Zhang et al. BMC Pediatrics(2024) 24:576
https://doi.org/10.1186/s12887-024-05045-x

BMC Pediatrics

RESEARCH

Open AccessPhenotypic and genetic characterizationof children with Wilson Disease
from Northeast China
Tianhe Zhang1†, Wenliang Song1† and Zhiqin Mao1*
Abstract
Background Wilson disease (WD) is an autosomal recessive inherited disease caused by ATP7B variants andcharacterized by copper metabolism defects. However, children with WD are often asymptomatic, making the clinicaldiagnosis difficult. Therefore, more accurate methods are required for clinical diagnosis. The objective of this study
was to highlight the phenotypic and genetic characteristics of children with WD in northeast China.
Methods We retrospectively analyzed the clinical data and gene sequencing results of 65 children with WD fromJanuary 1, 2014, to December 31, 2022, at the Shengjing Hospital of China Medical University. All data refer to the timeof diagnosis before treatment.
Results The median age at diagnosis was 5 years (range 1.2–15 years). In 50 cases (50/65, 76.9%) patients, routine
physical examinations revealed only abnormal liver function. However, they had a significantly negative (p < 0.05)
Kayser–Fleischer ring (KF). Children with acute liver failure had significantly increased 24 h urinary copper excretion(p < 0.05). We detected 46 genetic variants of ATP7B, including seven novel variants. The most frequent variant was
p.R778L with an allele frequency of 38.7%. Phenotype-genotype correlation analysis suggested that p.R778L wassignificantly associated with lower serum ceruloplasmin levels and higher zinc levels (p < 0.05). The loss-of-function(LOF) variant was associated with significantly lower albumin levels (p < 0.05).
Conclusion Most children with WD are asymptomatic, which makes early diagnosis of WD difficult. Therefore, clinicaland laboratory characteristics as well as genetic testing are essential. p.R778L is the most frequent variant of ATP7B in
China and may play an important role in lowering serum ceruloplasmin levels.
Keywords Wilson disease, Gene, Children, China†

Tianhe Zhang and Wenliang Song contributed equally to this work.

*Correspondence:
Zhiqin Maomaozq6590@126.com
1
Department of Pediatrics, Shengjing Hospital of China Medical
University, Shenyang City, Liaoning Province, People’s Republic of China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Zhang et al. BMC Pediatrics(2024) 24:576

BackgroundWilson disease (WD) is a rare autosomal recessive disorder caused by mutations in ATP7B [1]. ATP7B islocated on the short arm of chromosome 13 and encodes
a copper-transporting P-type ATPase [2, 3]. ATP7B is
responsible for transporting copper from intracellularchaperone proteins into the secretory pathway, both for
excretion into bile and incorporation into apo-ceruloplasmin for the synthesis of functional ceruloplasmin [3].
The absence or reduced function of the ATP7B protein
leads to copper accumulation and liver injury. Eventually,
copper is released into the bloodstream and deposited inother organs, notably the brain, kidneys, and cornea [4].
WD can develop at any age and often occurs between 5
and 35 years of age [3]. WD is increasingly diagnosed inchildren younger than 5 years of age, and the clinical findings may be nonspecific in children younger than 2 years[5]. Most children present with liver dysfunction, ranging
from incidental findings of elevated serum transaminases
in otherwise asymptomatic patients, acute hepatitis, andhepatomegaly, to acute liver failure (ALF) or cirrhosis[6]. However, children with WD are often asymptomatic,
making the clinical diagnosis difficult. Therefore, moreaccurate methods are required for clinical diagnosis.
Given these diagnostic challenges, genetic testing has
become increasingly important for early, rapid, and accurate diagnosis of WD. To date, more than 700 ATP7B
variants have been identified [7]. The most common variant in patients from Europe is the point variant H1069Q(exon 14), and that in Asia is R778L (exon 8) [8]. Morethan 50% of the variants are missense and nonsense, andthe rest may be insertion/deletion or splice-site variants [9, 10]. Studies have shown that variants affecting
critical portions of the protein, including copper-binding domains or the ATPase loop, may lead to the early
onset of liver disease [11]. Other studies have indicatedthat p.H1069Q is more frequently associated with neurological presentation and later symptom onset [12, 13].
Loss-of-function (LOF) of ATP7B gene variants has been
associated with ALF and an earlier age of onset [14, 15].
However, there are no confirmed genotype–phenotype
correlations [16].
The objective of our study was to summarize the clinical manifestations and laboratory and genetic characteristics of children with WD in northeast China. We aimed
to determine the spectrum and frequency of ATP7B variants and the genotype–phenotype correlation in pediatric patients with WD.
Methods
Patients and groups

