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COMMENTARY
Year : 2015  |  Volume : 141  |  Issue : 2  |  Page : 145-147

Low-density oligonucleotide microarrays - A major step in Wilson's disease diagnosis


Department of Cell Biology, Johns Hopkins University, School of Medicine, Baltimore MD, USA

Date of Web Publication21-Apr-2015

Correspondence Address:
Arnab Gupta
Department of Cell Biology, Johns Hopkins University, School of Medicine, Baltimore MD
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0971-5916.155534

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How to cite this article:
Gupta A. Low-density oligonucleotide microarrays - A major step in Wilson's disease diagnosis. Indian J Med Res 2015;141:145-7

How to cite this URL:
Gupta A. Low-density oligonucleotide microarrays - A major step in Wilson's disease diagnosis. Indian J Med Res [serial online] 2015 [cited 2019 Jul 24];141:145-7. Available from: http://www.ijmr.org.in/text.asp?2015/141/2/145/155534

Wilson's disease (WD) is a monogenic disorder of copper accumulation caused due to mutation in the copper transporting ATPase gene ATP7B[1],[2] . WD follows an autosomal recessive mode of inheritance with a world prevalence of 1 in 5000 to 1 in 30,000 live births and a carrier frequency of 1 in 90 3 . Though a single gene disorder, mutation detection in WD has been difficult, mainly due to two reasons: (i) size of the ATP7B gene is large (>6.5 kb) with 21 exons, and (ii) besides the prevalent mutations, extensive presence of rare mutations throughout the entire gene. To screen the entire gene one has to undertake amplification and sequencing strategy encompassing 24-26 exonic regions including intron-exon boundaries [4],[5] . This requires substantial time and effort. The second problem is heterogeneity of mutations i.e. the occurrence of hundreds of rare (non-prevalent) mutations in various world populations. Moreover, there also exists a huge variability in the prevalent mutations in these populations.

Indian population vs major world populations

Among the data available for a very few major world populations, H1069Q and E1064A are the major founder mutants in Caucasians [6] , R778L and P992L among the East Asians that includes Japanese, Korean and Chinese populations [7],[8],[9] . Among patients from Brazil, c.3402delC mutation had the highest frequency (30.8%), followed by the missense change, c.2123T>C (p.L708P) (16.7%) [10] . The Indian population is highly heterogeneous, comprising four major linguistic groups i.e. Indo-European, Dravidian, Tibeto-Burman and Austro-Asiatic, and 4693 communities with several thousand endogamous groups (The Indian Genome Variation Database) [11] . Due to high genetic heterogeneity in the Indian population, prevalent mutations for a specific sub-population might not be present in others, even if they are subgroups of the same larger linguistic group.

The data for WD mutations from India are quite divergent reflecting the ethno-genetic diversity of this large country. None of the common mutations detected has a prevalence over 25 per cent of all ATP7B mutations. In patients from southern India, c.G3182A (p.G1061E) in 16 per cent and c.C813A (p.C271X) in 12 per cent accounted for major ATP7B mutations [12] , whereas p.E122fs (10.6%) and C271X detected in western Indian patients were the prevalent ones [13] . Patients from eastern and northern India harboured five prevalent mutations. Three mutations c.813C>A (Cys271Stop), c.1708-1G>C (IVS4-G>C) (splice site mutation) and c.3182 GGG>GAG (Gly1061Glu) were found to be associated with 16, 8.5 and 8 per cent of all the mutant chromosomes [5],[14] .

To summarize, multiple studies from different zones of India suggest that detecting WD by screening an individual for one major founder mutation would not be fruitful. A comprehensive and efficient strategy to screen a panel of multiple mutations (both common and rare) in a single multiplex reaction would address the issue.

