3 例Menkes病患儿的临床与ATP7A基因分析及1例产前诊断研究

Menkes 病是一种罕见的X 连锁隐性遗传病,由于ATP7A 基因突变导致铜吸收障碍,铜相关酶功能缺陷,引起多系统功能障碍。该文拟通过对3 例Menkes 病患儿的临床经过和ATP7A 基因突变分析对该症进行研究,并对1 例再孕母亲进行产前诊断研究。3 例男婴于8~9 个月时来院就诊,均为婴儿期起病,主要表现为抽搐和智力运动落后,抗癫癎治疗无效,面色苍白,毛发稀疏、卷曲,小头,MRI 扫描显示脑萎缩、白质异常、基底节损害和脑血管形态改变,血浆铜蓝蛋白均显著降低,分别为70.2、73.5、81.0 mg/L（参考值210~530 mg/L）,符合经典型Menkes 病临床表型。例1 和2 的ATP7A 基因存在c.3914A>G（p. D1305G）突变,例3 为c.3265G>T（p.G1089X）突变,均为新生突变。c.3914A>G（p. D1305G）为已知突变,c.3265G>T（p.G1089X）为新突变,均为我国首次报道。例1 患儿的母亲再孕,于妊娠20 周时抽取羊水细胞,通过胎儿ATP7A 基因突变分析,进行产前诊断。羊水细胞ATP7A 基因未见c.3914A>G,提示胎儿未患与先证者相同的疾病。胎儿出生后发育正常。     

ADad 6: The Predictive Factors for Anxiety Disorders Among Adolescents in a Rural Community Population in India

Menkes disease is a rare inherited disorder of copper metabolism caused by mutations in the ATP7A gene. Its clinical course is characterized by early neurological regression, seizures, hypotonia and kinky friable hair. Neuroimaging typically reveals severe brain atrophy with subdural fluid collections and excessive tortuosity of cerebral arteries. The authors describe a case of Menkes disease with unusual imaging findings. The patient had macrocephaly and symmetrical bilateral confluent white matter changes with temporal cystic areas, reminiscent of megalencephalic leukodystrophy.     

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.     

Identification of three novel mutations in Japanese patients with Menkes disease and mutation screening by denaturing high performance liquid chromatography

BACKGROUND: Menkes disease is an X-linked recessive disorder resulting in a connective-tissue disturbance and profound neurodegeneration in early childhood. The gene for Menkes disease has been isolated and predicted to code for copper transporting ATPase. In this study, a mutation analysis in Japanese patients with Menkes disease was performed, as was a mutation screening by denaturing high performance liquid chromatography (DHPLC). METHODS: A mutation analysis on five Japanese patients with Menkes disease was performed using a direct sequencing method and DHPLC. RESULTS: Two nonsense mutations, two missense mutations and one splice donor site mutation were found. The DHPLC analysis showed differences in the peaks between the DNA fragments of wild type and heteroduplex (wild type and mutant). CONCLUSIONS: Three novel mutations (Asp1044Gly, Pro1279Leu and IVS21+1 g to a) were detected. The Asp1044Gly mutation destroys the highly conserved phosphorylation domain in exon 16. The splice site abnormality leads to a skipping of exon 21 coding for part of the seventh transmembrane domain. These two mutations could cause a severe protein dysfunction. Another missense mutation, Pro1279Leu, in exon 20 was found in a patient with a mild type of Menkes disease. It is speculated that this mutation partially maintains the ATP7A function is. A DHPLC analysis could detect these mutations. It is concluded that the best way to make a molecular diagnosis for Menkes disease is to first screen DNA samples for all exons using DHPLC, and thereafter perform direct sequencing for exons which have an abnormal elution profile in order to rapidly detect such mutations.     

Molecular genetics and pathophysiology of Menkes disease

The molecular genetics and pathophysiology of Menkes disease and an animal model for this disease are reviewed. The Menkes gene, located on chromosome X13.3, encodes a copper-transporting ATPase, as shown by the sequencing of a cDNA of 4500 bp. Mutations in the Menkes gene in patients with Menkes disease show great variety, including missense, nonsense, deletion and insertion mutations. Mutations in the Menkes gene have also been identified in patients with mild Menkes disease or occipital horn syndrome, showing that these diseases are allelic variants of Menkes disease. Mutations in the mottled gene, the murine homolog of the Menkes gene, have been demonstrated in mottled mutant mice that display biochemical and phenotypic abnormalities similar to those observed in patients with Menkes disease. In affected cells, copper significantly accumulates as metallothionein-bound copper in the cytosol and copper transport to the organelles, as well as copper efflux, is disturbed. As a result, cuproenzymes cannot receive the copper necessary for their normal function. Thus, the objective in treatment of Menkes disease and occipital horn syndrome is to deliver copper to the intracellular compartments where cuproenzymes are synthesized.     

