Call for participation in the neurogenetics consortium within the Human Variome Project.

The rate of DNA variation discovery has accelerated the need to collate, store and interpret the data in a standardised coherent way and is becoming a critical step in maximising the impact of discovery on the understanding and treatment of human disease. This particularly applies to the field of neurology as neurological function is impaired in many human disorders. Furthermore, the field of neurogenetics has been proven to show remarkably complex genotype-to-phenotype relationships. To facilitate the collection of DNA sequence variation pertaining to neurogenetic disorders, we have initiated the "Neurogenetics Consortium" under the umbrella of the Human Variome Project. The Consortium's founding group consisted of basic researchers, clinicians, informaticians and database creators. This report outlines the strategic aims established at the preliminary meetings of the Neurogenetics Consortium and calls for the involvement of the wider neurogenetic community in enabling the development of this important resource.

Functional analysis of mutations in the ATP loop of the Wilson disease copper transporter, ATP7B.

Wilson disease (WND) is an autosomal recessive disorder resulting from mutation of ATP7B. Transport of copper by ATP7B from the trans-Golgi of hepatocytes into apical membrane-trafficked vesicles for excretion in the bile is the major means of copper elimination from the body. Although copper is an essential nutrient, homeostasis must be carefully maintained. If homeostasis is disrupted, copper can accumulate within the liver, kidney, cornea, and/or brain. The range of organs affected leads to clinical heterogeneity and difficulty in WND diagnosis. Sequencing of ATP7B is an important adjunct for diagnosis but has led to the discovery of many novel missense variants. Although prediction programs are available, functional characterization is essential for determining the consequence of novel variants. We have tested 12 missense variants localized to the ATP loop of ATP7B and compared three predictive programs (SIFT, PolyPhen, and Align-GVGD). We found p.L1043P, p.G1000R, p.G1101R, p.I1102T, p.V1239G, and p.D1267V deleterious; p.G1176E and p.G1287S intermediate; p.E1173G temperature sensitive; p.T991M and p.I1148T mild; and p.R1228T functioning as wild type. We found that SIFT most often agreed with functional data (92%), compared with PolyPhen (83%) and Align-GVGD (67%). We conclude that variants found to negatively affect function likely contribute to the WND phenotype in patients.

A child with terminal 14q deletion syndrome: consideration of genotype-phenotype correlations.

Patients with terminal deletions of chromosome 14 usually share a number of clinical features. The syndrome is thought not to be associated with multiple congenital anomalies. We report on a patient having a terminal deletion of about 3.2 Mb, with the breakpoint in 14q32.32. Multiple health problems led to his early death. By molecular techniques (array comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH)), we identified two previously reported patients with deletions in the terminal part of chromosome 14 of almost exactly the same size and compare the phenotypes of all three children. The phenotype of the current patient is much more severe than the phenotypes of the two patients reported previously. The patients also present different sets of dysmorphic features described previously as characteristic for 14q deletion syndrome. Molecular cytogenetic mapping showed that the breakpoints in all three patients were clustered within a 240 kb interval. The possibility of recurrent breakpoint location in terminal 14q deletion syndrome, as well as detailed characterization of the spectrum of phenotypes associated with the syndrome, will require the investigation of multiple patients with similar deletions in 14q.

Sequence variation in the ATP-binding domain of the Wilson disease transporter, ATP7B, affects copper transport in a yeast model system.

ATP7B is a copper transporting P-type ATPase defective in the autosomal recessive copper storage disorder, Wilson disease (WND). Functional assessment of variants helps to distinguish normal from disease-causing variants and provides information on important amino acid residues. A total of 11 missense variants of ATP7B, originally identified in WND patients, were examined for their capacity to functionally complement a yeast mutant strain in which the yeast gene ortholog, CCC2, was disrupted. Solution structures of ATP7B domains were used to predict the effects of each variant on ATP7B structure. Three variants lie within the copper-binding domain and eight within the ATP-binding domain of ATP7B. All three ATP7B variants within the copper-binding domain and four within the ATP-binding domain showed full complementation of the yeast ccc2 phenotype. For the remaining four located in the ATP-binding domain, p.Glu1064Lys and p.Val1106Asp were unable to complement the yeast ccc2 high-affinity iron uptake deficiency phenotype, apparently due to mislocalization and/or change in conformation of the variant protein. p.Leu1083Phe exhibited a temperature-sensitive phenotype with partial complementation at 30 degrees C and a severe deficit at 37 degrees C. p.Met1169Val only partially complemented the ccc2 phenotype at 30 degrees C and 37 degrees C. Therefore, four variant positions were identified as important for copper transport and as disease-causing changes. Since the yeast assay specifically evaluates copper transport function, variants with normal transport could be defective in some other aspect of ATP7B function, particularly trafficking in mammalian cells. Functional assessment is critical for reliable use of mutation analysis as an aid to diagnosis of this clinically variable condition.

