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.

A common benign intronic deletion masking a pathogenic deep intronic PCCB variant - genome sequencing and RNA studies to the rescue.

Propionic acidemia (PA) is an autosomal recessive metabolic disorder caused by variants in PCCA or PCCB, both sub-units of the propionyl-CoA carboxylase (PCC) enzyme. PCC is required for the catabolism of certain amino acids and odd-chain fatty acids. In its absence, the accumulated toxic metabolites cause metabolic acidosis, neurologic symptoms, multi-organ dysfunction and possible death. The clinical presentation of PA is highly variable, with typical onset in the neonatal or early infantile period. We encountered two families, whose children were diagnosed with PA. Exome sequencing (ES) failed to identify a pathogenic variant, and we proceeded with genome sequencing (GS), demonstrating homozygosity to a deep intronic PCCB variant. RNA analysis established that this variant creates a pseudoexon with a premature stop codon. The parents are variant carriers, though three of them display pseudo-homozygosity due to a common large benign intronic deletion on the second allele. The parental presumed homozygosity merits special attention, as it masked the causative variant at first, which was resolved only by RNA studies. Arriving at a rapid diagnosis, whether biochemical or genetic, can be crucial in directing lifesaving care, concluding the diagnostic odyssey, and allowing the family prenatal testing in subsequent pregnancies. This study demonstrates the power of integrative genetic studies in reaching a diagnosis, utilizing GS and RNA analysis to overcome ES limitations and define pathogenicity. Importantly, it highlights that intronic deletions should be taken into consideration when analyzing genomic data, so that pseudo-homozygosity would not be misinterpreted as true homozygosity, and pathogenic variants will not be mislabeled as benign.

Hereditary orotic aciduria identified by newborn screening.

Introduction: Hereditary orotic aciduria is an extremely rare, autosomal recessive disease caused by deficiency of uridine monophosphate synthase. Untreated, affected individuals may develop refractory megaloblastic anemia, neurodevelopmental disabilities, and crystalluria. Newborn screening has the potential to identify and enable treatment of affected individuals before they become significantly ill. Methods: Measuring orotic acid as part of expanded newborn screening using flow injection analysis tandem mass spectrometry. Results: Since the addition of orotic acid measurement to the Israeli routine newborn screening program, 1,492,439 neonates have been screened. The screen has identified ten Muslim Arab newborns that remain asymptomatic so far, with DBS orotic acid elevated up to 10 times the upper reference limit. Urine organic acid testing confirmed the presence of orotic aciduria along with homozygous variations in the UMPS gene. Conclusion: Newborn screening measuring of orotic acid, now integrated into the routine tandem mass spectrometry panel, is capable of identifying neonates with hereditary orotic aciduria.

Addition of galactose-1-phosphate measurement enhances newborn screening for classical galactosemia.

Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.

Homozygous variant in MADD, encoding a Rab guanine nucleotide exchange factor, results in pleiotropic effects and a multisystemic disorder.

Rab proteins coordinate inter-organellar vesicle-mediated transport, facilitating intracellular communication, protein recycling, and signaling processes. Dysfunction of Rab proteins or their direct interactors leads to a wide range of diseases with diverse manifestations. We describe seven individuals from four consanguineous Arab Muslim families with an infantile-lethal syndrome, including failure to thrive (FTT), chronic diarrhea, neonatal respiratory distress, variable pituitary dysfunction, and distal arthrogryposis. Exome sequencing analysis in the independent families, followed by an internal gene-matching process using a local exome database, identified a homozygous splice-site variant in MADD (c.2816 + 1 G > A) on a common haplotype. The variant segregated with the disease in all available family members. Determination of cDNA sequence verified single exon skipping, resulting in an out-of-frame deletion. MADD encodes a Rab guanine nucleotide exchange factor (GEF), which activates RAB3 and RAB27A/27B and is thus a crucial regulator of neuromuscular junctions and endocrine secretory granule release. Moreover, MADD protects cells from caspase-mediated TNF-α-induced apoptosis. The combined roles of MADD and its downstream effectors correlate with the phenotypic spectrum of disease, and call for additional studies to confirm the pathogenic mechanism and to investigate possible therapeutic avenues through modulation of TNF-α signaling.

The role of orotic acid measurement in routine newborn screening for urea cycle disorders.

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.

Missense Mutations of the Pro65 Residue of PCGF2 Cause a Recognizable Syndrome Associated with Craniofacial, Neurological, Cardiovascular, and Skeletal Features.

PCGF2 encodes the polycomb group ring finger 2 protein, a transcriptional repressor involved in cell proliferation, differentiation, and embryogenesis. PCGF2 is a component of the polycomb repressive complex 1 (PRC1), a multiprotein complex which controls gene silencing through histone modification and chromatin remodelling. We report the phenotypic characterization of 13 patients (11 unrelated individuals and a pair of monozygotic twins) with missense mutations in PCGF2. All the mutations affected the same highly conserved proline in PCGF2 and were de novo, excepting maternal mosaicism in one. The patients demonstrated a recognizable facial gestalt, intellectual disability, feeding problems, impaired growth, and a range of brain, cardiovascular, and skeletal abnormalities. Computer structural modeling suggests the substitutions alter an N-terminal loop of PCGF2 critical for histone biding. Mutant PCGF2 may have dominant-negative effects, sequestering PRC1 components into complexes that lack the ability to interact efficiently with histones. These findings demonstrate the important role of PCGF2 in human development and confirm that heterozygous substitutions of the Pro65 residue of PCGF2 cause a recognizable syndrome characterized by distinctive craniofacial, neurological, cardiovascular, and skeletal features.