Connective tissue presentation in two families expands the phenotypic spectrum of PYROXD1 disorders

Recessive variants in the oxidoreductase PYROXD1 are reported to cause a myopathy in 22 affected individuals from 15 families. Here, we describe two female probands from unrelated families presenting with features of a congenital connective tissue disorder including osteopenia, blue sclera, soft skin, joint hypermobility and neuromuscular junction dysfunction in addition to known features of PYROXD1 myopathy including respiratory difficulties, weakness, hypotonia and oromotor dysfunction. Proband AII:1 is compound heterozygous for the recurrent PYROXD1 variant Chr12(GRCh38):g.21452130A\u3eG;NM_024854.5:c.464A\u3eG;p.(N155S) and Chr12(GRCh38):g.21462019_21462022del;NM_024854.5:c.892_895del;p.(V298Mfs*4) and proband BII:1 is compound heterozygous for Chr12(GRCh38):g.21468739-21468741del;NM_024854.5:c.1488_1490del;p.(E496del) and Chr12(GRCh38):g.21467619del;NM_024854.5:c.1254+1del. RNA studies demonstrate c.892_895del;p.(V298Mfs*4) is targeted by nonsense mediated decay and c.1254+1delG elicits in-frame skipping of exon-11. Western blot from cultured fibroblasts shows reduced PYROXD1 protein levels in both probands. Testing urine from BII:1 and six individuals with PYROXD1 myopathy showed elevated levels of deoxypyridinoline, a mature collagen crosslink, correlating with PYROXD1-disorder severity. Urine and serum amino acid testing of the same individuals revealed no reportable changes. In contrast to PYROXD1 knock-out, we find no evidence for disrupted tRNA ligase activity, as measured via XBP1 splicing, in fibroblasts expressing PYROXD1 variants. In summary, we expand the clinical spectrum of PYROXD1-related disorders to include an overlapping connective tissue and myopathy presentation, identify three novel, pathogenic PYROXD1 variants, and provide preliminary evidence that elevated urine DPD crosslinks may provide a clinical biomarker for PYROXD1 disorders. Our results advocate consideration of PYROXD1 variants in the differential diagnosis for undiagnosed individuals presenting with a connective tissue disorder and myopathy

Mutagenic and structural studies of the active center of oligoribonuclease, an essential enzyme for Escherichia coli viability

Maturation and degradation of RNA molecules are essential processes in both prokaryotic and eukaryotic cells. Oligoribonuclease (ORN) is the only exoribonuclease able to efficiently degrade small oligonucleotides. Upon loss of oligoribonuclease activity, E. coli cells accumulate short oligonucleotides and lose viability.ORN is a DEDDh type exoribonuclease. The importance of the conserved histidine in ORN activity is investigated using mutants of the enzyme. Kinetic analyses of these mutants show that histidine is essential and cannot be replaced by either tyrosine or asparagine.ORN activity is inhibited by zinc like RNase T, another DEDDh exoribonuclease. To investigate the role of His157 in attenuation of ORN activity by zinc, this residue was mutated to alanine. Activity of H157A mutant compared to that of wild type enzyme shows that His157 residue is responsible for the sensitivity of ORN to zinc, and its removal abolishes zinc inhibition. A metal preference of DEDDh versus DEDDy exonucleases is hypothesized.ORN structure reveals a basic patch near the active center, possibly involved in binding and orientation of the substrate at the active center. Two of the basic residues located in this patch, Arg129 and Lys153, were analyzed in detail using mutagenesis. Both residues were determined to be important for activity of the enzyme and productive binding of the substrate. The Arg129 residue is probably involved in correctly orienting the substrate at the active center, likely through charge interactions. A model of substrate binding by ORN, with special emphasis on Arg129, is proposed.X-ray crystal structures of two ORN mutants, H157A and H157Y, were solved. The overall folds of these mutants are similar to that of the wild type ORN. An approximate shift of 2 A was observed in the H157A structure at position 157 which provides further evidence for the flexible nature of this region and suggests that the ORN active center may be dynamic in nature.These studies advance our understanding of ORN active center and how ORN selects and binds substrates. This research has direct relevance to understanding the molecular mechanism of RNA processing and metabolism

Keeping you on your toes: Smith–Lemli–Opitz Syndrome is an easily missed cause of developmental delays

Abstract Smith‐Lemli‐Opitz syndrome (SLOS) is a relatively common genetic cause of developmental delay and may only present in conjunction with 2,3 toe syndactyly. This case series illustrates a milder phenotype of SLOS, where the predominant findings are neurocognitive in the presence of 2,3 toe syndactyly

Challenges in Diagnosing Intermediate Maple Syrup Urine Disease by Newborn Screening and Functional Validation of Genomic Results Imperative for Reproductive Family Planning

Maple syrup urine disease is caused by a deficiency of branched-chain alpha-ketoacid dehydrogenase, responsible for degradation of leucine, isoleucine, and valine. Biallelic pathogenic variants in BCKDHA, BCKDHB, or DBT genes result in enzyme deficiency. We report the case of a female infant who presented with mild gross motor delay at 4 months, and seizures with hypoglycaemia at 5 months. Newborn screening returned total leucine/isoleucine at the 99.5th centile of the population; however, as second-tier testing reported minimal alloisoleucine, the results were considered inconsistent with MSUD. Plasma amino acid and urine organic acid analyses at 5 months were, however, consistent with a diagnosis of MSUD. A brain MRI showed bilateral symmetrical T2 hyperintense signal abnormalities involving white matter, globus pallidus, thalamus, brainstem, and dentate nuclei with restricted diffusion. A repeat MRI 10 months post-dietary-intervention showed the resolution of these changes and progression in myelination. Her clinical phenotype, including protein tolerance, correlated with intermediate MSUD. Molecular analysis of all three genes identified two variants of uncertain significance, c.434-15_434-4del and c.365A>G (p. Tyr122Cys) in the DBT gene. The rate of leucine decarboxylation in fibroblasts was reduced, but not to the extent observed in classical MSUD patients, supporting an intermediate form of MSUD. Previously reported mRNA splicing studies supported a deleterious effect of the c.434-15_434-4del variant. This functional evidence and confirmation that the variants were in trans, permitted their reclassification as pathogenic and likely pathogenic, respectively, facilitating subsequent prenatal testing. This report highlights the challenges in identifying intermediate MSUD by newborn screening, reinforcing the importance of functional studies to confirm variant pathogenicity in this era of molecular diagnostics