mTORC1 functional assay reveals SZT2 loss-of-function variants and a founder in-frame deletion.

Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.

Case report of homozygous deletion involving the first coding exons of GCNT2 isoforms A and B and part of the upstream region of TFAP2A in congenital cataract.

BACKGROUND: Congenital cataracts affect 3-6 per 10,000 live births and represent one of the leading causes of blindness in children. Congenital cataracts have a strong genetic component with high heterogeneity and variability. CASE PRESENTATION: Analysis of whole exome sequencing data in a patient affected with congenital cataracts identified a pathogenic deletion which was further defined by other techniques. A ~98-kb homozygous deletion of 6p24.3 involving the first three exons (two non-coding and one coding) of GCNT2 isoform A, the first exon (coding) of GCNT2 isoform B, and part of the intergenic region between GCNT2 and TFAP2A was identified in the patient and her brother while both parents were found to be heterozygous carriers of the deletion. The exact breakpoints were identified and revealed the presence of Alu elements at both sides of the deletion, thus indicating Alu-mediated non-homologous end-joining as the most plausible mechanism for this rearrangement. Recessive mutations in GCNT2 are known to cause an adult i blood group phenotype with congenital cataracts in some cases. The GCNT2 gene has three differentially expressed transcripts, with GCNT2B being the only isoform associated with lens function and GCNT2C being the only isoform expressed in red blood cells based on earlier studies; previously reported mutations/deletions have either affected all three isoforms (causing blood group and cataract phenotype) or the C isoform only (causing blood group phenotype only). Dominant mutations in TFAP2A are associated with syndromic anophthalmia/microphthalmia and other ocular phenotypes as part of Branchio-Ocular-Facial-Syndrome (BOFS). While the patients do not fit a diagnosis of BOFS, one sibling demonstrates mild overlap with the phenotypic spectrum, and therefore an effect of this deletion on the function of TFAP2A cannot be ruled out. CONCLUSIONS: To the best of our knowledge, this is the first case reported in which disruption of the GCNT2 gene does not involve the C isoform. The congenital cataracts phenotype in the affected patients is consistent with the previously defined isoform-specific roles of this gene. The GCNT2-TFAP2A region may be prone to rearrangements through Alu-mediated non-homologous end-joining.

8q21.11 microdeletion in two patients with syndromic peters anomaly.

Peters anomaly is a form of anterior segment dysgenesis characterized by central ocular opacity and corneo-lenticular adhesions. Isolated and syndromic Peters anomaly can be observed and demonstrate significant genetic heterogeneity. We report the identification of overlapping 8q21.11 deletions in two patients with syndromic Peters anomaly via whole exome sequencing and chromosomal microarray analyses. Microdeletions of 8q21.11 were recently reported in 10 patients with highly variable phenotypes involving craniofacial features, ptosis, intellectual disability, abnormalities of the hands/feet and other defects; sclerocornea and/or microphthalmia were reported in three cases. The two additional cases presented in this report expand the phenotypic spectrum of 8q21.11 microdeletions to include Peters anomaly (seen in both patients) and persistent primary dentition (seen in one patient with a larger deletion). The two novel deletions include the ZFHX4 and PEX2 genes, which were also affected in all three previous cases involving ocular anomalies. Screening of the remaining alleles of ZFHX4 and PEX2 did not identify any additional likely pathogenic variants in either patient, suggesting a dominant mechanism (haploinsufficiency) for the identified deletion. This report provides further insight into the phenotypes associated with 8q21.11 deletions and, for the first time, reports Peters anomaly as an additional ocular feature; screening for copy number variations of the 8q21.11 region should be considered in patients with Peters anomaly and related syndromic features. © 2016 Wiley Periodicals, Inc.

Whole exome sequence analysis of Peters anomaly.

Peters anomaly is a rare form of anterior segment ocular dysgenesis, which can also be associated with additional systemic defects. At this time, the majority of cases of Peters anomaly lack a genetic diagnosis. We performed whole exome sequencing of 27 patients with syndromic or isolated Peters anomaly to search for pathogenic mutations in currently known ocular genes. Among the eight previously recognized Peters anomaly genes, we identified a de novo missense mutation in PAX6, c.155G>A, p.(Cys52Tyr), in one patient. Analysis of 691 additional genes currently associated with a different ocular phenotype identified a heterozygous splicing mutation c.1025+2T>A in TFAP2A, a de novo heterozygous nonsense mutation c.715C>T, p.(Gln239*) in HCCS, a hemizygous mutation c.385G>A, p.(Glu129Lys) in NDP, a hemizygous mutation c.3446C>T, p.(Pro1149Leu) in FLNA, and compound heterozygous mutations c.1422T>A, p.(Tyr474*) and c.2544G>A, p.(Met848Ile) in SLC4A11; all mutations, except for the FLNA and SLC4A11 c.2544G>A alleles, are novel. This is the first study to use whole exome sequencing to discern the genetic etiology of a large cohort of patients with syndromic or isolated Peters anomaly. We report five new genes associated with this condition and suggest screening of TFAP2A and FLNA in patients with Peters anomaly and relevant syndromic features and HCCS, NDP and SLC4A11 in patients with isolated Peters anomaly.

