Copper toxicosis in non-COMMD1 Bedlington terriers is associated with metal transport gene ABCA12

Wilson’s disease, caused by a mutation in the ATP-ase 7B gene, is the only genetically characterised human disease with inhibition of biliary copper excretion and toxic copper accumulation in liver and occasionally brain. A similar copper toxicosis occurs in Bedlington terriers (CT) with liver damage only. Although CT has been associated with a defect in the COMMD1 gene (COMMD1 ^del/del), Bedlington terriers with CT and lacking this mutation are also recognised (non-COMMD1 ^del/del).A study was designed to identify any other gene polymorphisms associated with copper toxicity in Bedlington terriers employing genome wide association studies (GWAS) followed by deep sequencing of the candidate region. Blood for DNA analysis and liver for confirmation of the diagnosis was obtained from 30 non-COMMD1 ^del/del Bedlington terriers comprising equal numbers of CT-affected dogs and controls. DNA was initially subjected to GWAS screening and then further sequencing to target the putative mutant gene.The study has identified a significant disease association with a region on chromosome 37 containing identified SNP’s which are highly significantly associated with non-COMMD1 ^del/del Bedlington terrier CT. This region contains the ABCA12 gene which bears a close functional relationship to ATP-ase 7B responsible for Wilson’s disease in man.     

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.     

Intestinal expression of metal transporters in Wilson’s disease

In Wilson’s disease (WND), biallelic ATP7B gene mutation is responsible for pathological copper accumulation in the liver, brain and other organs. It has been proposed that copper transporter 1 (CTR1) and the divalent metal transporter 1 (DMT1) translocate copper across the human intestinal epithelium, while Cu-ATPases: ATP7A and ATP7B serve as copper efflux pumps. In this study, we investigated the expression of CTR1, DMT1 and ATP7A in the intestines of both WND patients and healthy controls to examine whether any adaptive mechanisms to systemic copper overload function in the enterocytes. Duodenal biopsy samples were taken from 108 patients with Wilson’s disease and from 90 controls. CTR1, DMT1, ATP7A and ATP7B expression was assessed by polymerase chain reaction and Western blot. Duodenal CTR1 mRNA and protein expression was decreased in WND patients in comparison to control subjects, while ATP7A mRNA and protein production was increased. The variable expression of copper transporters may serve as a defense mechanism against systemic copper overload resulting from functional impairment of ATP7B.     

The role of compensatory neutral mutations in molecular evolution

A pair of mutations at different loci (or sites) which are singly deleterious but restore normal fitness in combination may be called compensatory neutral mutations. Population dynamics concerning evolutionary substitutions of such mutants was developed by making use of the diffusion equation method. Based on this theory and, also, by the help of Monte Carlo simulation experiments, a remarkable phenomenon was disclosed that the double mutants can easily become fixed in the population by random drift under continued mutation pressure if the loci arc tightly linked, even when the single mutants are definitely deleterious. More specifically, I consider two loci with allelesA andA’ in the first locus, and allelesB andB’in the second locus, and assign relative fitnesses 1, 1-s’, 1-s’ and 1 respectively to the four gene combinationsAB, A’B, AB’ andA’B’, wheres’ is the selection coefficient against the single mutants (s’ > 0). Letv be the mutation rate per locus per generation and assume that mutation occurs irreversibly fromA toA’ at the first locus, and fromB toB’ at the second locus, whereA andB are wild type genes, andA’ andB’ are their mutant alleles. In a diploid population of effective size N _e (or a haploid population of 2N _e breeding individuals), it was shown that the average time (T) until joint fixation of the double mutant (A’B’) starting from the state in which the population consists exclusively of the wild type genes (AB) is not excessively long even for large 4N _e s’ values. In fact, assuming2N _e v = 1 we have -T = 54Ne for 4Nes’ = 400, and -T = 128Ne for 4N _e s’ = 1000. These values are not unrealistically long as compared with -T~ 5N _e obtained for 4N _e s’ = 0. The approximate analytical treatment has also been extended to estimate the effect of low rate crossing over in retarding fixation. The bearing of these findings on molecular evolution is discussed with special reference to coupled substitutions at interacting amino acid (or nucleotide) sites within a folded protein (orrna) molecule. It is concluded that compensatory neutral mutants may play an important role in molecular evolution.     

