Wilson's Disease (WD; OMIM #277900) is a rare autosomal recessive disorder of copper metabolism, affecting approximately 1 individual from 30,000 in the general population [9]. Clinical manifestations are highly heterogeneous, the age of onset varies from 2 to 70 years [10, 11]. The main involvements are most commonly hepatic, neurological, and psychiatric, which can range from asymptomatic to fatal form if left untreated [12, 13]. Hepatic involvements occur in 43% of WD patients and vary from clinically asymptomatic patients with mild elevations in levels of the liver enzymes alanine transaminase and aspartate transaminase to hepatic failure and cirrhosis [2]. Neurologic symptoms occur in 35% of WD patients and may range from mild disturbances, dysarthria and trouble articulating to a severe and rapid neurologic and cognitive dysfunction [1]. Because of the progressive copper deposition in the cornea, ophthalmic symptoms can also associated with this disease and lead to formation of a dark rings that appear to encircle the cornea. The KF rings are easily detected by slit lamp examination, but it may present in only 50% of the patients with isolated hepatic involvement but often present in neurological forms in 95% [14]. Thus, WD patients may develop combinations of hepatic and neurological manifestations at the same time [14, 15].

Initially, diagnosis of WD depends primarily on clinical and biochemical parameters which include low ceruloplasmin and serum copper levels and increased 24-h urine copper and liver copper content; but none of the commonly used parameters are pathognomonic and cannot distinguish it from the common liver diseases.

Mutations are distributed throughout the ATP7B gene. More than 800 mutations of ATP7B gene are reported in the Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff (HGMD; http://www.hgmd.cf.ac.uk/ac/gene.php?gene=ATP7B). However, some mutations hotspots have been highly mentioned by several authors and are considered population specific. Herein some examples, the p.His1069Gln is the most worldwide mutations that occur in 37–63% of Caucasians populations and is often associated with late and neurologic presentation of WD [1]. The p.Arg778Leu and p.Pro992Leu are the most common mutation in Asiatic population which occurs in 50.43% of patients with WD [16]. The p.Cys271Ter is relatively frequent in China's population and touch approximately 19% of patients with WD [17]. However, the spectrum of ATP7B mutations in Moroccan patients with WD has never been studied before. As a department of medical genetics providing genetic services and testing for a variety of diseases in low-income countries, molecular diagnostic strategies through NGS for heterogeneous diseases pose a major challenge. The recent introduction of NGS technology in our laboratory has prompted us to adopt a personalized multigene panel approach for genes involved in the most common genetic diseases from our consultation. This strategy might be the best suited for molecular testing and remains our molecular approach in the public health system, facilitating recruitment of NGS candidates, increasing the availability of multiple diagnostic tests and reduce turnaround time and costs of molecular tests. We choose to include in this panel beside 20 other genes required for others Moroccan patients, the ATP7B gene causative of WD to analyze the 4 unrelated patients reported in this study. As result, we identified causal mutations in the 4 cases of WD, three at homozygous state and one at heterozygous state.

In the present study, the diagnosis of WD was based on combination of characteristics symptoms, liver biochemical tests and mutation analysis of the ATP7B gene according on the Leipzig score established in 2003 by Ferenci et al. [18] and re-evaluated in 2010 [5].

In WD-P1, Next-Generation Sequencing gene panel allowed to detect two pathogenic variants in the ATP7B gene, described in compound heterozygosity: c.3244-2A>G and c.3664delG (p.Asp1222fs) in exon 15 and exon 17, respectively. The two variants were once recorded in Clinvar as likely pathogenic [https://www.ncbi.nlm.nih.gov/clinvar/variation/282915/]. The c.3244-2A>G was firstly reported in one Italian patient with WD [18]. Another pathogenic variant in the same splice site mutation, c.3244-2A>C, was also reported in one Chinese patient with early onset age of severe hepatic manifestations and poor prognosis [19].

The missense homozygous pathogenic variant c.2507G>A; p.Gly836Glu of ATP7B gene found in patient WD-P2 was firstly reported in 2012 as deleterious in one WD Moroccan patient [18].

The third non-sense homozygous pathogenic variant c.865C>T; p.Gln289Ter of ATP7B gene found in patient WD-P3 was recorded in Clinvar as pathogenic/ likely pathogenic in patients with WD [https://www.ncbi.nlm.nih.gov/clinvar/variation/3864/]. This mutation is highly frequent in a small mountain village next to Heraklion city in the island of Crete [20]. The c.865C>T was firstly reported in 2005 in one girl at the age of 6 years, wish rapidly progressing to acute liver failure that required urgent liver transplantation [20]. The patient WD-P3 is clinically quite distinct from the patient described by Dedoussis GV in 2005. Our patient was clinically asymptomatic at 8-year-old and addressed to our consultation for mild elevations in levels of the liver enzymes alanine transaminase and aspartate transaminase, but the patient described in 2005 had already developed hepatic failure and cirrhosis at 6-year-old. This case suggests that diagnosis of WD is a real challenge, and molecular analysis of ATP7B gene should be recommended in patients with minimal alterations to establish the final diagnosis of Wilson disease.

The missense homozygous pathogenic variant c.3059A>G; p.Lys1020Arg of ATP7B gene found in patient WD-P4 with severe liver and neurological symptoms, was firstly reported in 2012 as deleterious in one French patient with mixed hepato-neurologic presentation of WD. This mutation p.Lys1020Arg was found to be frequent in French patients with two others mutations, Ile1148Thr and Thr1220Met with a frequency of 19% of the total mutations [21].

In our patients, mutations have been found distributed throughout the ATP7B gene. The highest reported mutation H1069Q was not observed in our patients. There are a few potential theories. Firstly, several studies showed that H1069Q is associated with late and neurologic presentation of Wilson Disease, but on the other side only one patient of our cohort has neurological signs. Another hypothesis is that H1069Q mutation was not detected in our cohort, probably due to the small sample sizes studied which requires subsequent genetic studies with a large sample sizes for possible identification of this mutation in Moroccan population. Finally, we could suggest that the epidemiology of Wilson’s disease in the Moroccan population may be different from those in other continents.