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.

Comprehensive phenotyping of fetuses with trisomy 18: a perinatal center experience.

Trisomy 18 is the second most common aneuploidy after trisomy 21. It presents with varying degrees of heterogeneous clinical phenotypes involving multiple organ systems, with a high mortality rate. Clinical assessment of fetal trisomy 18 is always challenging. In this study, we describe the phenotypes of the fetuses with trisomy 18 from a perinatal cohort. We reviewed fetuses with trisomy 18 in referrals for perinatal autopsy over the period of 15 years. A detailed phenotyping of the fetuses with trisomy 18 was executed by perinatal autopsy. Appropriate fetal tissues were obtained to perform genomic testing. We observed trisomy 18 in 16 fetuses (2%) in our cohort of 784 fetal/neonatal losses and a perinatal autopsy was performed on all of them. Abnormal facial profile was the most frequent anomaly (10/16, 62%) followed by anomalies of the extremities (9/16, 56%), and cardiac defects (6/16, 37%). We also observed esophageal atresia, diaphragmatic hernia, and neural tube defect. The study represents one of the largest cohorts of trisomy 18 from a perinatal center from a developing country and highlights the clinical heterogeneity attributed to trisomy 18. We also report a recurrence of trisomy 18 in a family.

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.

Early and severe tricuspid valve dysplasia in a fetus with cardiospondylocarpofacial syndrome due to a variant c.616T>G p.(Tyr206Asp) in MAP3K7.

Cardiospondylocarpofacial syndrome (CSCF; MIM#157800) is a rare condition caused by monoallelic variants in the MAP3K7 gene. The characteristic features of CSCF include growth retardation, facial dysmorphism, carpal-tarsal fusion, dorsal spine synostosis, deafness, inner ear malformation, cardiac septal defect and valve dysplasia. We present here a 20-week-old fetus with cardiospondylocarpofacial syndrome arising from a de novo variant c.616T>G p.(Tyr206Asp) in the MAP3K7 (NM_145331.3) gene with early and severe tricuspid valve dysplasia as a prenatal manifestation. Fetal echocardiography revealed tricuspid regurgitation with valve prolapse. Fetus had facial dysmorphism and dilated right atrium and right ventricle with tricuspid valve dysplasia on perinatal evaluation. To the best of our knowledge, this is the first report mentioning the prenatal manifestation of cardiospondylocarpofacial syndrome.

Deep intronic mutation in CRTAP results in unstable isoforms of the protein to induce type I collagen aggregation in a lethal type of osteogenesis imperfecta type VII.

Genetic mutations are involved in Mendelian disorders. Unbuffered intronic mutations in gene variants can generate aberrant splice sites in mutant transcripts, resulting in mutant isoforms of proteins with modulated expression, stability, and function in diseased cells. Here, we identify a deep intronic variant, c.794_1403A>G, in CRTAP by genome sequencing of a male fetus with osteogenesis imperfecta (OI) type VII. The mutation introduces cryptic splice sites in intron-3 of CRTAP, resulting in two mature mutant transcripts with cryptic exons. While transcript-1 translates to a truncated isoform (277 amino acids) with thirteen C-terminal non-wild-type amino acids, transcript-2 translates to a wild-type protein sequence, except that this isoform contains an in-frame fusion of non-wild-type twenty-five amino acids in a tetratricopeptide repeat sequence. Both mutant isoforms of CRTAP are unstable due to the presence of a unique 'GWxxI' degron, which finally leads to loss of proline hydroxylation and aggregation of type I collagen. Although type I collagen aggregates undergo autophagy, the overall proteotoxicity resulted in death of the proband cells by senescence. In summary, we present a genetic disease pathomechanism by linking a novel deep intronic mutation in CRTAP to unstable mutant isoforms of the protein in lethal OI type VII.

Clinically diverse and perinatally lethal syndromes with urorectal septum malformation sequence.

