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。

Mutation and Copy Number Variation Analysis of ATP7A Gene in Patients with Menkes Disease

Objective To analyze ATP7A gene mutations and copy number variation（CNV）in 13 Chinese patients with classical Menkes disease（MD） and discuss the relationship of genotype and phenotype. Methods Thirteen patients clinically diagnosed Menkes disease were included.All 23 exons and exon-intron boundaries of ATP7A gene were amplified by polymerase chain reaction（PCR） and directly sequenced for genomic DNA.The mutation had been proved by DNA restriction enzyme digestion of PCR-amplified fragments.Multiplex ligation-dependent probe amplification（MLPA） assays were performed to detect ATP7A copy number variation. Results All of 13 patients were boys.The symptoms onset occurred between 3 days to 7 months.The manifestations were typical hair changes,seizures,hypotonia,neurodevelo-pmental delays and inguinal hernias.Twelve cases（100%,n=12）patients had low serum ceruloplasmin concentration;6 cases（75%,n=8）patients had low serum copper concentration;4 cases（100%,n=4）patients had "corkscrew" appearance of cerebral vessels in MR angiography.Five patients had been found dead before 14 months old during follow-up.One patient was 47XXY/46XY chimerism;Six mutations were identified in 8 of 13 patients through DNA sequencing,including 2 missense mutations（c.3028A>C,c.2179G>A）;2 frame-shift mutations（one complicated deletion mutation:c.2589delTGAAGGA+c.2598delTT+ c.2601delT +c.2603delT+c.2605delGTAGATGA,one simple deletion:c.3045delT） and 2 splicing mutations（c.4005+1G>C,c.3658+1G>A）;three of these were novel.MLPA assays identified one case with deletion covered whole exon 10 which was proved by long-fragment PCR as c.2173-346₂407-346del,p.F725_K802 del.With the same mutations c.2179G>A（p.G727R）,P1 which was 47XXY/46XY,had earlier onset and heavier clinical symptoms than P2 which was the normal karyotype. Conclusions MLPA is a rapid and reliable technique to detect the big fragment or whole gene deletion of ATP7A gene.The phenotype of patient may be correlated with the dosage of ATP7A gene.More patients are needed to analyze the phenotype-genotype relation in future.