SEARCH

SEARCH BY CITATION

Keywords:

  • Wilson disease;
  • ATP7B;
  • genotype;
  • phenotype;
  • India;
  • GAS for WD

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Wilson disease (WD, MIM 277900) is an autosomal recessive disorder of copper metabolism resulting in excessive copper deposition, primarily in the liver and the brain (Ferenci et al., 2003). There is marked clinical heterogeneity with patients presenting with liver impairment, cognitive and behavioral disturbance, movement disorders, and osseomuscular symptoms in variable combinations (Dastur et al., 1968; Pfeiffer 2007; Aggarwal et al., 2009). Recently, multisystemic WD-specific scales have been developed and validated to grade and track the diverse clinical manifestation of WD (Leinweber et al., 2008; Aggarwal et al., 2009).

WD results from mutation of the copper transporting P-type ATPase gene, ATP7B, located on chromosome 13. ATP7B is a large gene, 80 kb in length spanning 21 exons and coding for a 1465 amino acid protein. It is primarily expressed in the liver with reduced expression seen in brain, kidney, and placenta (Bull et al., 1993; Tanzi et al., 1993). WD has a worldwide prevalence of around 1:30,000 and a carrier rate of 1 in 90.

Diagnosing WD is challenging and diagnostic delay remains a leading cause of disease associated morbidity and mortality (Scheinberg & Sternlieb, 1984). Molecular genetic analysis can improve the accuracy of diagnosis of patients with WD (Schmidt, 2009). However, over 600 WD mutations have been described, making routine genetic diagnosis of WD time consuming and expensive. Identification of high-frequency mutations in certain populations, like the p.H1069Q mutation (exon 14) in Northern, Central, and Eastern Europe and, the p.R778L and p.R778G mutations (exon 8) among the Chinese and Taiwanese populations, enables rapid genetic analysis of WD in these populations (Thomas et al., 1995; Chuang et al., 1996).

India has an ethnically diverse population and studies from Northwestern, Eastern, and Southern India have revealed a largely nonoverlapping mutational pattern of the ATP7B gene, distinct from the other Asian and European populations (Gupta et al., 2005; Kumar et al., 2005; Santhosh et al., 2006; Gupta et al., 2007b; Kumar et al., 2007). Common mutations and hotspots have yet to be identified in large parts of the Indian population and the country as a whole. Understanding the genotypic pattern of WD in India could pave the way for offering diagnostic mutational analysis of WD in the future.

This is the first study analyzing the mutational pattern of WD among the Western Indian population. Our study significantly extends the previously established spectrum of ATP7B polymorphisms and mutations observed in India. A previously recognized mutation, p.C271* and the novel mutation p.E122fs, accounted for WD in over one-third of the patient cohort. Our study suggests that mutation p.C271* is a high-frequency mutation in the Indian peninsula and may be the most common mutation in the country. The high frequency of homozygous mutations and the characterization of clinical phenotype by a multidimensional scoring system [Global Assessment Scale for Wilson Disease (GAS for WD)] allowed insights into the genotype–phenotype correlation in the patient cohort.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Study Population

Patients with WD from Western India visiting the Wilson Disease Clinic at Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, India, and their first-degree relatives (parents, siblings, and children) participated in the study. Fifty-two patients and their first-degree relatives (126 individuals) were subjected to sequencing of ATP7B. Patients were recruited from a large area of Western India (states of Maharashtra and Gujarat) that has not yet been included in genetic surveys for WD. Forty-nine patients from 46 unrelated families had WD based on established clinical diagnosis. WD was diagnosed in a further three asymptomatic participants based on genetic screening. Of the total 52 patients with WD, 22 patients were female and 30 were male with mean age at presentation being 11.6 ± 6.5 years. The study was approved by the Institutional Scientific and Ethics Review Boards and informed consent was obtained from all participants.

