A neurodevelopmental disorder associated with a loss-of-function missense mutation in RAB35.

Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.

USP27X variants underlying X-linked intellectual disability disrupt protein function via distinct mechanisms.

Neurodevelopmental disorders with intellectual disability (ND/ID) are a heterogeneous group of diseases driving lifelong deficits in cognition and behavior with no definitive cure. X-linked intellectual disability disorder 105 (XLID105, #300984; OMIM) is a ND/ID driven by hemizygous variants in the USP27X gene encoding a protein deubiquitylase with a role in cell proliferation and neural development. Currently, only four genetically diagnosed individuals from two unrelated families have been described with limited clinical data. Furthermore, the mechanisms underlying the disorder are unknown. Here, we report 10 new XLID105 individuals from nine families and determine the impact of gene variants on USP27X protein function. Using a combination of clinical genetics, bioinformatics, biochemical, and cell biology approaches, we determined that XLID105 variants alter USP27X protein biology via distinct mechanisms including changes in developmentally relevant protein-protein interactions and deubiquitylating activity. Our data better define the phenotypic spectrum of XLID105 and suggest that XLID105 is driven by USP27X functional disruption. Understanding the pathogenic mechanisms of XLID105 variants will provide molecular insight into USP27X biology and may create the potential for therapy development.

Unbiased phenotype and genotype matching maximizes gene discovery and diagnostic yield.

PURPOSE: Widespread application of next-generation sequencing, combined with data exchange platforms, has provided molecular diagnoses for countless families. To maximize diagnostic yield, we implemented an unbiased semi-automated genematching algorithm based on genotype and phenotype matching. METHODS: Rare homozygous variants identified in 2 or more affected individuals, but not in healthy individuals, were extracted from our local database of ∼12,000 exomes. Phenotype similarity scores (PSS), based on human phenotype ontology terms, were assigned to each pair of individuals matched at the genotype level using HPOsim. RESULTS: 33,792 genotype-matched pairs were discovered, representing variants in 7567 unique genes. There was an enrichment of PSS ≥0.1 among pathogenic/likely pathogenic variant-level pairs (94.3% in pathogenic/likely pathogenic variant-level matches vs 34.75% in all matches). We highlighted founder or region-specific variants as an internal positive control and proceeded to identify candidate disease genes. Variant-level matches were particularly helpful in cases involving inframe indels and splice region variants beyond the canonical splice sites, which may otherwise have been disregarded, allowing for detection of candidate disease genes, such as KAT2A, RPAIN, and LAMP3. CONCLUSION: Semi-automated genotype matching combined with PSS is a powerful tool to resolve variants of uncertain significance and to identify candidate disease genes.

Complex rearrangement in TBC1D4 in an individual with diabetes due to severe insulin resistance syndrome.

Severe insulin resistance syndromes result from primary insulin signaling defects, adipose tissue abnormalities or other complex syndromes. Mutations in TBC1D4 lead to partial insulin signaling defects, characterized mainly by postprandial insulin resistance. We describe an individual with severe insulin-resistant diabetes unresponsive to multiple therapies, in whom exome and genome analyses identified a complex rearrangement in TBC1D4. The rearrangement was of the pattern DUP-TRP/INV-DUP, with mutational signatures suggestive of replicative repair and Alu-Alu recombination as the underlying mechanisms. TBC1D4 encodes the TBC1D4/AS160 RabGTPase activating protein (RabGAP) involved in the translocation of glucose transporter 4 (GLUT4) from the cytosol to the cell membrane. Although the precise functional mechanism underlying insulin resistance in the proband is yet to be determined, this case provides further support for the link between TBC1D4 and hereditary insulin-resistant diabetes.

Bi-allelic loss-of-function variants in WBP4, encoding a spliceosome protein, result in a variable neurodevelopmental syndrome.

Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WW domain-binding protein 4 (WBP4) is part of the early spliceosomal complex and has not been previously associated with human pathologies in the Online Mendelian Inheritance in Man (OMIM) database. Through GeneMatcher, we identified ten individuals from eight families with a severe neurodevelopmental syndrome featuring variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal, and gastrointestinal abnormalities. Genetic analysis revealed five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated a complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including in genes associated with abnormalities of the nervous system, potentially underlying the phenotypes of the probands. We conclude that bi-allelic variants in WBP4 cause a developmental disorder with variable presentations, adding to the growing list of human spliceosomopathies.

