ATP7B Gene Mutations in Croatian Patients with Wilson Disease

Abstract

Aims:

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, characterized by its accumulation in tissues which results in hepatic, neurological, and/or psychiatric symptoms. The aim of this study was to investigate the genetics of WD in Croatian patients.

Methods:

Correlation of the clinical presentation subtype and the age at onset of the diagnosis of WD with the ATP7B genotype was investigated in a group of Croatian WD patients. DNA from peripheral blood samples was tested for the p.His1069Gln by direct mutational analysis and other polymorphisms were identified by sequence analysis of coding and flanking intronic regions of ATP7B gene.

Results:

In the group of 75 WD patients of Croatian origin, 18 different mutations in ATP7B gene were detected, three of which were novel. The p.His1069Gln mutation was most frequent, being detected in 44 Croatian WD patients (58.7%). Most ATP7B mutations (90.4%) were located in exons 5, 8, 13, 14, and 15.

Conclusions:

Clinical diagnosis of WD was confirmed in 59 patients by detecting mutations on both ATP7B alleles. The age at onset of WD and the type of WD clinical presentation showed no significant correlation with the ATP7B genotype.

Introduction

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, characterized by a decreased biliary copper excretion. Consequently, accumulation of copper occurs in various organs, most prominently in the liver and the brain, causing liver disease and neurological and/or psychiatric symptoms. The incidence of WD is estimated at 1 in 30,000 individuals in the general population (Reilly et al., 1993). The age at onset of symptoms varies from 3 to more than 40 years of age (Wilson et al., 2000; Ferenci et al., 2007).

The ATP7B gene, coding the copper transporting P-type ATPase (Bull et al., 1993), has been reported to have 380 variants that were proven to affect the cellular pathways of copper metabolism, causing WD (EASL, 2012). The most frequent mutation found in populations of European origin is the p.His1069Gln mutation, located in the ATP loop of the ATP7B protein (Caca et al., 2001). The low incidence of WD, in addition to its genetic heterogeneity, makes the molecular characterization and comparison between genotype and phenotype difficult.

According to a meta-analysis conducted by Stapelbroek et al. (2004), the homozygous p.His1069Gln mutation genotype gives rise to neurological symptoms and later onset of the disease more frequently than both the heterozygous and the non-p.His1069Gln WD genotypes. Gromadzka et al. (2006) presented similar results although Vrabelova et al. (2005) did not find such correlations. It is hypothesized that missense mutations cause later onset of disease and less severe clinical presentation and disturbances of copper metabolism compared to mutations which have a profound effect on ATP7B protein product (Merle et al., 2010; Okada et al., 2010; Lee et al., 2011).

As no data about the genetic basis of WD in the Croatian population have been published thus far, the aim of this study is to investigate mutations in the ATP7B gene in Croatian WD patients, as well as to determine possible correlations of the type of clinical presentation and the age at onset of WD with the ATP7B genotype.

Materials and Methods

Subjects

DNA samples were collected from a Croatian cohort of 75 unrelated WD patients (40 males, 35 females). The diagnosis of WD was based on hepatic, neurological, and/or psychiatric symptoms, the presence of Kayser-Fleischer rings, and copper test results such as high hepatic copper concentration (>250 μg/g dry weight), low serum “free” copper, serum ceruloplasmin levels (<0.17 g/L), and increased 24-h urinary copper excretion (>1.7 μmol/24 h). WD presentation was classified according to Ferenci et al. (2003). Solely hepatic symptoms were present in 46 patients, including 43 patients with chronic liver symptoms (H2 type of WD) and 3 with acute liver symptoms (H1 type). Solely neurological presentation of WD (N2) was found in 14 patients, while 15 patients presented with both hepatic and neurological symptoms (N1) (Table 1).

Table 1.

