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Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers.

Wilson disease (WD)[4] is an autosomal recessive disorder caused by defects in the copper-transporting P-type ATPase gene (ATP7B)[5] resulting in accumulation of copper in the liver and the brain (1, 2). The disease is diagnosed on the basis of typical symptoms and conventional biochemical indicators, which include low serum concentrations of ceruloplasmin, increased excretion of urinary copper, and presence of the Kayser-Fleischer (K-F) ring (3). Although the biochemical defects are present from birth, manifestation of WD appears at a median age of 12 to 23 years. Thus, the patients usually remain untreated until they manifest the disease, although adverse effects can be thwarted by the use of chelating agents such as penicillamine and zinc acetate (4,5).

Unlike most other genetic diseases, WD has an available treatment regimen that provides hope to those pre' disposed to the disease if it is diagnosed at a preclinical stage. Thus, in addition to carrier detection as in other genetic diseases, identification of presymptomatic sibs of the proband in WD-affected families is an important step in management of the disease. Although biochemical assays are well defined for WD, the overlapping range of the parameters between noncarriers and carriers of the mutant allele typical of most of the genetic diseases argues in favor of a molecular diagnostic test to allay any confusion. Without appropriate molecular genetic diagnosis, predisposition for the disease would remain unnoticed in WD-mutant children until they manifest signs of the disease. On the other hand, if these presymptomatic individuals are identified, progression of the disease could be monitored by regular checkups of biochemical indicators for therapeutic intervention at an appropriate time. In addition, carrier information can be used for genetic counseling, which is particularly useful for population groups that encourage and practice consanguinity.

Progress in identifying affected individuals has been made by locating the causal gene (ATP7B) on the long arm of chromosome 13 (6), enabling the use of flanking microsatellite markers to study the transmission of the mutant alleles in siblings of affected individuals by linkage analysis (7,8). We used polymorphic dinucleotide repeat markers (D13S314, D13S133, and D13S316) to identify 4 unrelated presymptomatic children in WD-affected families (9). Incorrect determination of the mutant allele may occur, however, because these extragenic markers reportedly can recombine with WD loci (e.g., D13S314 at 917 kb upstream and D13S316 at 375 kb downstream of ATP7B) (10). Hence entragenic markers would be the ideal choice to reduce the chances of recombination.

Prevalent mutations in ATP7B have been identified in many population groups (http://www.medicalgenetics.med.ualberta.ca/wilson/index.php). Identifying the mutation in a WD patient, followed by genotyping in the sibs for the same mutation, is the ideal strategy for unequivocal determination of the genotype of the sibs. Most WD mutations are rare, however, with a very low frequency that varies greatly from population to population. Hence, to determine genotype in sibs who do not harbor the prevalent mutations, the optimal choice is to use intragenic SNP markers with high heterozygosity values in the study population. New opportunities are opening up with the discovery of large numbers of single-nucleotide polymorphisms (SNPs) in the human genome that could be used for tracking disease loci, for association studies, or simply as markers for monogenic disorders. To date, limited information is available on the SNPs in ATP7B in any population. In the Indian population, SNPs have been used to construct haplotypes and identify prevalent mutations (11). Recently, an SNP profile for the ATP7B gene became available as part of the HapMap project (http://www.hapmap.org), but no attempt has yet been made to evaluate SNPs as markers for the WD locus. In addition, HapMap data do not yet include the Indian population (one-sixth of the world population; planning commission report of India: http://planningcommission.nic.in/data/dt_pophsd.pdf).

We report a comprehensive strategy for determining presymptomatic and carrier sibs of indexed WD patients by analyzing population-specific prevalent mutations and SNP markers, taking the Indian population as a model. In addition to prevalent mutations, we have identified and evaluated the SNP profile of the WD gene in the population of India across different regions, selected SNPs with high heterozygosity, determined the extent of linkage disequilibrium (LD) between SNPs, compared the data with the information available for other population groups in the HapMap project, and assessed the utility of these markers for genotyping carriers and presymptomatic sibs in WD-affected families.

