Synonymous mutation in adenosine triphosphatase copper-transporting beta causes enhanced exon skipping in Wilson disease.

Wilson disease (WD) is caused by biallelic pathogenic variants in adenosine triphosphatase copper-transporting beta (ATP7B); however, genetic testing identifies only one or no pathogenic ATP7B variant in a number of patients with WD. Synonymous single-nucleotide sequence variants have been recognized as pathogenic in individual families. The aim of the present study was to evaluate the prevalence and disease mechanism of the synonymous variant c.2292C>T (p.Phe764=) in WD. A cohort of 280 patients with WD heterozygous for a single ATP7B variant was investigated for the presence of c.2292C>T (p.Phe764=). In this cohort of otherwise genetically unexplained WD, the allele frequency of c.2292C>T (p.Phe764=) was 2.5% (14 of 560) compared to 7.1 × 10-6 in the general population (2 of 280,964 in the Genome Aggregation Database; p < 10-5 ; Fisher exact test). In an independent United Kingdom (UK) cohort, 2 patients with WD homozygous for p.Phe764= were identified. RNA analysis of ATP7B transcripts from patients homozygous or heterozygous for c.2292C>T and control fibroblasts showed that this variant caused high expression of an ATP7B transcript variant lacking exon 8. Conclusion: The synonymous ATP7B variant c.2292C>T (p.Phe764=) causes abnormal messenger RNA processing of ATP7B transcripts and is associated with WD in compound heterozygotes and homozygotes.

Intragenic Deletions in ATP7B as an Unusual Molecular Genetics Mechanism of Wilson's Disease Pathogenesis.

Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B resulting in copper overload in the liver and brain. Direct sequencing is routinely used to confirm WD diagnosis; however, partial and whole gene deletions in the heterozygous state cannot be detected by exon amplification since the normal allele will mask its presence. The aim of the present work was to search for unusual mutational events in the unexplained WD cases and to provide insight into the mechanisms. Out of 1420 clinically and biochemically confirmed WD samples received between 2000 and 2014 for routine mutation analysis, we were unable to detect mutant alleles in 142 samples, after extensive sequencing analysis. We used selective amplification and MLPA to identify the partial gene deletions and identified three different partial gene deletions in seven different families. All three deletions were fully characterized at the DNA sequence level. We report the first hemizygous case with WD due to intragenic deletion in the ATP7B (c.3134_3556+689del). This novel deletion resulted from an excision event mediated by consensus sequences in an AluSq2 repeat element and could be traced to micro homologous end joining (MMEJ). Finally, we determined the prevalence of the three deletions in DNA samples from a multinational group of WD patients. Our results emphasize the need for searching mutant alleles beyond routine methods and highlight that large ATP7B deletions are rare, but account for a detectable proportion in some WD patients. Screening for gene aberrations will further improve mutation detection in patients with unidentified ATP7B mutations presenting with clinical manifestations of WD.

Wilson disease mutation pattern with genotype-phenotype correlations from Western India: confirmation of p.C271* as a common Indian mutation and identification of 14 novel mutations.

Wilson disease (WD) is an autosomal recessive disorder resulting from mutations in the ATP7B gene, with over 600 mutations described. Identification of mutations has made genetic diagnosis of WD feasible in many countries. The heterogeneity of ATP7B mutants is, however, yet to be identified in the Indian population. We analyzed the mutational pattern of WD in a large region of Western India. We studied patients (n = 52) for ATP7B gene mutations in a cohort of families with WD and also in first-degree relatives (n = 126). All 21 exon-intron boundaries of the WD gene were amplified and directly sequenced. We identified 36 different disease-causing mutations (31 exonic and five intronic splice site variants). Fourteen novel mutations were identified. Exons 2, 8, 13, 14, and 18 accounted for the majority of mutations (86.4%). A previously recognized mutation, p.C271*, and the novel mutation p.E122fs, were the most common mutations with allelic frequencies of 20.2% and 10.6%, respectively. Frequent homozygous mutations (58.9%) and disease severity assessments allowed analysis of genotype-phenotype correlations. Our study significantly adds to the emerging data from other parts of India suggesting that p.C271* may be the most frequent mutation across India, and may harbor a moderate to severely disabling phenotype with limited variability.

Overexpressed ATP7B protects mesenchymal stem cells from toxic copper.

Wilson's disease (WD) is characterized by accumulation of high levels of copper in liver due to malfunction of copper transporter ATP7B which is central for copper homeostasis. Here we report for the first time that mesenchymal stem cells (MSC) derived from bone marrow express detectable levels of ATP7B. The role of ATP7B overexpression for MSC survival and selection in high copper was investigated. Hepatoma cell line HepG2 that has a high intrinsic expression of ATP7B served as a control. Using retroviral vector a significant higher expression level of ATP7B could be achieved in MSCs. Whereas copper treatment resulted in cell death in untransduced MSCs, viability assays demonstrated a unique copper resistance of ATP7B overexpressing MSCs that outcompeted HepG2. In long-term cell culture stable transgene expression for up to 9weeks was shown for ATP7B overexpressing MSCs which rapidly overgrew untransduced cells. Our findings suggest that ATP7B overexpression provides an important selection advantage to MSCs in high copper microenvironments, and may represent novel cell transplants for therapy of WD.