Genotype-Phenotype Correlation

Patient	Sex	Age at onset	Age at diagnosis	Symptoms at onset	KF	CP	Cu (serum)	Cu (urine)	Cu (liver)	Cu (urine) with penicillamine	ATP7B genotype
P1	M	30	44	N1	−	0.09	3.4	23.8			His1069Gln/His1069Gln
P2	M	31	32	H2	+	0.12	10.12	6.9			His1069Gln/His1069Gln
P3	F	21	21.5	H2	+	0.1	5.6	16.1			His1069Gln/His1069Gln
P4	M	20	24	N1	−	0.16	10.4			4.14	His1069Gln/His1069Gln
P5	M	29	47	N1	+	0.1	7.1	18.2			His1069Gln/His1069Gln
P6	F	15	16	H2	−						His1069Gln/His1069Gln
P7	F	26	26.5	N1	+						His1069Gln/His1069Gln
P8	M	20	20	H2	−	0.2	15.2	2.52			His1069Gln/His1069Gln
P9	F	30	36	N1	+	0.24	17	2.4	735	20.48	His1069Gln/His1069Gln
P10	F	37	39	N1	+	0.11	8	3.4			His1069Gln/His1069Gln
P11	F	20	28	H2	−	0.1		5.7			His1069Gln/His1069Gln
P12	F	24	24	H2	−	0.18	12.6	3.33	1466.8		His1069Gln/His1069Gln
P13	F	6	7	H2	−	0.237		1.85	933.5	5.91	His1069Gln/His1069Gln
P14	M	13	13.5	H2	−	0.12	4.2	0.77	700		His1069Gln/His1069Gln
P15	F	35	37	H2	+			51.2			His1069Gln/His1069Gln
P16	M	8	8.5	H2		0.27	14.5	1.4	596.9	9	His1069Gln/His1069Gln
P17	M	8	9	H2	−	0.11	5.7	1.53	778.4		His1069Gln/His1069Gln
P18	M	16	17	H2	−						His1069Gln/His1069Gln
P19	F	34	36	N1		0.18	14.8	4.26			His1069Gln/His1069Gln
P20	M	39	40	N2	+	0.17	9.3	4.16			His1069Gln/His1069Gln
P21	M	30	40	N2	+	0.09	9.9	7.68			His1069Gln/His1069Gln
P22	F	20	20.5	N1	+	0.14	7.8	18.2	577.8		His1069Gln/His1069Gln
P23	M	7	19	H2	−	0.12	7.7	3.5		15.56	His1069Gln/His1069Gln
P24	M	25	25.5	H2	−	0.19	16.2	6.86			His1069Gln/Ala1003Thr
P25	F	27	29	N1	+	0.08		5.32		4.43	His1069Gln/Ala1003Thr
P26	M	19	19.5	N2	+	0.08	9.5	7.5		7.5	His1069Gln/Ala1003Thr
P27	F	10	17	H2	−	0.19	14.7	8.1		26.1	His1069Gln/Ala1003Thr
P28	F	17	18	H2	−	0.13	11.6	6.47	1578.4		His1069Gln/Ala1003Thr
P29	F	18	19	H2	−						His1069Gln/Ala1003Thr
P30	M	14	19	N2	−	0.17	10.1	14.9			His1069Gln/2304dupC
P31	F	29	29	H2	−	0.17		22.3	1035.2		His1069Gln/2304dupC
P32	M	15	20	N1	−	0.09		2			His1069Gln/2304dupC
P33	M	30	31	N2	+	0.19	11.7	19			His1069Gln/2304dupC
P34	F			H2	−	0.1	11.2	2.46	2582	66.4	His1069Gln/2304dupC
P35	F	35	35.2	H1	−	0.17	12				His1069Gln/Arg616Gln
P36	M	36	56	N1	+	0.22	12.6	1.71			His1069Gln/Arg616Gln