Stability and ATP binding of the nucleotide-binding domain of the Wilson disease protein: effect of the common H1069Q mutation

Perturbation of the human copper-transporter Wilson disease protein (ATP7B) causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the nucleotide-binding subdomain (N-domain), including the most common mutation, H1069Q. To gain insight into the biophysical behavior of the N-domain under normal and disease conditions, we have characterized wild-type and H1069Q recombinant N-domains in vitro and in silico. The mutant has only twofold lower ATP affinity compared to that of the wild-type N-domain. Both proteins unfold in an apparent two-state reaction at 20 °C and ATP stabilizes the folded state. The thermal unfolding reactions are irreversible and, for the same scan rate, the wild-type protein is more resistant to perturbation than the mutant. For both proteins, ATP increases the activation barrier towards thermal denaturation. Molecular dynamics simulations identify specific differences in both ATP orientation and protein structure that can explain the absence of catalytic activity for the mutant N-domain. Taken together, our results provide biophysical characteristics that may be general to N-domains in other P_1B-ATPases as well as identify changes that may be responsible for the H1069Q WD phenotype in vivo.     

Mutations in the NRG1 gene are associated with Hirschsprung disease

Hirschsprung disease (HSCR, congenital colon aganglionosis) is a relatively common complex genetic condition caused by abnormal development of the enteric nervous system (ENS). Through a recent genome-wide association study conducted on Chinese HSCR patients, we identified a new HSCR contributing locus, neuregulin 1 (NRG1; 8p12), a gene known to be involved in the development of the ENS. As genes in which disease-associated common variants are found are to be considered as candidates for the search of deleterious rare variants (RVs) in the coding sequences, we sequenced the NRG1 exons of 358 sporadic HSCR patients and 333 controls. We identified a total of 13 different heterozygous RVs including 8 non-synonymous (A28G, E134K, V266L, H347Y, P356L, V486M, A511T, P608A) and 3 synonymous amino acid substitutions (P24P, T169T, L483L), a frameshift (E239fsX10), and a c.503-4insT insertion. Functional analysis of the most conserved non-synonymous substitutions, H347Y and P356L, showed uneven intracellular distribution and aberrant expression of the mutant proteins. Except for T169T and V486M, all variants were exclusive to HSCR patients. Overall, there was a statistically significant over-representation of NRG1 RVs in HSCR patients (p?=?0.008). We show here that not only common, but also rare variants of the NRG1 gene contribute to HSCR. This strengthens the role of NRG1.     

The spectrum of pathogenic variants of the ATP7B gene in Wilson disease in the Russian Federation

Background. Wilson’s disease (WD) is a rare inherited disorder caused by mutations in the ATP7B gene resulting in copper accumulation in different organs. However, data on ATP7B mutation spectrum in Russia and worldwide are insufficient and contradictory. The objective of the present study was estimation of the frequency of ATP7B gene mutations in the Russian population of WD patients. Materials and methods. 75 WDpatients were examined by next-generation sequencing (NGS). A targeted panel NimbleGen SeqCap EZ Choice: 151012_HG38_CysFib_EZ_HX3 (ROCHE)was designed for analysis of ATP7B gene and possible modifier genes. Retrospective assessment of a diagnostic WD score (Leipzig, 2001) was also performed. Results. 31 mutations in ATP7B gene were detected. Two most frequent mutations were c.3207C > A (51,85% of alleles) and c.3190 G > A (8,64% of alleles). Single rare mutations were detected in 29% of cases. In 96% cases mutations of both copies of the ATP7B were revealed. We also observed 3 novel potentially pathogenic variants which were not previously described (c.1870-8A > G, c.3655A > T (p.Ile1219Phe), c.3036dupC (p.Lys1013fs). For 25% of patients at the time of the manifestation the diagnosis WD could not be established using the earlier proposed diagnostic score. There was a remarkable delay in diagnosis for the majority of patients. Only 33% of patients WD was diagnosed in three months after the first symptoms, 29%patients - in 3–12 months, 30% – in 1–10 years, in 8% – more than 10 years. Generally, clinical appearance of WD may be rather variable at manifestation and genetic profiling at this step is the only way to confirm the presence of WD.     

In Silico profiling of deleterious amino acid substitutions of potential pathological importance in haemophlia A and haemophlia B

Background

In this study, instead of current biochemical methods, the effects of deleterious amino acid substitutions in F8 and F9 gene upon protein structure and function were assayed by means of computational methods and information from the databases. Deleterious substitutions of F8 and F9 are responsible for Haemophilia A and Haemophilia B which is the most common genetic disease of coagulation disorders in blood. Yet, distinguishing deleterious variants of F8 and F9 from the massive amount of nonfunctional variants that occur within a single genome is a significant challenge.

Methods

We performed an in silico analysis of deleterious mutations and their protein structure changes in order to analyze the correlation between mutation and disease. Deleterious nsSNPs were categorized based on empirical based and support vector machine based methods to predict the impact on protein functions. Furthermore, we modeled mutant proteins and compared them with the native protein for analysis of protein structure stability.

