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Misfolding of mutant enzymes may play an important role in the pathogenesis of cystathionine beta-synthase (CBS) deficiency. We examined properties of a series of 27 mutant variants, which together represent 70% of known alleles observed in patients with homocystinuria due to CBS deficiency. The median amount of SDS-soluble mutant CBS polypeptides in the pellet after centrifugation of bacterial extracts was increased by 50% compared to the wild type. Moreover, mutants formed on average only 12% of tetramers and their median activity reached only 3% of the wild-type enzyme. In contrast to the wild-type CBS about half of mutants were not activated by S-adenosylmethionine. Expression at 18 degrees C substantially increased the activity of five mutants in parallel with increasing the amounts of tetramers. We further analyzed the role of solvent accessibility of mutants as a determinant of their folding and activity. Buried mutations formed on average less tetramers and exhibited 23 times lower activity than the solvent exposed mutations. In summary, our results show that topology of mutations predicts in part the behavior of mutant CBS, and that misfolding may be an important and frequent pathogenic mechanism in CBS deficiency., ((c) 2010 Wiley-Liss, Inc.)

Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA mis-splicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5' splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported., ((c) 2010 Wiley-Liss, Inc.)

Objectives: To estimate the frequency of the cystathionine beta-synthase deficiency caused by c.1105C>T mutation in Central Europe compared to Norway, and to examine the pathogenicity of the corresponding p.R369C mutant enzyme., Study Design: Mutation c.1105C>T was analyzed in 600 anonymous Czech newborn blood spots. Catalytic activity and quaternary structure of the p.R369C mutant was evaluated after expression in 2 cellular systems., Results: Population frequency of the c.1105C>T mutation was 0.005, predicting the birth prevalence of homocystinuria of 1:40000, which increased to 1:15500 in a model including 10 additional mutations. In Escherichia coli the p.R369C mutant misfolded, and its activity was severely reduced, and expression in Chinese hamster ovary cells enabled proper folding with activity decreased to 63% of the wild-type enzyme. This decreased activity was not due to impaired K(m) for both substrates but resulted from V(max) lowered to 55% of the normal cystathionine beta-synthase enzyme., Conclusions: The c.1105C>T (p.R369C) allele is common also in the Czech population. Although the p.R369C mutation impairs folding and decreases velocity of the enzymatic reaction, our data are congruent with rather mild clinical phenotype in homozygotes or compound heterozygotes carrying this mutation.

Missense mutations in the cystathionine beta-synthase (CBS) gene are the most common cause of clinical homocystinuria in humans. The p.S466L mutation was identified in a homocystinuric patient, but enzymatic studies with recombinant protein show this mutant to be highly active. To understand how this mutation causes disease in vivo, we have created mice lacking endogenous mouse CBS and expressing either wild-type (Tg-hCBS) or p.S466L (Tg-S466L) human CBS under control of zinc inducible metallothionein promoter. In the presence of zinc, we found that the mean serum total homocysteine (tHcy) of Tg-S466L mice was 142+/-55 microM compared to 16+/-13 microM for hCBS mice. Tg-S466L mice also had significantly higher levels of total free homocysteine and S-adenosylhomocysteine in liver and kidney. Only 48% of Tg-S466L mice had detectable CBS protein in the liver, whereas all the Tg-hCBS animals had detectable protein. Surprisingly, CBS mRNA was significantly elevated in Tg-S466L animals compared to Tg-hCBS, implying that the reduction in p.S466L protein was occurring due to posttranscriptional mechanisms. In Tg-S466L animals with detectable liver CBS, the enzyme formed tetramers and was active, but lacked inducibility by S-adenosylmethionine (AdoMet). However, even in Tg-S466L animals that had in vitro liver CBS activity equivalent to Tg-hCBS animals there was significant elevation of serum tHcy. Our results show that p.S466L causes homocystinuria by affecting both the steady state level of CBS protein and by reducing the efficiency of the enzyme in vivo.

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is an inherited disorder of homocysteine transsulfuration, which manifests by neurological, vascular and connective tissue involvement. So far, 130 pathogenic mutations have been recognized in the CBS gene. We examined 10 independent alleles in Polish patients suffering from CBS deficiency, and we detected four already described mutations (c.1224-2A>C, c.684C>A, c.833T>C, and c.442G>A) and two novel mutations (c.429C>G and c.1039+1G>T). The pathogenicity of the novel mutations was demonstrated by expression in E.coli. This is the first published communication on mutations leading to CBS deficiency in Poland., (Copyright 2004 Wiley-Liss, Inc.)