Materials and Methods

PATIENTS AND CONTROLS

WD patients, mostly with neurological problems, were examined at the Bangur Institute of Neurology, Kolkata, India, with referrals being made to the Regional Institute of Ophthalmology and the Gastroenterology and Paediatrics Departments, Seth Sukhlal Karnani Memorial Hospital, for ophthalmologic and hepatic cases, respectively. The diagnosis was based (a) clinically on the presence of neurological features such as dystonia, tremor, rigidity, and bradykinesia and/or hepatic features such as signs and symptoms of acute or chronic liver failure, and K-F ring in the cornea and (b) biochemically on low serum ceruloplasmin (<200 mg/L) and high 24-h urinary copper excretion (>100 [micro]g) (3), plus computed tomography and MRI scanning where applicable. Diagnosis was based on the entire profile, and presence or absence of any one feature did not contribute toward diagnosis or exclusion. We collected 323 blood samples from 87 families that included at least 1 patient; 30 were multisibling families. The sibs were also examined clinically for possible sub-clinical or apparently mild manifestations of the disease, i.e., presence of tremor, rigidity, and bradykinesia and/or hepatic features such as signs and symptoms of jaundice, liver enlargement, and K-F ring. General intelligence levels were examined and any apparent abnormality (such as failing grades, forgetfulness, irrational behavior) noted. The sibs who were suspected to carry the disease in the preclinical stage based on the above examination underwent biochemical tests.

To assess the heterozygosity of the SNPs and evaluate the LD status among those in the general population, we included 1871 samples collected as a part of the Indian Genome Variation project (12). These individuals belonged to 55 distinct ethnic groups inhabiting 6 different geographical regions (north, northeast, east, south, west, and central) of mainland India. Moreover, 54 of the ethnic groups comprise the 4 major linguistic families of the Indian population, namely Indo-European, Dravidian, Tibeto-Burman, and Austro-Asiatic; the remaining 1 group represents a population known to have negroid origin and therefore was treated as an outgroup. We collected samples from individuals unrelated at least to the 1st-cousin level. The internal review committee on research using humans cleared the project after proper review as per the regulations of the Indian Council of Medical Research. A list of the populations, with sample sizes and brief notes on their linguistic and sociocultural backgrounds, is provided in Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue9.

COLLECTION OF BLOOD SAMPLES AND GENOMIC DNA

PREPARATION

We collected approximately 10.0-mL peripheral blood samples with informed consent from study participants who included WD patients, their family members, and the general population included under the Indian Genome Variation project. We prepared genomic DNA from fresh whole blood using the conventional phenol-chloroform method, followed by ethanol precipitation, after which the DNA was dissolved in TE buffer (10 mmol/L TrisHCl, 0.1 mmol/L EDTA, pH 8.0) (13).

AMPLIFICATION OF EXONS AND FLANKING REGIONS, DNA SEQUENCING, AND GENOTYPING

We performed PCR to amplify the exons and flanking regions of the WD gene from the DNA of patients using primers (14) and PCR conditions as described (11).

The PCR products that were free of contaminating bands due to nonspecific amplification were column-purified using Qiagen PCR-purification reagent sets (Qiagen). We performed bidirectional sequencing by use of an ABI 3100 DNA sequencer with dye-termination chemistry. We confirmed the nucleotide changes in the DNA samples from one of the parents of the proband. We carried out genotyping of the SNPs in the general population by allele-specific primer extension followed by MALDI-TCF mass spectrometry using the Sequenom mass array system, as a part of the Indian Genome Variation project at the Centre for Genomic Application (New Delhi, India).

STATISTICAL ANALYSIS

We detected novel nucleotide changes by comparing the sequence obtained in the chromatogram with the normal gene sequence (NT 024524; Homo sapiens chromosome 13 genomic contig) using pairwise BLAST (15). We computed allele frequencies and heterozygosities at each variant site by the genotype-counting method. Coefficients of pairwise LD (r z) were estimated using Haploview version 3.32 (16).