Clinical Data

WD was diagnosed based on a combination of characteristic clinical features, Kayser-Fleischer rings, low serum ceruloplasmin, high urinary copper, high liver copper, and abnormal brain magnetic resonance imaging. All patients with WD met the Ferenci and American Association for Study of Liver Diseases criterion for WD (Ferenci et al., 2003; Roberts & Schilsky, 2008). All participants underwent a standardized clinical interview and examination. Age at onset of WD and the initial symptom were recorded for all patients. The multisystemic clinical burden of WD at presentation was quantified by the GAS for WD (Aggarwal et al., 2009). Tier 1 of GAS measures WD disability across the domains liver (L), cognition and behavior (C), motor (M), and osseomuscular (O). Each domain is scored on an ascending six-point scale (0 to 5). Tier 2 assesses WD related neurological dysfunction across 14 items. Each item is graded on an ascending five-point scale (0 to 4) and summed to obtain the total tier 2 score (0 to 56).

Molecular Genetic Analysis

Genomic DNA was isolated from peripheral blood of the participants in ethylenediaminetetraacetic acid using QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). All 21 exons of the WD gene were amplified using primers flanking the exon–intron boundaries. The polymerase chain reaction products were sequenced using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany) with an ABI Prism 3100 Genetic Analyzer and the sequence was analyzed using Variant Reporter to characterize the molecular defect in the ATP7B gene. The reference sequence with GenBank accession number NM_00053.1 was used. The new sequence variants were confirmed as possibly disease causing by sequencing the affected exons in the parents and siblings of the index patient.

Statistical Analysis

To study the genotype–phenotype correlations, age at onset and various GAS for WD scores are presented as mean with standard deviation (SD). SPSS 18.0 software was used to formulate a box plot for age at onset and initial symptom of WD. Analysis was performed by Kruskal–Wallis test using Bonferroni corrections. A P value < 0.05 was considered as statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Patient Cohort

Patients were characterized by the GAS for WD (Aggarwal et al., 2009). Overall, Tier 1 GAS score showed the highest values for liver (mean 3.0 ± 1.0) with L3 scores representing irreversible but compensated liver cirrhosis. In contrast to Tier 1 scores for liver disease, overall lower values were observed for cognition and behavior (mean 2.1 ± 1.6), motor (mean 1.9 ± 1.4), and osseomuscular (mean 0.8 ± 1.2) domains. Overall GAS for WD Tier 2 scores indicated variable neurological dysfunction (mean 16.4 ± 10.6), with significant neurological disability in a majority of patients (Supporting Information Table S1).

Molecular Genetic Analysis

Direct sequence analysis identified disease-causing mutations in both alleles of 51 out of 52 patients (98%). In one patient with clinically definite WD who is improving with copper chelation treatment, we were unable to detect a WD mutation. Of the 51 patients with WD mutations, 30 (58.8%) were homozygous and 21 (41.2%) were compound heterozygous. Of the 126 unaffected first-degree relatives, 102 (80.9%) were carriers of a WD mutation and 24 (19.1%) had the wild-type ATP7B sequence (normal alleles).

The study identified 36 different WD mutations among the 51 patients; 31 were exonic mutations and five were intronic splice site variants (Table 1). Fourteen of the 34 mutations identified were novel and have not been described elsewhere. Mutations p.C271* and p.E122fs both located on exon 2 were the most frequent mutations among the patient cohort with an allelic frequency of 20.1% and 10.5%, respectively. The identified mutations were mostly located in exons 2, 8, 13, 14, and 18 of ATP7B (Fig. 1).

Table 1. Allelic frequency of mutations in Western Indian cohort (n = 52)
     Allelic
TypeNucleotideAmino acidExon/intronProtein domainfrequency (%)
  1. a

    Novel mutations (shaded gray).

  2. b

    India only.

  3. c

    Europe and India.

  4. d

    Asia only.

  5. e

    Suspected disease-causing mutation located on second allele.

