Genetic Screening of ATP7B Gene in Iranian Wilson Disease Patients: A diverse landscape of pathogenic variants

doi:10.21203/rs.3.rs-4957273/v1

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Research Article

Genetic Screening of ATP7B Gene in Iranian Wilson Disease Patients: A diverse landscape of pathogenic variants

https://doi.org/10.21203/rs.3.rs-4957273/v1

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Background/Objective:

Wilson's disease (WD) is an autosomal recessive condition caused by mutations in the ATP7B gene, leading to the copper accumulation in various organs. Data on the ATP7B mutation spectrum in Iran and the Middle East is insufficient. This study aims to screen the ATP7B gene in unrelated Iranian families (n = 23) from northeastern Iran.

Methods

DNA was extracted from peripheral blood, and variant screening was performed using direct sequencing of the entire ATP7B gene coding region. The full 3D structure of the defective proteins was determined using the I-TASSER software.

Results

The overall frequency of causative variant detection was 84.7% (39/46). Among the 23 patients with WD, we identified 13 different causative variants: eight missense, two nonsense, one splicing, one deletion, and one deletion/insertion changes. Two of which were novel: c.3431delTinsAGA (p.Phe1144Ter) and c.1156G > A (p.Gly386Arg). The c.2807T > A (p.Leu936Ter) variant at exon 12 was the most prevalent in our study, with an allelic frequency of 17.39%, followed by c.3188C > T (p.Ala1063Val) at exon 14, exhibiting an allelic frequency of 13.04%. Exons 12, 13, and 14 were identified as mutation hot spots, with detection rate of 50% (23/46). Ten out of the 13 variants identified in our study were reported for the first time in Iran (this report).

Conclusion

We reported two novel variants that broaden the known spectrum of mutations associated with the ATP7B gene. The variants identified in this study can facilitate carrier screening and presymptomatic detection and can be used in prenatal genetic diagnosis in affected families.

Wilson’s Disease

ATP7B

Molecular diagnosis

Targeted Sequencing

Mutation

Wilson's disease (WD) is a rare autosomal recessive disorder of copper metabolism that leads to the pathological accumulation of copper in the brain, liver, and other organs. WD is associated with mutations in the ATP7B gene, located on chromosome 13q14.3, which is the causative gene. The ATP7B gene encodes a P-type transmembrane ATPase responsible for transporting copper to the trans-Golgi network of hepatocytes and is highly expressed in the liver. This protein plays a crucial role in maintaining hepatic copper homeostasis. It comprises eight transmembrane domains (TMs), six metal-binding domains (MBDs), a nucleotide-binding domain (N-domain), a phosphorylation domain (P-domain), and an actuator domain (A-domain). (Bitter, Oh et al. 2022). In hepatocytes, ATP7B facilitates the incorporation of copper into ceruloplasmin and its excretion in bile. When ATP7B is dysfunctional, excess copper accumulates, leading to hepatocyte injury. Additionally, free copper enters the bloodstream, causing damage to other organs (Roberts and Schilsky 2008, 2012).

The prevalence of WD is estimated to be between 1 in 30,000 and 1 in 100,000 individuals worldwide (Ala, Walker et al. 2007). Since WD is autosomal recessive, an increase in cases may occur due to founder effects and consanguineous marriages.

WD is a clinically heterogeneous disorder characterized by a wide spectrum of manifestations, including hepatic, neurological, and psychiatric symptoms (Ferenci, Caca et al. 2003). Early in life, patients are asymptomatic, but the gradual accumulation of copper leads to varying degrees of subclinical liver disease (Brewer 1992). Assessing WD patients shows variability in the age of onset, ranging from early childhood to the sixth decade. However, WD generally occurs in children and young adults (Lorincz 2010).

The ATP7B gene consists of 21 exons, and to date, more than 1,300 different mutations throughout this gene have been reported in the Human Gene Mutation Database (HGMD professional 2023.4). Most of these mutations are rare, with a few hotspot mutations exhibiting racial and regional differences. There are notable differences in the spectrum of ATP7B mutations between populations (Gomes and Dedoussis 2016). Most of the mutations in the ATP7B gene are missense mutations, and their distribution is heterogeneous. However, certain hot spots have been identified, including exons 8 and 14 (Ferenci 2006).

