pathogenic variant in the MMADHC gene, known to cause homocystinuria, cblD type (MIM #277410). We describe significant clinical improvement with targeted therapy and emphasise the relevance of genomic testing in accurate management of inherited metabolic disorders.

Clinical and Mutation Spectra of Cockayne Syndrome in India.

BACKGROUND: Cockayne syndrome is an autosomal recessive disorder caused by biallelic mutations in ERCC6 or ERCC8 genes. AIMS: To study the clinical and mutation spectrum of Cockayne syndrome. SETTING AND DESIGN: Medical Genetics Outpatient Department of Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow. This was a prospective study from 2007 to 2015. MATERIALS AND METHODS: Clinical details were recorded, and sequencing of ERCC6 and ERCC8 were performed. RESULTS AND CONCLUSIONS: Of the six families, one family had a homozygous mutation in ERCC8 and the other five families had homozygous mutations in ERCC6. Novel variants in ERCC6 were identified in four families. Phenotypic features may vary from severe to mild, and a strong clinical suspicion is needed for diagnosis during infancy or early childhood. Hence, molecular diagnosis is needed for confirmation of diagnosis in a child with a suspicion of Cockayne syndrome. Prenatal diagnosis can be provided only if molecular diagnosis is established in the proband.