Apparent homozygosity due to compound heterozygosity of one point mutation and an overlapping exon deletion mutation in ABCA12: A genetic diagnostic pitfall.

BACKGROUND: Harlequin ichthyosis (HI), one of the most severe genetic skin disorders, is autosomal recessively inherited. Mutations in ABCA12, which encodes ATP-binding cassette transporter A12 (ABCA12), are known to be the cause of HI. It is very difficult to make precise genetic diagnosis when an exon deletion mutation overlaps the site of another causative point mutation. This combination of mutations may lead us to conclude incorrectly that the patient has the point mutation homozygously, a phenomenon called "apparent homozygosity". OBJECTIVE: To demonstrate that the present HI patient has apparent homozygosity in ABCA12 mutations. METHODS: We performed direct sequencing of gDNA in the entire coding region, including exon-intron boundaries, of ABCA12 in the HI patient and her parents. To further elucidate the mutations in the patient, parental mutation segregation study was done and SNP analysis was conducted on the region flanking ABCA12 in the patients and her parents. Quantitative PCR of gDNA in exon 11 of ABCA12 was also performed. Direct sequencing of cDNA from exon 9 to exon 13 and of gDNA between intron 9 and intron 11 of ABCA12 was done in the HI patient and her parents. RESULTS: Direct sequencing of gDNA in the entire coding region, including exon-intron boundaries, of ABCA12 seemed to indicate that the patient had the novel homozygous nonsense mutation c.1216A>T (p.Lys406X) in exon 11. However, mutation segregation analysis, SNP analysis, qRTPCR of gDNA in exon 11 of ABCA12 and direct sequencing of cDNA from exon 9 to exon 12 of ABCA12 and of gDNA between intron 9 and intron 11 of ABCA12 in the HI patient and her parents demonstrated that the present patient was compound heterozygous for two ABCA12 mutations: c.1216A>T (p.Lys406X) in exon 11 and g.111346_113217del1872 (p.Leu355_Lys428del, Gln354fs7*) which was overlapping exon deletion mutations involving exons 10 and 11. CONCLUSION: When direct sequencing indicates that a patient from a non-consanguineous family has an apparently homozygous non-founder point mutation, the homozygosity may be "apparent homozygosity", and we should keep in mind the possibility of overlapping exon deletion mutation.

Liver structures of a patient with idiopathic copper toxicosis.

This is the first report describing the liver structures of a Japanese patient with idiopathic copper toxicosis, which should be differentiated from hepatolenticular degeneration of Wilson disease. An 11-year-old Japanese boy presented with ascites associated with biochemical liver damage. Involvement of hepatitis virus was ruled out by laboratory tests. Because urinary copper excretion was increased, Wilson disease was highly suspected, but the serum level of ceruloplasmin was normal, and Kayser-Fleischer rings were not detected by slit lamp examination. Brain images were within normal limits. ATP7B analysis was negative for mutations. Liver specimen showed cirrhosis associated with chronic active hepatitis. Almost all hepatocytes were positive for orcein-stained granules. Mallory bodies were found in some hepatocytes. Fatty change was minimal, and there were no glycogenated nuclei in the parenchyma. Combined regimens of trientine and zinc for 6 months improved the decompensated state of liver function. After 2.5 years of treatment, a second liver biopsy was performed. The post-treatment liver showed complete disappearance of portal inflammation and remarkable decrease in cuprothionein granules. Mallory bodies disappeared from the parenchyma. An abundance of hepatocellular Mallory bodies and heavy copper loading limited to the liver may be specific to idiopathic copper toxicosis.

Potential of the international scoring system for the diagnosis of Wilson disease to differentiate Japanese patients who need anti-copper treatment.

AIM: Patients with Wilson disease show complex clinical features. Accurate diagnosis at the initial clinical manifestation is important for patients to receive effective treatment with anti-copper agents. In this study, we assessed whether the international scoring system for the diagnosis of Wilson disease is a reliable tool for screening Japanese patients with primary copper toxicosis requiring anti-copper treatment. METHODS: Twenty-three Japanese patients suspected of Wilson disease were enrolled in this study. We performed long-range polymerase chain reaction to detect ATP7B mutations in this series. Finally, we retrospectively assessed the reliability of using a diagnostic score of 4 or more points as the cut-off for this scoring system. RESULTS: Ten patients were homozygous or compound heterozygous for ATP7B mutations including a novel mutation of 3837 bp deletion including 3 exons. The mutation would have been missed by the traditional analysis. Six patients were heterozygous for ATP7B mutations. Three of these six patients had additional diagnostic points. The other three patients were diagnosed as carriers of a mutant gene based on their low scores. One of the seven patients free from ATP7B mutation was affected by copper toxicosis. Though the score was 3 points based on increased urinary copper and copper-positive cirrhosis, anti-copper treatment promptly improved liver failure, which was likely due to idiopathic copper toxicosis. CONCLUSION: The international scoring system for diagnosis of Wilson disease is a fairly reliable tool for screening Japanese patients who need anti-copper treatment. Caution is needed for patients with possible idiopathic copper toxicosis because the maximal score is 4 points.