A child with deletion (14)(q24.3q32.13) and auditory neuropathy.

An interstitial deletion in the middle and distal part of chromosome 14 is a rare chromosomal abnormality characterized by a wide spectrum of phenotypic manifestations. We present a patient with a nearly 20 Mb interstitial deletion of chromosome 14q24.3q32.13 determined by FISH, that is associated with minor dysmorphic features, developmental delay, absent speech and auditory neuropathy. The deleted region contains 130 known genes, among them 48 with reported function or association with human disease. The patient's phenotype is compared with interstitial deletions of the distal part of chromosome 14 reported previously. We hypothesize, that there is (are) a gene (genes) in the 14q32.11-q32.13 that is (are) important for the hearing process and for which haploinsufficiency can cause auditory neuropathy. Several genes in the region, among them calmodulin, chromogranin A, the goosecoid and FOXN3, can contribute to the observed phenotype. Detailed mapping in additional patients with 14q32 deletions and hearing loss could further define the candidate region.

New mutations in the Wilson disease gene, ATP7B: implications for molecular testing.

Wilson disease (WND), an autosomal recessive disorder of copper transport with a broad range of genotypic and phenotypic characteristics, results from mutations in the ATP7B gene. ATP7B encodes a copper transporting P-type ATPase involved in the transport of copper into the plasma protein ceruloplasmin, and for excretion of copper from the liver. Defects in ATP7B lead to copper storage in liver, brain and kidney. Mutation analysis was carried out on 300 WND patients of various origins, and new mutations not previously reported were identified: European white (p.L217X, c.918_931, c.1073delG, c.3082_3085delAAGAinsCG, p.V536A, p.S657R, p.A971V, p.T974M, p.Q1004P, p.D1164N, p.E1173G, p.I1230V, p.M1359I, c.2355+4A>G), Sephardic Jewish (p.Q286X), Filipino (p.G1149A), Lebanese (p.R1228T), Japanese (p.D1267V) and Taiwanese (p.A1328T). All but one missense variant have strong evidence for classification as disease-causing mutations. In the patients reported here, we also identified 20 nucleotide substitutions, six not previously reported, which cause silent amino acid changes or intronic changes. Documentation and characterization of all variants is essential for accurate DNA diagnosis in WND because of the wide range of clinical and biochemical variability.

Polymorphisms in canine ATP7B: candidate modifier of copper toxicosis in the Bedlington terrier.

A COMMD1(MURR1) deletion has been reported as the cause of copper toxicosis (CT) in Bedlington terriers. Recent studies identified Bedlington terriers with copper accumulation without homozygous COMMD1 deletions. Wilson disease in humans is a copper storage disorder similar to CT caused by mutations in ATP7B, and COMMD1 has been shown to interact with the ATP7B protein. ATP7B may act as a modifier in CT, allowing for copper accumulation in Bedlington terriers with one deletion or other variations in COMMD1. In this study, ATP7B was cloned and sequence analysis conducted in a subset of Bedlington terriers from a pedigree that does not show complete association between the COMMD1 deletion and CT. Eleven polymorphisms, two in the coding region, were identified in the Bedlington terrier ATP7B gene. However, these are not unique to the Bedlington terrier and pedigree analysis suggests that ATP7B is not a modifier of COMMD1 in this subset of dogs.

Sequence variation database for the Wilson disease copper transporter, ATP7B.

Wilson disease (WND) is a disorder of copper transport resulting in copper accumulation in liver, kidney, and brain. This recessive disorder expresses variable clinical symptoms affecting liver, brain, and/or kidney. The age of onset of symptoms varies from 3 to almost 70 years, so the diagnosis for this treatable disorder is easily missed. The defective gene is a membrane P-type ATPase, with similar structure to the other metal transporting ATPases. Most patients with Wilson disease are compound heterozygotes. This report describes the database we have developed for reporting of mutations in ATP7B, the gene defective in WND. The database includes more than 518 variants (379 probable disease-causing and the remainder possible normal variants) from populations worldwide (Available at: www.medicalgenetics.med.ualberta.ca/wilson/index.php; Last accessed: 20 June 2007). The tables in this database are a valuable resource for the study of population variation and the function of the transporter, and will assist in the identification of disease and non-disease-causing sequence variants.