WD was traditionally diagnosed based on biochemical indices, including low serum ceruloplasmin levels, elevated 24-hour urinary copper excretion, and increased liver copper content. Clinical signs, such as the presence of Kayser-Fleischer (KF) rings, were also considered in the diagnostic process (Mak and Lam 2008). Despite their utility, biochemical markers can sometimes be misleading, posing challenges in the diagnosis of WD. Therefore, molecular examination is recommended for an accurate and definitive diagnosis of WD patients. This becomes particularly crucial in diagnosing siblings of the proband within a WD-affected family and asymptomatic individuals (Kasztelan-Szczerbinska and Cichoz-Lach 2021). However, the available data regarding observed mutations in the Iranian population is insufficient, and no study has been conducted on the genetic status of WD in the Northeastern region of Iran. We, therefore aimed to investigate the ATP7B gene mutations in Iranian patients with WD in the Northeast region.

1 Subjects

This research was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Mashhad University of Medical Science (MUMS) (ethical code: IR.MUMS.MEDICAL.REC.1401.358). All patients and their family members were informed about the study's purpose, and consent forms were signed. Included in this study were 23 unrelated Iranian families affected by WD were referred to Akbar Hospital, a Children's Teaching Hospital in Mashhad, for genetic analysis. Clinical data of all probands and their parents were collected when available. The diagnosis of WD was confirmed by a combination of biochemical and clinical features, including 24-hour urine copper (> 100 µg/24 h), serum ceruloplasmin (< 20 mg/dL), hepatic copper content (> 250 µg/g dry weight), and the presence of Kayser-Fleischer (KF) rings.

2 Genetic testing

2.1 DNA extraction and Polymerase chain reaction (PCR)

Genomic DNA was extracted from whole blood samples using the salting-out method. Manual primer design was used to design 19 PCR amplicons that cover all 21 exons of the ATP7B Gene. Flanking intronic sequences were covered as much as possible, with at least 60 bp (Table 1). The amplification was carried out in a total volume of 25 µL, with the following steps: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 59–66°C for 40 s (with primers having different annealing temperatures), extension at 72°C for 40 s, and a final extension at 72°C for 10 min.

Table 1

Primer sequences used for PCR amplification of 21 exons of ATP7B gene
Exon Nomber	Primer Sequence (5’ > 3’)		Size (bp)	Annealing temperature (˚C)
1	F	CGCAACTTTGAATCATCCGTG	459	62
1	R	CAAACATCAGTTGACGGCAC	459	62
2A	F	CTACCCTTGGGATATTTTGACACC	844	66
2A	R	TGACCACATGGCTTCCTTGG	844	66
2B	F	CATTCAGCCCGAAGACCTCAG	828	65
2B	R	CCACTGTTGACATGGGAGGC	828	65
3	F	TGGGAGCCGGGACAATGAAC	475	63
3	R	GCTACCTGGTTATCAGGGCTACTG	475	63
4	F	TGGGTAAGAGACCAGACATCG	473	65
4	R	ACAAACCAGACACGTCCAAG	473	65
5	F	GTCCAGGGTCTTGAGAGCAG	499	66
5	R	ACCCATTCACTGATATCCTCCC	499	66
6	F	CCCACAAAGTCTACTGAGGCAC	335	63
6	R	CAAGGGTAAAGGCAGCTAATCC	335	63
7	F	CTAGATGCTCCCTCAGATGGC	447	65
7	R	GGAAAGCTGCAATAAAGTGCC	447	65
8	F	GACTGTGCACAAAGCTAGAGG	421	59
8	R	CTAAACATGGTGTTCAGAGGAAG	421	59
9	F	TAGCAAGTAACGCCCACCTG	357	66
9	R	CTGCCCACACTCACAAGGTC	357	66
10,11,12	F	CTATTGTAACAGCTGGCCTAGAAC	955	64
10,11,12	R	GCTGTCAATAAGAGAAGCAAGC	955	64
13	F	TGACTCTGCTCCTGTAATGC	612	61
13	R	TATGACTGGTGGCTACTCTG	612	61
14	F	GAACGACAGAGGATCACGTTAG	401	59
14	R	TAGGAGAGAAGGACATGGTGAG	401	59
15	F	CCTTTCCTATCTGTTCCACCTC	447	59
15	R	CCTTAGCCATGAACCGTCTG	447	59
16	F	GAGGTGCTTACAAGGTTACAG	507	60
16	R	TTCCAAGGTCAAGGAGACTG	507	60
17	F	CCTATTCCTTGGGGAGCCACTG	472	65
17	R	AGCAGGAGTACAGCTCAGTGC	472	65
18,19	F	CAGGAGCCAGGGATAAACTGG	694	59
18,19	R	GCCTTTCTAAAACGCCTCTAGC	694	59
20	F	ACATCAGGGCGAGTGGAAGAG	542	66
20	R	GAATTGCCTGCTCATGGTGC	542	66
21	F	AGATGGATGAGAGGCCTTCACC	797	66
21	R	CACACAGACAGGCGTCATCAG	797	66