A familial, telomere-to-telomere reference for human de novo mutation and recombination from a four-generation pedigree.

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess de novo mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 de novo single-nucleotide variants (SNVs), 7.4 non-tandem repeat indels, 79.6 de novo indels or structural variants (SVs) originating from tandem repeats, 7.7 centromeric de novo SVs and SNVs, and 12.4 de novo Y chromosome events per generation. STRs and VNTRs are the most mutable with 32 loci exhibiting recurrent mutation through the generations. We accurately assemble 288 centromeres and six Y chromosomes across the generations, documenting de novo SVs, and demonstrate that the DNM rate varies by an order of magnitude depending on repeat content, length, and sequence identity. We show a strong paternal bias (75-81%) for all forms of germline DNM, yet we estimate that 17% of de novo SNVs are postzygotic in origin with no paternal bias. We place all this variation in the context of a high-resolution recombination map (~3.5 kbp breakpoint resolution). We observe a strong maternal recombination bias (1.36 maternal:paternal ratio) with a consistent reduction in the number of crossovers with increasing paternal (r=0.85) and maternal (r=0.65) age. However, we observe no correlation between meiotic crossover locations and de novo SVs, arguing against non-allelic homologous recombination as a predominant mechanism. The use of multiple orthogonal technologies, near-telomere-to-telomere phased genome assemblies, and a multi-generation family to assess transmission has created the most comprehensive, publicly available "truth set" of all classes of genomic variants. The resource can be used to test and benchmark new algorithms and technologies to understand the most fundamental processes underlying human genetic variation.

Long-read sequencing and profiling of RNA-binding proteins reveals the pathogenic mechanism of aberrant splicing of an SCN1A poison exon in epilepsy.

Pathogenic loss-of-function SCN1A variants cause a spectrum of seizure disorders. We previously identified variants in individuals with SCN1A -related epilepsy that fall in or near a poison exon (PE) in SCN1A intron 20 (20N). We hypothesized these variants lead to increased PE inclusion, which introduces a premature stop codon, and, therefore, reduced abundance of the full-length SCN1A transcript and Na v 1.1 protein. We used a splicing reporter assay to interrogate PE inclusion in HEK293T cells. In addition, we used patient-specific induced pluripotent stem cells (iPSCs) differentiated into neurons to quantify 20N inclusion by long and short-read sequencing and Na v 1.1 abundance by western blot. We performed RNA-antisense purification with mass spectrometry to identify RNA-binding proteins (RBPs) that could account for the aberrant PE splicing. We demonstrate that variants in/near 20N lead to increased 20N inclusion by long-read sequencing or splicing reporter assay and decreased Na v 1.1 abundance. We also identified 28 RBPs that differentially interact with variant constructs compared to wild-type, including SRSF1 and HNRNPL. We propose a model whereby 20N variants disrupt RBP binding to splicing enhancers (SRSF1) and suppressors (HNRNPL), to favor PE inclusion. Overall, we demonstrate that SCN1A 20N variants cause haploinsufficiency and SCN1A -related epilepsies. This work provides insights into the complex control of RBP-mediated PE alternative splicing, with broader implications for PE discovery and identification of pathogenic PE variants in other genetic conditions.

Neurodevelopmental and Epilepsy Phenotypes in Individuals With Missense Variants in the Voltage-Sensing and Pore Domains of KCNH5.