  6. Cu, copper binding domain; Tm, transmembrane domain; Td, transduction domain.

Nonsensec.813C > Acp.Cys271*2Cu320
Insertion/deletionc.365_366delinsTTCGAAGCap.Glu122fs2Cu111
 c.1716delGap.Met573fs5Cu64
 c.678delGap.Leu227fs2Cu2/Cu32
 c.3147delCbp.Thr1050fs14ATP N-binding2
 c.2304_2305insCbp.Met769fs8Tm42
 c.2736_2746del11ap.Ile913fs12Td/Tm52
 c.2227delTap.Tyr743fs8Tm31
 c.2697_2723del27dp.Ile899_Gln907del11Td/Tm51
 c.3157_c.3158insCdp.Leu1053fs14ATP N-binding1
Missensec.2930C > Tcp.Thr977Met13Tm66
 c.2383C > Tcp.Leu795Phe9Tm4/Td6
 c.3809A > Gp.Asn1270Ser18ATP-binding3
 c.3895C > Tbp.Leu1299Phe18ATP-binding3
 c.3182G > Acp.Gly1061Glu14ATP N-binding3
 c.3301G > Abp.Gly1101Arg15ATP N-binding3
 c.3007G > Acp.Ala1003Thr13ATP-binding/Tm63
 c.4070C > Tbp.Ala1357Val20Tm82
 c.1544G > Aap.Gly515Asp4Cu52
 c.3089G > Aap.Gly1030Asp14ATP N-binding2
 c.3458G > Aap.Trp1153*16ATP N-binding2
 c.3818C > Tp.Pro1273Leu18ATP-binding2
 c.2071G > Ccp.Gly691Arg7Tm1/Tm21
 c.2189A > Gap.Asp730Gly8Tm31
 c.2333G > Acp.Arg778Gln8Tm41
 c.4021G > Adp.Gly1341Ser19Tm71
 c.2524G > Tap.Asp842Tyr10Td1
 c.3305T > Ccp.Ile1102Thr15ATP N-binding1
 c.442C > Tdp.Arg148Trp2Cu21
 c.2968G > Ccp.Ala990Pro13ATP-binding/Tm61
Intronic splice sitec.2866-2A > Ga 13 2
 c.1869+1_4delGT AAa 5 2
 c.3243+5G > Aa 14 2
 c.2865+1G > Ac 13 1
 c.1946+2T > Ga 6 1
Silentc.2145C > Tb, ep.Tyr715Tyr8Tm21
image

Figure 1. Schematic representation of the ATP7B gene indicating percentage of observed mutations attributable to individual exons, in the Western Indian cohort. Hot-spot exons are indicated as hatched. Three regions harboring mutations at the exon/intron boundary are indicated in italics.

Download figure to PowerPoint

In a total of 178 individuals, 13 single nucleotide polymorphisms (SNPs) and sequence variants were identified, of which p.P922P has been described only in India (Table 2). The novel nonpathogenic variant p.G859Q was found in cis with p.R969Q.

Table 2. Polymorphisms and sequence variants of ATP7B in a Western Indian cohort
TypeNucleotideAmino acidExon/intronProtein domain
  1. a

    Common.

  2. b

    Western India only.

  3. c

    India only.

  4. d

    Europe (Britain, Germany) and India.

Missensec.1216T > Gap.Ser406Ala2Cu4
 c.1366G > Cap.Val456Leu3Cu4/Cu5
 c.2495G > Aap.Arg832Lys10Tm4/Td
 c.2855G > Aap.Lys952Arg12Tm5/Tm6
 c.3419T > Cap.Ala1140Val16ATP N-binding
 c.2576G > Abp.Gly859Glu11Td
Silentc.3009G > Aap.Ala1003Ala13ATP binding/Tm6
 c.2973G > Aap.Thr991Thr13ATP binding/Tm6
 c.2484C > Tdp.Gly828Gly10Tm4/Td
 c.2976C > Acp.Pro992Pro13ATP binding/Tm6
No changec.2866-13G > CaIVS12-13G > C12 
 c.2866-90G > TaIVS12-90G > T12 
 c.3903+6T > CaIVS18+6T > C18 

As a result of our study a major area of India has now been screened for ATP7B mutations (Fig. 2).

image

Figure 2. Regions of India studied for WD genotype (marked in gray) and distribution of high frequency (>5%) WD mutations. Hatched areas indicate regions where mutation p.C271* has been identified.

Data for mutations were obtained from (Gupta et al., 2005; Kumar et al., 2005; Santhosh et al., 2006; Gupta et al., 2007b; Kumar et al., 2007).

