Study Of An X-linked Recessive Hereditary Menkes Disease In A Chinese Family

BackgroundMenkes disease (Menkes disease, MD)also known as curly hair syndrome is a rare multisystemic disorder of copper metabolism.It is first described by Menkes et al in1962. It is caused by the defect in ATP7A, a copper-transporting ATPase, localized in the trans-Golgi membrane of cells. MD is a progressive and systematic disease, the phenotypic features of are dominated by severe neurological degeneration and connective-tissue abnormalities,and most MD patients die usually before the third year of life. The incidence of Menkes disease is calculated as lin50,000-250,000in abroad.,and a prevalence of lin2,800,000(4.9in1,000,000male baby’s)in Japan from1993to2003reported by Japanese scholars. Now there is no incidence calculated in domestic,and only a very few cases are reported. The typical clinical features:psychomotor development backwards, failure to thrive, growth retardation and seizures. Hair resembles and feels like steel wool; it is lusterless and friable, especially in the areas of the scalp subjected to friction, skeletal changes, including pectus excavatum or pectus carrinatum, and spontaneous fractures, and loose and dry skin may be observed very early. Routine ophthalmoscopy is usually normal, but in later stages patients frequently fail to follow visual stimulus. Late manifestations of the disease are blindness, subdural hematoma,and respiratory failure. Most patients die within the third year of life due to infection, vascular complications (such as sudden and massive cerebral hemorrhage due to vascular rupture), or from the neurological degeneration itself. Neurological examination shows severe lower body muscle tone, and poor head control. Electroencephalogram (EEG) is often moderate to severe abnormal with multifocal spines slow wave, sometimes for peak performance lost law. Magnetic resonance angiography (MRA) shows vascular thickening, contorted arteries,. X ray shows bone metaphysis with bone spurs, bone dysplasia, congenital skull fractures and so on. Corneal dysplasia, optic disc pale, retinal pigment reduction, no KF ring. Laboratory tests:low levels of serum copper and ceruloplasmin. The present study showed that MD occurs due to mutations in the ATP7A gene. ATP7A expresses widely in brain, kidney, lung and muscle but not liver tissues. ATP7A, is mapped to Xq13.3. It consists of23exons, spanning a genomic region of about141kb. The normal gene product consists of1500amino acids (P-type copper transporter ATP enzyme) called for the MNK protein. At the N-terminus ATP7A has six metal-binding domains, the C-terminus ATP7A has ATP binding domain, phosphorylation domain (P-domain), and activation domain (A-domain). MNK protein is a Cu2+transmembrane pump which is formed by eight transmembrane pore region and export surplus copper from cells. Cu2+is anessential microelement for body metabolism, serving as a cofactor to many enzymes and participating in numerous biochemical reactions. In Menkes disease, malfunction and abnormal structure of MNK protein caused by mutations in the ATP7A gene results in insufficient absorption of Cu2+from intestine, and copper can not be transported to the cells in the fluid or cells into the blood.As a result, the biochemical changes in this disease is decreased Cu2+concentration in plasma and brain,and is secondary to a variety of copper dependent enzymes dysfunction and clinical symptoms.Menkes disease is a serious genetic disease and the prognosis is extremely bad.Now the treatment is still at an experimental stage and only is effective for the mild Patients. Therefore, earlier diagnosis is particularly important.Now initial diagnosis of MD is supported by demonstration of reduced levels of serum copper and ceruloplasmin(serum copper<11μmol/L, ceruloplasmin <200mg/L). However, in the neonatal period these markers should be interpreted with caution, as their levels are low also in healthy newborns.Abnormalities of placental copper concentration and the plasma catecholamine concentration (increase in the MD) in Newborn and fetal may be the choice as a rapid diagnostic test. Copper exportation of cultured fibroblasts discharge is the gold standard in the diagnosis,but the skin biopsy tissue cells need to be reproduced at least12weeks before the laboratory. As the mutations of ATP7A gene has being amplified by PCR, the gene sequencing offers a rapid diagnosis method for the MD and opened the way for early diagnosis and genetic counseling. Genetic testing of MD has been carried out,two novel mutations of ATP7A are reported:discontinuous missing bases in exonl2and missing bases in exon15.It laid the foundation for further development of prenatal diagnosis and genetic consultation. Now the numbers of accumulated cases is little, and can not provide for the gene mutation hot spots of ATP7A in China.The cases of MD in China should be accumulated and ATP7A gene analysis should be carried out. Clear and definite the gene mutation spectrum of ATP7A,and summed up the gene mutations hots of ATP7A in China.It will help us carry out rapid gene mutation detection of ATP7A in China in the future,and it will laid the foundation for the study of the functional characteristics,pathogenesis mechanism of ATP7A gene,and exploration to find new treatment program.Real-time fluorescence quantitative PCR is a method that refered to add the fluorophores in the PCR reaction system, then use the accumulated fluorescent signal to monitor the entire PCR process in real time, and finally analysis the unknown template through a standard curve. The copy number of DNA in the PCR reaction increased exponentially.And following the increase of reaction cycle number,the final PCR reaction is no longer exponentially generated template and enter to the platform stage. In conventional PCR, it commonly use gel electrophoresis and