2.2 Targeted Sanger sequencing

Direct sequencing of the obtained PCR products was performed to detect disease-causing variants and single nucleotide variants (SNVs) using an ABI Prism 3500XL system (Applied Biosystems, Foster City, CA). The exons were sequenced in the following order, exon 8, exon 14, exons 18–19, exon 15, exon 13, exon 2, exons 10-11-12, exon 16, exons 3-7-9-20-21, and exons 1,4,5,6,17. Sequenced reads were analyzed with Sequencher 5.0 software, and detected variants were evaluated with the reference sequences available at NCBI: NM_000053.4, NC_000013.11, NP_000044.2. Co-segregation analysis of detected variants in affected families was performed by sequencing corresponding exons in the parents.

2.3 In silico analysis

The pathogenic effects of variants on encoded proteins were predicted using several bioinformatic tools, including Mutation Taster, Polyphen2-HDIV, SIFT, PROVEAN, FATHMM, DANN, EIGEN, BayesDel, MetaLR, LRT, SpliceAI, and varSEAK databases. Clinical evaluation of the variants was carried out in the Franklin and Varsome databases based on the American College of Medical Genetics (ACMG) guidelines (Richards, Aziz et al. 2015). Furthermore, we utilized the ConSurf database (https://consurfdb.tau.ac.il/) to evaluate the evolutionary conservation of the amino acids affected by the majority of variants identified in this study.

2.4 Protein modeling

We modeled the three-dimensional structure of ATP7B wild-type protein based on NM_000053.4 transcript using I-TASSER protein structure prediction software (https://zhanggroup.org/I-TASSER/). Additionally, to identify structural changes, we predicted the defective protein resulting from the novel nonsense variant c.3431delTinsAGA. Furthermore, for the eight missense variants, we illustrated the alterations in the 3D structure of the protein and the interaction of amino acids before and after the variation. The models of the wild-type and mutant ATP7B proteins were visualized using the PyMOL molecular graphics system and animated using the ChimeraX program (video files are included in supplementary file 1).

All patients met the paraclinical and biochemical criteria for inclusion in the study, as outlined in the methods section. A total of 23 probands, comprising 14 females and nine males, were categorized into three different clinical conditions: hepatic (14 cases), neurological (5 cases), and combined (4 cases). The mean age at presentation was 7.9 years, ranging from 3.5 to 14. Among these, 19 probands were born to unaffected consanguineous parents, while in four cases, the parents were non-consanguineous. Kayser-Fleischer (KF) rings were detected in 17 out of 23 (74%) probands. The median ceruloplasmin level was 6.8 mg/dL (range 1.6–15 mg/dL). Table 2 summarizes the variously identified genotypes and clinical characteristics of the patients, including their age of disease onset, presence of KF rings, form of the disease, and the involved organs (liver, brain, or both).

