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Abstract: Recently, S-adenosylhomocysteine hydrolase deficiency was confirmed for the first time in an adult. Two missense mutations in codons 89 (A>V) and 143 (Y>C) in the AdoHcyase gene were identified [N.R.M. Buist, B. Glenn, O. Vugrek, C. Wagner, S. Stabler, R.H. Allen, I. Pogribny, A. Schulze, S.H. Zeisel, I. Barić, S.H. Mudd, S-Adenosylhomocysteine hydrolase deficiency in a 26-year-old man, J. Inh. Metab. Dis. 29 (2006) 538–545]. Accordingly, we have proven the Y143C mutation to be highly inactivating [R. Belužić, M. Ćuk, T. Pavkov, K. Fumić, I. Barić, S.H. Mudd, I. Jurak, O. Vugrek, A single mutation at tyrosine 143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound co-factor NAD, Biochem. J. 400 (2006) 245–253]. Now we report that the A89V exchange leads to a ⩾70% loss of enzymatic activity, respectively. Circular dichroism analysis of recombinant p.A89V protein shows a significantly reduced unfolding temperature by 5.5°C compared to wild-type. Gel filtration of mutant protein is almost identical to wild-type indicating assembly of subunits into the tetrameric complex. However, electrophoretic mobility of p.A89V is notably faster as shown by native polyacrylamide gel electrophoresis implicating changes to the overall charge of the mutant complex. ‘Bioinformatics’ analysis indicates that Val89 collides with Thr84 causing sterical incompatibility. Performing site-directed mutagenesis changing Thr84 to ‘smaller’ Ser84 but preserving similar physico-chemical properties restores most of the catalytic capabilities of the mutant p.A89V enzyme. On the other hand, substitution of Thr84 with Lys84 or Gln84, thereby introducing residues with higher volume in proximity to Ala89 results in inactivation of wild-type protein. In view of our mutational analysis, we consider changes in charge and the sterical incompatibility in mutant p.A89V protein as main reason for enzyme malfunction with AdoHcyase deficiency as consequence. [Copyright &y& Elsevier]

Abstract: Severe combined immunodeficiency (SCID) may result from a variety of genetic defects that impair the development of T cells. Signaling mediated by the cytokine interleukin-7 is essential for the differentiation of T cells from lymphoid progenitors, and mutations of either the interleukin-7 receptor α chain (IL-7Rα) or its associated cytokine receptor chain, the common γ chain (γc), result in SCID. Here we report a case of SCID due to heterozygous mutations of the IL7R gene encoding IL-7Rα. A previously unrecognized mutation found within intron 3 created a new exon between exons 3 and 4 in the mRNA transcribed from this allele, producing a truncated, unstable mRNA. This mutation illustrates the necessity of evaluating both coding and non-coding regions of genes when searching for pathogenic mutations. Following hematopoietic stem cell transplantation of our patient, immune reconstitution was accompanied by two unusual complications, immune-mediated myositis and myasthenia gravis. [Copyright &y& Elsevier]

Abstract: It has been argued that the molecular chaperone Hsp90 guards the organism against genetic variations by stabilizing variant Hsp90 substrate proteins. However, little is known about polymorphisms affecting its own functions. We have followed up on a recent study describing two polymorphisms that alter the amino acid sequences of the two Hsp90 isoforms Hsp90α and Hsp90β. Hsp90 is essential for cell proliferation in the budding yeast Saccharomyces cerevisiae, but the human proteins can replace the endogenous ones. In this growth assay, the variant V656M of Hsp90β was indistinguishable from wild-type. In contrast, the Hsp90α variant Q488H, which carries an alteration of a very highly conserved residue, was severely defective for growth compared to wild-type Hsp90α. Hence, the characteristics of this yeast-based system—simplicity, rapidity, low cost—make it ideal for phenotype screening of polymorphisms in HSP90 and possibly many other human genes. [Copyright &y& Elsevier]

Fabry disease is a lysosomal storage disease caused by deficiency in the enzyme α-galactosidase (α-GAL). To understand the molecular defects responsible for Fabry disease, we have collected more than 190 reported point and stop mutations and mapped them onto a model of human α-GAL based on the X-ray structure of the closely related enzyme α-N-acetylgalactosaminidase (α-NAGAL). The locations of the human α-GAL point mutations reveal two major classes of Fabry disease protein defects: active site mutations and folding mutations. Active site mutations reduce enzymatic activity by perturbing the active site without necessarily affecting the overall α-GAL structure. Folding mutations reduce the stability of α-GAL by disrupting its hydrophobic core. Examining the frequency of mutation around each α-GAL residue identifies the active site as a hotspot for mutations leading to Fabry disease. This study furthers our understanding of the structural basis for mutations leading to Fabry disease, from which new avenues for the treatment of lysosomal storage diseases may be developed. [Copyright &y& Elsevier]