A total of 65 pediatric patients with WD admitted toShengjing Hospital of China Medical University between
January 1, 2014, and December 31, 2022, were included inPage 2 of 9this study. The diagnosis of WD was established according to the scoring system provided by the Eighth International Meeting on Wilson Disease [17] and a Leipzig
score of > 3 was considered appropriate for the diagnosis of WD. The clinical data included medical history,
physical examination results, laboratory examination
results, and imaging findings. All data refer to the timeof diagnosis before treatment. This study was conductedin accordance with the protocols of the Ethics Committee of Shengjing Hospital of China Medical University
for a clinical retrospective study (No. 2022PS107K). Verbal consent was obtained from the legal guardians in thisstudy. The study was conducted in accordance with theprinciples of the Declaration of Helsinki.
According to the main clinical symptoms of WD inchildhood, all children with WD were divided into fourgroups according to the type of presentations as follows: Group 1 (n = 50): Asymptomatic cases with only an
increase in serum transaminases diagnosed during theroutine physical examination. Group 2 (n = 5): Patientswith biochemical abnormalities and abnormal haptic
manifestations including jaundice, ascites, or liver ultrasound findings suggestive of cirrhosis. Group 3 (n = 4):
Patients with ALF were diagnosed according to the criteria of the Pediatric Acute Liver Failure Study [18, 19].
Group 4 (n = 6): Patients with neurological, behavioral, or
psychiatric disorders.
LOF variants were classified as nonsense (substitution
in the coding base pair predicted to result in a prematurestop codon), frameshift (insertion or deletion predicted
to result in reading frame shift), and splice site (substitutions predicted to involve a splice site), which produce a
nonfunctional protein product [20, 21].
Variant analysis of the ATP7B gene

Genomic DNA was extracted from the peripheral blood
samples. All patients were tested using ATP7B-targeted
gene panel sequencing or whole-exome sequencing. All
testing protocols, including DNA extraction, gene library
construction, high-throughput sequencing, data analysis,
Sanger sequencing verification, and bioinformatics analysis, were performed by the Clinical Genetics Laboratory
at Shengjing Hospital of China Medical University andcommercial companies, including My Genostic (Beijing,
China), Chigene Corp (Beijing, China), and Kingmed(The Guangzhou Genomics Institute, Shenyang, China).
Statistical analysis

All continuous numerical values are expressed as medians with ranges. Discrete variables were presented as
patient numbers or percentages (%). Statistical comparisons between groups were performed using analysis of variance (ANOVA) for parametric variables andthe Kruskal–Wallis test for non-parametric variables.

Zhang et al. BMC Pediatrics(2024) 24:576

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Analyses were conducted using Statistical Product andService Solutions (SPSS 22.0). A p-value of less than 0.05
was considered statistically significant.