Urorectal septum malformation sequence (URSMS) is characterized by a spectrum of anomalies of the urogenital system, hindgut and perineum. It is presumed to be a constellation of an embryonic defect. Herein, we analyzed the clinically diverse syndromes associated with URSMS in our perinatal evaluation unit. We reviewed fetuses with URSMS in referrals for perinatal autopsy over a period of 3 years. Chromosomal microarray and genome sequencing were performed whenever feasible. Literature was reviewed for syndromes or malformations with URSMS. We ascertained URSMS in 12 of the 215 (5%) fetuses. Nine fetuses (75%) had complete URSMS and remainder had partial/intermediate URSMS. Eleven fetuses had malformations of other systems that included: cerebral ventriculomegaly; right aortic arch with double outlet right ventricle; microcephaly with fetal akinesia deformation sequence; ventricular septal defect and radial ray anomaly; thoraco-abdominoschisis and limb defects; myelomeningocele; spina bifida and fused iliac bones; omphalocele; occipital encephalocele; lower limb amelia and cleft foot. We report on six fetuses with recurrent and five fetuses with unique malformations/patterns where URSMS is a component. Exome sequencing (one family) and genome sequencing (eight families) were performed and were nondiagnostic. Additionally, we review the literature for genetic basis of this condition. URMS is a clinically heterogeneous condition and is a component of several multiple malformation syndromes. We describe several unique and recurrent malformations associated with URSMS.

Expanding the spectrum of syndromic PPP2R3C-related XY gonadal dysgenesis to XX gonadal dysgenesis.

Homozygous variants in PPP2R3C have been reported to cause a syndromic 46,XY complete gonadal dysgenesis phenotype with extragonadal manifestations (GDRM, MIM# 618419) in patients from four unrelated families, whereas heterozygous variants have been linked to reduced fertility with teratozoospermia (SPGF36, MIM# 618420) in male carriers. We present eight patients from four unrelated families of Turkish and Indian descent with three different germline homozygous PPP2R3C variants including a novel in-frame duplication (c.639_647dupTTTCTACTC, p.Ser216_Tyr218dup). All patients exhibit recognizable facial dysmorphisms allowing gestalt diagnosis. In two 46,XX patients with hypergonadotropic hypogonadism and nonvisualized gonads, primary amenorrhea along with absence of secondary sexual characteristics and/or unique facial gestalt led to the diagnosis. 46,XY affected individuals displayed a spectrum of external genital phenotypes from ambiguous genitalia to complete female. We expand the spectrum of syndromic PPP2R3C-related XY gonadal dysgenesis to both XY and XX gonadal dysgenesis. Our findings supported neither ocular nor muscular involvement as major criteria of the syndrome. We also did not encounter infertility problems in the carriers. Since both XX and XY individuals were affected, we hypothesize that PPP2R3C is essential in the early signaling cascades controlling sex determination in humans.

Biallelic deep intronic variant c.5457+81T>A in TRIP11 causes loss of function and results in achondrogenesis 1A.

Biallelic loss of function variants in TRIP11 encoding for the Golgi microtubule-associated protein 210 (GMAP-210) causes the lethal chondrodysplasia achondrogenesis type 1A (ACG1A). Loss of TRIP11 activity has been shown to impair Golgi structure, vesicular transport, and results in loss of IFT20 anchorage to the Golgi that is vital for ciliary trafficking and ciliogenesis. Here, we report four fetuses, two each from two families, who were ascertained antenatally with ACG1A. Affected fetuses in both families are homozygous for the deep intronic TRIP11 variant, c.5457+81T>A, which was found in a shared region of homozygosity. This variant was found to cause aberrant transcript splicing and the retention of 77 base pairs of intron 18. The TRIP11 messenger RNA and protein levels were drastically reduced in fibroblast cells derived from one of the affected fetuses. Using immunofluorescence we also detected highly compacted Golgi apparatus in affected fibroblasts. Further, we observed a significant reduction in the frequency of ciliated cells and in the length of primary cilia in subject-derived cell lines, not reported so far in patient cells with TRIP11 null or hypomorphic variants. Our findings illustrate how pathogenic variants in intronic regions of TRIP11 can impact transcript splicing, expression, and activity, resulting in ACG1A.

Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.

High diagnostic yield in skeletal ciliopathies using massively parallel genome sequencing, structural variant screening and RNA analyses.

