Detection of a novel deletion in the cystathionine beta-synthase (CBS) gene using an improved genomic DNA based method.

We elucidated the intron-exon boundaries of the 15 coding exons of the human cystathionine beta-synthase (CBS) gene in order to establish an improved method based on PCR and direct sequencing for detection of CBS mutations. Using this method we identified the pathogenic mutations in two Danish siblings with CBS deficiency. Patients were compound heterozygotes: we detected the 833T-->C mutation and a novel 22 bp deletion of exon 4 (493-514del) that introduces a frameshift and a stop codon immediately after the deletion. The deletion resulted in no detectable mRNA from this allele, as assessed by sequencing of cDNA. The established method represents an improvement of the existing method based on sequencing of cDNA because it permits the detection of mutations within the entire coding region of the CBS gene from a peripheral blood sample, including splice mutations and mutations resulting in the lack or a reduced amount of transcript.

Intermediate and severe hyperhomocysteinemia with thrombosis: a study of genetic determinants.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. In search of genetic factors causing elevated levels of total homocysteine in plasma (tHcy), we investigated a cohort of consecutively identified, unrelated thrombosis patients (n = 28) having intermediate or severe hyperhomocysteinemia (30 micromol/l 100 micromol/l, respectively). The methylene-tetrahydrofolate reductase (MTHFR) 677C-->T genotype, and the complete cystathionine beta-synthase (CBS) genotype was determined in all patients. We found that the MTHFR T/T genotype was strongly correlated with intermediate hyperhomocysteinemia, being present in 73.9% of those cases (17 of 23). In three of five patients with severe hyperhomocysteinemia, compound heterozygosity for CBS mutations was detected. Among the mutations, two novel missense mutations: 1265C-->T (S422L) and 1397C-->T (S466L) were detected. The phenotype in those patients was quite mild, thromboembolism apart. This indicates that a search for CBS mutations in patients with severe hyperhomocysteinemia is important to ensure the detection of a possible CBS deficiency, thus enabling treatment. Co-existence of the MTHFR T/T genotype and the common CBS 844ins68 variant was significantly higher among patients (10.7%) as compared to controls (1.2%), indicating that this genotype combination is a thrombotic risk factor (P <0.05). In a few patients, hyperhomocysteinemia could not be explained by this genetic approach, suggesting that other genetic risk factors were implicated.

Sudden infant death syndrome, childhood thrombosis, and presence of genetic risk factors for thrombosis.

Sudden infant death syndrome or "cot death" has until the late eighties been a significant cause of death in children between the ages of 1 month and 1 year. Approximately two per 1000 children born alive dies of sudden infant death syndrome each year in Western Europe, North America, and Australia. The vulnerability of the infant brain stem to ischemia has been suggested to be a conceivable cause of sudden infant death syndrome. This is compatible with a hypothesis that genetic risk factors for cerebral thrombosis could cause microinfarction in the brain stem during the first month of life, affecting vital centers or their blood supply. The presence of three common point mutations seen in families with thrombophilia (1691G-->A in the coagulation factor V gene, 677C-->T in the methylenetetrahydrofolate reductase gene, and the 20210G-->A mutation in the prothrombin gene) could increase the risk for thrombosis in the child. This prompted us to investigate these genetic markers of thromboembolic disease in 121 cases of sudden infant death syndrome and in relevant controls, in the expectation of a more frequent occurrence of these markers if thrombosis is an etiological factor in sudden infant death syndrome. The frequency of homozygous 1691G-->A mutation in SIDS cases was higher than expected (odds ratio: 7.3, 95% confidence interval, 1.2-45.8). The allele frequencies (theta;) in cases of sudden infant death syndrome of the 1691G-->A, 677C-->T, and 20210G-->A alleles was 2.6% (1.0-5.5), 32.6% (26.8-38.9), and 0.9% (0.1-3.4), respectively. None of the allele frequencies found in the background population (3.4% for the 1691G-->A allele, 29% for the 677C-->T allele, and 1% for the 20210G-->A allele) differed significantly from that in cases of sudden infant death syndrome. In 5,251,027 inhabitants in Denmark, the incidence of venous thromboembolism was 0.9 per 1000 per year in the background population, and less than one-thousandth of these were children. Consequently it is not likely that venous thrombosis is a major cause of sudden infant death syndrome. On the other hand, this does not exclude other known or unknown risk factors for thrombosis as possible etiological factors for sudden infant death syndrome. It is likely that we must continuously employ the exclusion principle on possible etiological causes in genetic material from a large group of victims of sudden infant death syndrome if the phenomenon of sudden infant death syndrome is to be ascribed to a specific hereditary disorder.

