Molecular analysis and diagnosis in Japanese patients with Wilson's disease

BACKGROUND: Wilson's disease is characterized by the toxic accumulation of copper in the liver, brain, cornea and other organs. It is caused by both impaired excretion via the bile and impaired incorporation of copper into ceruloplasmin in the liver. The Wilson's disease gene (ATP7B) has been cloned as a putative copper-transporting P-type ATPase gene. We therefore analysed mutations of ATP7B in Japanese patients with Wilson's disease. METHODS: Twenty-three Japanese patients with Wilson's disease were investigated. In all patients, the ATP7B coding sequence, including exon-intron junctions, was analysed by restriction endonuclease digestion, mutation detected enhancement gel electrophoresis and/or direct sequencing analysis of amplified fragments. RESULTS: Thirteen mutations were identified, including seven missense mutations, four detections, one insertion and one exon skipping in the coding region. The most common mutations were 2874deletion(del)C in exon 13 and arginine (Arg)778 leucine (Leu) in exon 8. DISCUSSION: None of the observed mutations, except for 2302insertion(ins)C, have been previously detected in either European or North American patients. We conclude that the mutation spectrum of Wilson's disease may thus indicate a population-dependent pattern. Based on the population-dependent manner of the occurrence of ATP7B gene mutations, it may be possible to establish a molecular diagnosis system. A molecular diagnosis system is considered to be very effective for making a definitive diagnosis in very young patients and for also detecting carriers.     

儿童肝豆状核变性临床表型及ATP7B基因突变关联性分析

目的 了解汉族儿童肝豆状核变性(WD) 患儿的临床表型与ATP7B基因突变的相关性。方法 以2005年7月至2012年12月就诊于复旦大学附属儿科医院确诊WD患儿为研究对象,按起病部位分为肝病型和神经型,以临床表现分为临床型和亚临床型。采用PCR技术扩增ATP7B基因全部外显子并直接测序。提取临床表型和基因型的信息;分析两者之间的相关性。结果 52个无亲缘关系家庭的53例WD患儿进行分析。肝脏损害52例（98.5%）。肝病型41例,神经型12例;临床型19例,亚临床型34例。①肝病型ALT升高明显;神经型24 h尿铜水平、胆汁酸水平和K-F环阳性率均显著高于肝病型;亚临床型起病年龄、ALT或AST异常率显著高于临床型;24 h尿铜水平、胆汁酸升高比例和K-F环阳性率显著低于临床型。②53例WD患儿ATP7B基因外显子序列分析发现致病性突变等位基因97个,突变频率为91.5%。纯合突变8例,复合杂合36例,杂合突变9例。错义突变23种,插入/缺失突变8种,无义突变2种,剪接突变3种。③错义突变与非错义突变,纯合突变与杂合突变患儿在起病年龄、K-F环阳性率、铜蓝蛋白水平、24 h 尿铜水平和ALT、AST异常率等方面差异无统计学意义。④3种高发突变p.Arg778Leu（35.0%,34/97）、p.Pro992Leu (15/97,15.5%)和p.Ala874Val/Pro(5/97,5.2%)在临床型和亚临床型、肝病型和神经型间的分布差异无统计学意义。⑤纯合、错义和错义+剪接突变在临床型和亚临床型、肝病型和神经型间的分布差异无统计学意义。结论 WD患儿几乎均有肝脏受累,多以肝病表现起病。ATP7B常见突变为p.Arg778Leu、p.Pro992Leu和p.Ala874Val,常见基因型和临床表型间未发现显著相关性。p.Ala874Val/Pro突变患儿起病年龄较低,剪接突变对血清铜蓝蛋白影响较小。     

Acute lymphoblastic leukemia in a child with Wilson disease

Wilson disease is an autosomal recessively inherited disease of copper metabolism and is characterized by liver and central nervous system dysfunction. The heterozygote carrier state rate is about one in 90 persons and the incidence of the disease is about 30 in 1,000,000. Although leukemia is the most common form of childhood malignancies, the probability of the presence of Wilson disease and acute lymphoblastic leukemia in the same patient is very low. We report an unusual case of a child with Wilson disease who developed acute lymphoblastic leukemia in three months.     

Intracellular localization of the Menkes and Wilson's disease proteins and their role in intracellular copper transport

Copper is a heavy metal ion essential for the activity of a variety of enzymes in the body. In excess, copper is a very toxic ion and therefore efficient regulation of its metabolism is required. This is dramatically illustrated by the genetic disorders X-linked Menkes disease and autosomal recessive Wilson's disease. In 1993, both the Menkes and Wilson's genes were isolated and it was found that these genes encode homologous cation copper transporting P-type ATPase proteins. The Menkes protein (ATP7A) is expressed in most tissues, except liver. In contrast, the Wilson's protein (ATP7B) is abundantly expressed in liver. Intracellular localization of those proteins was investigated. Both ATP7A and ATP7B are localized in the trans-Golgi network and post-Golgi vesicular compartment (PGVC) in the cell. This intracellular localization was altered by the copper content present in the cell. This result may support the hypothesis that ATP7A and ATP7B are involved in cellular copper transport and those proteins could be suitable models for elucidating intracellular copper metabolism.     

