Gastroenterology Research and Practice

Gastroenterology Research and Practice / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 4548039 | 6 pages | https://doi.org/10.1155/2016/4548039

Clinical Use of Next-Generation Sequencing in the Diagnosis of Wilson’s Disease

Academic Editor: Alfred Gangl
Received08 Jul 2015
Revised15 Sep 2015
Accepted20 Sep 2015
Published24 Dec 2015

Abstract

Objective. Wilson’s disease is a disorder of copper metabolism which is fatal without treatment. The great number of disease-causing ATP7B gene mutations and the variable clinical presentation of WD may cause a real diagnostic challenge. The emergence of next-generation sequencing provides a time-saving, cost-effective method for full sequencing of the whole ATP7B gene compared to the traditional Sanger sequencing. This is the first report on the clinical use of NGS to examine ATP7B gene. Materials and Methods. We used Ion Torrent Personal Genome Machine in four heterozygous patients for the identification of the other mutations and also in two patients with no known mutation. One patient with acute on chronic liver failure was a candidate for acute liver transplantation. The results were validated by Sanger sequencing. Results. In each case, the diagnosis of Wilson’s disease was confirmed by identifying the mutations in both alleles within 48 hours. One novel mutation (p.Ala1270Ile) was found beyond the eight other known ones. The rapid detection of the mutations made possible the prompt diagnosis of WD in a patient with acute liver failure. Conclusions. According to our results we found next-generation sequencing a very useful, reliable, time-saving, and cost-effective method for diagnosing Wilson’s disease in selected cases.

1. Introduction

Wilson’s disease (WD) is a rare autosomal recessive disorder of copper metabolism. ATP7B gene mutation is in the background of the excessive copper accumulation which is fatal without treatment. More than 550 disease-causing mutations of the gene located at chromosome 13q14.3-q21.1 consisting of 21 exons have been identified [1].

The geographical distribution of the mutations of the ATP7B gene is inhomogeneous [25]. In Hungary the p.His1069Gln mutation is the most frequent one with 71% prevalence among the patients [6].

The variable clinical presentation of WD may cause a real diagnostic challenge. The suspicion of the disease usually arises when hepatic or neurologic-psychiatric symptoms appear. Low ceruloplasmin level and presence of Kayser-Fleischer ring could support the diagnosis, but in many cases only genetic testing could confirm it. Genetic investigation of asymptomatic siblings has an extreme importance, since the early treatment could prevent the manifestation of the disease [7]. In acute liver failure urgent genetic testing of all known mutations may strengthen the diagnosis of Wilson’s disease.

The emergence of next-generation sequencing (NGS) provides a time-saving, cost-effective method for full sequencing of the whole ATP7B coding sequence compared to the traditional Sanger sequencing. The NGS technology is based on the detection of a signal during the synthesis of the DNA strand, and therefore the synthesis does not need to be terminated for the perception. On the other hand, several DNA strands can be examined simultaneously [8].

This is the first report on the clinical use of NGS to examine ATP7B gene in WD patients including doubtful cases. We used Ion Torrent Personal Genome Machine in heterozygous patients for the identification of the other mutations and also in patients with no known mutation including one with acute on chronic liver failure.

2. Materials and Methods

The method we used for the genetic testing has been previously published by our group for screening of neurofibromatosis type 1 gene [9].

2.1. Biological Samples and DNA Isolation

Six (five male and one female) WD patients, four heterozygous for ATP7B p.His1069Gln mutation identified by fast PCR test and two with unknown mutation, were selected for this study. The patients were diagnosed and treated at the 1st Department of Internal Medicine, Semmelweis University, Budapest. The diagnosis was based on the international WD score system published in 2003 [10], and each patient had 4 or more scores. The study was approved by the Semmelweis University’s Committee of Research Ethics and was conducted in accordance with the Helsinki Declaration. All patients gave written informed consent.

Genomic DNA was isolated from 200 μL of peripheral blood using ReliaPrep Blood gDNA Miniprep System (Promega, Madison, WI). Briefly, the blood samples were digested with Proteinase K solution in the presence of Cell Lysis Buffer, and, after 10 min of incubation at 56°C, DNA was bound to ReliaPrep Binding Column. After three washes, DNA was eluted into 50 μL of nuclease-free water. The concentration of the isolated DNA was determined with Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA).