Cervical spine anomalies in Menkes disease: a radiologic finding potentially confused with child abuse

Background

Menkes disease is an X-linked recessive disorder of copper transport caused by mutations in ATP7A, a copper-transporting ATPase. Certain radiologic findings reported in this condition overlap with those caused by child abuse. However, cervical spine defects simulating cervical spine fracture, a known result of nonaccidental pediatric trauma, have not been reported previously in this illness.

Objective

To assess the frequency of cervical spine anomalies in Menkes disease after discovery of an apparent C2 posterior arch defect in a child participating in a clinical trial.

Materials and methods

We examined cervical spine radiographs obtained in 35 children with Menkes disease enrolled in a clinical trial at the National Institutes of Health Clinical Center.

Results

Four of the 35 children with Menkes disease had apparent C2 posterior arch defects consistent with spondylolysis or incomplete/delayed ossification.

Conclusion

Defects in C2 were found in 11% of infants and young children with Menkes disease. Discovery of cervical spine defects expands the spectrum of radiologic findings associated with this condition. As with other skeletal abnormalities, this feature simulates nonaccidental trauma. In the context of Menkes disease, suspicions of child abuse should be considered cautiously and tempered by these findings to avoid unwarranted accusations.     

Menkes病患儿ATP7A基因突变分析

目的分析并确定2例Menkes病（MD）患儿的ATP7A基因突变及遗传特征。方法收集并分析2例Menkes病患儿的临床资料，提取其外周静脉血DNA，采用PCR法扩增ATPTA基因的23个外显子及其与内含子的连接区，并行DNA直接测序，异常者用DNA限制性内切酶酶切PCR扩增片段方法进行验证。结果发现2个半合子突变，1例在第12外显子检测到不连续的多处碱基缺失c．2589delTGAAGGA＋c．2598delTT＋c．2601delT＋c．2603delT＋c．2605delGTAGATGA（P．E864fsX883），导致移码突变。另1例在第15外显子检测到单个碱基缺失c．3045delT（p．T1016fsX1018），并发现移码突变。2例患儿的母亲均为相应突变但表型正常的携带者。结论发现2个国际上尚未见报道的新ATPTA基因突变（p．E864fsX883与p．T1016fsX1018），首次在国内明确了2例Menkes病患儿的基因诊断。     

Clinical manifestations and treatment of Menkes disease and its variants

The clinical manifestations of classical Menkes disease, mild Menkes disease and occipital horn syndrome are reviewed. Menkes disease is a neurodegenerative disease with X-linked recessive inheritance. Orally administered copper accumulates in the intestine, resulting in the failure of copper absorption. The primary metabolic defect that causes copper accumulation in the intestine is present in almost all extrahepatic tissues. The blood, liver and brain are in a state of copper deficiency, which is due to defective copper absorption. The characteristic features, including neurological disturbances, arterial degeneration and hair abnormalities, can be explained by the decrease in cuproenzyme activities. DNA-based diagnosis is now possible. Mild Menkes disease and occipital horn syndrome, which show milder forms than Menkes disease, have been identified as genetic disorders resulting from mutations in the Menkes disease gene. Because the clinical spectrum of Menkes disease is wide, males with mental retardation and connective tissue abnormalities should be evaluated for biochemical evidence of defective copper transport. The treatment accepted currently is parenteral administration of copper. When treatment is started in patients with classical Menkes disease above the age of 2 months, it does not improve the neurological degeneration. When the treatment is initiated in newborn babies affected with this disease, the neurological degeneration can be prevented in some, but not all, cases. Moreover, early treatment cannot improve non-neurological problems, such as connective tissue laxity. Therefore, alternative therapies for Menkes disease and occipital horn syndrome should be studied.     

ATP7A基因内含子突变致Menkes病1家系2例报告

目的探讨Menkes病的临床和实验室特点。方法对1家系2例确诊为Menkes病患儿的临床、实验室检查、影像学资料及基因表现进行回顾性分析。结果2例患儿均为男童，先证者生后4个月起病，肤色白、脸颊饱满、皮肤松弛、毛发细、色淡卷曲，有漏斗胸，有癫、发育迟滞。先证者哥哥自幼发育落后，康复治疗无效，1年前死亡。2例患儿血浆铜蓝蛋白分别为 80 mg·L-1及 92.4 mg·L-1。患儿的头发在光镜下观察有扭曲、串珠样改变；先证者颅脑MRI髓鞘化延迟，双侧大脑、小脑萎缩样改变，脑表面血管迂曲增多，双侧基底节及大脑脚异常信号，颅脑MRA+MRV示：大动脉走行迂曲，动脉分支及表浅静脉扭曲成团。先证者基因学检查未发现ATP7A基因存在大片段变异，但ATP7A基因c.2172+5_2172TGAAT（编码区第2172号核苷酸后内含子中第5与第6位核苷酸间插入TGAAT）的微小剪切变异，先证者哥哥与其变异位点相同，母亲为表型正常的携带者。结论Menkes病为遗传性的铜代谢障碍性疾病，以进行性加重的神经损害为主要表现，有特殊面容和毛发改变，脑萎缩和脑血管的形态改变，结合实验室检查、颅脑影像及基因检测可确诊。     