Prenatal diagnosis of lanosterol synthase deficiency: Fetal ultrasound findings as a window on family genetics.

Cholesterol is essential in the brain from the earliest stages of embryonic development. Disruption of cholesterol synthesis pathways that leads to cholesterol deficiency underlies a few syndromes, including desmosterolosis and Smith-Lemli-Opitz syndrome. In both syndromes, brain anomalies can occur. The LSS gene encodes lanosterol synthase (LSS), an important enzyme in the cholesterol biosynthesis pathway. Biallelic pathogenic variants in this gene cause alopecia-intellectual disability type 4 syndrome (APMR4, MIM 618840), a rare autosomal recessive disorder. Here, we describe two new LSS variants (c.1016C > T; p. Ser339Leu and c.1522G > C; p. Gly508Arg) found in a compound heterozygous fetus diagnosed prenatally with brain abnormalities by ultrasound scanning. Two of his siblings from the same parents also harbored these variants. Both siblings had alopecia, mild intellectual disability, autism spectrum disorder, and cataracts. To the best of our knowledge, this case represents the first prenatal diagnosis of APMR4 first suspected by ultrasound. In addition, the phenotypic features of the siblings are extensive compared with those described in previous reports and include abnormal corpus callosum, cataracts, alopecia, and developmental delay.

Neurodevelopmental disorder mutations in the purine biosynthetic enzyme IMPDH2 disrupt its allosteric regulation.

Inosine 5' monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report the identification of two additional missense variants in IMPDH2 from affected individuals and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation.

[NEXT-GENERATION SEQUENCING PERFORMED IN PATIENTS RAISING THE SUSPICION OF AN INBORN ERROR OF METABOLISM UNCOVERED A HOMOZYGOUS VARIANT IN YARS1 ALLOWING A NOVEL THERAPEUTIC TRIAL].

INTRODUCTION: Inborn-Errors of Metabolism (IEM) are genetic disorders resulting from mutations in genes encoding proteins involved in biochemical-metabolic pathways. However, some IEMs lack specific biochemical markers. Early incorporation of next-generation-sequencing (NGS) including whole exome sequencing (WES) into the diagnostic algorithm of IEMs herein provided, increases diagnostic accuracy, permits genetic counseling and improves therapeutic options. This is exemplified by diseases affecting aminoacyl-tRNA synthetases (ARSs), enzymes involved in protein translation. Recent studies showed that supplementing amino-acids to cell-culture and patients with ARSs deficiencies resulted in improvement of biochemical and clinical parameters, respectively.

Biallelic loss of function variants in WBP4, encoding a spliceosome protein, result in a variable neurodevelopmental delay syndrome.

Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WBP4 (WW Domain Binding Protein 4) is part of the early spliceosomal complex, and was not described before in the context of human pathologies. Ascertained through GeneMatcher we identified eleven patients from eight families, with a severe neurodevelopmental syndrome with variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal and gastrointestinal abnormalities. Genetic analysis revealed overall five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including enrichment for abnormalities of the nervous system and musculoskeletal system genes, suggesting that the overlapping differentially spliced genes are related to the common phenotypes of the probands. We conclude that biallelic variants in WBP4 cause a spliceosomopathy. Further functional studies are called for better understanding of the mechanism of pathogenicity.

A recurrent de novo variant in NUSAP1 escapes nonsense-mediated decay and leads to microcephaly, epilepsy, and developmental delay.

NUSAP1 encodes a cell cycle-dependent protein with key roles in mitotic progression, spindle formation, and microtubule stability. Both over- and under-expression of NUSAP1 lead to dysregulation of mitosis and impaired cell proliferation. Through exome sequencing and Matchmaker Exchange, we identified two unrelated individuals with the same recurrent, de novo heterozygous variant (NM_016359.5 c.1209C > A; p.(Tyr403Ter)) in NUSAP1. Both individuals had microcephaly, severe developmental delay, brain abnormalities, and seizures. The gene is predicted to be tolerant of heterozygous loss-of-function mutations, and we show that the mutant transcript escapes nonsense mediated decay, suggesting that the mechanism is likely dominant-negative or toxic gain of function. Single-cell RNA-sequencing of an affected individual's post-mortem brain tissue indicated that the NUSAP1 mutant brain contains all main cell lineages, and that the microcephaly could not be attributed to loss of a specific cell type. We hypothesize that pathogenic variants in NUSAP1 lead to microcephaly possibly by an underlying defect in neural progenitor cells.