Rearrangement in the PITX2 and MIPOL1 genes in a patient with a t(4;14) chromosome.

We report the molecular characterization of a patient with mild craniofacial and acallosal central nervous system midline defects and a t(4;14)(q25)(q13) chromosome. With the use of flow sorted chromosomes, the translocation breakpoint junction was defined within a 100 kb region with markers mapping to chromosomes 4q25 and 14q13. Analysis of genomic sequences demonstrated that the breakpoint junction at 14q13 was within the third intron of the 5' untranslated region of the MIPOL1 gene (GI: 22048098). On chromosome 4q25, two breakpoint junctions were found. One was about 47 kb distal to the 5' end of a putative gene (GI: 8923996) with unknown function but with partial similarity to kinases, and a second breakpoint was within the 3' end of the PITX2 gene (GI: 21361182) that resulted in the deletion of exons 6 and 7 of this gene. We also searched for microdeletions in a panel of candidate genes mapping within 2 Mb of the translocation breakpoint junction on chromosomes 4 and 14, however, no evidence for deletions or rearrangements was found. The finding of two breaks on chromosome 4q25 suggests a complex microrearrangement, such as an inversion, in addition to a translocation in this patient.

Association of NPAS3 exonic variation with schizophrenia.

BACKGROUND: We previously identified the neuronal PAS3 (NPAS3) gene as a candidate gene for schizophrenia. A mother and daughter, both with schizophrenia, were carriers of a translocation, t(9;14)(q34;q13), that disrupts the NPAS3 gene. The gene is located at 14q13, a region implicated in schizophrenia and bipolar disorder in various linkage studies. NPAS3 belongs to the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor family, involved in diverse processes including the regulation of cell differentiation and circadian rhythms, and the development and function of the nervous system. METHODS: The 12 exons encoding NPAS3 were sequenced in DNA from individuals with schizophrenia. NPAS3 variants were identified in exons 6 and 12, initially in 12 patients only. These two exons were then sequenced in 83 patients and 83 controls. RESULTS AND CONCLUSION: Three common variants of NPAS3, also found in controls, showed a positive association with schizophrenia (NM_001164749: rs12434716, c.1654G>C, p=0.009; rs10141940, c.2208C>T, p=0.01; rs10142034, c.2262C>G, p=0.01). The c.1654G>C variant, results in an p.Ala552Pro change and may affect NPAS3 protein function directly. Alternatively, the three SNPs may affect the splicing of NPAS3 transcripts, as they are each located within putative exonic splicing enhancer (ESE) motifs (ESEFinder). A c.726C>T variant, identified in three patients, is located in an ESE element and is predicted to reduce the function of the motif. Other variants, identified in controls, included c.2089G>A (p.Gly697Ser) and c.2097T>C. Our identification of potentially defective NPAS3 variants supports recent studies that implicate perturbations in NPAS3 pathways in impaired neurogenesis and psychosis.

A minigene approach for analysis of ATP7B splice variants in patients with Wilson disease.

Wilson disease (WND) is an autosomal recessive condition that results in accumulation of copper in the liver and brain when a membrane bound copper transporter, ATP7B, is defective. ATP7B is expressed in hepatic, brain and kidney cells, and a defect can lead to liver, neurological and renal damage in WND patients. Presentation is variable with a broad range of age of onset and symptoms, and not all biochemical signs used in diagnosis are found in every patient. Therefore, diagnosis by mutation analysis is particularly important. To date, there are approximately 380 probable disease-causing variants in ATP7B, 33 of which are splice site variants that are predicted to affect splicing, based on their location. Few of these splice site variants have been analyzed in vivo. Some exonic variations also have the potential to affect splicing. The aim of this project was to use minigenes for transcript analysis. We have chosen exon 8 as our focus and have cloned a wild-type three-exon minigene into a mammalian expression vector. After transfection, extracted RNA was analyzed by reverse transcription PCR and accurate splicing was detected. This minigene will facilitate the analysis of the numerous potential splice variants identified in exon 8 of ATP7B, with the advantage that patient cell lines are not required for each variant.

Three atypical cases of Wilson disease: assessment of the Leipzig scoring system in making a diagnosis.