2.2. Ion Torrent Sequencing

ATP7B (21 coding exons) amplicons were designed using the AmpliSeq Designer software (Life Technologies, CA, USA), targeting the complete coding sequence of ATP7B gene, resulting in a total of 55 amplicons. To gain a higher coverage of the coding exons, we designed the primers to also flank some parts of the introns. Amplicon library was prepared using the Ion AmpliSeq Library Kit 2.0 (Life Technologies, CA, USA); briefly, multiplex primer pools were added to 10 ng of genomic DNA and amplified with the following PCR cycles: at 99°C for 2 min, at 99°C for 15 s, and at 60°C for 4 min (18 cycles), and holding on at 10°C. Primers were partially digested using a FuPa reagent, and then sequencing adapters were ligated to the amplicons. The library was purified in multiple times using the Agencourt AMPure XP Reagent (Beckmann Coulter, CA, USA). The concentration of the final library was determined by fluorescent measurement on Qubit 2.0 instrument (Life Technologies, CA, USA). Template preparation was performed with Ion OneTouch kit (Life Technologies, CA, USA) on semiautomatic Ion OneTouch instrument using an emPCR method. After breaking the emulsion, the nontemplated beads were removed from the solution during the semiautomatic enrichment process on Ion OneTouch ES (Life Technologies, CA, USA) instrument. After adding the sequencing primer and polymerase, the fully prepared Ion Sphere Particle (ISP) beads were loaded into an Ion 314 v2 sequencing chip, and the sequencing runs were performed using the Ion PGM 200 Sequencing kit v2 (Life Technologies, CA, USA) with 500 flows.

2.3. Sanger Sequencing Validation

The PCR primers were designed using Primer3Plus (http://primer3plus.com/) software. Roche FastStart TaqMan Probe Master (Roche) kit was used to amplify the target regions and the PCR program was as follows: 95°C for 10 min, 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and the final step was 72°C for 5 min. PCR products were enzymatically cleaned using ExoSAP IT (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. Sanger sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) using an ABI 3130 instrument (Life Technologies).

2.4. Data Analysis

Data from the Ion Torrent runs were analyzed using the platform-specific pipeline software Torrent Suite v3.6 for base calling, trim adapter and primer sequences, filter out poor quality reads, and demultiplex the reads according to the barcode sequences. Briefly, TMAP (https://github.com/iontorrent/TMAP) algorithm was used to align the reads to the hg19 human reference genome, and then the variant caller plug-in was selected to run to search for germ line variants in the targeted regions. The variant caller algorithm parameters were more relaxed to avoid false negative cases. Integrative Genomics Viewer was used for visualization of the mapped reads. Variants were reviewed and annotated using dsSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) and Wilson Disease Mutation Database (http://www.wilsondisease.med.ualberta.ca/index.asp). For variant interpretation, Ingenuity Variant Analysis Pipeline (Ingenuity, Rewood City, CA) was also used. Pathogenic status of the variant was stated if it was a missense variant with <1% minor allele frequency and/or the variant was listed in the literature or in the databases as a pathogenic alteration. All of the deleterious variants were confirmed by Sanger sequencing. The Sanger sequence data were investigated using ABI Sequence Scanner 1.0 (Life Technologies) and BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) software.

3. Results

The demographic and clinical characteristics of the patients are shown in Table 1. One patient without known mutation was critically ill with acute on chronic liver disease. The typical laboratory findings (ALT 90, AST 178, ALP 88, and bilirubin 247) proposed Wilson’s disease. The diagnosis was strengthened by genetic testing making possible the liver transplantation via Eurotransplant program in Patient 5 with acute on chronic liver failure.


GenderAge at onset (year)KFRNeuHAUrin CuBiopsyCerul (g/L)ATP7B statusWD scorePhenotype

Patient 1 Female12PAA++ND0.18p.Met769-fs/p.His1069Gln6S
Patient 2Male17APA+ND0.05p.Ala1063Val/p.His1069Gln6N1
Patient 3Male8PAA++0.06p.His1069Gln/p.Gln1351Stop8H2
Patient 4Male17PAA+ND0.03p.Ala1135-fs/p.Leu1305Pro5H2
Patient 5Male44AAA++ND0.08p.Ala1270Ile/c.1707+2dupT4H1
Patient 6Male14PPANDND0.04p.Arg969Gln/p.His1069Gln7N2

KFR: Kayser-Fleischer ring; Neu: neurological signs and/or CT/MRI alterations; HA: hemolytic anemia; Urin Cu: urinary copper, 1-2X ULN: +, >2x ULN or positive D-penicillamine challenge: ++; Cerul: ceruloplasmin, P: present; A: absent; ND: not done; S: sibling; H1: acute liver failure; H2: chronic liver disease; N1: neurological symptoms with liver disease; N2: only neurological symptoms.
According to the international score system, 4 or more scores, diagnosis of WD is highly likely. Rhodanine positivity.

