Identification of a novel Wilson disease gene mutation frequent in Upper Austria: a genetic and clinical study.

Wilson disease (WD), a disorder of copper metabolism is caused by mutations in the ATP7B gene, a copper transporting ATPase. In the present study we describe a novel mutation in exon 9 of the ATP7B gene. The ATP7B gene was analyzed for mutations by denaturing HPLC and direct sequencing. DNA from 100 healthy blood donors from the same geographic area was examined as control. Sixteen (7.4%) out of the 216 patients diagnosed with WD in Austria carried the newly identified R816S(c.2448G>T) point mutation in exon 9 (4 male, age: 19 (6-30) years, median (range)). One patient was homozygous for R816S(c.2448G>T). Thirteen patients were compound heterozygotes (p.H1069Q(c.3207C>A)/R816S(c.2448G>T) (N=6), P539L/R816S(c.2448G>T) (N=3), each one G710S/R816S(c.2448G>T), P767P(2299insC)/R816S(c.2448G>T), W779G/R816S(c.2448G>T), T1220M/R816S(c.2448G>T)). In two patients no second mutation was identified. Interestingly, all but three of the patients originated within a distinct geographical area in Austria. Eleven patients presented with hepatic disease, 3 patients with neurological disease and 2 were asymptomatic sisters of an index case. A liver biopsy was available in 14 patients. Three patients showed advanced liver disease with liver transplantation for acute hepatic failure in two. The remaining patients had only mild histological changes, most commonly steatosis. Chronic hepatitis was described in five patients. Kayser-Fleischer ring was present in five patients. None of the 100 healthy controls carried the mutation. We describe a novel mutation in the ATP7B gene, occurring in patients originated from a distinct geographical area in Austria associated with a variable course of the disease.

Evolution of Crohn's disease-associated Nod2 mutations.

Several lines of evidence have confirmed the importance of Nod2 mutations for disease susceptibility in Crohn's disease. For tracing Nod2 evolution, exons 4a, 4e, 8, and 12 mutations were screened in a collection of 1,064 DNA samples from 52 worldwide populations. The overall allele frequency was 7.5% for single nucleotide polymorphism (SNP)5, 0.2% for SNP8, 0.3% for SNP12, and 0.4% for SNP13. Nod2 mutations are mainly Caucasian alleles with strong distribution dissimilarity between single populations and major geographical regions. This regional diversity of Nod2 mutations within Europe points to the regional existence of selection pressure (possibly through dairy-associated bacterial infections within Neolithic cattle farming populations). The SNP5 gradient between Africa and the Middle East and its absence in Asian and Native American populations indicate that the evolution of this variant occurred in the Middle East. As mutations in exons 4e, 8, and 12 were only found in association with SNP5, this variant may have allowed selection pressure to arise.

Novel mutations of the ATP7B gene among 109 Hungarian patients with Wilson's disease.

BACKGROUND/AIMS: Diagnosis of Wilson's disease may be difficult in patients presenting with liver disease and in asymptomatic siblings. The aim of the present study was to assess the impact of genetic testing for diagnosis of the disease in a large cohort (n=109) from Hungary. PATIENTS/METHODS: One hundred and nine patients with Wilson's disease were studied (65 men and 44 women; mean age at onset of symptoms: 20+/-9 years). Diagnosis of the disease was based on typical clinical and laboratory features (all had a Wilson's disease score of >or=4). H1069Q was assessed by the semi-nested polymerase chain reaction-based restriction fragment length polymorphism assay. H1069Q heterozygotes and H1069Q negative samples were then screened for mutations (on exons 6 to 20) by denaturating high-performance liquid chromatography and than sequenced on a genetic analyser. RESULTS: Twenty-three different mutations were found. H1069Q was the most frequent mutation in Hungary, detected in 77 patients (71%). Fourteen further known mutations were found by sequencing. We identified eight new mis-sense mutations not described before: N676I, S693Y, Y715H, M769L, W939C, P1273S, G1281D and G1341V. In 36/109 patients (33%) the diagnosis of Wilson's disease was established by adding mutational analysis. The Kayser-Fleischer ring was more frequent in H1069Q homozygous patients and their mean age at the time of diagnosis was higher than in patients heterozygous or negative for H1069Q. CONCLUSION: Eight novel mutations in addition to the 15 that are already known were found in Hungarian patients with Wilson's disease. Our results underline the importance and usefulness of genetic testing for patients presenting with liver disease and for family screening.

Toll-like receptor 4 and NOD2/CARD15 mutations in Hungarian patients with Crohn's disease: phenotype-genotype correlations.