Skeletal ciliopathies are a heterogenous group of disorders with overlapping clinical and radiographic features including bone dysplasia and internal abnormalities. To date, pathogenic variants in at least 30 genes, coding for different structural cilia proteins, are reported to cause skeletal ciliopathies. Here, we summarize genetic and phenotypic features of 34 affected individuals from 29 families with skeletal ciliopathies. Molecular diagnostic testing was performed using massively parallel sequencing (MPS) in combination with copy number variant (CNV) analyses and in silico filtering for variants in known skeletal ciliopathy genes. We identified biallelic disease-causing variants in seven genes: DYNC2H1, KIAA0753, WDR19, C2CD3, TTC21B, EVC, and EVC2. Four variants located in non-canonical splice sites of DYNC2H1, EVC, and KIAA0753 led to aberrant splicing that was shown by sequencing of cDNA. Furthermore, CNV analyses showed an intragenic deletion of DYNC2H1 in one individual and a 6.7 Mb de novo deletion on chromosome 1q24q25 in another. In five unsolved cases, MPS was performed in family setting. In one proband we identified a de novo variant in PRKACA and in another we found a homozygous intragenic deletion of IFT74, removing the first coding exon and leading to expression of a shorter message predicted to result in loss of 40 amino acids at the N-terminus. These findings establish IFT74 as a new skeletal ciliopathy gene. In conclusion, combined single nucleotide variant, CNV and cDNA analyses lead to a high yield of genetic diagnoses (90%) in a cohort of patients with skeletal ciliopathies.

Clinically relevant variants in a large cohort of Indian patients with Marfan syndrome and related disorders identified by next-generation sequencing.

Marfan syndrome and related disorders are a group of heritable connective tissue disorders and share many clinical features that involve cardiovascular, skeletal, craniofacial, ocular, and cutaneous abnormalities. The majority of affected individuals have aortopathies associated with early mortality and morbidity. Implementation of targeted gene panel next-generation sequencing in these individuals is a powerful tool to obtain a genetic diagnosis. Here, we report on clinical and genetic spectrum of 53 families from India with a total of 83 patients who had a clinical diagnosis suggestive of Marfan syndrome or related disorders. We obtained a molecular diagnosis in 45/53 (85%) index patients, in which 36/53 (68%) had rare variants in FBN1 (Marfan syndrome; 63 patients in total), seven (13.3%) in TGFBR1/TGFBR2 (Loeys-Dietz syndrome; nine patients in total) and two patients (3.7%) in SKI (Shprintzen-Goldberg syndrome). 21 of 41 rare variants (51.2%) were novel. We did not detect a disease-associated variant in 8 (15%) index patients, and none of them met the Ghent Marfan diagnostic criteria. We found the homozygous FBN1 variant p.(Arg954His) in a boy with typical features of Marfan syndrome. Our study is the first reporting on the spectrum of variants in FBN1, TGFBR1, TGFBR2, and SKI in Indian individuals.

Roberts syndrome in an Indian patient with humeroradial synostosis, congenital elbow contractures and a novel homozygous splice variant in ESCO2.

Roberts syndrome (also known as Roberts-SC phocomelia syndrome) is an autosomal recessive developmental disorder, characterized by pre- and postnatal growth retardation, limb malformations including bilateral symmetric tetraphocomelia or mesomelia, and craniofacial dysmorphism. Biallelic loss-of-function variants in ESCO2, which codes for establishment of sister chromatid cohesion N-acetyltransferase 2, cause Roberts syndrome. Phenotypic spectrum among patients is broad, challenging clinical diagnosis in mildly affected individuals. Here we report a 3-year-old boy with a mild phenotype of Roberts syndrome with bilateral elbow contractures, humeroradial synostosis, mild lower limb disparity, and facial dysmorphism. Trio whole-exome sequencing identified the novel biallelic splice variant c.1673+1G>A in ESCO2 in the patient. Aberrant ESCO2 pre-mRNA splicing, reduced relative ESCO2 mRNA amount, and characteristic cytogenetic defects, such as premature centromere separation, heterochromatin repulsion, and chromosome breaks, in patient cells strongly supported pathogenicity of the ESCO2 variant affecting one of the highly conserved guanine-thymine dinucleotide of the donor splice site. Our case highlights the difficulty in establishing a clinical diagnosis in individuals with minor clinical features of Roberts syndrome and normal intellectual and social development. However, next-generation sequencing tools allow for molecular diagnosis in cases presenting with mild developmental defects.

Mutations in MYLPF Cause a Novel Segmental Amyoplasia that Manifests as Distal Arthrogryposis.

We identified ten persons in six consanguineous families with distal arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T [p.Cys157Phe] or c.469T>C [p.Cys157Arg]) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A (p.Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly inherited DA, a c.98C>T (p.Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

Digital clubbing as the predominant manifestation of hypertrophic osteoarthropathy caused by pathogenic variants in HPGD in three Indian families.

