Gastroenterology Research and Practice
Volume 2016 (2016), Article ID 4548039, 6 pages
http://dx.doi.org/10.1155/2016/4548039

Research Article

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

Dániel Németh,¹ Kristóf Árvai,² Péter Horváth,¹ János Pál Kósa,1,2 Bálint Tobiás,² Bernadett Balla,² Anikó Folhoffer,¹ Anna Krolopp,¹ Péter András Lakatos,¹ and Ferenc Szalay¹

¹1st Department of Internal Medicine, Semmelweis University, Koranyi Sandor Street 2/a, Budapest 1083, Hungary
²PentaCore Lab, Koranyi Sandor Street 2/a, Budapest 1083, Hungary

Received 8 July 2015; Revised 15 September 2015; Accepted 20 September 2015

Academic Editor: Alfred Gangl

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.

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 [2–5]. 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.

Table 1: Demographic and clinical characteristics of the patients.

Gender
Age at onset (year)
KFR
Neu
HA
Urin Cu
Biopsy
Cerul (g/L)
ATP7B status
WD score
Phenotype

Patient 1
 Female
12
P
A
A
++
ND
0.18
p.Met769-fs/p.His1069Gln
6
S

Patient 2
Male
17
A
P
A
+
ND
0.05
p.Ala1063Val/p.His1069Gln
6
N1

Patient 3
Male
8
P
A
A
++

0.06
p.His1069Gln/p.Gln1351Stop
8
H2

Patient 4
Male
17
P
A
A
+
ND
0.03
p.Ala1135-fs/p.Leu1305Pro
5
H2

Patient 5
Male
44
A
A
A
++
ND
0.08
p.Ala1270Ile/c.1707+2dupT
4
H1

Patient 6
Male
14
P
P
A
ND
ND
0.04
p.Arg969Gln/p.His1069Gln
7
N2

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.

Table 2: Per sample and per amplicon coverage data.

Chromosome
Amplicon start
Amplicon end
Amplicon ID
Gene ID
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6