Download figure to PowerPoint

Genotype Correlation to GAS for WD Scores and Age at Onset

Various mutations identified were compared with respect to age of WD onset, initial symptom (liver, neurological, or osseomuscular), disease burden at onset assessed by the GAS for WD Tier 1 (L, C, M, and O), and by Tier 2 scores (Supporting Information Table S1). Overall, age of WD onset was significantly different (P < 0.001) in patients with initial liver symptoms (n = 15) compared to patients with initial neurological/osseomuscular symptoms (n = 34) (Fig. 3). In order to assess the genotype–phenotype correlations, five of the 30 homozygous mutations seen in the cohort were selected (Fig. 4A). Mutations were selected if they were present in at least two patients in order to minimize bias in computing the phenotype. Overall, similar high GAS for WD Tier 1 liver scores (mean 3.1 ± 0.7) were observed in all five selected homozygous mutations. However, other Tier 1 domains (C, M, and O) and Tier 2 scores as well as age of disease onset showed high variability between the five selected homozygous genotypes. Eight patients could be studied for mutation p.C271*. Apart from one asymptomatic patient, onset of disease was mostly early (mean 10.7 ± 3.5 years; range 3–15 years) and most of the patients had a relatively high neurological disease burden [high Tier 1 C and/or M scores and high (mean 22.3 ± 9.9; range 9–33) total Tier 2 scores]. A significant later onset of disease (mean 15.0 ± 3.9 years; range 11–21 years) with high overall disease burden was observed for four patients with mutation p.E122fs. The p.E122fs group differed from the other mutations by absence (mean 0.0 ± 0) of osseomuscular disease. Patients with homozygous mutation p.M573fs displayed an early onset disease (mean 7.5 ± 0.5 years) and showed the lowest Tier 1 scores for C, M, and O domains (score ≤1) and total Tier 2 score (score 0 and 8), indicating significantly reduced neurological and osseomuscular disease burden. Patients with homozygous mutation p.L795F had the latest WD onset of the group (mean 17.5 ± 0.5 years) and had the overall highest total Tier 2 scores (mean 30.0 ± 5). This mutation was also unique in having the lowest cognition/behavior scores (mean 0.0 ± 0.5). The earliest onset of disease (mean 4.0 ± 0.5 years) was observed in the two patients with mutation p.T977M who showed moderate Tier 1 and Tier 2 scores.

image

Figure 3. Box plot demonstrating younger age of onset of patients with initial liver disease (n = 15) compared to patients with neurological/ osseomuscular symptoms (n = 34), in a Western Indian cohort. Asterisk (*) indicates significance.

Download figure to PowerPoint

Heterozygous mutations were chosen to investigate a modulation of the phenotype observed for the homozygous mutations p.C271* and p.L795F (Fig. 4B). A marked reduction of Tier 1 (C, M, and O) as well as of total Tier 2 scores were observed in the two patients with mutations p.C271*/p.G1030D as compared to p.C271* homozygotes. The two patients with heterozygous mutations p.L795F/p.M769fs were different from the overall cohort as they had osseomuscular symptoms at presentation. Further, disease onset in these two patients was significantly earlier (by almost 4 years) as compared to the p.L795F homozygotes.

image

Figure 4. Genotype and phenotype correlation of patients with homozygous (A) and heterozygous (B) WD mutations present in two or more patients. Mean GAS for WD scores and mean age of onset are shown.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This is the first study exploring the mutational pattern of WD in Western India (states of Gujarat and Maharashtra), which represents a significant percentage of the Indian population. The study identified 36 disease-causing mutations among 52 patients, 14 of 36 mutations being novel. The study demonstrated that the region had marked genetic heterogeneity with a mutational pattern quite distinct from other parts of India and Asia. Two mutations, p.C271* and the novel mutation p.E122fs located in exon 2 of ATP7B, accounted for WD in ∼40% of the patient cohort. Our study significantly adds to the genotype data for WD in India and suggests that mutation p.C271* is likely a common mutation throughout the country. Interestingly, systematic and quantitative analysis of the phenotypic characteristics of patients with a shared homozygous mutations showed that each genotype manifested with a distinct phenotype.