P37	M	15	16	H2	−	0.12	11.2	10.6	1387		His1069Gln/3402delC
P38	F	27	27.5	N2	+	0.09	5.8	14.82			His1069Gln/3402delC
P39	F	25	27	N2	+						His1069Gln/Gly626Ala
P40	F	20	21	N2	+	0.1	4.6	8.1			His1069Gln/2304delC
P41	M	26	26	H2	−	0.08	9.1	6.27		25.48	His1069Gln/Asp1027Asn
P42	F	16	17	N1	−						His1069Gln/4374_4375delCA
P43	M	20	29	N2	−	0.15	11.3	9.45	1241		Gly591Asp/Gly1030Ser
P44	F	7	10	H2				1.84	636.6		Arg616Gln/Ala1003Thr
P45	M	10	19	H2	+	0.09	11.1	12			Arg616Gln/Asn1270Thr
P46	F	25	25	H1	−						2304dupC/4374_4375delCA
P47	F	27	27	H1	+	0.08		53.6			Arg969Gln/3402delC
P48	F	21	23	H2	−	0.06	3.4	2.33			Arg969Gln/3556 + 1G>A
P49	F	6.5	7	H2		0.01					Arg969Gln/4374_4375delCA
P50	M	22	23	N2	−	0.08	2.2	3.15	1190.6	23.9	Ala1003Thr/3402delC
P51	M	7	12	H2	−	0.19	8.2	5.94			Ala1003Thr/4374_4375delCA
P52	F	17	17.5	H2	+		14.9	22.4	63.5	53.2	Gly1030Ser/3402delC
P53	M	16	17	H2	−	0.01	3.6	3.53			3402delC/Gln1351^*
P54	F	12	14	H2	−	0.06	4.3	1.6	718	23.38	Gly591Asp/Gly591Asp
P55	M	35	39	N1	+	0.25	20	2.6			Arg616Gln/Arg616Gln
P56	M	30	35	H2	−						2304dupC/2304dupC
P57	M	16	18	H2	−	0.07	5.5	4.01		33.99	2648_2649delTG/2648_2649delTG
P58	F	45	45	N1	+	0.18	13.1	1.82	806.8		Ala1003Thr/Ala1003Thr
P59	M	15	16	H2	+	0.17	7.8	6.48		24.4	Ala1003Thr/Ala1003Thr
P60	M	32	38	H2	+	0.23	15.3	4.01			Arg616Gln/Unknown
P61	F	21	21.5	H2	−	0.05	6.3	1.62	1725.6	7.07	2304dupC/Unknown
P62	M	19	30	H2	−	0.11	7.5	0.58	151		His1069Gln/Unknown
P63	M	29		N2	+	0.147	14	0.78			His1069Gln/Unknown
P64	F	16	16	H2	−	0.08		2.97	549		3402delC/Unknown
P65	M	17	18	H2		0.08	10.8	1.7	1273.3		Asn1270Ser/Unknown
P66	M	31	32	N2	+	0.23	11.1	5.1			Ser1432Phe/Unknown
P67	M	10	12	N1	−	0.18	10.2		80.7	9.4	Unknown/Unknown
P68	M	20	25	H2	−	0.17	11	0.6	70.2		Unknown/Unknown
P69	M	29	31	H2	−	0.08	7	0.8	1190.6	2.5	Unknown/Unknown
P70	M	16	23	H2	−	0.18	13.6	0.77	996.5	4.43	Unknown/Unknown
P71	F	20	24	H2	+	0.08	11.3	13.7	932.5	33.45	Unknown/Unknown
P72	M			H2	−	0.2	11.7	0.51	37.3	16.43	Unknown/Unknown
P73	F	23	32	N2	−	0.1	7.7	1.5	76.2	2.9	Unknown/Unknown
P74	M	25	26	N2	−	0.13	10.5	0.53	381	2.96	Unknown/Unknown
P75	F	32	32	H2	−	0.14	7.6	0.39	99.1	4.17	Unknown/Unknown