chr13
52520358
52520440
AMPL1102520485
ATP7B
812
484
378
9467
3557
3275

chr13
52542530
52542653
AMPL1102672008
ATP7B
951
390
145
3149
5510
3183

chr13
52538909
52539030
AMPL1404436522
ATP7B
9
2
2
2
4
46

chr13
52523735
52523838
AMPL561308261
ATP7B
417
285
89
245
683
2611

chr13
52518244
52518356
AMPL561308388
ATP7B
1140
520
339
7934
6166
3595

chr13
52516492
52516613
AMPL561312436
ATP7B
600
309
70
1510
3218
2152

chr13
52515134
52515253
AMPL561312709
ATP7B
799
287
74
1340
3258
2733

chr13
52520435
52520563
AMPL561312859
ATP7B
622
308
149
409
906
2130

chr13
52511616
52511742
AMPL561313522
ATP7B
343
233
58
1462
2168
1069

chr13
52548475
52548562
AMPL561315457
ATP7B
740
557
248
660
1251
3869

chr13
52548014
52548135
AMPL561316164
ATP7B
506
109
93
244
614
3077

chr13
52535952
52536073
AMPL561319098
ATP7B
345
332
62
1845
3919
1350

chr13
52532445
52532575
AMPL561319439
ATP7B
495
382
75
260
477
2421

chr13
52518332
52518433
AMPL561320185
ATP7B
445
574
270
852
1305
2147

chr13
52511401
52511536
AMPL561321003
ATP7B
899
422
181
3036
4216
2756

chr13
52523839
52523934
AMPL561322321
ATP7B
802
389
181
3872
3781
2867

chr13
52520556
52520638
AMPL561322791
ATP7B
836
630
362
9187
4465
3540

chr13
52534277
52534394
AMPL561324803
ATP7B
670
295
126
3402
3937
2458

chr13
52542654
52542747
AMPL561326888
ATP7B
755
503
272
811
1791
3775

chr13
52524407
52524535
AMPL561327019
ATP7B
215
139
38
126
250
1023

chr13
52516614
52516708
AMPL561328057
ATP7B
546
401
206
591
1259
2779

chr13
52515254
52515365
AMPL561328062
ATP7B
768
466
191
508
1239
3144

chr13
52513223
52513345
AMPL561328524
ATP7B
454
382
113
239
732
1771

chr13
52548563
52548672
AMPL561329883
ATP7B
836
384
149
3334
4618
3056

chr13
52511743
52511824
AMPL561330934
ATP7B
718
556
323
946
1461
3040

chr13
52532576
52532683
AMPL561335846
ATP7B
671
433
227
5882
4370
2749

chr13
52511497
52511615
AMPL561335849
ATP7B
813
494
353
857
1761
3297

chr13
52549016
52549114
AMPL561337443
ATP7B
453
347
167
3818
3121
2050

chr13
52534395
52534476
AMPL561338245
ATP7B
734
542
476
1282
2378
2732

chr13
52539048
52539119
AMPL561339230
ATP7B
532
448
394
9393
3563
2527

chr13
52544567
52544690
AMPL561342268
ATP7B
806
449
130
2523
3519
2674

chr13
52548673
52548782
AMPL561343055
ATP7B
512
446
158
513
1052
3022

chr13
52548136
52548260
AMPL561345064
ATP7B
826
442
128
2692
4578
2624

chr13
52549115
52549227
AMPL561347059
ATP7B
469
369
144
398
771
2612

chr13
52539120
52539203
AMPL561347740
ATP7B
787
737
877
3031
2553
4243

chr13
52509711
52509847
AMPL561353128
ATP7B
329
280
70
208
669
2063

chr13
52549228
52549346
AMPL561354995
ATP7B
720
232
59
900
2717
2418

chr13
52508853
52508964
AMPL561358361
ATP7B
660
455
181
4930
3832
2325

chr13
52548783
52548894
AMPL561361353
ATP7B
1286
639
267
7815
7277
3588

chr13
52544691
52544813
AMPL561365512
ATP7B
542
354
150
336
898
2919

chr13
52508959
52509084
AMPL561366430
ATP7B
694
574
217
630
1344
2208

chr13
52524093
52524178
AMPL561367088
ATP7B
368
315
5
31
82
2778

chr13
52524179
52524298
AMPL561373391
ATP7B
725
390
108
2697
4100
2724

chr13
52544814
52544931
AMPL561373418
ATP7B
235
52
34
618
1093
1651

chr13
52548893
52549018
AMPL561375011
ATP7B
498
471
103
239
550
2166

chr13
52509084
52509181
AMPL561375394
ATP7B
719
461
144
5037
3879
2163

chr13
52531644
52531756
AMPL561379526
ATP7B
512
234
80
1601
2656
2155

chr13
52548255
52548381
AMPL561399016
ATP7B
467
446
98
286
676
2965

chr13
52548382
52548474
AMPL561401395
ATP7B
247
124
69
1000
357
2414

chr13
52513106
52513229
AMPL561308165
c.3699+27T>C, ATP7B
560
372
88
1778
3799
2176

chr13
52585387
52585514
AMPL561308108
c.-36C>T, c.-75A>C, ATP7B
294
287
110
313
616
2859

chr13
52585831
52585931
AMPL1275480480
ATP7B
493
397
129
807
780
1389

chr13
52585851
52585971
AMPL1275480698
ATP7B
458
140
55
1205
1642
811

chr13
52534093
52534223
AMPL1275484758
ATP7B
140
230
35
69
187
2149

chr13
52585478
52585613
AMPL561317674
ATP7B
560
186
58
997
1619
1979

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.

Figure 1: The identified mutations of patient 5. Both c.3809A>T (causing amino acid change p.Ala1270Ile) and c.1707+2dupT mutations are confirmed by Sanger-sequencing. The 3809A>C and A>G mutations are known, but the A>T substitution is a novel alteration at this position. (a1) Visualizing the alignment of the sequencing reads covering the ATP7B c.3809A>T heterozygous point mutation. The coverage was 400-fold (211-fold reference and 189-fold variant coverage). (b1) Validating our finding with Sanger sequencing, red arrow indicates the position of the point mutation. The mutation is present in both directions. (a2) Visualizing the alignment of the sequencing reads covering the ATP7B c.1707+2dupT heterozygous insertion mutation. The coverage was 399-fold (188-fold reference and 211-fold variant coverage). (b2) Validating our finding with Sanger-sequencing, red arrow indicates the position of the insertion. The mutation is present in both directions.

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, 12–14]. 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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