Results
Clinical features of pediatric WD patientsAt the time of diagnosis, 65 children had WD, including
34 males and 31 females, with a median age of 5 years(range 1.2–15 years). Five patients had family histories of
WD.
In 50 cases (50/65, 76.9%), routine physical examinations revealed only abnormal liver function. Casesof children with symptoms included neuropsychiatric
symptoms (6/65, 9.2%), ascites (3/65, 4.6%), jaundice(2/65, 3.1%), lower limb edema (2/65, 3.1%), and hemolytic anemia (1/65,1.5%). Fourteen patients (14/65, 21.5%)
showed a positive Kayser–Fleischer ring (KF) under a slit
lamp, and the youngest child with a positive KF ring was
6 years old. All asymptomatic patients exhibited a negative KF ring, which was significantly different from that in
the other three groups (p < 0.05). Of all 65 patients, 92%of children had a 24 h urinary Cu value > 40 µg, and 64.4%
of children had a 24 h urinary Cu value > 100 µg. Patientswith WD and ALF in Group 3 exhibited increased total
bilirubin, decreased albumin, increased white blood cellcount, and decreased hemoglobin. Group 3 also had significantly increased 24 h urinary Cu excretion comparedto the other three groups (p < 0.05). Groups 3 and 4 hadsignificantly lower blood platelets than the other twogroups (p < 0.05) (Table 1).
Brain MRI abnormalities were observed in six patientswith neurological symptoms in Group 4, including
abnormal signal intensity in the basal ganglia, thalamus, brainstem, and corpus callosum (6/65, 9.2%).
Liver ultrasonographic abnormalities included hepatomegaly, splenomegaly, hepatic steatosis, fibrosis, andcirrhosis. Patients in Group 3 had liver cirrhosis on
ultrasonography.
Gene variant analysis

ATP7B sequencing was performed in 65 patients. Fourpatients had only one allelic variant, eight had homozygous variants, and 53 had compound heterozygous variants. In total, 126 alleles were included in the analysis.
We identified 46 gene variants in ATP7B (Table 2; Fig. 1),
seven of which were novel. Novel variants included
c.2572 A > C, c.-362G > A, c.51 + 4 A > C, c.1543 + 40G > A,c.3903 + 2T > C,
c.2664-2665delCC,
andc.35243528delAAGGA. The most frequent variant was p.R778L,
with an allele frequency of 38.7%, followed by p.P992L

Table 1 Clinical and laboratory characteristics of 65 pediatric WD patients at diagnosis
Parameter
Male/female
Medianageofonset (yrs) (Range)
Family history
Jaundice
Edema
AscitesHemolytic anemia
Neuropsychiatric deficit
KF ring (+)
Brain involvement(MRI)
Hepatomegaly(USG)
Splenomegaly(USG)
Hepatosteatosis(USG)
Fibrosis(USG)
Cirrhosis(USG)
Ceruloplasmin (mg/dl)
Urinary copper (µg/24 h)
ALT (IU/L)
AST (IU/L)
TB (mg/dL)
ALB (g/L)
WBC (103/ml)
Hb (g/dL)
PLT(103/ml)
Zinc (mg/dl)

G1(n = 50)
26/24
5(3–6)
5.0(10.0)0(0.0)
0(0.0)
0(0.0)
0(0.0)
0(0.0)
0(0.0)
0(0.0)
14(28.0)1(2.0)
14(28.0)0(0.0)
0(0.0)
2.6(2.0-5.8)
109.6(75.5-152.9)
227.0(135.8–308.0)
110.5(62.0-151.8)
6.4(5.3–8.2)
42.9(40.8–44.1)
6.8(5.6–8.1)
125.5(120.5-133.3)
314.5(279.0-386.3)
73.4(61.2–85.6)

G2(n = 5)
3/2
8(4–10)0(0.0)
0(0.0)
0(0.0)
0(0.0)
0(0.0)
0(0.0)
4(80.0)0(0.0)
3(60.0)1(20.0)0(0.0)
3(60.0)2(40.0)3.8(2.2–12.3)
221.0(104.0-461.6)
131.0(69.0-173.0)
72.0(68.0-117.5)
7.4(5.2–34.3)
42.9(30.0-44.3)
6.0(4.7–6.4)
122.0(113.5-132.5)
116.0(84.5–272.0)
63.0(74.1–85.2)

G3(n = 4)
2/2
12(9–13)0(0.0)
2(50.0)3(75.0)2(50.0)1(25.0)0(0.0)
4(100.0)1(25.0)1(25.0)3(75.0)0(0.0)
0(0.0)
4(100.0)
4.1(3.4–10.4)
440.4(224.1-907.2)
110.0(62.5-221.3)
131.0(71.3-303.3)
64.7(57.0-104.8)
25.8(23.9–28.6)
4.8(3.2–7.1)
105.5(93.7-109.8)
73.5(53.3-104.3)
62.3(56.9–78.6)

