Using a new analytic approach for genotyping and phenotyping chromosome 9p deletion syndrome.

Using a new analytic method ("unique non-overlapping region" (UNOR) analysis), we characterized the genotypes and phenotypes of a large cohort of individuals diagnosed with chromosome 9p deletion syndrome (9PMS) and defined critical genomic regions. We extracted phenotypic information from 48 individuals with 9PMS from medical records and used a guided interview with caregivers to clarify ambiguities. Using high-resolution whole-genome sequencing for breakpoint definition, we aligned deletions and drew virtual breakpoints to obtain UNORs associated with phenotypic characteristics. We next extracted genotype and phenotype data for 57 individuals identified from a systematic review of the 9PMS literature and analyzed these as above. Common phenotypic features included developmental delay/intellectual disability, dysmorphic features, hypotonia, genital defects in XY individuals, psychiatric diagnoses, chronic constipation, atopic disease, vision problems, autism spectrum disorder, gastroesophageal reflux disease, trigonocephaly, congenital heart disease, and neonatal hypoglycemia. Our approach confirmed previous literature reports of an association of FREM1 with trigonocephaly and suggested a possible modifier element for this phenotype. In conclusion, the UNOR approach delineated phenotypic characteristics for 9PMS and confirmed the critical role of FREM1 and a possible long-distance regulatory element in pathogenesis of trigonocephaly that will need to be replicated in future studies.

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features.

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

De novo missense variants in exon 9 of SEPHS1 cause a neurodevelopmental condition with developmental delay, poor growth, hypotonia, and dysmorphic features.

Selenophosphate synthetase (SEPHS) plays an essential role in selenium metabolism. Two mammalian SEPHS paralogues, SEPHS1 and SEPHS2, share high sequence identity and structural homology with SEPHS. Here, we report nine individuals from eight families with developmental delay, growth and feeding problems, hypotonia, and dysmorphic features, all with heterozygous missense variants in SEPHS1. Eight of these individuals had a recurrent variant at amino acid position 371 of SEPHS1 (p.Arg371Trp, p.Arg371Gln, and p.Arg371Gly); seven of these variants were known to be de novo. Structural modeling and biochemical assays were used to understand the effect of these variants on SEPHS1 function. We found that a variant at residue Trp352 results in local structural changes of the C-terminal region of SEPHS1 that decrease the overall thermal stability of the enzyme. In contrast, variants of a solvent-exposed residue Arg371 do not impact enzyme stability and folding but could modulate direct protein-protein interactions of SEPSH1 with cellular factors in promoting cell proliferation and development. In neuronal SH-SY5Y cells, we assessed the impact of SEPHS1 variants on cell proliferation and ROS production and investigated the mRNA expression levels of genes encoding stress-related selenoproteins. Our findings provided evidence that the identified SEPHS1 variants enhance cell proliferation by modulating ROS homeostasis. Our study supports the hypothesis that SEPHS1 plays a critical role during human development and provides a basis for further investigation into the molecular mechanisms employed by SEPHS1. Furthermore, our data suggest that variants in SEPHS1 are associated with a neurodevelopmental disorder.

PTPN4 germline variants result in aberrant neurodevelopment and growth.

Protein-tyrosine phosphatases (PTPs) are pleomorphic regulators of eukaryotic cellular responses to extracellular signals that function by modulating the phosphotyrosine of specific proteins. A handful of PTPs have been implicated in germline and somatic human disease. Using exome sequencing, we identified missense and truncating variants in PTPN4 in six unrelated individuals with varying degrees of intellectual disability or developmental delay. The variants occurred de novo in all five subjects in whom segregation analysis was possible. Recurring features include postnatal growth deficiency or excess, seizures, and, less commonly, structural CNS, heart, or skeletal anomalies. PTPN4 is a widely expressed protein tyrosine phosphatase that regulates neuronal cell homeostasis by protecting neurons against apoptosis. We suggest that pathogenic variants in PTPN4 confer risk for growth and cognitive abnormalities in humans.