Table 2

Clinical, biochemical, and mutation characteristics of the 23 Iranian WD patients harboring the mutations in the ATP7B gene.
#	Mutation(s)	Zygosity	Gender	Consanguinity	Age at examination (yrs)	Age at onset (yrs)	Hepatic form	Neurological form	Ceruloplasmin level (mg/dL)	KF rings
WD1	c.3431delTinsAGA	Homozygous	F	+	11	5	+	-	5	+
WD2	c.2648_2649del	Homozygous	F	+	20	4	-	+	1.6	+
WD3	c.3895C > T & c.4103T > C	Compound heterozygous	M	-	14	11	-	+	4.3	+
WD4	c.2807T > A	Homozygous	F	+	15	3.5	+	-	8.6	-
WD5	c.3188C > T	Homozygous	F	+	9	7	+	-	10	-
WD6	c.813C > A & c.2621C > T	Compound Heterozygous	F	-	23	10	+	-	1.9	+
WD7	c.3188C > T	Homozygous	F	+	35	6	+	+	4.3	+
WD8	c.3207C > A	Homozygous	F	+	12	7	+	-	15	+
WD9	c.2930C > T	Homozygous	F	+	16	8	+	-	13.6	+
WD10	c.813C > A	Homozygous	F	+	15	12	-	+	7.3	+
WD11	c.4103T > C	Homozygous	M	+	26	13	-	+	5.1	+
WD12	c.2866-2A > C	Homozygous	F	+	13	8	+	+	4	+
WD13	c.2807T > A	Homozygous	M	+	18	7	+	+	8.8	+
WD14	c.2807T > A	Homozygous	M	+	17	9	+	-	9.6	+
WD15	c.1924G > C	Homozygous	M	+	8	6	+	-	11	-
WD16	c.2866-2A > C & ?	Heterozygous	M	-	13	8	+	-	6.1	+
WD17	-	-	F	+	14	10	+	-	11.2	-
WD18	c.3188C > T	Homozygous	F	+	34	7	+	+	3.5	+
WD19	-	-	M	-	16	11	+	-	2.5	-
WD20	c.1156G > A	Homozygous	M	+	31?	14	-	+	1.9	+
WD21	c.2930C > T	Homozygous	F	+	19	5	+	-	9.1	+
WD22	c.2807T > A	Homozygous	F	+	13	6	+	-	7	-
WD23	-	-	M	+	12	4	+	-	6.7	+
Abbreviations: F, Female; M, Male

Targeted Sequencing of the entire coding region of the ATP7B gene in 23 WD families found a total of 13 different causative variants in 20/23 families, with a detection rate of 84.7% (39/46) (Table 3). These variants included missense, nonsense, splicing, deletion, and deletion/insertion mutations. Among these, 10 variants including p.Cys271Ter, p.Gly386Arg, p.Val883AlafsTer3, p.Leu936Ter, c.2866-2A > C, p.Thr977Met, p.Ala1063Val, p.Phe1144Ter, p.Leu1299Phe, and p.Leu1368Pro were detected for the first time in the Iranian population. Two of the 13 variants were novel p.Phe1144Ter, and c.1156G > A p.Gly386Arg (Fig. 1). However, eight out of 13 (61.5%) variants were missense mutations, indicating that they were the most commonly observed variants in this study. Deletion, deletion/insertion, and splicing mutations were each observed once, while two variants were nonsense mutations (Table 3). Of the 20 families with variants, one family had a single variant in a heterozygous state, and no pathogenic variant was found for the second allele.

Table 3

Characteristics of the detected variants in the ATP7B gene in WD patients during present study (NM_000053.4).
#	Exon (E)/ Intron (I)	Mutation type	Domain	Allelic frequency in genomAD Exomes	Allelic frequency in this cohort	Reported/ Novel	ACMG criteria
c.813C > A p.Cys271Ter	E2	Nonsense	MBD3	0.0000376	6.52% (3/46)	Reported	Pathogenic/ PM3, PVS1, PM2
c.1156G > A p.Gly386Arg	E2	Missense	MBD4	0.00000137	4.34% (2/46)	Novel	Likely Pathogenic/ PM2, PP3, PP2
c.1924G > C p.Asp642His	E6	Missense	bet MBD6/TM1	0.000012	4.34% (2/46)	Reported	Likely Pathogenic/ PM2, PP2, PM5, PP3, PP5
c.2621C > T p.Ala874Val	E11	Missense	TM5	0.0000588	2.17% (1/46)	Reported	Pathogenic/ PM3, PP1, PS3, PM1, PP2, PM2, PM5, PP3
c.2648_2649del p.Val883AlafsTer3	E11	Deletion	TM5	NA	4.34% (2/46)	Reported	Likely Pathogenic/ PVS1, PM2, PM3
c.2807T > A p.Leu936Ter	E12	Nonsense	TM5	0.00000205	17.39% (8/46)	Reported	Pathogenic/ PM3, PVS1, PM2
c.2866-2A > C	I12	Splicing	TM6	0.00000954	6.52% (3/46)	Reported	Pathogenic/ PM3, PVS1, PM2
c.2930C > T p.Thr977Met	E13	Missense	TM6	0.000182	8.69% (4/46)	Reported	Pathogenic/ PM3, PP1, PS3, PM1, PP2, PM2, PP3
c.3188C > T p.Ala1063Val	E14	Missense	P-domain	0.0000041	13.04% (6/46)	Reported	Pathogenic/ PM3, PM1, PP2, PP2, PM2, PP3
c.3207C > A p.His1069Gln	E14	Missense	P-domain	0.000945	4.34% (2/46)	Reported	Pathogenic/ PM3, PP1, PS3, PM1, PP2, PM2, PM5, PP3
c.3431delTinsAGA p.Phe1144Ter	E16	Deletion/Insertion	N-domain	NA	4.34% (2/46)	Novel	Likely Pathogenic/ PVS1, PM2
c.3895C > T p.Leu1299Phe	E18	Missense	bet N-domain/ TM7	0.0000157	2.17% (1/46)	Reported	Pathogenic/ PM3, PM2, PM5, PP3, PP2
c.4103T > C p.Leu1368Pro	E20	Missense	TM8	0.0000081	6.52% (3/46)	Reported	Likely Pathogenic/ PM1, PP2, PM2, PP3, PP5
Abbreviations: TM, Transmembrane; MBD, Metal Binding Domains; NA, Not Applicable