The mutation p.C271* was the most common mutation observed in our study from Western India with an allelic frequency of 20.2%. It was also the predominant mutation in the reports from Southern (allelic frequency 10.0%) and Eastern (allelic frequency 16.0%) India (Gupta et al., 2005; Kumar et al., 2005; Santhosh et al., 2006). However, the mutation p.C271* has not been identified in Northwestern India. The identification of a common mutation, p.C271*, at a high frequency in geographically distant areas of the Indian peninsula suggests an early founder effect and/or genetic drift in the Indian population that represents a culturally and linguistically diverse, largely nested population (Consortium Indian Genome Variation, 2008). The mutation p.C271* has also been described in Turks living in Turkey and England, as well as in Germany (Curtis et al., 1999; Gupta et al., 2007a). Consistent with studies of other Asian populations, the mutation p.H1069Q that is frequent in central European population was absent in Western India.

Except for the shared high-frequency mutation p.C271*, the mutational pattern described for Western India was distinct from other regions of India with only some overlap. The shared mutations were as follows: (1) mutation p.G1061E (Santhosh et al., 2006; Gupta et al., 2007a), (2) mutation p.I1102T (Kumar et al., 2005; Gupta et al., 2007a), (3) mutations p.L795F, p.D1270S, p.L1299F, p.L1299L-fs (Santhosh et al., 2006), (4) mutation p.T977M (Gupta et al., 2007a), and (5) mutation A1003T (Kumar et al., 2005). The Western and the Southern Indian cohorts showed maximal overlap in shared mutations. Notably, some mutations with allelic frequency of over 5% were observed in other parts of India but not in the Western Indian cohort suggesting that there is a significant regional variation of WD genotypes in India (Gupta et al., 2005; Kumar et al., 2005; Santhosh et al., 2006; Gupta et al., 2007b; Kumar et al., 2007).

Screening of three asymptomatic siblings led to a genetic diagnosis of WD in three unrelated families corroborating previous results that genetic screening of ATP7B is excellently suited to identify affected individuals who can be commenced on treatment before they develop WD related disability (Schmidt 2009). Exons 2, 8, 13, 14, and 18 were the exonic hotspots identified in the Western Indian cohort and these accounted for WD in almost 90% of all patients. Mutations in exon 2 alone were responsible for WD in about 40% of patients in the Western Indian cohort. Although frequencies of hotspots in ATP7B found in other Indian regions differed to some extent, all hotspots except for the exon 8 harboring mutation p.M769fs were also observed in the other reports from India (Gupta et al., 2005; Kumar et al., 2005; Santhosh et al., 2006; Gupta et al., 2007b). Further exploration of India for WD genotypes may reveal a pool of shared exonic hotspots, enabling rapid screening for WD mutations in India (Schmidt 2009).

The high degree of homozygosity (∼60%) observed in this study may reflect a cultural practice of people preferring to marry within their own or related ethnic group. A similar high degree of homozygosity has also been observed in Southern India (Santhosh et al., 2006) while elsewhere in the world rates of homozygosity are generally lower (Barada et al., 2010). As pedigree analysis has not been performed, it cannot be ruled out that some of the families that seemed to be unrelated have a common origin. The large number of mutations in the relatively small cohort of patients suggests a high degree of mutational heterogeneity in the studied Western Indian population.

Interestingly, analysis of homozygous mutations present in two or more patients revealed that each genotype was largely associated with a characteristic clinical pattern of WD including age of onset, system affected, and disease severity. Quantification of WD burden across multiple systems at disease onset enabled us to understand the impact of genotype on phenotype and revealed some useful insights. Independent of the genotype, high Tier 1 GAS for WD liver scores were observed in most patients in the Western Indian cohort, indicating that advanced or irreversible liver injury was already established at the time of diagnosis in most of the patients. While a third of the patients with WD presented with symptoms of liver dysfunction, subclinical or clinical liver impairment was also present in all patients with initial neurological or osseomuscular symptoms. Therefore, GAS for WD Tier 1 liver scores are in line with the observation that the liver is the primary target organ of WD and is universally affected in patients with initial neurological or osseomuscular symptoms (Scheinberg and Sternlieb, 1984). Liver disease was mostly seen within the first decade while homozygous mutation p.T977M, located in transmembrane region 6, was associated with very early onset liver dysfunction (<5 years of age) suggesting a predominant liver-specific disease manifestation of this mutation as also observed in children from Egypt (Abdel Ghaffar et al., 2011).