Identification of two novel mutations in the <Emphasis Type="Italic">ATP7B</Emphasis> gene that cause Wilson’s disease

Background:Wilson's disease is an autosomal recessive disorder characterized by liver disease and/or neurologic deficits due to copper accumulation and is caused by pathogenic mutations in the ATP7B gene.Methods:Two unrelated Chinese patients born to nonconsanguineous parents who were diagnosed with earlyonset Wiison's disease.DNA sequencing and bioinformation analysis were conducted.Results:We have identified four mutations in two family trios,of which two were novel,namely,c.3028A＞G(p.K1010E) and c3992T＞G (p.Y1331X),in each patient.Conclusions:Gene testing is playing an important role in diagnosis of Wilson's disease.The early-onset of Wilson's disease is apparently not associated with P-ATPase domain in the ATP7B protein.Our findings further widen the spectrum of mutations involving the ATP7B gene.     

Menkes病临床及ATP7A基因突变和拷贝数改变分析

目的通过ATP7A基因突变及拷贝数改变（CNV）分析,了解13例临床诊断为经典型Menkes病患儿的临床表型以及ATP7A基因型特征,探讨其基因型、表型的相关性。方法收集并分析13例Menkes病患者的临床资料,采用DNA直接测序进行ATP7A基因突变检测,发现突变后用DNA限制性内切酶酶切进行验证;应用多重连接依赖的探针扩增技术（MLPA）对未发现突变的患儿行CNV分析,阳性患者用长片段PCR进行验证,并进行基因型、表型相关性分析。结果临床特点:（1）13例均为男性,有典型的毛发改变、癫发作、肌张力减低、智力运动发育迟缓和结缔组织异常,3例有阳性家族史;（2）起病年龄早,均在出生7个月内（3 d～7个月）,除1例患儿以智力运动发育落后起病外均以癫起病,伴严重发育停滞和倒退;（3）均呈进行性加重,随访5例患儿,均于14个月内死亡;（4）12/12例患儿（100%）铜蓝蛋白降低;6/8例患儿（75%）血清铜降低;4/4例患儿（100%）脑血管成像见血管走行紊乱,呈"螺丝锥样"改变,1例为47XXY/46XY嵌合体。基因突变确诊9例:（1）DNA直接测序检测到6种突变,包括2种缺失突变,2种错义突变及2种剪切位点突变,其中3种为国际上未报道过的新突变,均位于高度保守区,100例健康男性对照中相应位点均未发现上述突变;（2）MLPA检测发现1例患儿为第10外显子缺失突变,长片段PCR验证其断点为c.2173-346₂407-346del,共1 783 bp,导致p.F725_K802del,其母亲为表型正常的携带者。基因型-表型关系:同为c.2179G>A（p.G727R）突变患儿,47XXY/46XY嵌合体起病年龄早于核型正常者,且临床症状较重。结论 Menkes病ATP7A基因大片段缺失或重复突变检测中ML-PA方便、快捷,Menkes病的致病机制可能与ATP7A基因剂量关系密切。     

Neonatal onset of Menkes disease: diagnosis interest of cupremia and microscopic examination of the hairs]

BACKGROUND: Menkes disease is a rare X-linked disorder due to a defect in intracellular copper transport. Clinical symptoms appear during the first months of life, with a progressive developmental delay leading to death within a few years. Diagnosis is confirmed by the demonstration of copper retention in fibroblasts and/or DNA testing. However, these investigations are complexes and time consuming. CASE REPORT: We report 1 case of Menkes disease with neonatal onset, diagnosed on multiple organ failure, hypothermia, and major central nervous system damage, leading to death in a few weeks. The diagnosis, suggested by the clinical features, was rapidly supported by the microscopic examination of the hairs, showing pili torti, and the demonstration of severely decreased levels of plasma copper and ceruloplasmin. Diagnosis was further confirmed by the demonstration of an increased copper uptake and retention in fibroblasts. CONCLUSION: This report highlights the clinical variability of Menkes disease with the possibility of a neonatal onset. Microscopic examination of the hairs and the determination of copper and ceruloplasmin plasma levels are simple and inexpensive investigations, which can provide rapidly valuable information to support this diagnosis.     

