Mono-allelic KCNB2 variants lead to a neurodevelopmental syndrome caused by altered channel inactivation.

Ion channels mediate voltage fluxes or action potentials that are central to the functioning of excitable cells such as neurons. The KCNB family of voltage-gated potassium channels (Kv) consists of two members (KCNB1 and KCNB2) encoded by KCNB1 and KCNB2, respectively. These channels are major contributors to delayed rectifier potassium currents arising from the neuronal soma which modulate overall excitability of neurons. In this study, we identified several mono-allelic pathogenic missense variants in KCNB2, in individuals with a neurodevelopmental syndrome with epilepsy and autism in some individuals. Recurrent dysmorphisms included a broad forehead, synophrys, and digital anomalies. Additionally, we selected three variants where genetic transmission has not been assessed, from two epilepsy studies, for inclusion in our experiments. We characterized channel properties of these variants by expressing them in oocytes of Xenopus laevis and conducting cut-open oocyte voltage clamp electrophysiology. Our datasets indicate no significant change in absolute conductance and conductance-voltage relationships of most disease variants as compared to wild type (WT), when expressed either alone or co-expressed with WT-KCNB2. However, variants c.1141A>G (p.Thr381Ala) and c.641C>T (p.Thr214Met) show complete abrogation of currents when expressed alone with the former exhibiting a left shift in activation midpoint when expressed alone or with WT-KCNB2. The variants we studied, nevertheless, show collective features of increased inactivation shifted to hyperpolarized potentials. We suggest that the effects of the variants on channel inactivation result in hyper-excitability of neurons, which contributes to disease manifestations.

Functional assessment of homozygous ALDH18A1 variants reveals alterations in amino acid and antioxidant metabolism.

Mono- and bi-allelic variants in ALDH18A1 cause a spectrum of human disorders associated with cutaneous and neurological findings that overlap with both cutis laxa and spastic paraplegia. ALDH18A1 encodes the bifunctional enzyme pyrroline-5-carboxylate synthetase (P5CS) that plays a role in the de novo biosynthesis of proline and ornithine. Here we characterize a previously unreported homozygous ALDH18A1 variant (p.Thr331Pro) in four affected probands from two unrelated families, and demonstrate broad-based alterations in amino acid and antioxidant metabolism. These four patients exhibit variable developmental delay, neurological deficits and loose skin. Functional characterization of the p.Thr331Pro variant demonstrated a lack of any impact on the steady-state level of the P5CS monomer or mitochondrial localization of the enzyme, but reduced incorporation of the monomer into P5CS oligomers. Using an unlabeled NMR-based metabolomics approach in patient fibroblasts and ALDH18A1-null human embryonic kidney cells expressing the variant P5CS, we identified reduced abundance of glutamate and several metabolites derived from glutamate, including proline and glutathione. Biosynthesis of the polyamine putrescine, derived from ornithine, was also decreased in patient fibroblasts, highlighting the functional consequence on another metabolic pathway involved in antioxidant responses in the cell. RNA sequencing of patient fibroblasts revealed transcript abundance changes in several metabolic and extracellular matrix-related genes, adding further insight into pathogenic processes associated with impaired P5CS function. Together these findings shed new light on amino acid and antioxidant pathways associated with ALDH18A1-related disorders, and underscore the value of metabolomic and transcriptomic profiling to discover new pathways that impact disease pathogenesis.

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature.

PURPOSE: Pathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD. METHODS: Through international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature. RESULTS: We recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism. CONCLUSION: Pathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood.

Expanding the clinical and metabolic phenotype of DPM2 deficient congenital disorders of glycosylation.

Pathogenic alterations in the DPM2 gene have been previously described in patients with hypotonia, progressive muscle weakness, absent psychomotor development, intractable seizures, and early death. We identified biallelic DPM2 variants in a 23-year-old male with truncal hypotonia, hypertonicity, congenital heart defects, intellectual disability, and generalized muscle wasting. His clinical presentation was much less severe than that of the three previously described patients. This is the second report on this ultra-rare disorder. Here we review the characteristics of previously reported individuals with a defect in the DPM complex while expanding the clinical phenotype of DPM2-Congenital Disorders of Glycosylation. In addition, we offer further insights into the pathomechanism of DPM2-CDG disorder by introducing glycomics and lipidomics analysis.

Cohesin complex-associated holoprosencephaly.

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.

Clinical utility of the X-chromosome array.

Previous studies have limited the use of specific X-chromosome array designed platforms to the evaluation of patients with intellectual disability. In this retrospective analysis, we reviewed the clinical utility of an X-chromosome array in a variety of scenarios. We divided patients according to the indication for the test into four defined categories: (1) autism spectrum disorders and/or developmental delay and/or intellectual disability (ASDs/DD/ID) with known family history of neurocognitive disorders; (2) ASDs/DD/ID without known family history of neurocognitive disorders; (3) breakpoint definition of an abnormality detected by a different cytogenetic test; and (4) evaluation of suspected or known X-linked conditions. A total of 59 studies were ordered with 27 copy number variants detected in 25 patients (25/59 = 42%). The findings were deemed pathogenic/likely pathogenic (16/59 = 27%), benign (4/59 = 7%) or uncertain (7/59 = 12%). We place particular emphasis on the utility of this test for the diagnostic evaluation of families affected with X-linked conditions and how it compares to whole genome arrays in this setting. In conclusion, the X-chromosome array frequently detects genomic alterations of the X chromosome and it has advantages when evaluating some specific X-linked conditions. However, careful interpretation and correlation with clinical findings is needed to determine the significance of such changes. When the X-chromosome array was used to confirm a suspected X-linked condition, it had a yield of 63% (12/19) and was useful in the evaluation and risk assessment of patients and families.

Unique cerebrovascular anomalies in Noonan syndrome with RAF1 mutation.

Noonan syndrome is a common autosomal dominant neurodevelopmental disorder caused by gain-of-function germline mutations affecting components of the Ras-MAPK pathway. The authors present the case of a 6-year-old male with Noonan syndrome, Chiari malformation type I, shunted benign external hydrocephalus in infancy, and unique cerebrovascular changes. A de novo heterozygous change in the RAF1 gene was identified. The patient underwent brain magnetic resonance imaging, computed tomography angiography, and magnetic resonance angiography to further clarify the nature of his abnormal brain vasculature. The authors compared his findings to the few cases of Noonan syndrome reported with cerebrovascular pathology.