Feasibility of RNA studies on illegitimate transcription for molecular characterization of splicing mutations in the ATP7B gene: a case report.

Approximately 520 Wilson disease-causing mutations in the ATP7B gene have been described to date. In this study we report DNA and RNA analyses carried out for molecular characterization of a consensus sequence splicing mutation found in homozygosity in a Swiss Wilson disease patient. RNA analysis of 1946 +6 T→C in both the peripheral lymphoblasts and liver resulted in the production in the propositus of only an alternative transcript lacking exons 6, 7, and 8 resulting most likely in alterations of cell biochemistry and disease. The patient presents an early form of severe hepatic disease characterized by hepatosplenomegaly, reduced hepatic function, anemia and thrombocytopenia indicating that 1946 +6 T→C is a severe mutation. Since identical results were obtained from both peripheral lymphoblasts and liver they also suggest that RNA studies of illegitimate transcripts can be safely used for molecular characterization of ATP7B splicing mutations, thus improving genetic counseling and diagnosis of Wilson disease. Moreover these studies, contribute to reveal the exact molecular mechanisms producing Wilson disease.

Mutation analysis of the ATP7B gene in a new group of Wilson's disease patients: contribution to diagnosis.

Wilson's disease (WD), an autosomal recessive disorder of copper transport with a broad range of genotypic and phenotypic characteristics, results from mutations in the ATP7B gene. Herein we report the results of mutation analysis of the ATP7B gene in a group of 118 Wilson disease families (236 chromosomes) prevalently of Italian origin. Using DNA sequencing we identified 83 disease-causing mutations. Eleven were novel, while twenty one already described mutations were identified in new populations in this study. In particular, mutation analysis of 13 families of Romanian origin showed a high prevalence of the p.H1069Q mutation (50%). Detection of new mutations in the ATP7B gene in new populations increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.

DNA and RNA studies for molecular characterization of a gross deletion detected in homozygosity in the NH2-terminal region of the ATP7B gene in a Wilson disease patient.

Wilson disease is an autosomal recessive disorder caused by defective function of the copper transporting protein ATP7B. Approximately 520 Wilson disease-causing mutations have been described to date. In this study we report the use of DNA and RNA analysis for molecular characterization of a gross deletion of the ATP7B gene detected in homozygosity in a Wilson disease patient. The c.51+384_1708-953del mutation spans an 8798 bp region of the ATP7B gene from exon 2 to intron 4. The results obtained suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counselling and diagnosis of Wilson disease. Moreover these studies, help to better establish the molecular mechanisms producing Wilson disease.

Development of TaqMan allelic specific discrimination assay for detection of the most common Sardinian Wilson's disease mutations. Implications for genetic screening.

Wilson's disease (WD) is an autosomal recessive disorder caused by a defective function of the copper transporting ATP7B protein. Analysis of ATP7B gene in the Sardinian population revealed the presence of six common mutations that together account for 85% of WD chromosomes. We have developed an automated approach for the detection of these 6 common Sardinian mutations based on TaqMan technology. Ten DNA samples of WD patients carrying different combinations of the six most common Sardinian mutations and normal controls previously analysed were used in triplicate to set up the allelic discrimination assays. The system was validated in 96 samples obtained from WD patients carrying different combinations of the most common mutations under investigation. The results showed that allelic discrimination is a valid method that could be used for efficient diagnosis of single cases but also for a mass screening.

High incidence and allelic homogeneity of Wilson disease in 2 isolated populations: a prerequisite for efficient disease prevention programs.

OBJECTIVES: Herein we report the results of mutation-based screening for Wilson disease (WD) in 2 isolated populations of Sardinia and the Greek island of Kalymnos. PATIENTS AND METHODS: Mutation analysis was performed in 110 and 9 WD families originating respectively from Sardinia and Kalymons using single-strand conformation polymorphism and sequencing methods. In Sardinia, a limited screening was performed for -441/-427del in 5290 newborns, whereas in Kalymnos 397 newborns underwent mutation screening for H1069Q and R969Q using appropriate methods. RESULTS: In Sardinia, mutation analysis showed the presence of 6 mutations accounting for 85% of chromosomes, 1 of which (-441/-427del) is present in 61.7% of alleles. The screening for -441/-427del in 5290 newborns revealed the presence of 122 heterozygotes, which is equal to an allelic frequency of 1.15%. Assuming the same distribution of WD mutations in the general Sardinian population, we also inferred an allelic frequency of 0.77% for mutations other than -441/-427del, which accounts for an overall frequency of any WD mutation of 1.92%. Assuming Hardy-Weinberg equilibrium, these data could be translated into a WD incidence of 1 in 2707 live births. In Kalymnos, mutation analysis in 9 WD families revealed the presence of only 2 mutations. The screening of 397 newborns revealed the presence of 18 heterozygotes for H1069Q, 9 for R969Q, and 1 compound heterozygote for these mutations, which is equal to an allele frequency of 3.7%. Assuming Hardy-Weinberg equilibrium, the expected carrier rate is 7%. CONCLUSIONS: These data indicate the need for health education for WD prevention in these isolated populations.

