SRD5A3-CDG: a patient with a novel mutation.

Congenital disorders of glycosylation (CDG) are genetic diseases with an extremely broad spectrum of clinical presentations due to defective glycosylation of glycoproteins and glycolipids. Some 45 CDG types have been reported since the first clinical description in 1980. Protein glycosylation disorders are defects in protein N- and/or O-glycosylation. Dolichol phosphate is the carrier of the N-glycan during their assembly first at the outside and subsequently at the inside of the endoplasmic reticulum (ER) membrane, and hence is a key molecule in protein glycosylation. Recently, defects have been identified in the last three steps of the dolichol phosphate biosynthesis: dolicholkinase deficiency (DK1-CDG), steroid 5alpha-reductase type 3 deficiency (SRD5A3-CDG), and dehydrodolichyl diphosphate synthase deficiency (DHDDS-CDG). We report on a patient with SRD5A3-CDG carrying a novel (homozygous) mutation. The diagnostic features of this novel inborn error of glycosylation are psychomotor retardation, nystagmus, visual impairment due to variable eye malformations, cerebellar abnormalities/ataxia, and often ichthyosiform skin lesions.

The occurrence of mutations in FUS in a Belgian cohort of patients with familial ALS.

BACKGROUND AND PURPOSE: Mutations in fused in sarcoma (FUS) were recently identified as a cause of familial amyotrophic lateral sclerosis (ALS). The frequency of occurrence of mutations in FUS in sets of patients with familial ALS remains to be established. METHODS: We sequenced the FUS gene in a cohort of patients with familial ALS seen at the neuromuscular clinic in Leuven. A total of 28 patients with SOD1-negative ALS from 22 families were analyzed. RESULTS: We identified a R521H mutation in 4 patients, belonging to a kindred of dominantly inherited classical ALS. The mutation segregated with disease. Mutations in FUS were observed in 2.9% of ALS pedigrees in our cohort. CONCLUSIONS: These results show that mutations in FUS are also a significant cause of familial ALS in Belgium.

Adenylosuccinate lyase deficiency: study of physiopathologic mechanism(s).

Nucleotide concentrations were normal in adenylosuccinate lyase-deficient fibroblasts, and the succinylpurines were not toxic for cultured neuronal cells.

New evidences for a regulation of deoxycytidine kinase activity by reversible phosphorylation.

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post-translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK-293 cells as a His-tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP lambda) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.

Mutation of a nuclear respiratory factor 2 binding site in the 5' untranslated region of the ADSL gene in three patients with adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL; also called "adenylosuccinase") catalyzes two steps in the synthesis of purine nucleotides: (1) the conversion of succinylaminoimidazolecarboxamide ribotide into aminoimidazolecarboxamide ribotide and (2) the conversion of adenylosuccinate into adenosine monophosphate. ADSL deficiency, a recessively inherited disorder, causes variable-but most often severe-mental retardation, frequently accompanied by epilepsy and/or autism. It is characterized by the accumulation, in body fluids, of succinylaminoimidazolecarboxamide riboside and succinyladenosine, the dephosphorylated derivatives of the two substrates of the enzyme. Analysis of the ADSL gene of three unrelated patients with ADSL deficiency, in whom one of the ADSL alleles displayed a normal coding sequence, revealed a -49T-->C mutation in the 5' untranslated region of this allele. Measurements of the amount of mRNA transcribed from the latter allele showed that it was reduced to approximately 33% of that transcribed from the alleles mutated in their coding sequence. Further investigations showed that the -49T-->C mutation provokes a reduction to 25% of wild-type control of promoter function, as evaluated by luciferase activity and mRNA level in transfection experiments. The mutation also affects the binding of nuclear respiratory factor 2 (NRF-2), a known activator of transcription, as assessed by gel-shift studies. Our findings indicate that a mutation of a regulatory region of the ADSL gene might be an unusually frequent cause of ADSL deficiency, and they suggest a role for NRF-2 in the gene regulation of the purine biosynthetic pathway.

Functional and molecular modelling studies of two hereditary fructose intolerance-causing mutations at arginine 303 in human liver aldolase.

We have identified a novel hereditary fructose intolerance mutation in the aldolase B gene (i.e. liver aldolase) that causes an arginine-to-glutamine substitution at residue 303 (Arg(303)-->Gln). We previously described another mutation (Arg(303)-->Trp) at the same residue. We have expressed the wild-type protein and the two mutated proteins and characterized their kinetic properties. The catalytic efficiency of protein Gln(303) is approx. 1/100 that of the wild-type for substrates fructose 1,6-bisphosphate and fructose 1-phosphate. The Trp(303) enzyme has a catalytic efficiency approx. 1/4800 that of the wild-type for fructose 1,6-bisphosphate; no activity was detected with fructose 1-phosphate. The mutation Arg(303)-->Trp thus substitution impairs enzyme activity more than Arg(303)-->Gln. Three-dimensional models of wild-type, Trp(303) and Gln(303) aldolase B generated by homology-modelling techniques suggest that, because of its larger size, tryptophan exerts a greater deranging effect than glutamine on the enzyme's three-dimensional structure. Our results show that the Arg(303)-->Gln substitution is a novel mutation causing hereditary fructose intolerance and provide a functional demonstration that Arg(303), a conserved residue in all vertebrate aldolases, has a dominant role in substrate binding during enzyme catalysis.

Clinical, biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency (MIM 103050) is an autosomal recessive inborn error of purine synthesis characterized by the accumulation in body fluids of succinylaminoimidazolecarboxamide (SAICA) riboside and succinyladenosine (S-Ado), the dephosphorylated derivatives of the two substrates of the enzyme. Because ADSL-deficient patients display widely variable degrees of psychomotor retardation, we have expressed eight mutated ADSL enzymes as thioredoxin fusions and compared their properties with the clinical and biochemical characteristics of 10 patients. Three expressed mutated ADSL enzymes (M26L, R426H and T450S) were thermolabile, four (A2V, R141W, R303C and S395R) were thermostable and one (del206-218), was inactive. Thermolabile mutations decreased activities with SAICA ribotide (SAICAR) and adenylosuccinate (S-AMP) in parallel, or more with SAICAR than with S-AMP. Patients homozygous for one of these mutations, R426H, displayed similarly decreased ADSL activities in their fibroblasts, S-Ado:SAICA riboside ratios of approximately 1 in their cerebrospinal fluid and were profoundly retarded. With the exception of A2V, thermostable mutations decreased activity with S-AMP to a much more marked extent than with SAICAR. Two unrelated patients homozygous for one of the thermostable mutations, R303C, also displayed a much more marked decrease in the activity of fibroblast ADSL with S-AMP than with SAICAR, had S-Ado:SAICA riboside ratios between 3 and 4 in their cerebrospinal fluid and were mildly retarded. These results suggest that, in some cases, the genetic lesion of ADSL determines the ratio of its activities with S-AMP versus SAICAR, which in turn defines the S-Ado:SAICA riboside ratio and the patients' mental status.