BACKGROUND/AIMS: The diagnosis of this condition in the absence of any neurological findings may pose a dilemma. In 2001, experts from The 8th International Conference on Wilson disease (WD) and Menkes disease in Leipzig, Germany proposed a scoring system that may facilitate diagnosis of WD. METHODS/RESULTS: Three patients were identified as having an atypical presentation of WD as they all presented after the age 40. Two of the three presented with established cirrhosis, and none had any neuropsychiatric manifestations. All three patients fulfilled the Leipzig diagnostic criteria proposed by EASL prior to confirmatory mutation analysis. Patient A died of liver failure despite treatment. Patients B and C have remained with stable liver disease on chelation therapy. CONCLUSIONS: We believe these patients represent a group most likely to be missed in the diagnostic work-up of liver disease due to a combination of atypical features such as older age of onset, presence of other confounders for liver disease, and sometimes absence of Kayser-Fleischer rings. The Leipzig scoring system proposed in 2003 was helpful in support of an initial diagnosis of Wilson disease in these patients, validated later by genetic testing.

Defining a holoprosencephaly locus on human chromosome 14q13 and characterization of potential candidate genes.

Holoprosencephaly (HPE) is the most common developmental field defect in patterning of the human prosencephalon and associated craniofacial structures. The genetics is complex, with 12 loci defined on 11 chromosomes. We defined a locus for HPE (HPE8) on human chromosome 14q13 between markers D14S49 and AFM205XG5, by mapping deletion intervals of affected subjects with proximal chromosome 14q interstitial cytogenetic deletions. A 35-BAC contig was built by chromosome walking. By annotation of the 2.82-Mb minimal critical region, we identified 28 possible genes. Seven genes were expressed in human fetal brain: NPAS3, SNX6, C14ORF11, C14ORF10, PAX9, NKX2.1, and C14ORF19, the last an apparent gene fragment. Molecular embryology, animal modeling, and human mutation studies were reported elsewhere for PAX9 and NKX2.1. We focused on three genes, SNX6, NPAS3, and C14ORF11, as potential candidates for HPE. Genomic structure, human expression patterns, protein cellular localization, and embryonic expression patterns of orthologous murine genes were determined, showing that the three genes have properties similar to those of known HPE genes.

FISH-mapping of telomeric 14q32 deletions: search for the cause of seizures.

Ring chromosome 14 is a rare cytogenetic disorder. Individuals with r(14) generally have developmental delay and seizures. Other features include hypotonia, microcephaly, mild facial dysmorphism, and retinal pigmentation. Most of these features are also found in patients with linear terminal deletions of chromosome 14, except for seizures and retinal abnormalities. The objective of the study was to determine if deletion of a specific chromosome region is a possible explanation for the occurrence of seizures in patients with ring chromosome 14. Patients diagnosed either with r(14) (six patients) or a deletion of distal 14q (three patients) were analyzed by FISH (fluorescence in situ hybridization) with BAC probes. We observed differences in the size of deletions in the studied group. In two r(14) patients, we did not detect any deletion; the four other patients had deletions of various sizes, ranging from 0.8 Mb to 5 Mb. Two linear deletions were 3.2 Mb and 5.3 Mb in length, respectively; the third case had an interstitial deletion that did not overlap with the others. The deleted regions in ring chromosomes showed overlap with those in the two linear terminal deletions. We conclude that there is unlikely to be a specific deleted locus in 14q32.3 that predisposes r(14) patients to seizures or retinal pigmentation. The cause is probably related to the formation of the ring itself and the effect this may have on local chromatin structure.

A comparison of the mutation spectra of Menkes disease and Wilson disease.

The genes for two copper-transporting ATPases, ATP7A and ATP7B, are defective in the heritable disorders of copper imbalance, Menkes disease (MNK) and Wilson disease (WND), respectively. A comparison of the two proteins shows extensive conservation in the signature domains, with amino acid identities outside of the conserved domains being limited. The mutation spectra of MNK and WND were compared to confirm and refine further regions critical for normal function. Mutations were found to be relatively widespread; however, the majority was concentrated within defined functional domains and membrane-spanning segments, reinforcing the importance of these regions for protein function. Of the total published point mutations in ATP7A, 23.0% are splice-site, 20.7% nonsense, 17.2% missense, and 39.1% small insertions/deletions. There is a high prevalence (58.2%) of missense mutations in ATP7B. For the other mutations in ATP7B, 7.4% are splice-site, 7.4% nonsense, and 27.0% small insertions/deletions. A region of possible importance is the intervening sequence between the last copper-binding domain and the first transmembrane helix, as this region has a high percentage of MNK mutations. Similarly, the region containing the ATP-binding domain has 24.6% of all WND mutations. The study of mutation locations is useful for defining critical regions or residues and for efficient molecular diagnosis.

Expression in mouse kidney of membrane copper transporters Atp7a and Atp7b.