Twenty-four novel mutations in Wilson disease patients of predominantly Italian origin.

Herein we report the results of mutation analysis of the ATP7B gene in a group of 134 Wilson disease (WD) families (268 chromosomes) prevalently of Italian origin. Using the SSCP and sequencing methods we identified 71 disease-causing mutations. Twenty-four were novel, while 19 more mutations already described, were identified in new populations in this study. A known mutation G591D showed a regional distribution, since it was only detected in 38.5% of the analyzed chromosomes in WD patients originating from Apulia, a region of South Italy. Detection of new mutations in the ATP7B gene increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.

Wilson's disease in an adult asymptomatic patient: a potential role for modifying factors of copper metabolism.

Diagnosis of Wilson's disease (WD) still remains a challenge since no single test has an accuracy of 100%. Molecular testing for ATP7B gene mutations can help reach the diagnosis when routine testing is equivocal. We herein report an asymptomatic WD patient diagnosed accidentally by genetic analysis. Th is case suggests that WD is a challenge even in particular contexts such as family screening. Genetic testing of ATP7B gene should be recommended in the family members of WD patients with minimal alterations of specific tests such as ceruloplasmin, and presence of steatosis or increased body mass index.

The homozygosity index (HI) approach reveals high allele frequency for Wilson disease in the Sardinian population.

Wilson disease (WD) is an autosomal recessive disorder resulting in pathological progressive copper accumulation in liver and other tissues. The worldwide prevalence (P) is about 30/million, while in Sardinia it is in the order of 1/10,000. However, all of these estimates are likely to suffer from an underdiagnosis bias. Indeed, a recent molecular neonatal screening in Sardinia reported a WD prevalence of 1:2707. In this study, we used a new approach that makes it possible to estimate the allelic frequency (q) of an autosomal recessive disorder if one knows the proportion between homozygous and compound heterozygous patients (the homozygosity index or HI) and the inbreeding coefficient (F) in a sample of affected individuals. We applied the method to a set of 178 Sardinian individuals (3 of whom born to consanguineous parents), each with a clinical and molecular diagnosis of WD. Taking into account the geographical provenance of the parents of every patient within Sardinia (to make F computation more precise), we obtained a q=0.0191 (F=7.8 × 10(-4), HI=0.476) and a corresponding prevalence P=1:2732. This result confirms that the prevalence of WD is largely underestimated in Sardinia. On the other hand, the general reliability and applicability of the HI approach to other autosomal recessive disorders is confirmed, especially if one is interested in the genetic epidemiology of populations with high frequency of consanguineous marriages.

Wilson's disease in two consecutive generations: the detection of three mutated alleles in the ATP7B gene in two Sardinian families.

BACKGROUND: Wilson's disease diagnosis is still a challenge for clinicians. AIM: To underline the importance of genetic testing in carrier detection and diagnosis of atypical Wilson's disease cases. METHODS: Two families with Wilson's disease in two consecutive generations were analysed with clinical, biochemical and genetic testing. RESULTS: In one family with triplet siblings, two of whom monozygotic, molecular screening of ATP7B, the gene responsible for Wilson's disease phenotype, allowed detection of 3 disease alleles, the discrimination between carrier and disease state and the postmortem diagnosis of Wilson's disease in the siblings' father. In the second family, molecular analysis detected 3 disease alleles and confirmed the diagnosis of Wilson's disease in two asymptomatic monozygotic twins. CONCLUSION: These results demonstrate that mutational analysis is determinant for carrier identification and diagnosis of atypical Wilson's disease patients.

RNA analysis of consensus sequence splicing mutations: implications for the diagnosis of Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder caused by a defective function of the copper-transporting ATP7B protein. This results in progressive copper overload and consequent liver, brain, and kidney damage. Approximately 300 WD-causing mutations have been described to date. Missense mutations are largely prevalent, while splice-site mutations are rarer. Of these, only a minority are detected in splicing consensus sequences. Further, few splicing mutations have been studied at the RNA level. In this study we report the RNA molecular characterization of three consensus splice-site mutations identified by DNA analysis in WD patients. One of them, c.51 + 4 A --> T, resides in the consensus sequence of the donor splice site of intron 1; the second, c. 2121 + 3 A --> G, occurred in position + 3 of intron 7; and the c.2447 + 5 G --> A is localized in the consensus sequence of the donor splice site of intron 9. Analysis revealed predominantly abnormal splicing in the samples carrying mutations compared to the normal controls. These results strongly suggest that consensus sequence splice-site mutations result in disease by interfering with the production of the normal WD protein. Our data contribute to understanding the mutational spectrum that affect splicing and improve our capability in WD diagnosis.