ATP7B Gene Mutations in Croatian Patients with Wilson Disease.

AIMS: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, characterized by its accumulation in tissues which results in hepatic, neurological, and/or psychiatric symptoms. The aim of this study was to investigate the genetics of WD in Croatian patients. METHODS: Correlation of the clinical presentation subtype and the age at onset of the diagnosis of WD with the ATP7B genotype was investigated in a group of Croatian WD patients. DNA from peripheral blood samples was tested for the p.His1069Gln by direct mutational analysis and other polymorphisms were identified by sequence analysis of coding and flanking intronic regions of ATP7B gene. RESULTS: In the group of 75 WD patients of Croatian origin, 18 different mutations in ATP7B gene were detected, three of which were novel. The p.His1069Gln mutation was most frequent, being detected in 44 Croatian WD patients (58.7%). Most ATP7B mutations (90.4%) were located in exons 5, 8, 13, 14, and 15. CONCLUSIONS: Clinical diagnosis of WD was confirmed in 59 patients by detecting mutations on both ATP7B alleles. The age at onset of WD and the type of WD clinical presentation showed no significant correlation with the ATP7B genotype.

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.

Wilson's disease caused by alternative splicing and Alu exonization due to a homozygous 3039-bp deletion spanning from intron 1 to exon 2 of the ATP7B gene.

We describe a case of Wilson's disease (WD) diagnosed at 5 years after routine biochemical test showed increased aminotransferases. Mutation analysis of the ATP7B gene revealed a 3039-bp deletion in the homozygous state spanning from the terminal part of intron 1 to nt position 368 of exon 2. This deletion results in the activation of 3 cryptic splice sites: an AG acceptor splice site in nt positions 578-579 producing a different breakpoint and removing the first 577 nts of exon 2, an acceptor and a donor splice site in nt positions 20363-4 and 20456-7, respectively, in intron 1, resulting in the activation of a 94-bp cryptic Alu exon being incorporated into the mature transcript. The resulting alternative transcript contains a TAG stop codon in the first amino acid position of the cryptic exon, likely producing a truncated, non-functional protein. This study shows that intron exonization can also occur in humans through naturally occurring gross deletions. The results suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counseling and diagnosis of WD. Moreover these studies help to better establish new molecular mechanisms producing Wilson's disease.

Wilson's disease.

Wilson's disease (WD) is a disorder of copper transport resulting from the defective function of a copper transporting P-type ATPase, ATP7B. The WD incidence is approximately 1/50-10,000 live births worldwide. Clinical manifestations of WD may be of any kind, but usually the symptoms of presentation are hepatic or neuropsychiatric, with a vast range of disturbances for both groups of symptoms. In children, however, clinical symptoms may be absent, making the diagnosis of the disease more difficult than in adults. Hepatic manifestations may range from asymptomatic minor biochemical disturbances, to acute, but mostly chronic, hepatitis, cirrhosis or severe fulminant hepatic failure. The spectrum of neurological manifestations is wide, including tremor, hypersalivation, Dysarthria, coordination defects, dystonia, ataxia. The spectrum of psychiatric manifestations is considerable and may include different disturbances such as altered working performance, anxiety, depression and antisocial behaviour. Kayser-Fleischer rings (KF) are present in 95% of patients with neurological symptoms and somewhat over half of those without neurological symptoms. In children presenting with liver disease, KF rings are usually absent. To obtain a more reliable diagnosis of WD, the Leipzig scoring system was proposed by an international consensus of experts. Wilson's disease copper overload is treated with chelating agents such as penicillamine, trientine and tetrathiomolybdate. Zinc is used mostly for mantainance therapy or the treatment of asymptomatic WD patients.

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.

Allan-Herndon-Dudley syndrome (AHDS) in two consecutive generations caused by a missense MCT8 gene mutation. Phenotypic variability with the presence of normal serum T3 levels.

Allan-Herndon-Dudley syndrome (AHDS), an X linked condition, is characterized by severe intellectual disability, dysarthria, athetoid movements, muscle hypoplasia and spastic paraplegia in combination with altered TH levels, in particular, high serum T3 levels. Mutations in the MCT8 gene coding for the monocarboxylate thyroid hormone transporter 8 have been associated with AHDS. Here we describe a family with the presence of a MCT8 gene mutation, p.A224T, in three consecutive generations. In two generations its presence was detected in the hemizygous state in two males with neurological abnormalities including mental retardation, axial hypotonia, hypertonia of arms and legs and athetoid movements. One of them presented normal thyroid hormone levels. Mutation was also detected, although in the heterozygous state, in three females showing thyroid hormone levels in the normal range. Our results show the difficulty of distinguishing AHDS from patients with X-linked intellectual disability solely on the basis of clinical features and biochemical tests, and we advise screening for MCT8 mutations in either young or older patients with severe intellectual disability, axial hypotonia/dystonia, poor head control, spastic paraplegia, and athetoid movements even when they have normal thyroid hormone profiles.

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.

A new deletion in 5'-end of dystrophin gene removing M and P promoters and dystrophin muscle enhancers.