G4(n = 6)
3/3
12(11–13)0(0.0)
0(0.0)
1(16.7)0(0.0)
0(0.0)
6(100.0)6(100.0)6(100.0)2(33.3)1(16.7)1(16.7)1(16.7)3(50.0)3.8(2.6–6.02)
242.1(158.8-383.8)30(22.3-152.5)
55.0(31.0-112.8)
14.1(7.0–22.0)
39.2(32.1–42.8)
4.0(3.0-6.1)
127.5(115.8-135.5)
110.5(224.8)
70.9(58.8–83.2)

p value
0.986< 0.010.654< 0.01< 0.01< 0.01< 0.01< 0.01< 0.01< 0.010.519< 0.010.428< 0.01< 0.010.686< 0.01
0.013
0.109
0.010
0.030
0.003
< 0.01< 0.01
0.114

G1: Asymptomatic cases with only increase in serum transaminases; G2: Patients with biochemical abnormalities and hepatic presentation; G3: WD patients withacute liver failure at diagnosis; G4: WD patients with neurological/psychiatric symptoms. Zinc values is not used before Zinc treatment.

Zhang et al. BMC Pediatrics(2024) 24:576

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Table 2 Spectrum of gene variants in the ATP7B gene of this study
Exon

Nucleotide variant

Amino acid change

1
2
2
2
3
4
6
8
8
8
8
8
8
8
10
11
11
11
11
11
12
12
12
13
13
13
13
14
14
15
16
16
16
16
17
17
18
18
18
18
18
19
19
20
20
20

c.51 + 4 A > C*
c.122 A > G
c.-362G > A*
c.525DupA*
c.1488 C > T*c.1543 + 40G > A*
c.1924G > T
c.2128G > A
c.2132G > A
c.2145 C > T
c.2252 C > A
c.2333G > T
c.2337G > A*c.2293G > A
c.2572 A > C
c.2621 C > T
c.2633 A > C
c.2662 A > C
c.2664-2665delCC*
c.2668G > A
c.2755 C > G
c.2785 A > G
c.2790-2792delCAT
c.2924 C > A
c.2930 C > T
c.2975 C > T
c.3029 A > C
c.3140 A > T
c.3168delT*c.3316G > A
c.3459G > T
c.3517G > A
c.3524-3528delAAGGA*
c.3532 A > G
c.3634 A > G
c.3646G > A
c.3797G > A
c.3809 A > G
c.3818 C > T
c.3851T > A
c.3903 + 2T > C*
c.3955 C > T*c.4006delA*
c.4064G > T
c.4112T > C
c.4114 C > T*

p. N41S
p. V176Sfs*28
p. G496=
p. D642Y
p. G710S
p. G711E
p. Y715=
p. A751D
p. R778L
p. W779X
p. D765N
p. T858P
p. A874V
p. N878T
p. T888P
p. H889fs
p. V890M
p. R919G
p. I929V
p. I930delp. S975Y
p. T977M
p. P992L
p. K1010T
p. D1047V
p. L1057fs
p. V1106I
p. Trp1153Cys
p. E1173K
p. T1178A
p. M1212V
p. V1216M
p. G1266E
p. N1270S
p. P1273L
p. I1284N
p. R1319X
p. I1336fs
p. G1355V
p. L1371P
p. Q1372X

Variation
type
splice site
Missense
Nonsense
Frameshift
Synonymous
splice site
Missense
Missense
Missense
Synonymous
Missense
Missense
Nonsense
Missense
Missense
Missense
Missense
Missense
Frameshift
Missense
Missense
Missense
In-frame deletion
Missense
Missense
Missense
Missense
Missense
Frameshift
Missense
Missense
Missense
Frameshift
Missense
Missense
Missense
Missense
Missense
Missense
Missense
splice site
Nonsense
Frameshift
Missense
Missense
Nonsense