AIM: To determine common NOD2/CARD15 mutations and TLR4 D299G polymorphism in Hungarian patients with CD. METHODS: A total of 527 unrelated patients with CD (male/female: 265/262, age: 37.1 (SD 7.6) years) and 200 healthy subjects were included. DNA was screened for possible NOD2/CARD15 mutations by denaturing high-performance liquid chromatography (confirmed by direct sequencing). TLR4 D299G was tested by PCR-RFLP. RESULTS: NOD2/CARD15 mutations were found in 185 patients (35.1%) and in 33 controls (16.5%, P<0.0001). SNP8/R702W (10.8% vs 6%, P = 0.02), SNP13/3020insC (19.4% vs 5%, P<0.0001) and exon4 R703C (2.1% vs 0%, P = 0.02) mutations were more frequent in CD, while the frequency of SNP12/G908R was not increased. The frequency of TLR4 D299G was not different (CD: 9.9% vs controls: 12.0%). Variant NOD2/CARD15 allele was associated with an increased risk for CD (OR(het) = 1.71, 95%CI = 1.12-2.6, P = 0.0001, OR(two-risk alleles) = 25.2, 95%CI = 4.37-8, P<0.0001), early disease onset (carrier: 26.4 years vs non-carrier: 29.8 years, P = 0.0006), ileal disease (81.9% vs 69.5%, OR = 1.99, 95%CI = 1.29-3.08, P = 0.02, presence of NOD2/CARD15 and TLR4: 86.7% vs 64.8%), stricturing behavior (OR = 1.69, 95%CI = 1.13-2.55, P = 0.026) and increased need for resection (OR=1.71, 95%CI: 1.13-2.62, P = 0.01), but not with duration, extra-intestinal manifestations, familial disease or smoking. TLR4 exhibited a modifier effect: age of onset in wt/TLR4 D299G carriers: 27.4 years vs NOD2mut/TLR D299G: 23 years (P = 0.06), in NOD2mut/wt: 26.7 years. CONCLUSION: These results confirm that variant NOD2/CARD15 (R702W, R703C and 3020insC) alleles are associated with earlier disease onset, ileal disease, stricturing disease behavior in Hungarian CD patients. In contrast, although the frequency of TLR4 D299G polymorphism was not different from controls, NOD2/TLR4 mutation carriers tended to present at earlier age.

[NOD2/CARD15 mutations and genotype-phenotype correlations in patients with Crohn's disease. Hungarian multicenter study]. / NOD2/CARD15 mutációk és a genotípus-fenotípus kapcsolatának multicentrikus vizsgálata Crohn-betegségben Magyarországon.

UNLABELLED: Recently mutations of the NOD2/CARD15 gene were identified as genetic markers for Crohn's disease (CD). It has also been suggested that the presence of mutation may influence the phenotype of the disease. The aim of this study was to determine the frequency of common NOD2/CARD15 mutations in Hungarian Crohn's patients. PATIENTS AND METHODS: DNA was obtained from 142 pts with Crohn's disease (70 male and 72 female, mean age: 36.2 yrs) and of 115 healthy subjects. Clinical data were obtained by filling in a questionnaire by the physician. DNA was screened for possible mutations by denaturing HPLC (WAVE Nucleic acid fragment analysis systems, Cheshire, UK) using published primers (Lesage et al, Am J Hum Gen 2002) for SNP's 8, 12, and 13. Mutations were then confirmed by direct sequencing on a ABI Prism 310 Genetic Analyzer (Perkin Elmer; Norwalk, USA). RESULTS: Mutations of NOD2/CARD15 were significantly more frequent in patients (n = 42, 29.6%) than in controls (11.3%, p = 0.0007, OR = 3.3, 95% CI = 1.7-6.5). R702W and 3020insC mutations were significantly more common in IBD compared to controls (4.3% and 2.6%); 18 patients had the R702W mutation (12.7%, p = 0.001, 13 homozygous, one homozygous for R703C) and 22 the 3020insC (15.9%, p = 0.001, 4 homozygous). 7 patients (4.9%) were heterozygous for the G908R, which was not different from the controls (4.3%). 5 patients were compound heterozygous. The allele frequency of R702W (11.6% vs. 4.3%, p = 0.0026, OR = 5.9, 95% CI: 2.3-14.9) and 3020insC (9.1% vs. 2.6% p = 0.004, OR = 7.6, 95% CI = 2.4-24.0) was significantly higher in Crohn's disease compared to the controls. Age at presentation was not different between carriers and non-carriers (26.7 +/- [SD] 10.3 yrs vs. 27.7 +/- 11.8 yrs). There was a tendency of ileal location to be more common in carriers of the mutation (40.5% vs. 29.0%), while colonic location was less frequent (16.7% vs. 44%). The presence of the mutation did not affect disease behaviour (carrier: inflammatory: 30.9%, stenosing: 26.2%, penetrating: 42.9%, non-carrier: inflammatory: 36%, stenosing: 27%, penetrating: 37%). Among the extraintestinal manifestations arthritis (30.1% vs. 47%, p = 0.05) and primary sclerosing cholangitis (0% vs. 9%, p = 0.047%) was significantly less frequent in carriers of the mutation. CONCLUSIONS: In concordance with European data we found a high number of NOD2/CARD15 R702W and 3020insC mutations in Hungarian patients with Crohn's disease. The G908R mutation was uncommon in Hungarian Crohn's patients. The presence of the mutation was associated with ileal but not with fibrostenosing disease and extraintestinal manifestations were less common in carriers of the mutation.