Human blood eosinophils produce and secrete interleukin 4.

Interleukin 4 (Il-4) is an immunoregulatory cytokine which induces T-cell proliferation and differentiation into a Th2 phenotype, and is of particular importance for the induction of IgE synthesis. In the present study, the capability of human peripheral blood eosinophils from allergic and non-allergic donors to produce Il-4 was examined. Using reverse transcribed polymerase chain reaction (RT-PCR), it was shown that highly purified eosinophils from allergic patients express mRNA for Il-4. Resting eosinophils also gave specific immunoreactivity with anti-Il-4 antibodies, consistent with translation of Il-4 mRNA. Light and electron microscopic immunocytochemistry revealed that Il-4 was prestored in the eosinophilic granules. These results were confirmed by Il-4 specific ELISA which showed that Il-4 production could be upregulated in the eosinophils and released from the eosinophils following stimulation with the calcium ionophore A23187. These data indicate that eosinophils may be an important source of Il-4 at sites of allergic inflammation. Thus, eosinophils may act as immunomodulatory cells enhancing the allergic response through formation of Th2-cells and inducing the isotype switching to IgE in human B-cells.

Thrombophilic predisposition in stroke and venous thromboembolism in Danish patients.

The present study describes 403 patients with thrombosis, from a uniform ethnic and geographical background. Two-hundred-and-seven individuals had suffered mild or moderate stroke and 196 individuals suffered venous thromboembolism. We recorded levels of antithrombin, protein C and protein S, plasminogen and plasma homocysteine, and the presence of the factor V Leiden mutation, the prothrombin 20210G-->A variant, and the methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism. Controls for the mutation frequencies consisted of Guthrie card blood spots from a cohort of new-born babies. The cumulative prevalence of deficiencies in antithrombin, protein C, protein S or plasminogen was 2.4% in patients with stroke and 11.2% in patients with venous thrombosis. The factor V Leiden mutation was present in 11.1% of patients with stroke and 26.5% of patients with venous thrombosis, compared with 6.6% of controls (n = 4188; P < 0.05 and P < 0.0001, respectively). The prevalence of the prothrombin 20210A variant was 3.1% in patients with venous thrombosis, 1.9% in patients with stroke and 2.0% in controls (n = 500; P > 0.05). Hyperhomocysteinemia was present in 16.0% of patients with stroke and 17.6% of patients with venous thrombosis. The prevalence of the MTHFR 677T/T genotype was no different in patients with stroke (10.6%) and venous thrombosis (8.7%) than in controls (8.3%; n = 1084; P > 0.05); thus, it apparently contributed to thrombosis only via its influence on total plasma homocysteine, which was significantly increased in patients with the T/T genotype (P < 0.001). The MTHFR T/T genotype did not further increase the risk for thrombosis in carriers of the factor V Leiden mutation. Overall, thrombotic events were associated with a known risk factor in 27% of patients with stroke and 55% of patients with venous thrombosis.

Intestinal interleukin-8 concentration and gene expression in inflammatory bowel disease.

BACKGROUND: Interleukin-8 (IL-8) is an important cytokine for recruitment and activation of polymorphonuclear neutrophils (PMNs), cells that are abundant in the intestinal lesions of ulcerative colitis (UC) and Crohn's disease (CD). The present investigation was conducted to evaluate intestinal IL-8 concentration and IL-8 gene expression in parallel in inflammatory bowel disease (IBD) patients and a non-inflammatory control group. METHODS: The intestinal concentration of IL-8 was measured with a sandwich enzyme-linked immunosorbent assay (ELISA) technique (detection limit, 17.4 pg/mg protein), and relative quantitation of IL-8 mRNA transcript levels was done with a reverse transcription polymerase chain reaction (RT-PCR)-based method. Biopsy specimens from 66 humans who underwent colonoscopy--28 with UC, 18 with CD and colonic involvement, and 20 non-inflammatory disease-specific controls who subsequently were found to fulfill the diagnostic criteria for irritable bowel syndrome (IBS)--were included. None had received glucocorticoids within 3 months. RESULTS: Using a one-tailed variance analysis, a significant concordance between increasing IL-8 protein concentrations and disease activity was found both in UC and CD (P < 0.001), and only trace amounts were detected in IBS biopsy specimens. No differences were found between the two groups of UC and CD patients (P > 0.05), and no differences were found between quiescent IBD and IBS (P > 0.05). However, the PCR method showed IL-8 mRNA in 8 of 18 CD patients (44.4%; 95% confidence limits, 21.5-69.2%) and 7 of 28 UC patients (25.9%; 95% confidence limits, 11.1-46.3%), as compared with 0 of 20 IBS (P < 0.005). Increased IL-8 mRNA levels were found only in active CD, which was not the case in UC. No correlation was found between intestinal IL-8 ELISA and IL-8-mRNA levels (r = 0.24, P > 0.05). CONCLUSIONS: The observed correlation between disease activity and expression of the IL-8 gene in active CD colitis but not in UC and the increased IL-8 protein concentrations in affected intestinal segments of IBD as compared with the non-inflamed IBS indicate a possible transient IL-8 gene expression or altered mRNA stability in UC and CD, as is well known for other cytokines, such as IL-2. If so, it may form the basis of new therapeutic regimens for IBD like IL-10.

