ATP6V0C variants impair V-ATPase function causing a neurodevelopmental disorder often associated with epilepsy.

The vacuolar H+-ATPase is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the vacuolar H+-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modelling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased vacuolar H+-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behaviour, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder and provides insight into disease mechanisms.

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals.

Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.

Enzymatic activity of methionine adenosyltransferase variants identified in patients with persistent hypermethioninemia.

Methionine adenosyltransferases (MAT's) are central enzymes in living organisms that have been conserved with a high degree of homology among species. In the liver, MAT I and III, tetrameric and dimeric isoforms of the same catalytic subunit encoded by the gene MAT1A, account for the predominant portion of total body synthesis of S-adenosylmethionine (SAM), a versatile sulfonium ion-containing molecule involved in a variety of vital metabolic reactions and in the control of hepatocyte proliferation and differentiation. During the past 15years 28 MAT1A mutations have been described in patients with elevated plasma methionines, total homocysteines at most only moderately elevated, and normal levels of tyrosine and other aminoacids. In this study we describe functional analyses that determine the MAT and tripolyphosphatase (PPPase) activities of 18 MAT1A variants, six of them novel, and none of them previously assayed for activity. With the exception of G69S and Y92H, all recombinant proteins showed impairment (usually severe) of MAT activity. Tripolyphosphate (PPPi) hydrolysis was decreased only in some mutant proteins but, when it was decreased MAT activity was always also impaired.

Increased risk for developmental delay in Saethre-Chotzen syndrome is associated with TWIST deletions: an improved strategy for TWIST mutation screening.

The majority of patients with Saethre-Chotzen syndrome have mutations in the TWIST gene, which codes for a basic helix-loop-helix transcription factor. Of the genetic alterations identified in TWIST, nonsense mutations, frameshifts secondary to small deletions or insertions, and large deletions implicate haploinsufficiency as the pathogenic mechanism. We identified three novel intragenic mutations and six deletions in our patients by using a new strategy to screen for TWIST mutations. We used polymerase chain reaction (PCR) amplification with subsequent sequencing to identify point mutations and small insertions or deletions in the coding region, and real-time PCR-based gene dosage analysis to identify large deletions encompassing the gene, with confirmation by microsatellite and fluorescence in situ hybridization (FISH) analyses. The size of the deletions can also be analyzed by using the gene dosage assay with "PCR walking" across the critical region. In 55 patients with features of Saethre-Chotzen syndrome, 11% were detected to have deletions by real-time gene dosage analysis. Two patients had a translocation or inversion at least 260 kb 3' of the gene, suggesting they had position-effect mutations. Of the 37 patients with classic features of Saethre-Chotzen syndrome, the overall detection rate for TWIST mutations was 68%. The risk for developmental delay in patients with deletions involving the TWIST gene is approximately 90% or eight times more common than in patients with intragenic mutations.