Results

We screened 87 unrelated WD patients for nucleotide variants in the ATP7B gene. In addition to 4 prevalent mutations previously identified (11), we detected a mutation (c.3182 G>A/p.G1y1061Glu) accounting for 11% of the WD chromosomes in our patient pool. A comprehensive list of the prevalent mutations in different populations is provided in Table 1.

In addition, we identified 24 innocuous allelic variants apparently not causal to the disease in 82 WD patients (see Table 2 in the online Data Supplement). First, we estimated the frequencies of the allelic variants for each SNP, and only those with relatively higher minor allele frequencies (>0.25) were selected. The 4 SNPs (c.1216 TCT>GCT/p.Ser406Ala, c.2495 AAG>AGG/p.Lys832Arg, c.2855 AGA>AAA/p. Arg952Lys, and c.1544-53A>C) thus picked were observed to have high heterozygosity across various linguistic groups of India (Table 2). Interestingly, although Austro-Asiatic groups are exclusively endogamous and tribes are dispersed in small inbreeding groups in central and eastern India, the heterozygosities of all the SNPs were high, ranging from 0.41 to 0.481.

To investigate the utility of the SNPs as markers in different populations, we also checked the allele frequency and heterozygosity of the variants in the 4 HapMap project populations, namely Utah residents with ancestry from northern and western Europe (CEU, CEPH), Han Chinese in Beijing, China (CHB), Japanese in Tokyo, Japan (JPT), and Yoruba in Ibadan, Nigeria (YRI). The allele frequencies of the 4 SNPs in these populations are illustrated in Fig. 1. It is evident that, for all the SNPs, the minor allele frequency is high enough to yield high heterozygosity in all the populations except YRI, in which the G allele for the SNP c.1216 TCT>GCT shows a low frequency of 0.09 and overall low heterozygosity (0.169; Table 2). Interestingly, the minor allele frequencies for the SNPs c.2495 AAG>AGG and c.2855 AGA>AAA are identical in 3 of the 4 HapMap populations, CEU, CHB, and JPT. Both Han Chinese and Japanese are members of the larger mongoloid population, but a difference was evident in the heterozygosity of 2 SNPs, c.1216 TCT>GCT (0.444 vs 0.568) and c.1544-53A>C (0.378 vs 0.545).

A set of SNPs would be most applicable as markers if the LD between them were low, i.e., the 2 SNPs independently segregate in the population and hence can exclusively serve as markers. In cases in which the 2 SNPs show high LD, either one suffices for the other. LD plots were constructed for the 4 different HapMap populations and the Indian population consisting of 55 ethnic groups. As shown in Fig. 2, the SNPs were denoted as follows: c.2855 AGA>AAA (p.Arg952Lys), SNP1; c.2495 AAG>AGG (p.Lys832Arg), SNP2; c.1544-53A>C, SNP3; and c.1216 TCT>GCT (p.Ser406Ala), SNP4. The best possible combinations for the SNPs to be used as markers were determined based on the [r.sup.2] value between them. In all population groups, SNP1 and SNP2 are in very high LD ([r.sup.2] value 0.90 to 1.00). LD is comparatively much lower between the other SNP pairs for all 5 populations. The Yoruban population shows minimum LD between the different SNP pairs. The LD pattern between the 4 SNPs was also determined for the 4 linguistic families and the outgroup population of India. Like the entire Indian population and other populations, SNPs 1 and 2 showed highest LD, with [r.sup.2] ranging from 0.79 to 1.00 in all 4 linguistic groups. The outgroup population known to have an African origin showed an LD pattern similar to that of YRI.