In contrast to the elevated GAS for WD Tier 1 liver scores, a high variability was observed in the impact of WD in other systems as graded by Tier 1 (C, M, and O scores) as well as total Tier 2 scores. The finding suggests that age at onset, neurological or osseomuscular manifestation of WD at the time of diagnosis showed a higher interpatient variability when compared to liver dysfunction. Homozygous mutations p.C271*, p.E122fs, p.L795F, and T977M were associated with significant neurological disease burden as reflected in the GAS for WD Tier 1 (C and/or M domain) and total Tier 2 scores. However, there was a variation in the overall clinical phenotype among the four mutations, including age at presentation. As exemplified for p.C271* and p.T977M, a heterozygous mutation may significantly affect the overall phenotype observed in association with the corresponding homozygous mutations p.C271* and p.T977M. Missense mutation p.G1030D, located in the ATP N-binding loop, when co-expressed with nonsense mutation p.C271*, seems to compensate the predominant neurological dysfunction caused by the latter mutation. However, whether such modulation of the phenotype is a result of the biological activity of the respective proteins awaits further functional analysis. In addition, heterozygous mutation p.M769fs remarkably reduced onset of disease by ∼4 years compared to disease onset in patients with homozygous mutation p.L795F, while overall disease burden remained similar in both genotypes.

A characteristic pattern was also observed with respect to the onset of WD in the Western Indian cohort. While the overall age of onset of WD was 11.6 ± 6.5 years, patients with initial neurological or osseomuscular symptoms presented on average ∼2 years later than those with initial liver disease, as was suggested before (Lee et al., 2011). This is in keeping with the understanding that liver is possibly the first organ affected by WD (Scheinberg & Sternlieb, 1984). Of note, the age of onset as reported here and elsewhere for Indian WD patients was earlier than that reported for patients in Europe, Korea, and South America (Deguti et al., 2004; Stapelbroek et al., 2004; Taly et al., 2009; Lee et al., 2011).

Compared to a relatively late onset (2nd to 3rd decade of life) and a predominantly neurological phenotype of mild to moderate disability that could be associated with the mutation p.H1069Q, mutations p.C271* and p.E122fs seem to lead to a more serious disease with earlier onset in the first and second decade, respectively. Of note, homozygous mutations p.C271*, p.E122fs, and p.M573fs which result in a premature stop codon are likely more deleterious as compared to the point mutation p.H1069Q. However, the phenotypic variability reported from homozygous and heterozygous patients suggests that other genetic, epigenetic, and environmental factors may also contribute to the clinical phenotype (Caca et al., 2001; Stapelbroek et al., 2004; Gromadzka et al., 2005; Gupta et al., 2005; Nicastro et al., 2009).