Prevalence of germline mutations in the TTR gene in a consecutive series of surgical pathology specimens with ATTR amyloid.

Transthyretin-derived amyloidosis (ATTR) amyloidosis is the third most prevalent amyloid type in surgical pathology and may occur as a hereditary disease with germline mutations in the TTR gene or as senile systemic amyloidosis (SSA) without mutations. Distinction between hereditary ATTR amyloidosis and SSA is of central importance, as the former necessitates genetic counseling and can be treated by liver transplantation. However, little is known about the prevalence of hereditary ATTR amyloidosis in surgical pathology specimens. We have examined the distribution of hereditary ATTR amyloidosis and SSA in a consecutive series of surgical pathology specimens with histologically and immunohistochemically confirmed ATTR amyloid. Thirty-three consecutive patients were retrieved from the Amyloid Registry of the Charité University Hospital. Genomic DNA was extracted from formalin-fixed and paraffin-embedded tissue or patient blood and examined by DNA sequencing. ATTR amyloid was found in the gastrointestinal tract, endomyocardium, peripheral nerve, carpal tunnel ligament, synovia, breast, and testicle. Amyloid fibrils were present as interstitial and vascular deposits, as evidenced by Congo red staining. TTR gene mutations were detected in 12 of 30 patients, with p.Val30Met being the most prevalent (5 patients). Furthermore, 2 novel mutations (p.Asp39Val and p.Glu54Asp) were found. In patients carrying a mutation, ATTR amyloid was found in the gastrointestinal tract, myocardium, nerve, and testicles. To conclude, the hereditary form of ATTR amyloid seems to be more common in elderly patients than previously thought. It is, therefore, important to genetically test every patient when diagnosing ATTR amyloidosis.

No association of the CARD8 (TUCAN) c.30T>A (p.C10X) variant with Crohn's disease: a study in 3 independent European cohorts.

BACKGROUND: A recent study reported that the c.30T>A (p.Cys10Ter; rs2043211) variant, in the CARD8 (TUCAN) gene, is associated with Crohn's disease (CD). The aim of this study was to analyze the frequency of p.C10X in 3 independent European (IBD) cohorts from Germany, Hungary, and the Netherlands. METHODS: We included a European IBD cohort of 921 patients and compared the p.C10X genotype frequency to 832 healthy controls. The 3 study populations analyzed were: (1) Germany [CD, n = 317; ulcerative colitis (UC), n = 180], (2) Hungary (CD, n = 149; UC, n = 119), and (3) the Netherlands (CD, n = 156). Subtyping analysis was performed in respect to NOD2 variants (p.Arg702Trp, p.Gly908Arg, c.3020insC) and to clinical characteristics. Ethnically matched controls were included (German, n = 413; Hungarian, n = 202; Dutch, n = 217). RESULTS: We observed no significant difference in p.C10X genotype frequency in either patients with CD or patients with UC compared with controls in all 3 cohorts. Conversely to the initial association study, we found a trend toward lower frequencies of the suggestive risk wild type in CD from the Netherlands compared with controls (P = 0.14). We found neither evidence for genetic interactions between p.C10X and NOD2 nor the C10X variant to be associated with a CD or UC phenotype. CONCLUSIONS: Analyzing 3 independent European IBD cohorts, we found no evidence that the C10X variant in CARD8 confers susceptibility for CD.

A study in three European IBD cohorts confirms that the ATG16L1 c.898A>G (p.Thr300Ala) variant is a susceptibility factor for Crohn's disease.

BACKGROUND AND AIMS: A recent study reported that a nonsynonymous SNP rs2241880 (c.898A>G, p.Thr300Ala) within ATG16L1 confers susceptibility to Crohn's disease (CD). We analyzed ATG16L1 c.898A>G in three independent European inflammatory bowel disease (IBD) cohorts from Germany, Hungary and the Netherlands. METHODS: In total, we included 910 European IBD patients and compared the ATG16L1 c.898A>G genotype frequency with 707 ethnically matched healthy controls. We included patients from 3 populations originating from Germany (CD n=310; ulcerative colitis [UC] n=179), Hungary (CD n=147; UC n=117), and the Netherlands (CD n=157). Subtyping analysis was performed in respect to CARD15 alterations and clinical characteristics. RESULTS: We found a highly significant association of c.898A>G to CD. The association was significant (p=0.0005) for the total CD cohort but also for the individual populations from Germany (p=0.02) and Netherlands (p=0.02) whereas in the Hungarian CD patients a clear trend was observed (p=0.19; OR 1.227, 95% CI 0.910; 1.654). No association was found between c.898A>G and UC. No statistical interactions were observed between ATG16L1 c.898A>G and CARD15 variants. Furthermore no association to a CD subphenotype was detected. CONCLUSIONS: We confirm that ATG16L1 variant c898A>G confers a risk variant for CD but is not associated with a distinct CD phenotype.