On the basis of the frequency of the prevalent mutations and the heterozygosity of the intragenic SNPs and LDs ([r.sup.2] values), we propose a comprehensive strategy for identification of presymptomatic and carrier sibs of WD patients, through which the potential problem of recombination due to large distance of the microsatellite markers (i.e., 375-917 kb flanking the ATP7B gene) can be avoided (Fig. 3). To test the usefulness of these SNP markers in our patient pool, we tested 45 offspring from 17 of the 30 multisibling families with 1 proband each. Ten of these 17 patients were found to harbor disease-causing Mutations--IVS4-1G>C, p.G1061E, p.Y187X, p.C271X, c.892delC, p.G710S, and c.448_452del5--in either homozygous or compound heterozygous condition before SNP genotyping. We determined the genotypes at the SNP markers in 28 individuals who were sibs to the 17 unrelated WD patients in addition to screening of prevalent mutations. We confirmed genotyping for the SNP markers by bidirectional sequencing and Mendelian pattern of inheritance within the family. Thus, as shown in Table 3, it was found that among the sibs of the patients, 14 were heterozygous for the mutant chromosome (i.e., carrier), 6 children lacking any sign or symptoms of the disease had 2 copies of the same mutant chromosomes as the affected sib (presymptomatic), and 5 children did not harbor a mutant chromosome (genotypically normal). In 3 children (approximately 11% of the test samples), SNP-based diagnosis could not distinguish between the carriers and those harboring no mutant allele (Table 3). This anomaly was resolved by using the microsatellite marker D13S133.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Discussion

The causal gene for WD, ATP7B, is fairly large in size (approximately 80 kb), and >300 mutations have been reported encompassing the entire gene. In the Indian population, although prevalent mutations account for approximately 41% of total mutations, rare mutations are numerous and do not cluster to any specific region of the gene (11). Hence, it is an arduous job to identify the mutations in WD patients and screen those mutations in sibs. The problem is even more acute in populations in which prevalent mutations are either not identified or account for a very low percentage of the total WD mutations. Intragenic SNPs can be ideal tools to identify and segregate the normal and mutant chromosomes. Although ATP7B is rich in SNPs, with 198 reported variants (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=540&chooseRs=all), reports on SNPs used as markers in WD are rare. The publication of the SNP HapMap data has enabled us to devise a generalized strategy that can be used in Indians and other populations for the identification of presymptomatic and carrier sibs in WD families.

India consists of ethnically, geographically, and genetically diverse populations consisting of 4693 communities with several thousand endogamous groups, 325 functioning languages, and 25 scripts (17). In general, the Indian population can be, to a large extent, substructured on the basis of ethnic origin as well as linguistic lineage. Of India's 4 major language families, Indo-European and Dravidian languages are spoken in the northern and southern parts of the subcontinent, respectively (18); Tibeto-Burman speakers, concentrated in the northern and northeastern parts of the country, are supposed to have immigrated to India from Burma (now, Myanmar) and Tibet (19); and Austro-Asiatic speakers are exclusively tribes and are dispersed mostly in the central and eastern parts of the country. We extended our study to find the heterozygosities and LDs among the selected SNP markers in 55 populations selected on the basis of their linguistic lineage and geographic location.

Therefore, the present study modeled on the Indian population has a worldwide applicability. The SNPs selected as markers in the Indian population have also been checked in all the HapMap populations. High heterozygosity of the 4 SNPs in all populations suggests that these SNPs are evolutionarily old enough and hence can probably be used as markers worldwide. The LD values ([r.sup.2]) among the 4 SNPs based on the data from 4 linguistic groups of India and HapMap populations suggest that SNPs 1 and 2 (i.e., c.2495 AAG>AGG and c.2855 AAA>AGA, respectively) share a high LD among them. The lower the [r.sup.2] value between 2 SNPs, the better are the chances of using them individually as markers. Hence either SNP1 or SNP2 can serve as a marker in combination with SNP3 and SNP4. Incidentally, SNPs 3 and 4 in ATP7B alter the restriction sites for BtsCI and AciI, respectively. Therefore, genotyping of these 2 SNPs can easily be done by restriction fragment length polymorphism analysis instead of DNA sequencing.

The general strategy to diagnose a sib of a WD patient as carrier, presymptomatic, or normal begins with screening for the WD mutations prevalent in the population, followed by typing the recommended SNPs in the family to understand the chromosomal segregation. In rare cases, if all the SNPs are found to be uninformative, microsatellite markers could be genotyped in the family to diagnose the sib of a WD patient. Once WD predisposition is identified in an individual, regular checkups, suitable nutrition, and preventive medication should be recommended to thwart disease progression and onset. Families affected with the disease should be encouraged to undergo carrier detection and given genetic counseling as appropriate.