In conclusion, we report on several novel and predominant mutations in a large area of Western India. The large group of homozygous patients allowed a detailed phenotypic classification for some of the genotypes including mutation p.C271* which is suggested to be a major genotype throughout India. However, as many patients were admitted to our hospital due to neurological symptoms, this bias may result in an overall overestimation of relatively mild neurological disease as opposed to severe hepatic disease. Also, regional peculiarities driven by genetic, social, and environmental factors that modulate the impact of disease manifestation cannot be excluded. Caution must therefore be taken when transferring our conclusions on mutation-associated phenotypes to other WD patient cohorts. Nevertheless, within the limits of our study, we propose that a limited phenotypic variability could be assigned to important genotypes found in WD patients of Western India. For the first time a validated WD clinical scale to measure disease disability has been applied to a large cohort of patients with definitive WD. This adds a new spectrum and allows for more robust comparison of genotype and phenotype.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank M. Mehta and O. Nadzemova for technical support. Funding of the study was in part by the Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, India (project number C3/13/2010). We would like to thank the study participants and their families. The authors have no conflict of interests.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Aggarwal, A., Aggarwal, N., Nagral, A., Jankharia, G., & Bhatt, M. (2009) A novel Global Assessment Scale for Wilson's Disease (GAS for WD). Mov Disord 24, 509518.
  • Barada, K., El-Atrache, M., El, H., II, Rida, K., El-Hajjar, J., Mahfoud, Z., & Usta, J. (2010) Homozygous mutations in the conserved ATP hinge region of the Wilson disease gene: Association with liver disease. J Clin Gastroenterol 44, 432439.
  • Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R., & Cox, D. W. (1993) The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 5, 327337.
  • Caca, K., Ferenci, P., Kuhn, H. J., Polli, C., Willgerodt, H., Kunath, B., Hermann, W., Mossner, J., & Berr, F. (2001) High prevalence of the H1069Q mutation in East German patients with Wilson disease: Rapid detection of mutations by limited sequencing and phenotype-genotype analysis. J Hepatol 35, 575581.
  • Chuang, L. M., Wu, H. P., Jang, M. H., Wang, T. R., Sue, W. C., Lin, B. J., Cox, D. W., & Tai, T. Y. (1996) High frequency of two mutations in codon 778 in exon 8 of the ATP7B gene in Taiwanese families with Wilson disease. J Med Genet 33, 521523.
  • Consortium Indian Genome Variation. (2008) Genetic landscape of the people of India: A canvas for disease gene exploration. J Genet 87, 320.
  • Curtis, D., Durkie, M., Balac, P., Sheard, D., Goodeve, A., Peake, I., Quarrell, O., & Tanner, S. (1999) A study of Wilson disease mutations in Britain. Hum Mutat 14, 304311.
  • Dastur, D. K., Manghani, D. K., & Wadia, N. H. (1968) Wilson's disease in India. I. Geographic, genetic, and clinical aspects in 16 families. Neurology 18, 2131.
  • Deguti, M. M., Genschel, J., Cancado, E. L., Barbosa, E. R., Bochow, B., Mucenic, M., Porta, G., Lochs, H., Carrilho, F. J., & Schmidt, H. H. (2004) Wilson disease: Novel mutations in the ATP7B gene and clinical correlation in Brazilian patients. Hum Mutat 23, 398406.
  • Ferenci, P., Caca, K., Loudianos, G., Mieli-Vergani, G., Tanner, S., Sternlieb, I., Schilsky, M., Cox, D., & Berr, F. (2003) Diagnosis and phenotypic classification of Wilson disease. Liver Int 23, 139142.
  • Abdel Ghaffar, T. Y., Elsayed, S. M., Elnaghy, S., Shadeed, A., Elsobky, E. S., & Schmidt, H. (2011) Phenotypic and genetic characterization of a cohort of pediatric Wilson disease patients. BMC Pediatr 11, 5666.
  • Gromadzka, G., Schmidt, H. H., Genschel, J., Bochow, B., Rodo, M., Tarnacka, B., Litwin, T., Chabik, G., & Czlonkowska, A. (2005) Frameshift and nonsense mutations in the gene for ATPase7B are associated with severe impairment of copper metabolism and with an early clinical manifestation of Wilson's disease. Clin Genet 68, 524532.