The distribution of the 13 variants identified in our study of 23 WD patients can be observed in Fig. 2. These variants were located within nine exons of the ATP7B gene, including exons 2, 6, 11, 12, 13, 14, 16, 18, and 20, as well as intron 12. The p.Leu936Ter in the exon 12 with an allelic frequency of 17.39 (8/46), was observed in 4 out of 23 probands and was considered the most common variant in the present study. The second most frequent variant, p.Ala1063Val in exon 14, was detected in 3 out of 23 patients, with an allelic frequency of 13.04% (6/46). In this research, the positioning of separate variants introduced exons 12, 13, and 14 as “hot spots” exons of the ATP7B gene in the Northeastern region of Iran. The total disease-causing variant detection rate on these three exons was 50% (23/46), suggesting their importance for molecular diagnosis of WD in this area.

The compound-heterozygous form was found only in 2 out of the 23 (9%) patients, while the homozygous form was identified in 17 out of 23 (74%) patients, consistent with the pattern of consanguineous marriages in families. Our novel variant p.Phe1144Ter was absent in many public databases such as 1,000 Genomes, ExAC, gnomAD, GME Variome Project, ABraOM, HEX database, and in Iranome or more than 100 in-house exomes of the unrelated Iranians affected with non-metabolic and non-neurological disorders, (minor allele frequency (MAF) = 0). The p.Phe1144Ter variant is a null variant that causes premature termination of translation and was predicted as "likely pathogenic" by default InterVar (ACMG). Alternatively, the absence of protein expression for the p.Phe1144Ter variant could be due to degradation of the mutant mRNA via the Nonsense-mediated mRNA decay (NMD) mechanism. At the protein level, the conservation analysis with the ConSurf database showed that phenylalanyl residue in the p.Phe1144Ter variant is located in an average region, and probably following that, a large number of highly conserved amino acids are removed from the protein structure (Fig. 3A). The p.Gly386Arg variant, another undescribed variant, is reported ultra-rarely in population databases, and we introduce it for the first time as a disease-causing variant. This variant is reported as deleterious by the majority of bioinformatic prediction tools (Supplementary Table 1). According to the ConSurf database, the p.Gly386Arg variant occurs in a conserved region (Fig. 3B). Changes in the 3D structure of the mutated proteins compared to intact ATP7B are depicted in Fig. 3C. The following parameters were obtained for the predicted model: C-score (a confidence score for estimating the quality of the predicted models by I-TASSER) was − 2.55. The estimated TM-score was 0.42 ± 0.14, and The Estimated RMSD (Root-Mean-Square Deviation that measures the average distance between atoms of superimposed proteins) was 16.5 ± 3.0.

The c.2866-2A > C variant detected in families WD12 and WD16 is located in the conserved acceptor splice site upstream of exon 13 and is considered pathogenic by the SpliceAl and varSEAK databases, which likely leads to exon skipping and the creation of a defective ATP7B protein. The results of in silico analysis using bioinformatic tools such as Mutation Taster, Polyphen2-HDIV, SIFT, PROVEAN, FATHMM, DANN, BayesDel, EIGEN, MetaLR, and LRT are shown in Supplementary Table 1. In addition to these variants, twelve single nucleotide polymorphisms (SNPs) that do not disrupt ATP7B gene function were identified (Table 4).