Novel variants are highlighted in bold*: LOF variants were defined as produced a non-functional protein productPathogenicity

DomainAllele frequency (%)

Uncertain
Likely Pathogenic
Uncertain
Pathogenic
Uncertain
Uncertain
Pathogenic
Pathogenic
Pathogenic
Uncertain
Likely Pathogenic
Pathogenic
Pathogenic
Pathogenic
Likely Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Likely Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Likely Pathogenic
Pathogenic
Likely Pathogenic
Pathogenic
Pathogenic
Likely Pathogenic
Likely Pathogenic
Pathogenic
Pathogenic
Likely Pathogenic
Pathogenic
Pathogenic

Before Cu1
Before Cu1
Cu1
Cu2
Cu2
Cu5
bet Cu6/TM1
TM2
TM2
TM4
TM4
TM4
TM4
TM4
Td
bet Td/TM5
bet Td/TM5
bet Td/TM5
bet Td/TM5
bet Td/TM5
bet Td/TM5
TM5
TM5
TM6
TM6
bet TM6/Ph
bet TM6/Ph
ATP loop
ATP loop
ATP loop
ATP loop
ATP loop
ATP loop
ATP loop
ATP bind
ATP bind
ATP hinge
ATP hinge
ATP hinge
ATP hinge
bet ATP hinge/TM7
bet ATP hinge/TM7
TM7
TM8
TM8
TM8

0.81
0.81
0.81
0.81
0.81
0.81
1.61
1.61
0.81
0.81
0.81
38.71
0.81
0.81
0.81
4.84
0.81
0.81
0.81
0.81
4.84
0.81
0.81
1.61
0.81
6.45
0.81
0.81
0.81
1.61
0.81
1.61
0.81
0.81
0.81
3.22
1.61
2.42
0.81
0.81
0.81
0.81
1.61
0.81
0.81
2.42

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Fig. 1 Spectrum of gene variants in ATP7B gene of this study. Novel variants detected in this study are in red

Fig. 2 Distribution of variants within 21 exons of ATP7B.

(6.5%), p.R919G, p.A874V (4.9% each), p.V1216M(3.2%),
p.N1270S, and p. Q1372Ter (2.4% each).
Of the 46 identified variants, 33 were classified as mild
variants, including 31 missense variants, one synonymous variant, and one in-frame deletion variant. Thirteen
were LOF variants, including five frameshift variants,
four nonsense variants, three splice-site variants, and one
synonymous splice-related variant.
The 46 variants were distributed throughout all exonsof ATP7B except for exons 5, 7, 9, and 21. The exons harboring the highest percentage of variants were exons 8,
11, 12, 13, and 18 (Fig. 2). The overall variant detection
rate for these five exons was 75%, suggesting that theseexons may be important regions for detecting variants.

Assessment of phenotype and genotype correlation

We compared the two most common variants,
c.2333G > T (p.R778L) and c.2975 C > T (p.P992L), withother variants in terms of the age at symptom onset andclinical parameters. Of the 65 patients, 41 (63.1%) had thep.R778L variant, eight were homozygous, and 33 were
heterozygous. We observed that patients carrying the
p.R778L variant had lower serum ceruloplasmin levels
and higher zinc levels than patients with other variantsof both alleles (p < 0.05) (Fig. 3a, b). Eight (12.3%) patients
had the p.P992L variant, all of whom were heterozygous,
and there was no significant association between thevariant types and phenotype.
Of the 65 patients, 16 (24.6%) harbored a LOF variant on their allelic chromosomes. We observed that

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Fig. 3 Correlation of phenotype and genotype of this study. (a) Correlation of 2333G > T (R778L) and serum ceruloplasmin level; (b) Correlation of
2333G > T (R778L) and zinc level; (c) Correlation of LOF variant and albumin protein level; (d) Correlation of LOF variant and clinical manifestations

patients carrying the LOF variant had lower albumin
levels for both alleles than patients without the LOF variant (p < 0.05) (Fig. 3b, c). Four patients with WD and ALF
were identified (Fig. 3d; Table 3). All patients had a LOF
variant on one allelic chromosome and a missense variant on the other.