  • Gupta, A., Aikath, D., Neogi, R., Datta, S., Basu, K., Maity, B., Trivedi, R., Ray, J., Das, S. K., Gangopadhyay, P. K., & Ray, K. (2005) Molecular pathogenesis of Wilson disease: Hplotype analysis, detection of prevalent mutations and genotype-phenotype correlation in Indian patients. Hum Genet 118, 4957.
  • Gupta, A., Chattopadhyay, I., Dey, S., Nasipuri, P., Das, S. K., Gangopadhyay, P. K., & Ray, K. (2007a) Molecular pathogenesis of Wilson disease among Indians: A perspective on mutation spectrum in ATP7B gene, prevalent defects, clinical heterogeneity and implication towards diagnosis. Cell Mol Neurobiol 27, 10231033.
  • Gupta, A., Maulik, M., Nasipuri, P., Chattopadhyay, I., Das, S. K., Gangopadhyay, P. K., & Ray, K. (2007b) Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers. Clin Chem 53, 16011608.
  • Kumar, S., Thapa, B. R., Kaur, G., & Prasad, R. (2005) Identification and molecular characterization of 18 novel mutations in the ATP7B gene from Indian Wilson disease patients: Genotype. Clin Genet 67, 443445.
  • Kumar, S., Thapa, B., Kaur, G., & Prasad, R. (2007) Analysis of most common mutations R778G, R778L, R778W, I1002T and H1069Q in Indian Wilson disease patients: Correlation between genotype/phenotype/copper ATPase activity. Mol Cell Biochemistry 294, 110.
  • Lee, B. H., Kim, J. H., Lee, S. Y., Jin, H. Y., Kim, K. J., Lee, J. J., Park, J. Y., Kim, G. H., Choi, J. H., Kim, K. M., & Yoo, H. W. (2011) Distinct clinical courses according to presenting phenotypes and their correlations to ATP7B mutations in a large Wilson's disease cohort. Liver Int 31, 831839.
  • Leinweber, B., Moller, J. C., Scherag, A., Reuner, U., Gunther, P., Lang, C. J., Schmidt, H. H., Schrader, C., Bandmann, O., Czlonkowska, A., Oertel, W. H., & Hefter, H. (2008) Evaluation of the Unified Wilson's Disease Rating Scale (UWDRS) in German patients with treated Wilson's disease. Mov Disord 23, 5462.
  • Nicastro, E., Loudianos, G., Zancan, L., D'Antiga, L., Maggiore, G., Marcellini, M., Barbera, C., Marazzi, M. G., Francavilla, R., Pastore, M., Vajro, P., D'Ambrosi, M., Vegnente, A., Ranucci, G., & Iorio, R. (2009) Genotype-phenotype correlation in Italian children with Wilson's disease. J Hepatol 50, 555561.
  • Pfeiffer, R. F. (2007) Wilson's Disease. Semin Neurol 27, 123132.
  • Roberts, E. A., & Schilsky, M. L. (2008) Diagnosis and treatment of Wilson disease: An update. Hepatology 47, 20892111.
  • Santhosh, S., Shaji, R. V., Eapen, C. E., Jayanthi, V., Malathi, S., Chandy, M., Stanley, M., Selvi, S., Kurian, G., & Chandy, G. M. (2006) ATP7B mutations in families in a predominantly Southern Indian cohort of Wilson's disease patients. Indian J Gastroenterol 25, 277282.
  • Scheinberg, I. H., & Sternlieb, I. (1984) Wilson's disease. In: Major problems in internal medicine. Philadelphia: Elsevier Publishing.
  • Schmidt, H. H. (2009) Role of genotyping in Wilson's disease. J Hepatol 50, 449452.
  • Stapelbroek, J. M., Bollen, C. W., van Amstel, J. K., van Erpecum, K. J., van Hattum, J., van den Berg, L. H., Klomp, L. W., & Houwen, R. H. (2004) The H1069Q mutation in ATP7B is associated with late and neurologic presentation in Wilson disease: results of a meta-analysis. J Hepatol 41, 758763.
  • Taly, A. B., Prashanth, L. K., & Sinha, S. (2009) Wilson's disease: An Indian perspective. Neurol India 57, 528540.
  • Tanzi, R. E., Petrukhin, K., Chernov, I., Pellequer, J. L., Wasco, W., Ross, B., Romano, D. M., Parano, E., Pavone, L., Brzustowicz, L. M., Devoto, M., Peppercorn, J., Bush, A. I., Sternleib, I., Pirastu, M., Gusells, J. F., Evgrafov, O., Penchaszadeh, G. K., Honig, B., Edelman, I. S., Soares, M., B., Scheinberg, I. H., & Gilliam, T. C. (1993) The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 5, 344350.
  • Thomas, G. R., Forbes, J. R., Roberts, E. A., Walshe, J. M., & Cox, D. W. (1995) The Wilson disease gene: Spectrum of mutations and their consequences. Nat Genet 9, 210217.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
ahg12024-sup-0001-TableS1.doc109KTable S1 Clinical characterization of the Western Indian patient cohort.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.