Phenotypic expansion of CACNA1C-associated disorders to include isolated neurological manifestations.

PURPOSE: CACNA1C encodes the alpha-1-subunit of a voltage-dependent L-type calcium channel expressed in human heart and brain. Heterozygous variants in CACNA1C have previously been reported in association with Timothy syndrome and long QT syndrome. Several case reports have suggested that CACNA1C variation may also be associated with a primarily neurological phenotype. METHODS: We describe 25 individuals from 22 families with heterozygous variants in CACNA1C, who present with predominantly neurological manifestations. RESULTS: Fourteen individuals have de novo, nontruncating variants and present variably with developmental delays, intellectual disability, autism, hypotonia, ataxia, and epilepsy. Functional studies of a subgroup of missense variants via patch clamp experiments demonstrated differential effects on channel function in vitro, including loss of function (p.Leu1408Val), neutral effect (p.Leu614Arg), and gain of function (p.Leu657Phe, p.Leu614Pro). The remaining 11 individuals from eight families have truncating variants in CACNA1C. The majority of these individuals have expressive language deficits, and half have autism. CONCLUSION: We expand the phenotype associated with CACNA1C variants to include neurodevelopmental abnormalities and epilepsy, in the absence of classic features of Timothy syndrome or long QT syndrome.

De novo variants of NR4A2 are associated with neurodevelopmental disorder and epilepsy.

PURPOSE: This study characterizes the clinical and genetic features of nine unrelated patients with de novo variants in the NR4A2 gene. METHODS: Variants were identified and de novo origins were confirmed through trio exome sequencing in all but one patient. Targeted RNA sequencing was performed for one variant to confirm its splicing effect. Independent discoveries were shared through GeneMatcher. RESULTS: Missense and loss-of-function variants in NR4A2 were identified in patients from eight unrelated families. One patient carried a larger deletion including adjacent genes. The cases presented with developmental delay, hypotonia (six cases), and epilepsy (six cases). De novo status was confirmed for eight patients. One variant was demonstrated to affect splicing and result in expression of abnormal transcripts likely subject to nonsense-mediated decay. CONCLUSION: Our study underscores the importance of NR4A2 as a disease gene for neurodevelopmental disorders and epilepsy. The identified variants are likely causative of the seizures and additional developmental phenotypes in these patients.

Mutations in CUL4B, which encodes a ubiquitin E3 ligase subunit, cause an X-linked mental retardation syndrome associated with aggressive outbursts, seizures, relative macrocephaly, central obesity, hypogonadism, pes cavus, and tremor.

We have identified three truncating, two splice-site, and three missense variants at conserved amino acids in the CUL4B gene on Xq24 in 8 of 250 families with X-linked mental retardation (XLMR). During affected subjects' adolescence, a syndrome emerged with delayed puberty, hypogonadism, relative macrocephaly, moderate short stature, central obesity, unprovoked aggressive outbursts, fine intention tremor, pes cavus, and abnormalities of the toes. This syndrome was first described by Cazebas et al., in a family that was included in our study and that carried a CUL4B missense variant. CUL4B is a ubiquitin E3 ligase subunit implicated in the regulation of several biological processes, and CUL4B is the first XLMR gene that encodes an E3 ubiquitin ligase. The relatively high frequency of CUL4B mutations in this series indicates that it is one of the most commonly mutated genes underlying XLMR and suggests that its introduction into clinical diagnostics should be a high priority.

Sialic acid storage disease of the Salla phenotype in American monozygous twin female sibs.

Salla disease, one of three disease phenotypes that manifest increased urinary excretion of unconjugated sialic acid, is an autosomal recessive condition caused by a mutation in SLC17A5. This gene encodes sialin, a lysosomal membrane transporter for sialic acid. Salla disease is rare outside of individuals of Finnish ancestry. In this report we describe the disorder in non-Finnish monozygous twin siblings, the first reported American cases of Salla disease.