Exome sequencing for structurally normal fetuses-yields and ethical issues.

The yield of chromosomal microarray analysis (CMA) is well established in structurally normal fetuses (0.4-1.4%). We aimed to determine the incremental yield of exome sequencing (ES) in this population. From February 2017 to April 2022, 1,526 fetuses were subjected to ES; 482 of them were structurally normal (31.6%). Only pathogenic and likely pathogenic (P/LP) variants, per the American College of Medical Genetics and Genomics (ACMG) classification, were reported. Additionally, ACMG secondary findings relevant to childhood were reported. Four fetuses (4/482; 0.8%) had P/LP variants indicating a moderate to severe disease in ATP7B, NR2E3, SPRED1 and FGFR3, causing Wilson disease, Enhanced S-cone syndrome, Legius and Muenke syndromes, respectively. Two fetuses had secondary findings, in RET and DSP. Our data suggest that offering only CMA for structurally normal fetuses may provide false reassurance. Prenatal ES mandates restrictive analysis and careful management combined with pre and post-test genetic counseling.

Expanding the phenotypic spectrum of COLEC10-Related 3MC syndrome: A glimpse into COLEC10-Related 3MC syndrome in the Ashkenazi Jewish population.

Bi-allelic variants in COLEC11 and MASP1 have been associated with 3MC syndrome, a clinical entity made of up four rare autosomal recessive disorders: Carnevale, Mingarelli, Malpuech, and Michels syndromes, characterized by variable expression of facial dysmorphia, cleft lip/palate, postnatal growth deficiency, hearing loss, cognitive impairment, craniosynostosis, radioulnar synostosis, and genital and vesicorenal anomalies. More recently, bi-allelic variants in COLEC10 have been described to be associated with 3MC syndrome. Syndromic features seen in 3MC syndrome are thought to be due to disruption of the chemoattractant properties that influence neural crest cell migration. We identified nine individuals from five families of Ashkenazi Jewish descent with homozygosity of the c.311G > T (p.Gly104Val) variant in COLEC10 and phenotype consistent with 3MC syndrome. Carrier frequency was calculated among 52,278 individuals of Jewish descent. Testing revealed 400 carriers out of 39,750 individuals of Ashkenazi Jewish descent, giving a carrier frequency of 1 in 99 or 1.01%. Molecular protein modeling suggested that the p.Gly104Val substitution alters local conformation. The c.311G > T (p.Gly104Val) variant likely represents a founder variant, and homozygosity is associated with features of 3MC syndrome. 3MC syndrome should be in the differential diagnosis for individuals with short stature, radioulnar synostosis, cleft lip and cleft palate.

Clinical and Functional Study of a De Novo Variant in the PVP Motif of Kv1.1 Channel Associated with Epilepsy, Developmental Delay and Ataxia.

Mutations in the KCNA1 gene, encoding the voltage-gated potassium channel Kv1.1, have been associated with a spectrum of neurological phenotypes, including episodic ataxia type 1 and developmental and epileptic encephalopathy. We have recently identified a de novo variant in KCNA1 in the highly conserved Pro-Val-Pro motif within the pore of the Kv1.1 channel in a girl affected by early onset epilepsy, ataxia and developmental delay. Other mutations causing severe epilepsy are located in Kv1.1 pore domain. The patient was initially treated with a combination of antiepileptic drugs with limited benefit. Finally, seizures and ataxia control were achieved with lacosamide and acetazolamide. The aim of this study was to functionally characterize Kv1.1 mutant channel to provide a genotype-phenotype correlation and discuss therapeutic options for KCNA1-related epilepsy. To this aim, we transfected HEK 293 cells with Kv1.1 or P403A cDNAs and recorded potassium currents through whole-cell patch-clamp. P403A channels showed smaller potassium currents, voltage-dependent activation shifted by +30 mV towards positive potentials and slower kinetics of activation compared with Kv1.1 wild-type. Heteromeric Kv1.1+P403A channels, resembling the condition of the heterozygous patient, confirmed a loss-of-function biophysical phenotype. Overall, the functional characterization of P403A channels correlates with the clinical symptoms of the patient and supports the observation that mutations associated with severe epileptic phenotype cluster in a highly conserved stretch of residues in Kv1.1 pore domain. This study also strengthens the beneficial effect of acetazolamide and sodium channel blockers in KCNA1 channelopathies.