KF, Kayser-Fleischer; CP, ceruloplasmin.

The median age (interquartile range [IQR]) at onset of WD symptoms in this cohort of patients was 20 years (IQR 16-29 years), median age (IQR) at diagnosis was 24 years (18-32 years), and the median difference between these two variables was 1 year (IQR 1-5 years). Patients were clinically characterized at the Department of Internal Medicine, Department of Neurology, and Department of Pediatrics, University Hospital Centre Zagreb. This study was approved by the Ethics Committee of the University Hospital Center Zagreb and an informed consent was obtained from all patients.

ATP7B gene mutation analysis

Genomic DNA was extracted from peripheral blood leukocytes by the salting-out method. Patients were first screened for the p.His1069Gln mutation, using primers and probes according to Witt and Landt (2001). DNA samples heterozygous for or without p.His1069Gln mutation were further analyzed by sequencing 21 exons and flanking intronic regions of the ATP7B gene according to Loudianos et al. (1998). PCR products were separated by capillary electrophoresis using the AB Genetic Analyzer 3130xl (Applied Biosystems; Life Technologies).

DNA variants were characterized using Wilson disease mutation database (Bugbee et al., 2001), and the effect of an amino acid substitution on a protein function for novel mutations was assessed using the following software tools: PolyPhen2 version 2.2.2 (Adzhubei et al., 2010), Provean version 1.1.3. (Choi and Chan, 2015), and SIFT (Kumar et al., 2009). The ATP7B gene mutations were named according to the GenBank NCBI reference sequences NM_000053.3 and NG_008806.1.

Statistical analysis

The Kolmogorov-Smirnov test was used to assess the data distribution and, according to its findings, appropriate nonparametric tests were used in the following analyses. Categorical variables were shown through frequencies and percentages, while medians and IQRs were used to describe the quantitative variables. Differences in quantitative variables were analyzed with the Kruskal-Wallis test. Chi-square test was used to analyze differences between investigated groups in categorical variables. All p-values below 0.05 were considered significant. Statistical package IBM SPSS Statistics version 19.0.0.1 (www.spss.com) was used for all statistical procedures.

Results

The analysis of ATP7B gene coding region and flanking intronic regions by sequencing method and/or direct mutation analysis was performed in a Croatian cohort of WD patients. We detected 18 different mutations, 11 of which were missense, 5 frameshift, 1 nonsense, and 1 splice-site mutation (Table 2). In most WD patients, segregation of variants in the ATP7B gene was tested and confirmed by analyzing their parents' DNA. The p.His1069Gln mutation proved to be the most prevalent in the Croatian population with a frequency of 44.7% among the total number of tested alleles, followed by p.Ala1003Thr (8.7%), c.2304dupC (6%), p.Arg616Gln (4.7%), and c.3402delC (4.7%).

Table 2.

Mutations in the ATP7B Gene in Croatian Patients with WD

No	DNA level	Protein level	Type	Exon	Alleles	Frequency (%)
1	c.3207C>A	p.His1069Gln	Missense	14	67	44.67
2	c.3007G>A	p.Ala1003Thr	Missense	13	13	8.67
3	c.2304dupC	p.Met769HisfsX26	Duplication	8	9	6
4	c.1847G>A	p.Arg616Gln	Missense	5	7	4.67
5	c.3402delC	p.Ala1135GlnfsX13	Deletion	15	7	4.67
6	c.4374_4375delCA	p.Arg1459GlyfsX2	Deletion	21	4	2.67
7	c.1772G>A	p.Gly591Asp	Missense	5	3	2
8	c.2906G>A	p.Arg969Gln	Missense	13	3	2
9	c.2648_2649delTG	p.Val883AlafsX3	Deletion	11	2	1.33
10	c.3088G>A	p.Gly1030Ser	Missense	14	2	1.33
11	c.1877G>C	p.Gly626Ala	Missense	6	1	0.67
12	c.2304delC	p.Met769CysfsX38	Deletion	8	1	0.67
13	c.3079G>A	p.Asp1027Asn	Missense	14	1	0.67
14	c.3556 + 1G>A	—	Splice-site	Intron 16	1	0.67
15	c.3809A>G	p.Asn1270Ser	Missense	18	1	0.67
16	c.3809A>C	p.Asn1270Thr	Missense	18	1	0.67
17	c.4051C>T	p.Gln1351^*	Nonsense	20	1	0.67
18	c.4295C>T	p.Ser1432Phe	Missense	21	1	0.67
19	Mutation not detected				25	16.67
Total number of alleles					150	100

Novel mutations are stated in bold letters.

WD, Wilson disease.

Three novel missense mutations, namely the p.Asp1027Asn, the p.Gly1030Ser, and the p.Ser1432Phe in the ATP7B gene were detected in the Croatian population. In patients in whom novel mutations were determined, all 21 exons of the ATP7B gene were analyzed to exclude the presence of other ATP7B mutations.