Table 4

Characteristics of the detected single nucleotide polymorphisms in the ATP7B gene during present study (NM_000053.4).
#	Exon (E)/ Intron (I)	type	Domain	Reported/ Novel
c.51 + 108G > A	I1	Intronic	-	Reported
c.1216T > G p.Ser406Ala	E2	Missense	MBD4	Reported
c.1366G > C p.Val456Leu	E3	Missense	bet MB4/ MBD5	Reported
c.2322T > C p.Ile774=	E8	Silent	TM4	Reported
c.2495A > G p.Lys832Arg	E10	Missense	bet TM4/ TM5	Reported
c.2855G > A p.Arg952Lys	E12	Missense	TM5	Reported
c.2866-13G > C	I12	Intronic	-	Reported
c.2866-90G > T	I12	Intronic	-	Reported
c.2973G > A p.Thr991=	E13	Silent	TM6	Reported
c.2976C > A p.Pro992=	E13	Silent	TM6	Reported
c.3009G > A p.Ala1003=	E13	Silent	TM6	Reported
c.3419T > C p.Val1140Ala	E16	Missense	N-domain	Reported
Abbreviations: TM, Transmembrane; MBD, Metal Binding Domains

The advent of DNA sequencing and targeted gene sequencing of the ATP7B gene made a breakthrough in the molecular diagnosis of WD, which exhibits significant allelic heterogeneity. In certain populations, the prevalence of specific mutations enables basic screening and rapid disease diagnosis. Consequently, numerous population-specific studies on WD are underway worldwide. In our study of WD patients, 74% (34/46) of causative alleles were identified through direct sequencing of five PCR amplicons, covering exons 2B, 11–12, 13, 14, and 20. This proves to be significantly cost-effective compared to sequencing all 19 PCR amplicons encompassing the entire 21 exons of the ATP7B gene coding region. Therefore, we recommend prioritizing screening of these exons in the Iranian population in the Northeast region.

Our findings are consistent with previous studies in Middle Eastern countries, confirming a wide range of pathogenic variants in the ATP7B gene, most of which are rare and of low frequency (Simsek Papur, Akman et al. 2013, Barada, El Haddad et al. 2017). The absence of the p.Arg778Leu mutation, the most common mutation in the Asian pedigree, in our cohort is noteworthy. Similarly, we did not detect any mutations in exon 8, which is one of the most frequent mutational sites in the ATP7B gene and was the first exon we examined. Additionally, The p.His1069Gln, the most common mutation in Europe affecting 15_70% European WD population (Chang and Hahn 2017), was only found in 4.34% (2/46) of our cohort, While the other mutation located in exon 14, p.Ala1063Val, which has lower frequency in Europe, was found in 13.04% (6/46) of our cohort. The frequency of p.Leu936Ter in our study was 17.39%, which is also common in the United Kingdom (10%), and Greece (7%) population (Butler, McIntyre et al. 2001, Panagiotakaki, Tzetis et al. 2004). Additionally, the p.Leu936Ter mutation was reported in India (1–2%) and Turkey (2%) (Simsek Papur, Akman et al. 2013, Kumari, Kumar et al. 2018). Indeed, it appears that this mutation is relatively common in European and Mediterranean populations. On the other hand, the p.Ala1063Val mutation has been reported as rare in most populations, such as China (0.73%), Czech Republic and Slovakia (0.25%), France (0.5%), and Italian/Turkish (0.4%) (Loudianos, Dessi et al. 1999, Vrabelova, Letocha et al. 2005, Bost, Piguet-Lacroix et al. 2012, Cheng, Wang et al. 2017), however, the frequency of this mutation (13.04%) was notably high in our population.