Patients with the cblE type of homocystinuria usually present with megaloblastic anaemia, feeding difficulties, developmental delay and cerebral atrophy. We present a 14-year-old Spanish girl (patient 1) and a 10-year-old Portuguese boy (patient 2) with cblE disease and mild clinical phenotype. The main clinical feature in both patients was persistent megaloblastic anaemia observed at 3 years and at 2 months of age, respectively. Diagnosis was made at the ages of 9 and 7 years, respectively, owing to persistent macrocytosis despite cobalamin treatment. Plasma total homocysteine values at diagnosis were 91 micromol/L and 44 micromol/L, respectively, in the absence of methylmalonic aciduria. Neurological and neurophysiological examinations were normal except for two small lesions on brain MRI suggestive of ischaemia and slight abnormalities in somatosensitive evoked potentials. Enzymatic analysis, complementation studies and clearly reduced production of methylcobalamin from 57Co-labelled cyanocobalamin indicated functional methionine synthase reductase deficiency due to the cblE defect. Genetic analysis confirmed that both patients are homozygous for a novel mutation c.1361C>T in the methionine synthase reductase gene leading to a replacement of serine by leucine (S454L) in a highly conserved FAD-binding domain. We propose that homozygosity for this novel mutation may be associated with a mild phenotype, although its long-term deleterious neurological consequences remain possible. Furthermore, we propose that even in the absence of apparent neurological involvement, total homocysteine should be investigated in patients with resistant megaloblastic anaemia to detect possible mild forms of the cblE type of homocystinuria.

During the past 20 years, cystathionine beta-synthase (CBS) deficiency has been detected in the former Czechoslovakia with a calculated frequency of 1:349,000. The clinical manifestation was typical of homocystinuria, and about half of the 21 patients were not responsive to pyridoxine. Twelve distinct mutations were detected in 30 independent homocystinuric alleles. One half of the alleles carried either the c.833 T-->C or the IVS11-2A-->C mutation; the remaining alleles contained private mutations. The abundance of five mutant mRNAs with premature stop codons was analyzed by PCR-RFLP. Two mRNAs, c.828_931ins104 (IVS7+1G-->A) and c.1226 G-->A, were severely reduced in the cytoplasm as a result of nonsense-mediated decay. In contrast, the other three mRNAs-c.19_20insC, c.28_29delG, and c.210_235del26 (IVS1-1G-->C)-were stable. Native western blot analysis of 14 mutant fibroblast lines showed a paucity of CBS antigen, which was detectable only in aggregates. Five mutations-A114V (c.341C-->T), A155T (c.463G-->A), E176K (c.526G-->A), I278T (c.833T-->C), and W409_G453del (IVS11-2A-->C)-were expressed in Escherichia coli. All five mutant proteins formed substantially more aggregates than did the wild-type CBS, and no aggregates contained heme. These data suggest that abnormal folding, impaired heme binding, and aggregation of mutant CBS polypeptides may be common pathogenic mechanisms in CBS deficiency.

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is frequently caused by missense mutations. In this article, we report four novel missense mutations in the CBS gene: 172C-->T (R58W) linked in cis with A114V; 376A-->G (M126V); 904G-->A (E302K); and 1006C-->T (R336C). The CBS activity of the corresponding mutant enzymes expressed in Escherichia coli was greatly diminished, confirming the pathogenicity of these mutations. Western analysis showed that the R58W+A114V and M126V mutant enzymes were unstable in E. coli, while the E302K subunits were partially degraded to shorter products. Using site-directed mutagenesis we found that CBS containing either the R58W or A114V as the only mutations demonstrated 18% and 46% of normal activity, respectively. Both mutant forms of CBS were stable in E. coli. When these two mutations were expressed in cis, the resultant mutant protein exhibited activity 1.3% that of a control. All these in vitro results were in good agreement with the clinical manifestation in these patients. The Italian patient 2241, an A114V+R58W/M126V compound heterozygote, exhibited severe pyridoxine nonresponsive homocystinuria, while another Italian patient 2242, with an A114V/E302K genotype, responded to pyridoxine treatment and had a much milder phenotype. The third patient 3064, an English compound heterozygote for two severe mutations R336C and G307S, was B6 nonresponsive. This report of a ninth homocystinuric allele carrying two mutations in cis raises the possibility that double mutant alleles may be underestimated in homocystinuric patients. In this context, a search for additional mutations in cis may sometimes be necessary to establish a good genotype-phenotype relationship.

Deficiency of cystathionine beta-synthase (CBS) causes the most common form of inherited homocystinuria. We developed a simple CBS expression system in E. coli to screen for pathogenic mutations in affected individuals. Portions of patient cDNAs were amplified by PCR and used to replace the corresponding segments of normal human CBS cDNA in the bacterial expression plasmid pHCS3. Hybrid CBS was expressed in E. coli and the segments of patient's cDNA which extinguished CBS activity were sequenced to identify the mutation. The first study of a pyridoxine-responsive patient using this screen revealed that of the clones which contained either the middle or the 3'-portion of his cDNA, about half were devoid of catalytic activity. Subsequent sequencing of the affected segments confirmed a compound heterozygosity for a maternal T833-->C transition (I278T) and for a paternal A-->C transversion in the intron 11 splice acceptor. The latter mutation leads to an in-frame deletion of exon 12 (nt 1224-1358, amino acids W408 to G453). This bacterial expression system proved to be a rapid screening method for localizing pathogenic mutations in CBS, allowing us to sequence the affected portions of mutant cDNA within 7-10 days of harvesting cultured fibroblasts.