Results

Out of 510 nsSNPs in F8, 378 nsSNPs (74%) were predicted to be ''intolerant'' by SIFT, 371 nsSNPs (73%) were predicted to be ''damaging'' by PolyPhen and 445 nsSNPs (87%) as ''less stable'' by I-Mutant2.0. In F9, 129 nsSNPs (78%) were predicted to be intolerant by SIFT, 131 nsSNPs (79%) were predicted to be damaging by PolyPhen and 150 nsSNPs (90%) as less stable by I-Mutant2.0. Overall, we found that I-Mutant which emphasizes support vector machine based method outperformed SIFT and PolyPhen in prediction of deleterious nsSNPs in both F8 and F9.

Conclusions

The models built in this work would be appropriate for predicting the deleterious amino acid substitutions and their functions in gene regulation which would be useful for further genotype-phenotype researches as well as the pharmacogenetics studies. These in silico tools, despite being helpful in providing information about the nature of mutations, may also function as a first-pass filter to determine the substitutions worth pursuing for further experimental research in other coagulation disorder causing genes.     

Large-scale analysis of non-synonymous coding region single nucleotide polymorphisms

MOTIVATION: Single nucleotide polymorphisms (SNPs) are the most common form of genetic variant in humans. SNPs causing amino acid substitutions are of particular interest as candidates for loci affecting susceptibility to complex diseases, such as diabetes and hypertension. To efficiently screen SNPs for disease association, it is important to distinguish neutral variants from deleterious ones. RESULTS: We describe the use of Pfam protein motif models and the HMMER program to predict whether amino acid changes in conserved domains are likely to affect protein function. We find that the magnitude of the change in the HMMER E-value caused by an amino acid substitution is a good predictor of whether it is deleterious. We provide internet-accessible display tools for a genomewide collection of SNPs, including 7391 distinct non-synonymous coding region SNPs in 2683 genes. AVAILABILITY: http://lpgws.nci.nih.gov/cgi-bin/GeneViewer.cgi     

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.     

A new ATP7B gene mutation with severe condition in two unrelated Iranian families with Wilson disease

Wilson disease is associated with a defect in copper metabolism and caused by different mutations in ATP7B gene. The aim of this study was to determine mutation frequency of ATP7B exons 8 and 14 in Wilson disease patients from the south of Iran. The exons 8 and 14 of ATP7B gene were analyzed in 65 unrelated Wilson disease patients by Denaturing High Performance Liquid Chromatography, and samples with abnormal peak profile were selected for direct DNA sequencing. Seven out of 65 (10.8%) patients had mutations at exon 14, including c.3061-1G>A in four and c.3207C>A in three patients. In addition, four different mutations were identified at exon 8 of six patients (9.2%). Three of these mutations have been previously reported, including c.2304delC in two patients, c.2293G>A and 2304dupC each in one patient. Furthermore, a novel mutation, c.2335T>G (p.Trp779Gly), was identified in two unrelated patients. The patients with this novel mutation demonstrated severe neuropsychiatric condition. All together, 13 out of 65 (20%) patients had mutations within exons 8 and 14. We also identified a lower frequency of the most common mutations of exons 8 and 14 in the southern Iranian population.     

Birth and rapid subcellular adaptation of a hominoid-specific CDC14 protein

Gene duplication was prevalent during hominoid evolution, yet little is known about the functional fate of new ape gene copies. We characterized the CDC14B cell cycle gene and the functional evolution of its hominoid-specific daughter gene, CDC14Bretro. We found that CDC14B encodes four different splice isoforms that show different subcellular localizations (nucleus or microtubule-associated) and functional properties. A microtubular CDC14B variant spawned CDC14Bretro through retroposition in the hominoid ancestor 18–25 million years ago (Mya). CDC14Bretro evolved brain-/testis-specific expression after the duplication event and experienced a short period of intense positive selection in the African ape ancestor 7–12 Mya. Using resurrected ancestral protein variants, we demonstrate that by virtue of amino acid substitutions in distinct protein regions during this time, the subcellular localization of CDC14Bretro progressively shifted from the association with microtubules (stabilizing them) to an association with the endoplasmic reticulum. CDC14Bretro evolution represents a paradigm example of rapid, selectively driven subcellular relocalization, thus revealing a novel mode for the emergence of new gene function.     

Computational refinement of functional single nucleotide polymorphisms associated with ATM gene

Background

Understanding and predicting molecular basis of disease is one of the major challenges in modern biology and medicine. SNPs associated with complex disorders can create, destroy, or modify protein coding sites. Single amino acid substitutions in the ATM gene are the most common forms of genetic variations that account for various forms of cancer. However, the extent to which SNPs interferes with the gene regulation and affects cancer susceptibility remains largely unknown.

Principal findings

We analyzed the deleterious nsSNPs associated with ATM gene based on different computational methods. An integrative scoring system and sequence conservation of amino acid residues was adapted for a priori nsSNP analysis of variants associated with cancer. We further extended our approach on SNPs that could potentially influence protein Post Translational Modifications in ATM gene.