In the present study, we report a high detection rate (84.7%) of ATP7B causative variants in our cohort. The direct sequencing detection rate in patients with WD that has been clinically proven has varied widely in previous studies. For example, Alison J. Coffey et al. showed that the rate of mutation discovery in their study was 98% (356/362) of alleles on direct sequencing of all exons of the ATP7B gene in the 181 unrelated WD patients from the United Kingdom (Coffey, Durkie et al. 2013). Mingming Li conducted a mutational investigation of 101 WD patients from China and found that the mutation detection rate was up to 80.7% (163/202) (Li, Ma et al. 2021). A similar study conducted in China by Gu YH on 40 patients recorded an 83.8% (67/80) mutation detection rate (Gu, Kodama et al. 2003). In Iran, two separate studies on 70 and 30 WD patients, found a mutational detection rate of 30% (42/140) and 76% (46/60), respectively (Zali, Mohebbi et al. 2011, Maleki, Zali et al. 2013). However, The detection rate of direct genetic analyses of ATP7B can vary depending on the accuracy of clinical diagnosis. In this study, out of the 13 variants we identified, 6 (46.1%) were recurrent, suggesting the possibility of founder mutations. Due to the high rate of same-caste marriages in Iran, the founder mutations can be as influential as consanguineous marriages in the occurrence of recessive disorders. In families WD12 and WD16, we identified an intronic variant c.2866-13G > C in association with causative c.2866-2A > C pathogenic variant, indicating a potential founder haplotype in these patients (Fig. 4). Previously, Mohammed al-tobi et al., found that the c.2866-2A > G mutation leads to the skipping of exon 13 (195 bp) from the mRNA transcript, likely rendering the protein non-functional (Al-Tobi, Kashoob et al. 2011). The c.2866-2A > C will probably have the same effect. Functional studies for this variant are required to authenticate our conjecture. In WD3 and WD11 probands, the c.2322T > C (p.Ile774=) polymorphism was detected in heterozygous and homozygous states, respectively, and is linked with the c.4103T > C (p.Leu1368Pro) causative variant in these patients. It is possible that the coexistence of c.2322T > C (p.Ile774=) polymorphism and c.4103T > C (p.Leu1368Pro) variant could have a particular effect on the ATP7B function. Further investigations are needed to confirm this claim. Additionally, the detection of the c.3009G > A (p.Ala1003=) polymorphism in all 4 patients with c.2807T > A (p.Leu936Ter) mutation was notable. This suggests that their linkage may affect the expression of the ATP7B protein.

In various studies, cases with no mutations in the coding sequence of ATP7B are always reported, with variable frequency among different populations. In our study, in 3 out of 23 Iranian patients with WD, We could not find any pathogenic variant in the exons of the ATP7B gene. In these cases, Failure to detect any variant can be explained by several reasons, including the occurrence of unknown mutations located outside the coding sequence and flanking regions, such as gene regulatory elements or deep intronic sequences (Woimant, Poujois et al. 2020, Collins, Yi et al. 2021). Having one of Wilson's mimicry disorders can also be considered a rare cause (Roberts 2018). However, further studies using high-throughput techniques such as Whole Exome Sequencing (WES) or Whole Genome Sequencing (WGS) are needed for molecular diagnosis of WD patients with no specific mutations in coding sequences of the ATP7B gene.

In conclusion, The spectrum of ATP7B gene variants in the Northeastern region of Iran is very diverse. Therefore, large-scale studies can provide more insights into the genetic status of the disease in the region. Most of the variants found in this study were previously unreported in Iran. Furthermore, we identified two novel undescribed variants in the Northeastern Iranian WD patients that could expand the previously defined spectrum of ATP7B gene mutations.

We acknowledge the Mashhad of the University of Medical Sciences for funding the research (Grant No.4010194) based on the PhD thesis of Seyyed-Saleh Hashemi and thank the patients and their family members for participating in the study.

Competing Interests

The authors have nothing to disclose.

Author Contributions

Seyyed-Saleh Hashemi: DNA extraction, primer design, analysis of sanger sequencing data, mutation screening of candidate variants, writing and editing of the manuscript; Seyed Ali Jafari: Clinical evaluations; Aida Gholoobi: Principal investigator, review and editing; Tayebeh Hamzehloei: Conceptualization, supervision, methodology, validation, review and editing. All authors read and approved the final version of the manuscript.

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No competing interests reported.

SupplementaryTable1.docx
Supplementary Table 1. The results of in silico analysis using several prediction tools.
Supplementaryfile1.rar
Supplementary file 1. Animated model of wild-type ATP7B protein (NP_000044.2), created using the ChimeraX program.

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Genetic Screening of ATP7B Gene in Iranian Wilson Disease Patients: A diverse landscape of pathogenic variants

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Abstract

Background/Objective:

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INTRODUCTION

PATIENTS AND METHODS

1 Subjects

2 Genetic testing

2.1 DNA extraction and Polymerase chain reaction (PCR)

2.2 Targeted Sanger sequencing

2.4 Protein modeling

RESULTS

DISCUSSION

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References

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Supplementary Files

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