fluorescent dyes to detect the final amplification products of PCR, so there is unreliable of quantitativing PCR products at this end point method.In real-time Q-PCR throughout the PCR amplification process, the amplified fluorescence signal is monitored in real-time and continuous analysised. With the reaction time, the fluorescence signal changes can be plotted as a curve. Early in the PCR reaction, the level of fluorescence can not be clearly distinguished from background, and then the generation of fluorescence enter to the exponential phase, linear phase and the final plateau phase, and therefore the PCR products at a point of the index in the PCR reaction can be detected and infered to the initial content of the template.Based on above theory, from2010July to2011March,3pediatric patients with Menkes disease were diagnosised in Zhujiang Hospital of Southern Medical University, the experiment aimed at investigate and clinical follow-up to these3patients and their family members,and made the diagnosis by the use of genetic methods.This study was designed by PCR amplification of the ATP7A gene exon23and intron connection region, then purified and sequenced,and detected the gene mutations. The abnormalities were determined copy number of template through real-time quantitative PCR, to verify the existence of exon deletion. This ATP7A gene mutation and genetic features of the family menmeber can further enrich the gene mutation spectrum of Menkes disease.It will contribute to the fast detection of ATP7A gene mutation in China, and lay the foundation for the study of the functional characteristics,pathogenesis mechanism of ATP7A gene,and exploration to find a new treatment program.Specific contents includes two parts as following:Part1:a clinical diagnosis analysis of an X-chromosome recessive inherited pedigree of Menkes diseaseObjective:In2010July,a children with recurrent lung infection went to pediatric department of Zhujiang Hospital, Southern Medical University,and was diagnosised of Menkes disease.By asking about the family history revealed that the children in the family with similar clinical presentation. To make diagnosis of the children with the Menkes disease,and make clear of the familial incidence, we plan to discuss the clinical features of Menkes disease with these family as an example.Methods:Survey was carried out into family members of the proband, including clinical history inquisition, physical examination, blood tests and ceruloplasmin, MR examination of brain, and the pedigree chart.Results:A total of10members in the family were divided into A, B, C three groups. Each subset of pedigrees had one patient with Menkes disease diagnosed.Their features are handed down from age to age with gender differences and consistent with the X-chromosome-1inked recessive.Conclusion:A pedigrees of Menkes disease was found and diagnosis. The inheritance type of this family is an X-chromosome-linked recessive. The features of this pedigree were consistent with the presently features of Menkes disease in world.Part2:Gene sequencing of the ATP7A gene in a X chromosome-linked recessive pedigreeObjective:On the basis of anterior work and combined with ATP7A which currently is accepted as the pathogenic gene for Menkes that is an X-autosomal recessive hereditary disease,we sequence the ATP7A genes to discover the mutation characteristics of ATP7A gene in the pedigrees with Menkes disease.Methods:Genomic DNA isolated from peripheral blood of the lOfamily members and1normal control child. According to the human genome database (UCSC) obtained in the ATP7A gene sequence, a total of23pairs of primers were designed to amplify all the encoding exons and their flanking intron sequences by the primer design software Primer3. According to the result of gene sequencing, the abnormal will be further verified by real-time fluorescence quantitative PCR to determine the copy number and the existence of exon deletion.Results1.The normal children and family members (Ⅰ generation:grandmother; Ⅱ generation:children’s mothers and aunt;Ⅲ generation:three cases of male children and their sister) are sequenced, and the ATP7A gene of three male children fails to get PCR product bands of exon2-12, while the remaining fragments get PCR products.ATP7A genes of other members of families and normal male child obtain PCR products. Again,the ATP7A gene of normal child get a clear PCR product bands, while the frist child is still unable to get PCR product. So we consider that the ATP7A gene of three cases children and family members exist exon2-12missing and two single nucleotide polymorphisms c.4048G> A and c.2299G> C that have been reported.2. The copy numbers of exon2-12of ATP7A gene of normal children and family members are verified by real-time fluorescence quantitative PCR. The results show the exon2-12missing of ATP7A gene of three cases children, their grandmother, mother and their aunt.Conclusion:1.2-12exon of ATP7A gene are minssing in the3male children,so they can be clearly diagnosed as Menkes disease,and other5family members are carriers.The exon2-12missing of ATP7A is the cause of the Menkes disease in the family,and genetic manner of the pedigree conform the X-linked recessive genetic characteristics.2.The exon2-12deletions of ATP7A gene has not been reported in Menkes disease. And it enrich gene mutation spectrum of Menkes disease in China.3. Real-time quantitative PCR is frist applied to detect the missing of the ATP7A gene fragment, it is a new method for comprehensive genetic diagnosis of Menkes disease.The main innovations1.The exon2-12deletions of ATP7A gene has not been reported in Menkes disease. And it enrich gene mutation spectrum of Menkes disease in China.2. Real-time quantitative PCR is frist applied to detect the missing of the ATP7A gene fragment, it is a new method for comprehensive genetic diagnosis of Menkcs disease.