Thirty-two different ATP7B genotypes were determined in this study (Table 1). Clinical diagnosis of WD was confirmed in 59 patients (78.7%) as mutations were identified in both ATP7B alleles. A total of 23 WD patients (30.7%) were homozygous for p.His1069Gln mutation, 6 patients (8%) were homozygous for other mutations, while 30 patients (40%) were compound heterozygotes for ATP7B mutations. In seven patients (9.3%), mutations in only one ATP7B allele were detected, while no mutations were detected in nine patients (12%). Using the sequencing method combined with direct mutation analysis for p.His1069Gln, we were able to identify disease-causing mutations on 125 (83.3%) out of 150 tested ATP7B alleles.

Polymorphisms, including one silent mutation unreported thus far, that were found in Croatian WD patients are stated in Table 3.

Table 3.

Polymorphisms in the ATP7B Gene in Croatian Patients with WD

DNA level	Protein level	Exon	Intron	Domain	Reference
c.-129_-125del		5′UTR		5′UTR	Figus et al. (1995)
c.-75A>C		5′UTR		5′UTR	Figus et al. (1995)
c.1216T>G	p.Ser406Ala	2		Cu4	Thomas et al. (1995)
c.1366C>G/G>C	p.Leu456Val/Val456Leu	3		Cu4/Cu5	Figus et al. (1995)
c.1543 + 51G>A			3	—	Gupta et al. (2005)
c.1544-53A>C/C>A			3	—	Shah et al. (1997)
c.1870-65G>A			5	—	Vrabelova et al. (2005)
c.2448-25G>A			9	—	Figus et al. (1995)
c.2495A>G	p.Lys832Arg	10		TM4/Td	Figus et al. (1995)
c.2855A>G	p.Lys952Arg	12		TM5/TM6	Thomas et al. (1995)
c.2866-13G>C			12	—	Thomas et al. (1995)
c.2973G>A	p.Thr991Thr	13		TM6/Ph	Figus et al. (1995)
c.2994C>T	p.Gly998Gly	13		TM6/Ph	—
c.3009G>A	p.Ala1003Ala	13		TM6/Ph	Figus et al. (1995)
c.3045G>A	p.Leu1015Leu	13		TM6/Ph	Figus et al. (1995)
c.3419C>T	p.Ala1140Val	16		ATP loop	Thomas et al. (1995)
c.3620A>G^a	p.His1207Arg^a	17			Loudianos et al. (1999)
c.3903 + 6T>C			18	—	Thomas et al. (1995)
c.4311G>A	p.Lys1437Lys	21		3COOH	Loudianos et al. (1999)

Novel polymorphism is stated in bold letters.

Possible deleterious effect.

Genotype-phenotype correlation in Croatian WD patients

First, WD patients with mutations detected in both ATP7B alleles were classified into three groups according to the effect of mutation: the first group was composed of patients with missense mutations on both ATP7B alleles; the second group comprised patients with one missense mutation with an addition of one other mutation that has significant effect on the protein, such as a frameshift, splice-site, or a nonsense mutation; the third group included patients with both such mutations. WD phenotypes were classified into four groups according to Ferenci et al. (2003): N1, N2, H1, and H2. Statistical analysis of the correlation between the same genotype classification groups and the age at onset of the disease was also performed. No statistically significant correlation between the ATP7B mutation genotype and type of disease presentation, or between genotype and age at onset of the disease was found (p = 0.124 and p = 0.531, respectively).

Second, WD patients were classified into three groups based on the p.His1069Gln mutation, as follows: the first group included patients homozygous for p.His1069Gln, the second group was composed of compound heterozygotes for p.His1069Gln and some other mutation, while patients with other mutations on both alleles comprised the third group. WD phenotype was divided into two groups, hepatic (including H1 and H2 patients) and neurological (including N1 and N2 type of presentation). These genotype groups were also analyzed to investigate the possible correlation with age at onset of the disease. No statistically significant correlation between p.His1069Gln-based mutation genotype classification and type of disease presentation, or between genotype and age at onset of the disease was found (p = 0.195 and p = 0.453, respectively).