In each case, the diagnosis of Wilson’s disease was confirmed by identifying the mutations in both alleles. The results were available within 48 hours.

The average read number per sample was 134386, with an average 1X on-target coverage of 99.46%. The mean raw accuracy was 99.2%. The average base coverage depth was 1883 (Table 2). The number of identified variants per sample was between 8 and 13; however most of them were known as non-disease-causing variants.


ChromosomeAmplicon startAmplicon endAmplicon IDGene IDPatient 1Patient 2Patient 3Patient 4Patient 5Patient 6

chr135252035852520440AMPL1102520485ATP7B812484378946735573275
chr135254253052542653AMPL1102672008ATP7B951390145314955103183
chr135253890952539030AMPL1404436522ATP7B9222446
chr135252373552523838AMPL561308261ATP7B417285892456832611
chr135251824452518356AMPL561308388ATP7B1140520339793461663595
chr135251649252516613AMPL561312436ATP7B60030970151032182152
chr135251513452515253AMPL561312709ATP7B79928774134032582733
chr135252043552520563AMPL561312859ATP7B6223081494099062130
chr135251161652511742AMPL561313522ATP7B34323358146221681069
chr135254847552548562AMPL561315457ATP7B74055724866012513869
chr135254801452548135AMPL561316164ATP7B506109932446143077
chr135253595252536073AMPL561319098ATP7B34533262184539191350
chr135253244552532575AMPL561319439ATP7B495382752604772421
chr135251833252518433AMPL561320185ATP7B44557427085213052147
chr135251140152511536AMPL561321003ATP7B899422181303642162756
chr135252383952523934AMPL561322321ATP7B802389181387237812867
chr135252055652520638AMPL561322791ATP7B836630362918744653540
chr135253427752534394AMPL561324803ATP7B670295126340239372458
chr135254265452542747AMPL561326888ATP7B75550327281117913775
chr135252440752524535AMPL561327019ATP7B215139381262501023
chr135251661452516708AMPL561328057ATP7B54640120659112592779
chr135251525452515365AMPL561328062ATP7B76846619150812393144
chr135251322352513345AMPL561328524ATP7B4543821132397321771
chr135254856352548672AMPL561329883ATP7B836384149333446183056
chr135251174352511824AMPL561330934ATP7B71855632394614613040
chr135253257652532683AMPL561335846ATP7B671433227588243702749
chr135251149752511615AMPL561335849ATP7B81349435385717613297
chr135254901652549114AMPL561337443ATP7B453347167381831212050
chr135253439552534476AMPL561338245ATP7B734542476128223782732
chr135253904852539119AMPL561339230ATP7B532448394939335632527
chr135254456752544690AMPL561342268ATP7B806449130252335192674
chr135254867352548782AMPL561343055ATP7B51244615851310523022
chr135254813652548260AMPL561345064ATP7B826442128269245782624
chr135254911552549227AMPL561347059ATP7B4693691443987712612
chr135253912052539203AMPL561347740ATP7B787737877303125534243
chr135250971152509847AMPL561353128ATP7B329280702086692063
chr135254922852549346AMPL561354995ATP7B7202325990027172418
chr135250885352508964AMPL561358361ATP7B660455181493038322325
chr135254878352548894AMPL561361353ATP7B1286639267781572773588
chr135254469152544813AMPL561365512ATP7B5423541503368982919
chr135250895952509084AMPL561366430ATP7B69457421763013442208
chr135252409352524178AMPL561367088ATP7B368315531822778
chr135252417952524298AMPL561373391ATP7B725390108269741002724
chr135254481452544931AMPL561373418ATP7B235523461810931651
chr135254889352549018AMPL561375011ATP7B4984711032395502166
chr135250908452509181AMPL561375394ATP7B719461144503738792163
chr135253164452531756AMPL561379526ATP7B51223480160126562155
chr135254825552548381AMPL561399016ATP7B467446982866762965
chr135254838252548474AMPL561401395ATP7B2471246910003572414
chr135251310652513229AMPL561308165c.3699+27T>C, ATP7B56037288177837992176
chr135258538752585514AMPL561308108c.-36C>T, c.-75A>C, ATP7B2942871103136162859
chr135258583152585931AMPL1275480480ATP7B4933971298077801389
chr135258585152585971AMPL1275480698ATP7B4581405512051642811
chr135253409352534223AMPL1275484758ATP7B14023035691872149
chr135258547852585613AMPL561317674ATP7B5601865899716191979

