A novel gross deletion and breakpoint junction sequence analysis of ATP7B in a Chinese family with Wilson disease using next‑generation sequencing and Sanger sequencing

Abstract

Wilson disease (WD) is a rare autosomal recessive genetic disorder that causes abnormal copper metabolism, resulting in pathological accumulation of copper in the liver, brain and other organs. Mutations in the ATPase copper transporter 7B (ATP7B) gene, which encodes a membrane P‑type adenosine triphosphatase, have been identified as being responsible for WD. The present study analyzed clinical data and collected DNA samples from a pediatric patient with WD and her healthy parents. Mutation screening for ATP7B was performed using direct sequencing, multiplex ligation‑dependent probe amplification(MLPA), next‑generation sequencing (NGS) and Sanger sequencing of the breakpoint junction sequence. The patient (age, 2.7 years) presented with early‑onset hepatic disease. The present study identified compound heterozygous mutations of ATP7B, including a heterozygous mutation (p.Arg1,041Trp) and a novel heterozygous gross deletion of a 57,771 bp fragment (chr13: 52490972‑52548742) (GRCh37) from partial exon2‑ exon21 to external ATP7B sequence (15.833bp) in the patient. Analysis of the family members of the patient showed that the missense mutation and the gross deletion mutation were inherited from her mother and father, respectively. Microhomology and inverted repeat sequences, which may mediate the deletion mutation, were identified through sequence analysis on both sides of the breakpoints of this deletion. The present study provided additional information on the genotypic spectrum of the ATP7B gene, particularly with regard to early onset hepatic disease, as observed in the present patient with WD. The identification of the precise breakpoint junction sequence warrants further investigation of DNA break and recombination mechanisms. In detecting precise deletions, the NGS associated with Sanger sequencing of breakpoint junction sequence have been found to have more advantages than MLPA.

Keywords: Wilson disease; child; aTPase copper transporter B; next-generation sequencing; mutation.

Figure 1.

CT scans of the patient and a heterozygous missense mutation analysis in the family. (A) Coronal CT of the abdomen of the patient. (B) Axial CT of the abdomen of the patient. Abdominal CT of the patient with WD showed an increased volume and decreased density of the liver. (C) Direct sequencing showed a heterozygous mutation (c.3121C>T; p.Arg1,041Trp) in exon 14 of ATP7B gene. c.3121 is indicated by arrows. (D) c.3121C>T mutation was inherited from the mother of the patient. (E) The father of the patient did not have the heterozygous mutation.

Figure 2.

A heterozygous deletion analysis in the patient and her father using MLPA. (A) MLPA result of the patient with WD showing a heterozygous deletion of the exons 3–21 of the ATP7B gene, as assessed by RPA ratios of 0.3–0.75. (Normal RPA ratio, 0.85–1.25 according to the manufacturer's recommendations). (B) The heterozygous gross deletion was inherited from the father of the patient. RPA, relative peak area; •represents the probe detection value.

Figure 2.

A heterozygous deletion analysis in the patient and her father using MLPA. (A) MLPA result of the patient with WD showing a heterozygous deletion of the exons 3–21 of the ATP7B gene, as assessed by RPA ratios of 0.3–0.75. (Normal RPA ratio, 0.85–1.25 according to the manufacturer's recommendations). (B) The heterozygous gross deletion was inherited from the father of the patient. RPA, relative peak area; •represents the probe detection value.

Figure 3.

Putative breakpoint junction sequence was amplified by PCR. The 868 bp band corresponding to the breakpoint junction sequence in the patient is presented in the second lane.

Figure 4.

Analysis of the breakpoints. (A) Sequence characteristics around the breakpoints. Nucleotides underlined by a dotted line in exon 2 are targeted by the probe used for multiplex ligation-dependent probe amplification. Underlined nucleotides represent the Microhomology part. Bold nucleotides are inverted repeat sequences. Small case letters indicate the extremities of the deleted sequence. (B) Direct sequencing showed the breakpoint junction sequence in the patient. Breakpoint junction is indicated by an arrow. (C) The father presented the same breakpoint junction, indicated by an arrow. (D) DNA secondary structure in the vicinity of the proximal breakpoint was predicted using bioinformatic tools. The breakpoint was predicted to be in the proximity of a hairpin structure and is indicated by an arrow. (E) Magnification of the proximal breakpoint, which is indicated by an arrow.