BACKGROUND AND OBJECTIVES: KCNH5 encodes the voltage-gated potassium channel EAG2/Kv10.2. We aimed to delineate the neurodevelopmental and epilepsy phenotypic spectrum associated with de novo KCNH5 variants. METHODS: We screened 893 individuals with developmental and epileptic encephalopathies for KCNH5 variants using targeted or exome sequencing. Additional individuals with KCNH5 variants were identified through an international collaboration. Clinical history, EEG, and imaging data were analyzed; seizure types and epilepsy syndromes were classified. We included 3 previously published individuals including additional phenotypic details. RESULTS: We report a cohort of 17 patients, including 9 with a recurrent de novo missense variant p.Arg327His, 4 with a recurrent missense variant p.Arg333His, and 4 additional novel missense variants. All variants were located in or near the functionally critical voltage-sensing or pore domains, absent in the general population, and classified as pathogenic or likely pathogenic using the American College of Medical Genetics and Genomics criteria. All individuals presented with epilepsy with a median seizure onset at 6 months. They had a wide range of seizure types, including focal and generalized seizures. Cognitive outcomes ranged from normal intellect to profound impairment. Individuals with the recurrent p.Arg333His variant had a self-limited drug-responsive focal or generalized epilepsy and normal intellect, whereas the recurrent p.Arg327His variant was associated with infantile-onset DEE. Two individuals with variants in the pore domain were more severely affected, with a neonatal-onset movement disorder, early-infantile DEE, profound disability, and childhood death. DISCUSSION: We describe a cohort of 17 individuals with pathogenic or likely pathogenic missense variants in the voltage-sensing and pore domains of Kv10.2, including 14 previously unreported individuals. We present evidence for a putative emerging genotype-phenotype correlation with a spectrum of epilepsy and cognitive outcomes. Overall, we expand the role of EAG proteins in human disease and establish KCNH5 as implicated in a spectrum of neurodevelopmental disorders and epilepsy.

A 2020 View on the Genetics of Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathies (DEEs) can be primarily attributed to genetic causes. The genetic landscape of DEEs has been largely shaped by the rise of high-throughput sequencing, which led to the discovery of new DEE-associated genes and helped identify de novo pathogenic variants. We discuss briefly the contribution of de novo variants to DEE and also focus on alternative inheritance models that contribute to DEE. First, autosomal recessive inheritance in outbred populations may have a larger contribution than previously appreciated, accounting for up to 13% of DEEs. A small subset of genes that typically harbor de novo variants have been associated with recessive inheritance, and often these individuals have more severe clinical presentations. Additionally, pathogenic variants in X-linked genes have been identified in both affected males and females, possibly due to a lack of X-chromosome inactivation skewing. Collectively, exome sequencing has resulted in a molecular diagnosis for many individuals with DEE, but this still leaves many cases unsolved. Multiple factors contribute to the missing etiology, including nonexonic variants, mosaicism, epigenetics, and oligogenic inheritance. Here, we focus on the first 2 factors. We discuss the promises and challenges of genome sequencing, which allows for a more comprehensive analysis of the genome, including interpretation of structural and noncoding variants and also yields a high number of de novo variants for interpretation. We also consider the contribution of genetic mosaicism, both what it means for a molecular diagnosis in mosaic individuals and the important implications for genetic counseling.

Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses.

Extracellular RNAs (exRNAs) can be released by numerous cell types in vitro, are often protected within vesicles, and can modify recipient cell function. To determine how the composition and cellular sources of exRNAs and the extracellular vesicles (EVs) that carry them change in vivo during tissue inflammation, we analyzed bronchoalveolar lavage fluid (BALF) from mice before and after lung allergen challenge. In the lung, extracellular microRNAs (ex-miRNAs) had a composition that was highly correlated with airway-lining epithelium. Using cell type-specific membrane tagging and single vesicle flow, we also found that 80% of detected vesicles were of epithelial origin. After the induction of allergic airway inflammation, miRNAs selectively expressed by immune cells, including miR-223 and miR-142a, increased and hematopoietic-cell-derived EVs also increased >2-fold. These data demonstrate that infiltrating immune cells release ex-miRNAs and EVs in inflamed tissues to alter the local extracellular environment.

MicroRNA regulation of type 2 innate lymphoid cell homeostasis and function in allergic inflammation.

MicroRNAs (miRNAs) exert powerful effects on immunity through coordinate regulation of multiple target genes in a wide variety of cells. Type 2 innate lymphoid cells (ILC2s) are tissue sentinel mediators of allergic inflammation. We established the physiological requirements for miRNAs in ILC2 homeostasis and immune function and compared the global miRNA repertoire of resting and activated ILC2s and T helper type 2 (TH2) cells. After exposure to the natural allergen papain, mice selectively lacking the miR-17∼92 cluster in ILC2s displayed reduced lung inflammation. Moreover, miR-17∼92-deficient ILC2s exhibited defective growth and cytokine expression in response to IL-33 and thymic stromal lymphopoietin in vitro. The miR-17∼92 cluster member miR-19a promoted IL-13 and IL-5 production and inhibited expression of several targets, including SOCS1 and A20, signaling inhibitors that limit IL-13 and IL-5 production. These findings establish miRNAs as important regulators of ILC2 biology, reveal overlapping but nonidentical miRNA-regulated gene expression networks in ILC2s and TH2 cells, and reinforce the therapeutic potential of targeting miR-19 to alleviate pathogenic allergic responses.