15-Hydroxyprostaglandin dehydrogenase is NAD-dependent catalytic enzyme involved in prostaglandin biosynthesis pathway encoded by HPGD. The pathogenic variations in HPGD cause primary hypertrophic osteoarthropathy (PHO). The objective of the present study is to identify the genetic basis in patients with digital clubbing due to PHO. We performed detailed clinical and radiographic evaluation and exome sequencing in patients from three unrelated Indian families with PHO. Exome sequencing revealed two novel, c.34G>A (p.Gly12Ser) and c.313C>T (p.Gln105*) and a known variant, c.418G>C (p.Ala140Pro) in HPGD. Herein, we add three Indian families to HPGD mutation spectrum and review the literature on variants in this gene.

Biallelic c.1263dupC in DOK7 results in fetal akinesia deformation sequence.

Fetal akinesia deformation sequence (FADS) is a clinically and genetically heterogeneous condition. Pathogenic variants in DOK7 are known to cause myasthenic syndrome, congenital, 10 (MIM#254300) and, rarely (reported in a single family) lethal FADS. Herein, we describe a biallelic variant c.1263dupC in DOK7, known to cause congenital myasthenic syndrome 10, causing lethal FADS in a consanguineous family. The present report illustrates wide phenotypic variability caused by biallelic pathogenic variants in DOK7. We also describe the second family with FADS due to pathogenic variants in DOK7.

Meckel syndrome: Clinical and mutation profile in six fetuses.

Meckel syndrome (MKS) is a perinatally lethal, genetically heterogeneous, autosomal recessive condition caused by defective primary cilium formation leading to polydactyly, multiple cysts in kidneys and malformations of nervous system. We performed exome sequencing in six fetuses from six unrelated families with MKS. We identified seven novel variants in B9D2, TNXDC15, CC2D2A, CEP290 and TMEM67. We describe the second family with MKS due to a homozygous variant in B9D2 and fifth family with bi-allelic variant in TXNDC15. Our data validates the causation of MKS by pathogenic variation in B9D2 and TXNDC15 and also adds novel variants in CC2D2A, CEP290 and TMEM67 to the literature.

Cornelia de Lange syndrome in diverse populations.

Cornelia de Lange syndrome (CdLS) is a dominant multisystemic malformation syndrome due to mutations in five genes-NIPBL, SMC1A, HDAC8, SMC3, and RAD21. The characteristic facial dysmorphisms include microcephaly, arched eyebrows, synophrys, short nose with depressed bridge and anteverted nares, long philtrum, thin lips, micrognathia, and hypertrichosis. Most affected individuals have intellectual disability, growth deficiency, and upper limb anomalies. This study looked at individuals from diverse populations with both clinical and molecularly confirmed diagnoses of CdLS by facial analysis technology. Clinical data and images from 246 individuals with CdLS were obtained from 15 countries. This cohort included 49% female patients and ages ranged from infancy to 37 years. Individuals were grouped into ancestry categories of African descent, Asian, Latin American, Middle Eastern, and Caucasian. Across these populations, 14 features showed a statistically significant difference. The most common facial features found in all ancestry groups included synophrys, short nose with anteverted nares, and a long philtrum with thin vermillion of the upper lip. Using facial analysis technology we compared 246 individuals with CdLS to 246 gender/age matched controls and found that sensitivity was equal or greater than 95% for all groups. Specificity was equal or greater than 91%. In conclusion, we present consistent clinical findings from global populations with CdLS while demonstrating how facial analysis technology can be a tool to support accurate diagnoses in the clinical setting. This work, along with prior studies in this arena, will assist in earlier detection, recognition, and treatment of CdLS worldwide.

Novel pathogenic variants in GBE1 causing fetal akinesia deformation sequence and severe neuromuscular form of glycogen storage disease type IV.

Glycogen storage disease IV (GSD IV), caused by a defect in GBE1, is a clinically heterogeneous disorder. A classical hepatic form and a neuromuscular form have been described. The severe neuromuscular form presents as a fetal akinesia deformation sequence or a congenital subtype. We ascertained three unrelated families with fetuses/neonates who presented with fetal akinesia deformation sequence to our clinic for genetic counseling. We performed a detailed clinical evaluation, exome sequencing, and histopathology examination of two fetuses and two neonates from three unrelated families presenting with these perinatally lethal neuromuscular forms of GSD IV. Exome sequencing in the affected fetuses/neonates identified four novel pathogenic variants (c.1459G>T, c.144-1G>A, c.1680C>G, and c.1843G>C) in GBE1 (NM_000158). Histopathology examination of tissues from the affected fetuses/neonate was consistent with the diagnosis. Here, we add three more families with the severe perinatally lethal neuromuscular forms of GSD IV to the GBE1 mutation spectrum.