The strategy using intragenic SNPs for diagnosis is highly specific because of the extremely low chances of recombination between the mutation and the SNP marker and is highly sensitive because of the high frequency of the selected markers throughout most world populations. A similar approach can be adopted for other inherited disorders for which prevalent mutations and informative markers are available in the public domain.

Grant/funding support: This study was supported by the Council of Scientific and Industrial Research, Government of India, through a research grant (CMM 0016), a predoctoral fellowship (to A.G. and P.N.), and a postdoctoral fellowship (to I.C.).

Financial disclosures: None declared.

Acknowledgments: We thank the patients and their family members for participating in the study.

Received January 19, 2007; accepted June 5, 2007. Previously published online at DOI: 10.1373/clinchem.2007.086066

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[4] Nonstandard abbreviations: WD, Wilson Disease; K-F, Kayser-Fleischer; SNP, single-nucleotide polymorphism; LD, linkage disequilibrium; CEU, CEPH: Utah residents with ancestry from northern and western Europe; CHB, Han Chinese in Beijing, China; JPT, Japanese in Tokyo, Japan; YRI, Yoruba in Ibadan, Nigeria.

[5] Human gene: ATP7B, ATPase, [Cu.sup.2[dagger]] transporting, [beta]-polypeptide.

ARNAB GUPTA, [1][dagger][double dagger] MAHUA MAULIK, [1][double dagger] POONAM NASIPURI, [1] ISHITA CHATTOPADHYAY, [1] SHYAMAL K. DAS, [2] PRASANTA K. GANGOPADHYAY, [2] THE INDIAN GENOME VARIATION CONSORTIUM, [3] and KUNAL RAY [1]*

[1] Molecular and Human Genetics Division, Indian Institute of Chemical Biology, Kolkata, India.

[2] Bangur Institute of Neurology, Kolkata, India.

[3] Nodal Laboratory, Institute of Genomics and Integrative Biology, New Delhi, India.

[dagger] Current affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR.

[double dagger] These authors contributed equally to this study.

* Address correspondence to this author at: Molecular and Human Genetics Division, Indian Institute of Chemical Biology, 4 Raja S C Mullick Rd., Jadavpur, Kolkata 700 032, India. Fax 91-33-2473-5197; e-mail kunalray@gmail.com.
Table 1. Prevalent mutations of ATP7B in world populations.

Population Mutation Chromosomes
 studied, n (%)
White
 North European c.3207C>A (p.H1069Q) 174 (21.2)
 British c.3207C>A (p.H1069Q) 84 (16.6)
 French, Italian c.3207C>A (p.H1069Q) 64 (11.0)
 Swedish c.3207C>A (p.H1069Q) 42 (38.0)
 German c.3207C>A (p.H1069Q) 66 (42.0)
 Continental Italian c.3207C>A (p.H1069Q) 73 (17.5)
 Bulgarian c.3207C>A (p.H1069Q) 178 (58.75)
 Czech, Slovakian c.3207C>A (p.H1069Q) 454 (57.0)
 Israeli c.3207C>A (p.H1069Q) 38 (13.1)
 Continental Italian c.2532delA 73(9.0)
 Sardinians c.2463deIC 92(8.5)
 Sardinians c.3436G>A (p.V1146M) 18(7.9)
 Turkish c.3659C>T (p.T1220M) 43 (10.0)
 Bulgarian c.2304 2305insC 178 (11.25)
 Spanish c.1934T>G (p.M645R) 80 (27.0)
Mongoloid
 Japanese c.2333G>T (p.R778L) 42 (12.0)
 Taiwanese c.2333G>T (p.R778L) 44 (27.0)
 Chinese (Han and Hui) c.2333G>T (p.R778L) 80 (33.8)
 Korean c.2333G>T (p.R778L) 16 (37.9)
 Chinese c.2333G>T (p.R778L) 108 (45.6)
 Taiwanese c.2333G>T (p.R778L) 58 (43.1)
South Asian
 Northwest Indian c.3350T>C (p.11102T) 86(6.1)
 Eastern Indian c.813C>A (p.C271X) 124 (16.0)
 Eastern Indian c.1708-1G>C 124 (8.5)
 Eastern Indian c.3182G>A (p.G1061E) 174 (11.0)
 Eastern Indian c.448 452del5 124 (5.6)
 Eastern Indian c.561T>A (p.Y187X) 124 (2.5)
Other populations
 Saudi Arabian c.4193deIC 32 (53.3)
 Brazilian c.3402deIC 120 (30.8)
 Canary Island c.2123T>C (p.L708P) 48 (64.5)