Copper is essential for activity of many enzymes, but is toxic in excess. Several copper proteins are required for copper homeostasis. ATP7A and ATP7B are genes encoding membrane copper transporters. ATP7A, defective in Menkes disease (MNK), is expressed in many tissues involved primarily in copper uptake from dietary sources. ATP7B, defective in Wilson disease (WND), is essential for copper excretion. Although MNK patients have a copper deficiency in most tissues, copper accumulates in proximal tubules in the kidney. WND patients also have copper accumulation in the proximal tubules. In some WND patients this copper accumulation may result in tubular dysfunction, resulting in the increased excretion of low molecular weight substances (e.g. amino acids and calcium). In mouse, we have demonstrated, by in situ hybridization, the expression pattern in the kidney of mouse orthologues, Atp7a and Atp7b, and have confirmed Atp7b expression by immunohistochemistry. Both Atp7a and Atp7b are expressed in glomeruli; however, Atp7b is also seen in the kidney medulla. This suggests that glomeruli are responsible for regulating copper levels in the filtrate. In WND patients, urinary copper levels are extremely high suggesting Atp7b in the loops of Henle may have a role in copper reabsorption.

Copper transporting P-type ATPases and human disease.

Copper transporting P-type ATPases, designated ATP7A and ATP7B, play an essential role in mammalian copper balance. Impaired intestinal transport of copper, resulting from mutations in the ATP7A gene, lead to Menkes disease in humans. Defects in a similar gene, the copper transporting ATPase ATP7B, result in Wilson disease. This ATP7B transporter has two functions: transport of copper into the plasma protein ceruloplasmin, and elimination of copper through the bile. Variants of ATP7B can be functionally assayed to identify defects in each of these functions. Tissue expression studies of the copper ATPases and their copper chaperone ATOX1 indicate that there is not complete overlap in expression. Other chaperones may be important for the transport of copper into ATP7A and ATP7B.

Value of an enzymatic assay for the determination of serum ceruloplasmin.

The serum concentration of the copper protein ceruloplasmin has been an important diagnostic indicator of Wilson's disease (WD). It is widely quoted that 95% of people with WD have low serum ceruloplasmin concentrations. Current evidence suggests that a normal serum ceruloplasmin concentration is more common in patients with WD, particularly those with liver disease, perhaps in part because of the routine use of an immunologic assay. This assay might indicate a normal level of ceruloplasmin when the enzymatic activity is lower. Enzymatic activity is the biologically relevant parameter. We compared the immunologic measurement with the enzymatic assessment of oxidase activity in patients with liver or neurologic symptoms of unknown origin in whom WD was considered in the differential diagnosis. Although a strong correlation of ceruloplasmin protein concentration with oxidase activity was observed in controls, this was not the case for these patients. Twelve patients, presenting with various types of hepatic disease, demonstrated a weak correlation between ceruloplasmin protein concentration and oxidase activity. Ten percent of patients with neurologic symptoms ( n = 41) had low ceruloplasmin concentrations and oxidase activity, and another 8% had normal ceruloplasmin concentrations associated with low oxidase activity. Although the enzymatic method is preferred for its biologic relevance, ceruloplasmin analysis is not a reliable diagnostic parameter for the diagnosis of WD in patients with liver disease. An important use of the ceruloplasmin oxidase assay is in the follow-up of patients with WD. Ceruloplasmin oxidase activity was undetectable in sera from patients with WD who were undergoing long-term chelation therapy, suggesting an early sign of copper depletion and a need for subsequent monitoring for symptoms of copper deficiency.

Functional assessment of the carboxy-terminus of the Wilson disease copper-transporting ATPase, ATP7B.

The carboxy-terminus of ATP7B, the protein defective in the copper-transport disorder Wilson disease, was investigated with respect to its role in copper delivery to the ferroxidase ceruloplasmin. We use yeast as a model system to assess the functional capabilities of ATP7B variants. The yeast ferroxidase, Fet3p, acquires copper from Ccc2p and cannot function if Ccc2p is impaired; expression of wild-type ATP7B in ccc2 yeast complements the iron-deficient phenotype. Our results demonstrate that the C-terminus of ATP7B is necessary for protein stability, as removal of the nonmembranous terminus leads to reduced protein levels and cessation of growth in iron-limited medium. Growth is partially restored when an additional three amino acids are present and is near wild-type levels when only one-third of the C-terminus is present. Measurement of ferroxidase activity is a more sensitive indicator of copper transport function and allowed identification of impaired variants not detected with the growth assay.