Trace element studies in three patients and a fetus with Menkes' disease. Effect of copper therapy

This paper reports the results of a multielement analysis of postmortem samples of Menkes patients, of which one was untreated and two had been treated for various lengths of time with intramuscular injections of copper-EDTA. The findings have been compared with data from a Menkes fetus and from controls. The results confirm that copper accumulates in various tissues and demonstrate a further increase in copper levels as a result of the treatment with copper-EDTA. Although no clinical improvement was observed, the levels of some copper-containing enzymes normalized during the copper-therapy. Furthermore, in agreement with the identification of the copper-binding protein in the kidney as metallothionein, it was found that not only copper, but also zinc, cadmium, and mercury are trapped in this tissue. A low copper concentration in the brain was also found in a Menkes fetus, indicating that brain damage might already have occurred before birth. Speculation Until recently, Menkes' disease was considered to be due to copper deficiency. However, the symptoms are more typical of a storage disease in which copper is irreversibly trapped in some tissues, in particular in the kidneys, by metallothionein. This abnormal storage pattern gives rise to copper deficiency elsewhere in the organism, particularly in the brain where it may cause irreversible damage in the foetus. Parenteral administration of copper does not lead to clinical improvement. The only "therapy" that seems feasible at present is tracing the carriers of the disease and advising abortion when prenatal diagnosis indicates a male fetus carrying the disease.     

Menkes' disease: heterozygosity testing by quantitative real-time PCR and the dilemma of therapeutic support

Menkes' disease is a rare X-linked multisystemic lethal disorder of copper transport metabolism. Failure of synthesis of several copper enzymes explains most of the clinical features, which were characterised by neurodegenerative symptoms and connective tissue manifestations. Most cases are still prone to rapidly progressive cerebral degeneration and early death in the first few years. Since CNS-dysfunction usually preceeds development of the pathognomonic "steely" hair, delay of clinical diagnosis and onset of therapeutic intervention precludes longlasting neurological benefit. This is particularly true for patients with large deletions or severe truncations of the responsible ATP7A gene. We report on our own experience with a patient, who was diagnosed to be affected by Menkes' syndrome at the age of one year, due to the specific hair texture and biochemical abnormalities. Molecular investigation revealed a total deletion of exon 15 of the ATP7A gene. Heterozygosity was confirmed by means of real-time PCR in the child's mother, but could be excluded in the grandmother and other female relatives at risk. Therapeutic support with subcutaneous injection of copper-histidinate normalised diminished copper and coeruloplasmin serum levels, but was unable to influence the clinical course and to prevent the fatal outcome at the age of two years. This observation is in line with the experience of the literature claiming that currently available medication will hardly be able to normalise brain copper levels. However, observations of clinical variants of Menkes' disease with quite a different outcome and, more importantly, emerging of alternative copper transport pathways might still justify this time-limited therapeutic intervention.     

Effects of Copper and Hydrocortisone on Cell Cycle of Cultured Fibroblasts from the Macular Mouse as a Model of Menkes Disease: DNA Analysis by Flow Cytometry

Abstract Effects of copper and hydrocortisone on the proliferation of cultured fibroblasts from macular mouse, a model of Menkes kinky hair disease, were analyzed by flow cytometry. In fibroblasts from the macular mouse, copper induced cytotoxicity and S-phase accumulation of cell cycle were observed at lower concentrations of copper than in fibroblasts from the controls. However, the fibroblasts cultured with a combination of copper and hydrocortisone revealed recruitment of cell cycle. These results suggest that the proliferative activity of the cells from a macular mouse are more vulnerable to an excess amount of copper than normal, and that supplementation of hydrocortisone is effective for the Menkes kinky hair disease.     

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.     

Excision of massive bladder diverticula in Menkes disease

Menkes disease is a rare disorder of copper metabolism. Massive bladder diverticula are a characteristic urological abnormality associated with this condition. In this case report, we describe excision of such diverticula with good postoperative results in terms of bladder function. There are only three previous instances reported of excision of these diverticula, and the postoperative outcome has not been described previously.     

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.     

Menkes病三例临床及实验室特点

目的 探讨Menkes病的临床和实验窜特点.方法 对3例确诊为Menkes病患儿的临床、实验室检查及影像学资料进行回顾性分析.结果 3例患儿均婴儿早期生后3～6个月起病,有皮肤白、头发黄短、稀疏卷曲、眼距宽和眼裂小等特殊面容及毛发改变,均表现智能发育落后,3例患儿的头发在光镜下观察均有扭曲、串珠样改变;血浆铜蓝蛋白分别为32.3 mg/L、72.5 mg/L和60.7mg/L,均明显减低;头颅影像学示弥漫性脑萎缩,脑白质髓鞘化落后,脑血管走行紊乱扭曲;例1和例2基因学检查示X染色体上14号外显子分别缺失或无义突变.结论 Menkes病为遗传性的铜代谢障碍性疾病,以进行性加重的神经损害为主要表现,有特殊面容和毛发改变,以及脑萎缩和脑血管的形态改变.