Familial thrombophilia associated with homozygosity for the cystathionine beta-synthase 833T-->C mutation.

Severe hyperhomocysteinemia due to cystathionine beta-synthase (CBS) deficiency is a strong risk factor for premature cardiovascular disease. Among untreated patients, approximately 50% have suffered a thromboembolic event by 30 years of age. We report on 3 sisters with severe hyperhomocysteinemia due to homozygosity for the CBS 833T-->C mutation. These patients, who displayed no other known thrombophilic predisposition, had suffered single or multiple venous thrombosis before CBS deficiency was diagnosed relatively late in life. In this family, homozygosity for the 833T-->C mutation was associated with a mild phenotype with respect to other sequelae of CBS deficiency. Consequently, our results indicate that most cases with this genotype may remain undiagnosed. Investigated family members heterozygous for the 833T-->C mutation displayed normal total homocysteine in plasma (tHcy) levels, even when they were homozygous for the methylenetetrahydrofolate reductase 677C-->T polymorphism. The prevalence of homozygosity for the 833T-->C mutation has previously been estimated at no less than 1:20 500 in our population. Because a reduction of the severely elevated levels of tHcy in CBS deficiency reduces cardiovascular risk and because homozygosity for the 833T-->C mutation is more prevalent than previously thought, our results emphasize the importance of measuring tHcy routinely in thrombophilia screening.

Regulation of human cystathionine beta-synthase by S-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region.

Cystathionine beta-synthase (CBS), condensing homocysteine and serine, represents a key regulatory point in the biosynthesis of cysteine via the transsulfuration pathway. Inherited deficiency of CBS causes homocystinuria. CBS is activated by S-adenosyl-L-methionine (AdoMet) by inducing a conformational change involving a noncatalytic C-terminal region spanning residues 414-551. We report the purification of two patient-derived C-terminal mutant forms of CBS, S466L and I435T, that provide new insight into the mechanism of CBS regulation and indicate a regulatory function for the "CBS domain". Both of these point mutations confer catalytically active proteins. The I435T protein is AdoMet inducible but is 10-fold less responsive than wild-type (WT) CBS to physiologically relevant concentrations of this compound. The S466L form does not respond to AdoMet but is constitutively activated to a level intermediate between those of WT CBS in the presence and absence of AdoMet. Both mutant proteins are able to bind AdoMet, indicating that their impairment is related to their ability to assume the fully activated conformation that AdoMet induces in WT CBS. We found that I435T and WT CBS can be activated by partial thermal denaturation but that the AdoMet-stimulated WT, S466L, and a truncated form of CBS lacking the C-terminal region cannot be further activated by this treatment. Tryptophan and PLP fluorescence data for these different forms of CBS indicate that activation by AdoMet, limited proteolysis, and thermal denaturation share a common mechanism involving the displacement of an autoinhibitory domain located in the C-terminal region of the protein.

Cystathionine beta-synthase mutations in homocystinuria.

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine beta-synthase (CBS). Deficiency of CBS activity results in elevated levels of homocysteine as well as methionine in plasma and urine and decreased levels of cystathionine and cysteine. Ninety-two different disease-associated mutations have been identified in the CBS gene in 310 examined homocystinuric alleles in more than a dozen laboratories around the world. Most of these mutations are missense, and the vast majority of these are private mutations. The two most frequently encountered of these mutations are the pyridoxine-responsive I278T and the pyridoxine-nonresponsive G307S. Mutations due to deaminations of methylcytosines represent 53% of all point substitutions in the coding region of the CBS gene.