Discussion

Results obtained from this study reveal the genetic background in the Croatian cohort of WD patients for the first time. Detailed ATP7B gene analysis results showed a significant number of different mutations and confirmed the genetic heterogeneity among WD patients in Croatia. As expected, p.His1069Gln showed to be the most frequent mutation, as is the case in central and eastern European populations. The following three most frequent mutations are similar to those present in the Serbian population (Loudianos et al., 2003), which is not surprising considering the geographic proximity of the two populations. Frameshift mutation c.4374_4375del in exon 21, which was detected in four patients in this study, was previously detected only in the Serbian population thus far, further facilitating the similarity of WD genetic basis between these two populations (Lepori et al., 2007).

Pathogenicity estimation of novel missense mutations was carried out by using the following software tools: PolyPhen2 version 2.2.2 (Adzhubei et al., 2010), Provean version 1.1.3. (Choi and Chan, 2015), and SIFT (Kumar et al., 2009), and it indicated that all three novel mutations are likely pathogenic. Both p.Asp1027Asn and p.Gly1030Ser are located in exon 14, in the phosphorylation domain of the ATP7B protein, and the highly conserved amino acid residue motif Asp-Lys-Thr-Gly-Thr, which is supposed to have a significant impact on the normal function of the protein by disturbing catalytic phosphorylation. Mutation p.Gly1030Ser was found in two unrelated patients, which indicates that this variant could be more frequent in the Croatian population. Exon 14 appears to be a hot spot for mutations in the Croatian cohort of WD patients.

The third novel mutation, detected in exon 21, was p.Ser1432Phe and it is located in the C-terminal domain of the protein which is not involved in the catalytic function, but might have a role in maintaining the protein's stability or localization during its normal transfer between cellular compartments.

The newly described p.Asp1027Asn mutation was detected in a 26-year-old male patient who had previously undergone liver transplantation due to decompensated liver cirrhosis. The p.Gly1030Ser mutation was detected in a 17-year-old female patient with decompensated liver cirrhosis and in a 30-year-old male patient with neurological manifestations. The p.Ser1432Phe mutation was found in a male patient who presented with early neurological manifestations followed by liver cirrhosis and decompensation due to noncompliance to therapy. The functional effect of novel mutations on the ATP7B protein should also be investigated using further studies.

As the frequency of p.His1069Gln mutation alone is 44.7%, diagnostic sensitivity of WD genetic testing by sequencing the entire coding region of ATP7B compared to direct mutation analysis method for p.His1069Gln was increased by 38.6%, which allowed for 36 more WD patients (48%) to establish their molecular diagnosis of WD. According to the results of this study, the exons of the ATP7B gene that should be analyzed first for mutations in Croatian patients with WD are 5, 8, 13, 14, and 15 where 113 of 125 detected molecular defects are located (90.4%). By analyzing these five exons only, WD diagnosis was confirmed for 50 patients (66.7%) on the molecular level.

Finally, the five most frequent mutations in the Croatian population were present on 103 ATP7B alleles (68.7%), which indicate that a simpler and faster screening method would also be useful for the diagnosis of WD in this population. A new polymorphism c.2994C>T, p.Gly998Gly, which has also been detected in this study, is probably not a pathogenic variant since it causes a silent mutation and is also sufficiently distant from the exon-intron border not to cause irregularities in mRNA splicing.

The lack of statistically significant correlation between WD genotype and the type of disease presentation, as well as correlation between WD genotype and the age at onset of the disease, can be explained by a too small number of subjects, as well as by other genetic and environmental factors, which have a significant impact on age at onset and disease presentation even among siblings with the same WD genetic background (Kegley et al., 2010; Ferenci, 2014).

In conclusion, new mutations in ATP7B gene were discovered, which corroborate its genetic heterogeneity. This study contributes to the enrichment of knowledge of the genetics of WD in Europe, adds to the present understanding of its genotype/phenotype relationship, and provides data for a genetic screening approach for the most frequent mutations in the Croatian population.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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