Discussion
Most children (50/65, 76.9%) in our study were asymptomatic with elevated serum transaminase levels
observed only on routine physical examination. The
mean age of onset in asymptomatic children was 5 (3–6)
years and it was therefore significantly younger thanthat of the other groups. Asymptomatic children only
manifested hepatomegaly and liver steatosis, which weredetected using liver ultrasonography. If left untreated,

asymptomatic patients with signs of organ damage typically develop symptomatic WD [22]. Therefore, the early
diagnosis and treatment of WD are crucial to achieve
better outcomes. However, the early diagnosis of asymptomatic children with WD is challenging. Therefore, clinical and laboratory characteristics, as well as support for
genetic testing, are essential.
ALF is the leading cause of death in children with
WD, with a mortality rate of approximately 95% if left
untreated [3]. In our study, four pediatric patients with
WD and ALF were identified, two of whom showed
improvement after appropriate treatment, one after livertransplantation, and one who died because she did notreceive a liver transplant in time. Prompt diagnosis is
crucial, as these patients urgently require liver transplantation to survive [4].

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Table 3 Clinical and variant data from four pediatric WD patients with ALF
Patient

ATP7B variants

1c.2333G > T
c.3524-3528delAAGGA*
c.2333G > T
c.3955 C > T*
c.1924G > T

Variantion
type
missense
frameshift
missense
nonsense
missense

c.3903 + 2T > C*
c.2128G > A
c.1488 C > T*

splice site
missense
Synonymous

2
3

4

Sex

KF ringM

Age at diagnosis (y)13Hemolytic
anemia-Outcome+

Neuropsychiatric deficit
-

F

11

+

-

-

Death

M

8

+

-

-

Liver
transplant

F

12

+

-

+

Survival

Survival*: LOF variants were defined as producing a nonfunctional protein product.

Six patients presented neurological symptoms. In addition to involuntary movements, tremors, and dysarthria
because of extrapyramidal involvement, they may be
associated with cognitive difficulties, depressive disorders, sleep disorders, and other psychiatric symptoms.
All patients had brain MRI abnormalities, including
abnormal signal intensities in the basal ganglia, thalamus,
brainstem, and corpus callosum. In addition, all patients
had abnormal liver ultrasound findings, and 50% of thepatients had liver cirrhosis on liver ultrasound. Therefore,
attention should be paid to the possibility of WD in children with neurological symptoms, particularly those withconcomitant ultrasound abnormalities of the liver.
Previous studies have suggested that KF rings are usually absent in children with WD and liver disease [23, 24].
In our study, they were absent in asymptomatic children
and were present in children with ALF and neurologicalsymptoms. This suggests that the positivity rate of the KF
ring is very low in the early stages, especially in asymptomatic children. The KF ring is often associated withdisease progression, particularly neurological and severe
hepatic manifestations, in WD. Therefore, the KF ringcannot be used as a basis for early diagnosis.
In our study, 92% of children had 24 h urinary Cu
excretion > 40 µg, and 64.4% of children had 24 h urinary
Cu excretion > 100 µg. Thus, these results suggest that> 40 µg appears to be more sensitive to the abnormalityof 24 h urinary Cu. This is consistent with previous studies [25]. A recent study showed that cholestasis prevents
biliary copper excretion and may lead to systemic copper
overload with markedly increased urinary copper excretion, particularly in children [26]. In our study, children
in Group 3 with ALF also had significantly increased 24 h
urinary Cu excretion compared to the other three groups(p < 0.05). We suspected that cholestasis occurred during
ALF, resulting in abnormally high 24 h urine levels.
Previous studies have mainly focused on southern
China, and there are few reports on northern China. In
our study, the high frequencies were p.R778L, p.P992L,
p.R919G, and p.A874V, which accounted for 54.8% of all
variants, which differs from previous studies in southern