Overall, we found nine disease-causing variants. The most frequent mutation was p.His1069Gln (exon 14, ATP loop) detected in four patients. One novel missense mutation (p.Ala1270Ile, exon 18, ATP hinge vide Figure 1) and three well-known missense mutations (p.Arg969Gln, exon 13, TM6; p.Ala1063Val, exon 14, ATP loop, and p.Leu1305Pro, exon 19 bet ATP hinge/TM7), three frame-shift mutations (c1707+2dupT, exon 4, Cu6; p.Met769-fs exon 8 TM4, and p.Ala1135-fs exon 15 ATP loop), and one nonsense mutation (p.Gln1351Stop, exon 20, TM8) were detected. All of these variants had been validated by Sanger sequencing.

4. Discussion

Although there is an international diagnostic score system for WD [10], the set-up of the diagnosis remains a great challenge in many cases. The signs and symptoms are very colorful, and most of the criteria have relatively low sensitivity and/or specificity. Although genetic testing in itself can ascertain the diagnosis, it is limited by the great variety of the mutations. It is also difficult to screen the siblings of a WD patient, especially of those who do not have identified mutations, since the abnormal laboratory results of copper metabolism may occur in heterozygous carriers. The tight observation of these siblings and the doubt if they are affected can make their life very stressful and uncomfortable. The detection of the mutations in the index patient and searching for the same in the siblings can resolve this problem.

The whole gene analysis of ATP7B by PCR and capillary sequencing in a large cohort of WD patients has been recently published [11]. According to our knowledge based on PubMed data this is the first report on next-generation sequencing of the ATP7B gene for genetic diagnosis of Wilson’s disease in a clinical setting. Since the disease-causing mutations may occur in the whole length of the gene and every exon could be affected, the genetic examination by classical methods is ponderous and time-consuming.

Our study clearly shows the great benefit of NGS. The compound heterozygosity has been proved in each patient within a very short examination time. Previously, we published that p.His1069Gln mutation is most common one in Hungary (71%) similar to other Central and Eastern European countries [6, 1214]. Results of this study are in concordance with the former epidemiological data, since this mutation was confirmed in the majority of the cases, in 4 out of 6. Among the eight other mutations we found, there is one novel mutation in exon 18 which is a missense mutation causing an asparagine-isoleucine change in the transporter. Interestingly, the mutations beyond p.His1069Gln occurred only in one allele of a single patient.

However, it is already well known that p.His1069Gln homozygous mutation tends to relate with neurological symptoms; the effect of other infrequent mutations on the phenotype is hard to be examined due to the low number of cases (vide Table 1) [15].

p.Ala1063Val mutation detected in one patient who has been diagnosed with WD prior to genetic testing is thought to be a non-disease-causing variant according to the Wilson Disease Mutation Database, although only one publication suggested that it might be a polymorphism [4]. On the other hand, subsequent data show that it might be a variant of unknown significance (VUS) [16]. Furthermore it was the one and only nucleotide change in a WD family analyzed by Loudianos et al. [17]. Overall it seems that p.Ala1063Val mutation still might be associated with Wilson’s disease.

NGS gave a tremendous benefit for a 47-year-old patient with acute on chronic liver failure. Although nearly all patients with ALF due to Wilson’s disease are potentially diagnosed (or suspicion is very high) with use of simple biochemical and laboratory criteria (ratio of alkaline phosphatase to bilirubin, ratio of AST to ALT, and Coombs negative hemolytic anemia) [18], the diagnosis may require an urgent genetic testing of all mutations. In some patients the laboratory data alone cannot give enough scores in the international score system [10], which is required by Eurotransplant program for donor liver allocation. In our case, the results of D-penicillamine test and the NGS arrived simultaneously, confirming Wilson’s disease. According to the actual regulation of Eurotransplant Organization in case of acute on chronic liver failure only WD and Budd-Chiari syndrome are accepted as indication for urgent transplantation. Identifying mutations in both alleles gave a clear-cut evidence of the disease despite lack of Kayser-Fleischer ring, lack of neurological symptoms, and p.His1069Gln mutation. Thanks to the quick diagnosis the patient has been transplanted within two days and survived, and he is still in good condition one year later.

5. Conclusion

According to our results we found next-generation sequencing to be a very useful, reliable, time-saving, and cost effective method for diagnosing Wilson’s disease in selected cases.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Dániel Németh and Kristóf Árvai have made an equal contribution.

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Copyright © 2016 Dániel Németh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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