Population Reference

White
 North European (20)
 British (21)
 French, Italian (22)
 Swedish (23)
 German (24)
 Continental Italian (25)
 Bulgarian (26)
 Czech, Slovakian (27)
 Israeli (28)
 Continental Italian (25)
 Sardinians (25)
 Sardinians (25)
 Turkish (25)
 Bulgarian (26)
 Spanish (29)
Mongoloid
 Japanese (30)
 Taiwanese (31)
 Chinese (Han and Hui) (32)
 Korean (33)
 Chinese (34)
 Taiwanese (35)
South Asian
 Northwest Indian (36)
 Eastern Indian (11)
 Eastern Indian (11)
 Eastern Indian This report
 Eastern Indian (11)
 Eastern Indian (11)
Other populations
 Saudi Arabian (37)
 Brazilian (38)
 Canary Island (39)

Table 2. Observed heterozygosity of the SNPs among
different populations.

 Ser406Ala
Population n (c.1216 T>G) c.1544-53A>C

World populations
 Europe (CEPH) 90 0.6 0.5
 East Asia (Han Chinese) 45 0.444 0.378
 East Asia (Japanese) 44 0.568 0.545
 West African (Yoruba) 90 0.169 0.367
 India 1871 0.444 0.502
Linguistic groups of the
Indian population
 Indo-European 1111 0.458 0.521
 Dravidian 369 0.38 0.461
 Tibeto-Burman 159 0.47 0.508
 Austro-Asiatic 209 0.471 0.481
 Outgroup 23 0.364 0.391

 Lys832Arg Arg952Lys
Population (c.2495 A>G) (c.2855 G >A)

World populations
 Europe (CEPH) 0.578 0.578
 East Asia (Han Chinese) 0.356 0.364
 East Asia (Japanese) 0.349 0.364
 West African (Yoruba) 0.6 0.633
 India 0.466 0.428
Linguistic groups of the
Indian population
 Indo-European 0.466 0.422
 Dravidian 0.466 0.461
 Tibeto-Burman 0.472 0.405
 Austro-Asiatic 0.464 0.41
 Outgroup 0.45 0.455

Table 3. SNP-based genotyping data for
WD-affected families.

Family No. of
no. sibs Patient Presymptomatic

1 4 1 1
2 2 1 1
3 3 1 1
4 3 1 1
5 3 1 1
6 4 1
7 2 1 1
8 3 1
9 2 1
10 2 1
11 2 1
12 4 1
13 2 1
14 2 1
15 2 1
16 2 1
17 3 1

Family Carrier/
no. Carrier Normal normal (a)

1 2
2
3 1
4 1
5 1
6 1 1 1
7
8 1 1
9 1
10 1
11 1
12 2 1
13 1
14 1
15 1
16 1
17 2

(a) In these samples, SNP-based genotyping could not
distinguish carrier from homozygous normal. The anomaly
was resolved by genotyping those families using D13S133
short tandem repeat (microsatellite) markers.
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Title Annotation:Molecular Diagnostic and Genetics
Author:Gupta, Arnab; Maulik, Mahua; Nasipuri, Poonam; Chattopadhyay, Ishita; Das, Shyamal K.; Gangopadhyay,
Publication:Clinical Chemistry
Date:Sep 1, 2007
Words:5122
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