Genetic diagnosis of WD has a tremendous advantage

Disease onset varies from as early as age of 2 years to as late as the 7 th decade of life [15] . Early or presymptomatic diagnosis can alleviate disease progression and in some cases entirely prevent onset of disease symptoms. However, there is no confirmatory biochemical or clinical tests available for early disease detection. In the last 10 years major effort has been made in genetic diagnosis of the disease. Roberts and Schilsky laid guidelines for WD diagnosis approved by the American Association for the Study of Liver Diseases (AASLD) [16] . Besides, typical clinical symptoms, such as presence of Keyser Fleischer ring, low serum ceruloplasmin (<20 mg/dl) and 24 h urinary copper excretion (>40 μg), molecular testing to determine the mutation has been considered crucial for disease diagnosis [16] . Also, genetic testing can be carried out to determine the mutation status in neonates, much before the onset of any clinical symptoms. Further, detection of carrier status of an individual can ensure proper genetic counselling. To date, major advances have been made to identify the disease status in siblings of WD patients. Gupta et at[14],[17] have utilized heterozygous microsatellite markers and informative single nucleotide polymorphisms (SNPs) to identify the mutant chromosome segregating within a WD family. The advantage of this study lies in rapid identification of presymptomatic individuals. However, in such studies the specific mutation often goes undetected. In some cases, high genetic homogeneity or consanguineous marriage results in non-informativeness of the SNP or microsatellite markers. The present study by Mathur et al[18] further improves WD diagnosis by identifying specific mutations and not relying on informativeness or heterozygosity of markers.

Introducing low-density microarray for WD detection

In this issue the study by Mathur et al[18] on microarray based diagnosis of WD is a major development not only in the area of genetic diagnosis of WD but also for monogenic disorders as a whole. The study attempts to establish detection and analysis procedures for microarray based diagnosis of WD. Presymptomatic identification by mutation detection would be now possible in neonates.

Mathur et al[18] describes designing and utilizing an oligonucleotide microarray for detecting WD mutations. A subset of 62 mutations that were specific to Indian population was identified in the Wilson disease database and was used to design oligonucleotide probes for a low-density WD microarray. The entire process is simple but effective and can be split into two parts. Part 1 involves selection of mutations from the WD database, designing oligonucleotide probes and printing them as microarrays. The second part involved designing DNA amplicons to be tested. The amplicons were DIG labelled. As a proof of principle, the test amplicons were hybridized on the microarrays containing the probes, washed and then analyzed for binding. Around 60 per cent of the mutations showed high detectability. On including weakly hybridized but reproducible amplicons, the detection rate rose to 80 per cent. Moreover, prevalent mutations in the Indian population, i.e. Tyr187Stop, Cys271Stop, Gly1061Glu, c.1708-1G>C were detected in the tests against controls. Authors also included ambivalent variants, e.g. c.2623G>A (Gly875Arg) that exhibit dual status in the WD database. Presence of this variant is linked to disease causation (DV) in southern Indian WD patients; however, is designated as a non-disease variant (NDV) in Han-Chinese population [19],[20] . In vitro studies carried out in cell lines have demonstrated that copper content of the cell dictates subcellular localization of the Arg 875 variant and hence affecting intracellular copper content [21] . Early detection of these variants of ambiguous status, early in life of individuals will certainly help to take proper lifestyle and nutritional measures and possibly prevent the disease.

In a nation like India with great genetic diversity, inexpensive genetic testing of common as well as rare mutations would prove to be a blessing. Efforts should be made to improve accuracy and efficiency of this WD microarray platform to make it available for the healthcare system of the Indian subcontinent. Also, similar studies should be extended towards other monogenic diseases that show high mutational heterogeneity.

 
   References Top

1.
Kinnier Wilson SA. Progressive lenticular degeneration: a familial nervous disease associated with cirrhosis of the liver. Brain 1912; 34 : 295-509.  Back to cited text no. 1
    
2.
Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 1993; 5 : 327-37.  Back to cited text no. 2
    
3.
Figus A, Angius A, Loudianos G, Bertini C, Dessi V, Loi A, et al. Molecular pathology and haplotype analysis of Wilson disease in Mediterranean populations. Am J Hum Genet 1995; 57 : 1318-24.  Back to cited text no. 3
    
4.
Waldenstrom E, Lagerkvist A, Dahlman T, Westermark K, Landegren U. Efficient detection of mutations in Wilson disease by manifold sequencing. Genomics 1996; 37 : 303-9.  Back to cited text no. 4
    
5.
Gupta A, Aikath D, Neogi R, Datta S, Basu K, Maity B, et al. Molecular pathogenesis of Wilson Disease: haplotype analysis, detection of prevalent mutations and genotype-phenotype correlation in Indian patients. Hum Genet 2005; 118 : 49-57.  Back to cited text no. 5
    