China, where the three most common variants were
p.R778L, p.P992L, and p.T935M, accounting for 50–60%
of all variants [24, 27]. Furthermore, in southern China,
the major ATP7B variants are concentrated in exons 8,
12, 13, and 16 [28, 29], whereas the variants detected inour study were mainly present in exons 8, 11, and 13. The
results regarding the exons are the same in Qingdao andare also prevalent in northern China [30], suggesting thatthese regions may be hotspots for variants in northern
China.
Seven novel variants have not been previously reported,
including c.2572 A > C, c.-362G > A, c.51 + 4 A > C,
c.1543 + 40G > A, c.3903 + 2T > C, c.2664-2665delCC, andc.3524-3528delAAGGA. These had not yet been includedin the Exome Aggregation Consortium or 1000 Genomes
Project databases. Bioinformatics software analysis
assumes that they are pathogenic or probably pathogenic.
The relationship between the clinical phenotypes andgenotypes is complex. Most studies suggest that thephenotypic variability in WD may be caused by additional genetic, epigenetic, and even environmental factors that play a role in the timing of disease onset andclinical phenotype [31–33]. LOF variants result in severe
impairment of ATP7B gene function owing to the lack ofa full-length gene product. European studies have suggested that patients with LOF variants have lower serum
ceruloplasmin oxidase activity and earlier onset of WD
than those with missense variants [14, 15, 34, 35]. The
current research suggests that the presence of at least
one LOF variant in patients receiving chelation therapy is
associated with poor transplant-free survival over a long
follow-up [20]. In our study, the LOF variant was significantly associated with lower albumin levels, which may
be associated with ALF, similar to previous studies [14,15]. Furthermore, patients carrying the p.R778L variant
had lower serum ceruloplasmin levels and higher zinclevels for both alleles than patients with other variants.
Recent studies showed that p.R778L was also associatedwith lower ceruloplasmin levels [27, 29]. Some observations suggest that serum Zn levels could be related to theclinical phenotype of patients with WD and its severity;Zhang et al. BMC Pediatrics(2024) 24:576

patients with excessive Cu overload have lower serum Znlevels [36]. The toxicity of Cu observed in WD might becaused by the disruption of numerous Zn-dependent and
Zn-responsive proteins, including transcription factors[37]. However, the role of Zn in WD remains unclear.
Our study had several limitations. The sample size ofour study was small. Therefore, errors and outliers may
have a greater impact on the statistical results. Therefore, multicenter and large-scale studies are needed totest these conclusions and investigate the relationshipbetween different genotypes and phenotypes of WD.

Conclusion
In conclusion, the diagnosis of WD in children can bechallenging, particularly during the early stages of liverdisease and neurological symptoms. Therefore, supportive clinical and laboratory characteristics and genetic
testing are essential.
Abbreviations
WD	Wilson Disease
KF ring	Kayser–Fleischer ring
ALF	acute liver failure
LOF variant	Loss of function variant
MRI	magnetic resonance imaging
USG	ultrasonogram
ALT	alanine aminotransferase
AST	aspartate aminotransferase
TB	total bilirubinDB	directly bilirubin
ALB	Albumin proteinWBC	white blood cellHb	hemoglobin
PLT	platelets
Acknowledgements
The author thank all the team members for corporation.
Author contributions
Tianhe Zhang (zth)contributed to conceptualization, methodology andoriginal draft preparation. Wenliang Song contributed to visualization,
software, data curation and editing. Zhiqin Mao contributed toconceptualization, validation, review and supervision, project administration,and funding acquisition. All authors have read and agreed to the publishedversion of the manuscript(All authors read and approved the final Manuscript).
Funding
This work has no funding.
Data availability
No datasets were generated or analysed during the current study.

Declarations
Ethics approval and consent to participate
This work was in accordance with the protocols of the Ethics Committeesof Shengjing Hospital of China Medical University for clinical retrospective
study (No. 2022PS107K). Verbal consent was obtained from legal guardians inthis study. This study was conducted in accordance with the Declaration of
Helsinki.
Consent for publication
Not applicable.

Page 8 of 9Competing interests
The authors declare no competing interests.
Received: 22 December 2023 / Accepted: 30 August 2024

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