The Long–Evans Cinnamon rat: An animal model for Wilson's disease

The Long-Evans Cinnamon (LEC) rat is known to develop hepatitis and liver cancer spontaneously, phenomena attributed to abnormal copper metabolism. This mutant strain of rat shows some clinical features that are similar to those of Wilson's disease, including excessive copper in the liver, reduced excretion of copper into bile, a reduced level of serum copper and a remarkable decrease in serum ceruloplasmin activity. Molecular studies have revealed that the copper transporting P-type ATPase, atp7b, which is the rat gene homologous to human ATP7B, was found to be defective in the LEC rat. These observations have confirmed that the LEC rat is a rodent model for Wilson's disease. In addition, recent studies have suggested that the ATP7B protein is involved in the intracellular transport of hepatic copper. The absence or diminution of ATP7B function results in abnormal copper metabolism in the LEC rat and in patients with Wilson's disease.     

Acute lymphoblastic leukemia presenting in fulminant hepatic failure

A previously healthy 4-year-old boy was admitted because of acute liver failure. He was icteric, lethargic, had elevated ammonia and abnormal liver function tests. Serology was negative for viral hepatitis. There was no history of hepatotoxic drugs. Family history was unremarkable. The child was taken to the operating room for a living-related hepatic transplant. Frozen section showed massive hepatic leukemic infiltration and hepatocellular necrosis. Bone marrow aspiration confirmed the diagnosis of acute lymphoblastic leukemia (ALL). Transplant was withheld and chemotherapy was attempted. He died the following day due to systemic leukemic infiltration, cerebral edema, and severe anoxic ischemic encephalopathy.     

Numb chin syndrome as initial manifestation in a child with acute lymphoblastic leukemia

Numb chin syndrome (NCS) describes the affection of the inferior alveolar nerve and is a purely sensory neuropathy. Its symptoms include numbness of the skin of the chin, the lip and the gingival mucosa. Mostly seen in adults, it has rarely been described in children. We report on an 11-year-old male who presented with NCS as initial manifestation of acute lymphoblastic leukemia of B-cell type.     

Common acute lymphoblastic leukemia in a girl with genetically confirmed LEOPARD syndrome

Germline mutations in PTPN11 gene cause Noonan syndrome and the clinically similar LEOPARD syndrome (LS). LS is a rare congenital developmental disorder characterized by multiple lentigines, cardiac abnormalities, facial dysmorphism, retardation of growth, and deafness. Mutations in exons 7 and 12 of the PTPN11 gene can be identified in nearly 90% of patients with LS. PTPN11 gene encodes for an ubiquitously expressed protein tyrosine phosphatase SHP-2 involved in a variety of intracellular signaling processes in development and hematopoiesis. Somatic PTPN11 mutations contribute to leukemogenesis in children with hematologic malignancies including juvenile myelomonocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, and myelodysplasia. Two cases of leukemia (acute myeloid leukemia) have been reported in children with LS. The authors describe for the first time a girl with genetically confirmed LEOPARD syndrome presenting with common acute lymphoblastic leukemia.     

Acute lymphoblastic leukemia in a patient with Miller-Dieker syndrome

A 15-month-old girl with Miller-Dieker syndrome, a contiguous gene deletion syndrome involving chromosome 17p13.3 and resulting in lissencephaly, was diagnosed with precursor B-cell acute lymphoblastic leukemia. Cytogenetic analysis identified both the previously detected 17p13.3 deletion and additional complex numerical and structural abnormalities, including loss of chromosome 9, isochromosome 9q and interstitial deletion of 20q. This is, to our knowledge, the first report of acute leukemia in the setting of Miller-Dieker syndrome. Herein we review the literature regarding Miller-Dieker syndrome, with particular attention to the presence of several candidate tumor suppressor genes within the deleted material.     

Acute lymphoblastic leukemia at relapse in a child with acute myeloblastic leukemia

We describe a child diagnosed as having acute myelogenous leukemia (AML) at 25 months of age who relapsed with acute lymphoblastic leukemia (ALL) 1 year later. The AML was morphologically M2 by the French-American-British classification, periodic acid Schiff (PAS) stain negative and peroxidase positive. The ALL was L1 by this classification, PAS positive, and peroxidase negative. The initial AML was associated with a small segment deletion of the long arm of chromosome 11 [46, XY, de (11) (q23)] not seen in the relapse ALL, which had a normal karyotype. The child was rapidly reinduced with vincristine and prednisone, and remains in remission on maintenance lymphoma type (LSA2-L2) therapy more than 2 years later. These findings suggest the development of a new leukemic clone, rather than a phenotypic modulation of the initial leukemia.     

Acute lymphoblastic leukemia and hepatoblastoma in a family

Acute lymphoblastic leukemia is the most common and hepatoblastoma is a rare malignancy diagnosed in children. Their report on an Iranian boy with acute lymphoblastic leukemia diagnosed at the age of 2 years; 20 months later his 10-month-old sister was referred to their hospital with hepatoblastoma. The occurrence of such two types of cancer is rare in a family.     

Laparoscopic adnexectomy of a persistent ovarian tumor in a girl with acute lymphoblastic leukemia relapse

The management of children with acute lymphoblastic leukemia (ALL) relapse and an ovarian tumor remains controversial. The authors report about a 4-year-old girl who developed a late bone marrow and cutaneous relapse of her pre-B-cell ALL and revealed an enlargement of her left ovary (4 ×3 ×2 cm). Chemotherapy (ALL-REZ-BFM pilot-protocol 2002) achieved effective remission, but the ovarian mass depicted no regression. Laparoscopic adnexectomy was performed and the tumor could be extracted in a specimen-bag through a 12-mm umbilical incision. Histology detected no viable lymphoblasts, but a fibrotic enlargement due to previous cellular infiltration. The authors conclude that in children with ALL relapse and an ovarian tumor, malignant infiltration as well as local response to chemotherapy can be judged only by surgical excision and histopathologic examination. Laparoscopic oophoradnexectomy is a valuable management option in these children.