6.
Duc HH, Hefter H, Stremmel W, Castaneda-Guillot C, Hernandez Hernandez A, Cox DW, et al. Auburger G. His1069Gln and six novel Wilson disease mutations: analysis of relevance for early diagnosis and phenotype. Eur J Hum Genet 1998; 6 : 616-23.  Back to cited text no. 6
    
7.
Kim EK, Yoo OJ, Song KY, Yoo HW, Choi SY, Cho SW, et al. Identification of three novel mutations and a high frequency of the Arg778Leu mutation in Korean patients with Wilson disease. Hum Mutat 1998; 11 : 275-8.  Back to cited text no. 7
    
8.
Nanji MS, Nguyen VT, Kawasoe JH, Inui K, Endo F, Nakajima T, et al. Haplotype and mutation analysis in Japanese patients with Wilson disease. Am J Hum Genet 1997; 60 : 1423-9.  Back to cited text no. 8
    
9.
Gu YH, Kodama H, Du SL, Gu QJ, Sun HJ, Ushijima H. Mutation spectrum and polymorphisms in ATP7B identified on direct sequencing of all exons in Chinese Han and Hui ethnic patients with Wilson's disease. Clin Genet 2003; 64 : 479-84.  Back to cited text no. 9
    
10.
Deguti MM, Genschel J, Cancado EL, Barbosa ER, Bochow B, Mucenic M, et al. Wilson disease: novel mutations in the ATP7B gene and clinical correlation in Brazilian patients. Hum Mutat 2004; 23 : 398.  Back to cited text no. 10
    
11.
Available from: www.igvdb.res.in, accessed on November 3, 2014.  Back to cited text no. 11
    
12.
Santhosh S, Shaji RV, Eapen CE, Jayanthi V, Malathi S, Chandy M, et al. ATP7B mutations in families in a predominantly Southern Indian cohort of Wilson's disease patients. Indian J Gastroenterol 2006; 25 : 277-82.  Back to cited text no. 12
    
13.
Aggarwal A, Chandhok G, Todorov T, Parekh S, Tilve S, Zibert A, et al. Wilson disease mutation pattern with genotype-phenotype correlations from Western India: confirmation of p.C271* as a common Indian mutation and identification of 14 novel mutations. Ann Hum Genet 2013; 77 : 299-307.  Back to cited text no. 13
    
14.
Gupta A, Maulik M, Nasipuri P, Chattopadhyay I, Das SK. Gangopadhyay PK; Indian Genome Variation Consortium. Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers. Clin Chem 2007; 53 : 1601-8.  Back to cited text no. 14
    
15.
Ala A, Borjigin J, Rochwarger A, Schilsky M. Wilson disease in septuagenarian siblings: raising the bar for diagnosis. Hepatology 2005; 41 : 668-70.  Back to cited text no. 15
    
16.
Roberts EA, Schilsky ML; American Association for Study of Liver Diseases (AASLD). Diagnosis and treatment of Wilson disease: an update. Hepatology 2008; 47 : 2089-111.  Back to cited text no. 16
    
17.
Gupta A, Neogi R, Mukherjea M, Mukhopadhyay A, Roychoudhury S, Senapati A, et al. DNA linkage based diagnosis of Wilson disease in asymptomatic siblings. Indian J Med Res 2003; 118 : 208-14.  Back to cited text no. 17
    
18.
Mathur M, Singh E, Poduval TB, Rao AVSSN. Development of low-density oligonucleotide microarrays for detecting mutations causing Wilson's disease. Indian J Med Res 2015; 141 : 175-86.  Back to cited text no. 18
    
19.
Wu ZY, Wang N, Lin MT, Fang L, Murong SX, Yu L. Mutation analysis and the correlation between genotype and phenotype of Arg778 Leu mutation in Chinese patients with Wilson disease. Arch Neurol 2001; 58 : 971-6.  Back to cited text no. 19
    
20.
Santosh S, Shaji RV, Eapen CE, Jayanthi V, Malathi S, Finny P. Genotype phenotype correlation in Wilson's disease within families - a report on four south Indian families. World J Gastroenterol 2008; 14 : 4672-6.  Back to cited text no. 20
    
21.
Gupta A, Bhattacharjee A, Dmitriev OY, Nokhrin S, Braiterman L, Hubbard A, et al. Cellular copper levels determine the phenotype of the Arg 875 variant of ATP7B/Wilson disease protein. Proc Natl Acad Sci USA 2011; 108 : 5390-5 .  Back to cited text no. 21
    



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