We describe a 3-year-old boy who, at age of 8 months, during investigations for upper respiratory tract infection was found to have an incidental grossly elevated CK of 20,000 UI/l. Investigations showed only mild calf hypertrophy and absent Gower's sign, normal cognitive function. Electromyography (EMG) showed myopathic features. Electrocardiography and echocardiography were normal. His muscle biopsy revealed myopathic features indicating Duchenne-type dystrophy. Immunohistochemistry for dystrophin N-terminal, C-terminal and mid-rod antibodies analysis showed the complete absence of dystrophin in the muscle fibers. Genetic studies showed a 141.1 Kb deletion removing muscle promoter, muscle exon 1, Purkinje promoter, Purkinje exon 1, dystrophin muscle enhancers similar to one previously reported in a DMD patient who exhibited some residual expression of dystrophin. The difference in dystrophin expression between these two patients might be due to the extension of deletions. The precise delimitation of the macrodeletion here described provides a better understanding of functional organization of the 5' end of the DMD gene.

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.

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.

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.

Allan-Herndon-Dudley syndrome (AHDS) caused by a novel SLC16A2 gene mutation showing severe neurologic features and unexpectedly low TRH-stimulated serum TSH.

Thyroid hormones are known to be essential for growth, development and metabolism. Recently mutations in the SLC16A2 gene coding for the monocarboxylate thyroid hormone transporter 8, MCT8, have been associated with Allan-Herndon-Dudley syndrome (AHDS), an X-linked condition characterized by severe mental retardation, dysarthria, athetoid movements, muscle hypoplasia and spastic paraplegia. Here we describe in detail the clinical and biochemical features in a boy affected by AHDS with severe neurological abnormalities and a novel de novo SLC16A2 gene insertion, 1343-1344insGCCC, resulting in a truncated protein lacking the last four transmembrane domains (TMDs) as well as the carboxyl cytoplasmic end. He presents mental retardation, axial hypotonia, hypertonia of arms and legs, paroxysmal dyskinesias, seizures. The endocrine phenotype showed low serum total and free thyroxine (T4), very elevated total and free triiodothyronine (T3) and normal thyrotropin (TSH) with blunted response to thyrotropin-releasing hormone (TRH). The latter finding was unexpected and suggested that the lack of functional MCT8 was counterbalanced at the thyrotrope cell level by high serum T3 concentration and/or by increased intrapituitary type 2 deiodinase (D2) activity. Our case constitutes a relevant contribution to better characterize this disorder and to elucidate the functional consequences of SLC16A2 gene mutations.

Molecular analysis of glycogen storage disease type Ib in Sardinian population: evidence for a founder effect.

We describe epidemiological, genetic, and clinical data of the 1124-2del mutation in the G6PT gene, detected in homozygosity in three glycogen storage disease type Ib patients of Sardinian origin. This mutation was found to be associated with four sequence variations: c.593 A>T (p.N198I), c.625+19 C>T, c.1062 C>T (N354N), and c.1224 G>A (p.T408T) in the G6PT gene. RNA studies were performed for c.1124-2del and c.625+19 C>T. The c.1124-1del2 acceptor splicing mutation showed skipping of 31 nucleotides of exon 9 due to the activation of a downstream cryptic acceptor splice site in 1154-1155 nucleotide positions, resulting in a downstream stop codon at aa position 402. RNA analysis of c.625+19 C>T variation showed a small amount of alternative splicing with skipping of exon 4, resulting in a stop codon at aa position 211. Our cases present most of features of the severe form of disease, including early onset with chronic neutropenia, frequent infections, and inflammatory bowel disease. Our results suggest a founder effect for glycogen storage disease type Ib that facilitates diagnosis using mutation analysis, sparing patients from liver biopsy. DNA-based diagnosis will enable us to make accurate determination of carrier status and prenatal diagnosis, thus improving genetic counseling.

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.

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.

Genotype-phenotype correlation in Italian children with Wilson's disease.

BACKGROUND/AIMS: Wilson's disease phenotype is very variable for clinical and laboratory features. Our aim was to assess the role of the type of ATP7B disease causing mutations on Wilson's disease phenotype. METHODS: We retrospectively evaluated the data of children with Wilson's disease from eight pediatric departments. RESULT: Fifty-eight patients (34 male, median age at diagnosis 7.4 years) from 47 unrelated families were studied, carrying 34 different mutations. The most common mutations were the missense p.H1069Q and p.M769V, the nonsense p.R1319X, the frameshift c.2299delC, c.2298_2299insC and c.2530delA, and the splice site mutation c.2447+5G>A. Serum ceruloplasmin and copper were lower among the patients' homozygotes for nonsense and frameshift mutations than in patients with missense mutations. A normalization of serum alanine aminotransferase after therapy was not achieved in 23.6% of patients with missense mutations versus 45.5% of patients with nonsense/frameshift mutations. A direct linear correlation was found between age at diagnosis and urinary copper excretion at diagnosis. CONCLUSIONS: The type of mutation explains at least a part of Wilson's disease phenotype, and mutation analysis should be considered as an integrative tool for such a challenging diagnosis. Urinary copper excretion appears to be correlated to the age at diagnosis rather than genotype.

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.