Significance

In the lack of adequate prior reports on the possible deleterious effects of nsSNPs, we have systematically analyzed and characterized the functional variants in both coding and non coding region that can alter the expression and function of ATM gene. In silico characterization of nsSNPs affecting ATM gene function can aid in better understanding of genetic differences in disease susceptibility.     

A Simple and Rapid Method for Detection of the 1069Gln Mutation in Wilson Disease

A simple and rapid method for detecting the 1069Gln mutation in gene ATP7B based on a PCR specific for this allele has been developed. The 1069Gln mutation is the main cause of Wilson disease (WD) in Russia and accounts for approximately 40% of all mutant alleles of gene ATP7B. Therefore, the method proposed makes the postnatal and prenatal diagnosis of Wilson disease in Russia considerably more rapid and less expensive.     

Predicting the Functional Effect of Amino Acid Substitutions and Indels

As next-generation sequencing projects generate massive genome-wide sequence variation data, bioinformatics tools are being developed to provide computational predictions on the functional effects of sequence variations and narrow down the search of casual variants for disease phenotypes. Different classes of sequence variations at the nucleotide level are involved in human diseases, including substitutions, insertions, deletions, frameshifts, and non-sense mutations. Frameshifts and non-sense mutations are likely to cause a negative effect on protein function. Existing prediction tools primarily focus on studying the deleterious effects of single amino acid substitutions through examining amino acid conservation at the position of interest among related sequences, an approach that is not directly applicable to insertions or deletions. Here, we introduce a versatile alignment-based score as a new metric to predict the damaging effects of variations not limited to single amino acid substitutions but also in-frame insertions, deletions, and multiple amino acid substitutions. This alignment-based score measures the change in sequence similarity of a query sequence to a protein sequence homolog before and after the introduction of an amino acid variation to the query sequence. Our results showed that the scoring scheme performs well in separating disease-associated variants (n = 21,662) from common polymorphisms (n = 37,022) for UniProt human protein variations, and also in separating deleterious variants (n = 15,179) from neutral variants (n = 17,891) for UniProt non-human protein variations. In our approach, the area under the receiver operating characteristic curve (AUC) for the human and non-human protein variation datasets is ∼0.85. We also observed that the alignment-based score correlates with the deleteriousness of a sequence variation. In summary, we have developed a new algorithm, PROVEAN (Protein Variation Effect Analyzer), which provides a generalized approach to predict the functional effects of protein sequence variations including single or multiple amino acid substitutions, and in-frame insertions and deletions. The PROVEAN tool is available online at http://provean.jcvi.org.     

Structural insight into mutations at 155 position of valosin containing protein (VCP) linked to inclusion body myopathy with Paget disease of bone and frontotemporal Dementia

Mutations in Valosin-containing protein (VCP) have been implicated in the pathology linked to inclusion body myopathy, paget disease of bone and frontotemporal dementia (IBMPFD). VCP is an essential component of AAA-ATPase superfamily involved in various cellular functions. Advanced In-silico analysis was performed using prediction based servers to determine the most deleterious mutation. Molecular dynamics simulation was used to study the protein dynamics at atomic level. Molecular docking was used to study the effect of mutation on ATP/ADP transition in the kinase domain. This ATPase of 806 amino acids has four domains: N-terminal domain, C-terminal domain and two ATPase domains D1 and D2 and each of these domains have a distinct role in its functioning. The mutations in VCP protein are distributed among regions known as hotspots, one such hotspot is codon 155. Three missense mutations reported in this hotspot are R155C, R155H and R155P. Potentiality of the deleteriousness calculated using server based prediction models reveal R155C mutation to be the most deleterious. The atomic insight into the effect of mutation by molecular dynamics simulation revealed major conformational changes in R155C variants ATP binding site in D1 domain. The nucleotide-binding mode at the catalytic pocket of VCP and its three variants at codon 155 showed change in the structure, which affects the ATP–ADP transition kinetics in all the three variants.     

Germ Line Variants of Human N-Methylpurine DNA Glycosylase Show Impaired DNA Repair Activity and Facilitate 1,N6-Ethenoadenine-induced Mutations

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N⁶-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (K_D) of hMPG toward 1,N⁶-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.     

Identification of deleterious non-synonymous single nucleotide polymorphisms using sequence-derived information

Background  

As the number of non-synonymous single nucleotide polymorphisms (nsSNPs), also known as single amino acid polymorphisms (SAPs), increases rapidly, computational methods that can distinguish disease-causing SAPs from neutral SAPs are needed. Many methods have been developed to distinguish disease-causing SAPs based on both structural and sequence features of the mutation point. One limitation of these methods is that they are not applicable to the cases where protein structures are not available. In this study, we explore the feasibility of classifying SAPs into disease-causing and neutral mutations using only information derived from protein sequence.