Combining cytogenetic and genomic technologies for deciphering challenging complex chromosomal rearrangements.

Complex chromosomal rearrangements (CCRs), a class of structural variants (SVs) involving more than two chromosome breaks, were classically thought to be extremely rare. As advanced technologies become more available, it has become apparent that CCRs are more common than formerly thought, and are a substantial cause of genetic disorders. We attempted a novel approach for solving the mechanism of challenging CCRs, which involve repetitive sequences, by precisely identifying sequence-level changes and their order. Chromosomal microarray (CMA) and FISH analyses were used for interpretation of SVs detected by whole exome sequencing (WES). Breakpoint junctions were analyzed by Nanopore sequencing, a novel long-read whole genome sequencing tool. A large deletion identified by WES, encompassing the FOXF1 enhancer, was the cause of alveolar capillary dysplasia and respiratory insufficiency, resulting in perinatal death. CMA analysis of the newborn's mother revealed two duplications encompassing the deleted region in the proband, raising our hypothesis that the deletion resulted from the mother's CCR. Breakpoint junctions of complex SVs were determined at the nucleotide level using Nanopore long-read sequencing. According to sequencing results of breakpoint junctions, the CCR in the newborn was considered the consequence of at least one double-strand break during meiosis, and reassembly of DNA fragments by intra-chromosomal homologous recombination. Our comprehensive approach, combining cytogenetics and long-read sequencing, enabled delineation of the exact breakpoints in a challenging CCR, and proposal of a mechanism in which it arises. We suggest applying our integrative approach combining technologies for deciphering future challenging CCRs, enabling risk assessment in families.

A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System.

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day-night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator's period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep-wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Bi-allelic PAGR1 variants are associated with microcephaly and a severe neurodevelopmental disorder: Genetic evidence from two families.

Exome and genome sequencing were used to identify the genetic etiology of a severe neurodevelopmental disorder in two unrelated Ashkenazi Jewish families with three affected individuals. The clinical findings included a prenatal presentation of microcephaly, polyhydramnios and clenched hands while postnatal findings included microcephaly, severe developmental delay, dysmorphism, neurologic deficits, and death in infancy. A shared rare homozygous, missense variant (c.274A > G; p.Ser92Gly, NM_024516.4) was identified in PAGR1, a gene currently not associated with a Mendelian disease. PAGR1 encodes a component of the histone methyltransferase MLL2/MLL3 complex and may function in the DNA damage response pathway. Complete knockout of the murine Pagr1a is embryonic-lethal. Given the available evidence, PAGR1 is a strong candidate gene for a novel autosomal recessive severe syndromic neurodevelopmental disorder.

Clinical presentation and analysis of genotype-phenotype correlations in patients with malignant infantile osteopetrosis.

Malignant infantile osteopetrosis (MIOP) is the autosomal recessive, severe form of osteopetrosis. This rare genetic syndrome usually presents soon after birth and is often fatal if left untreated. Early diagnosis is key for proper management but clinical presentation is diverse, and oftentimes diagnosis may be challenging. In this study, we retrospectively collected data of genetic mutations and phenotypic characteristics at the initial presentation of 81 MIOP patients and analyzed genotype-phenotype correlations. The most common genetic mutation was in the TCIRG1 gene (n = 46, 56.8%), followed by SNX10 (n = 20, 25%). Other genetic mutations included RANK (n = 7, 8.7%), CLCN7 (n = 5, 6.2%) and CA2 (n = 3, 3.7%). More than half of the patients presented with growth retardation (n = 46, 56.8%). Twenty-one of the patients were blind (26%) and thirty-seven patients had other neurological deficits (45.7%) at the time of initial presentation. Most patients presented with hematological signs of bone marrow failure including anemia (n = 69, 85.2%) and thrombocytopenia (n = 33, 40.7%). Thrombocytopenia at initial presentation was significantly more prevalent in patients with mutations in the TCIRG1 gene (p = 0.036). Other phenotypic presenting features were not found to be significantly correlated to specific gene mutations. In conclusion, the initial presentation of MIOP is variable, but some features are common such as growth retardation, visual impairment, and cytopenias. High awareness of MIOP presenting signs is essential for prompt diagnosis of this challenging disease.