【作者】 周明星；

【导师】 邬玲仟；

【作者基本信息】 中南大学 ， 遗传学， 2010， 硕士

【摘要】 背景：丙二酰辅酶A脱羧酶(Malonyl Coenzyme A decarboxylase, MCD)缺乏症(OMIM 248360)是极其罕见的常染色体隐性遗传代谢性疾病,亦称丙二酸尿症(malonic aciduria)。1984年Brown首报了该疾病,1999年K.A.Sacksteder, D.R.FitzPatrick, Jim Gao三人分别克隆了导致MCD缺乏症的MLYCD基因,迄今为止,全世界共报导32例病例包括23种不同的基因突变(见附表1),其临床表现多变：常见有中枢神经系统异常如儿童早期智障,发育迟缓,癫痫,张力减退,脑部器质性病变；腹泻,呕吐,肥厚型心肌病等,甚至导致新生儿死亡。其他少见症状有身材矮小,嗜睡,厌食,胃肠炎,听力障碍,面容畸形等。实验室检查常见尿有机酸代谢异常(如丙二酰尿,甲基丙二酰尿),低血糖,代谢性酸中毒,酮症酸中毒,乳酸血症等。国内暂无该疾病的研究报导。目的：运用细胞和多种分子遗传学方法对1例丙二酰辅酶A脱羧酶(MCD)缺乏症患者及其家系进行研究,为患者及其家庭提供准确的诊断和遗传咨询,为临床上MCD缺乏患者的基因诊断提供指导。方法：①对患者行尿有机酸代谢筛查；②对患者行常规细胞遗传学分析；③对患者及其父母外周血和患者皮肤gDNA的MLYCD基因的5个外显子及其侧翼序列行PCR扩增并测序；④对患者及其父母外周血和患者皮肤cDNA的MLYCD基因行PCR扩增并测序；⑤对患者外周血gDNA行全基因组拷贝数基因芯片检测；⑥对患者及其父母外周血gDNA的MLYCD基因行MLPA检测；⑦对50个无关个体的外周血gDNA针对发现的MLYCD基因的新错义突变位点行PCR扩增并测序；结果：1、患者尿有机酸代谢筛查结果：09年8月24日发现尿丙二酸增高；09年9月24日复查示尿丙二酸更增高,甲基丙二酸轻度增高；2、患者外周血淋巴细胞染色体G带、高分辨核型发现550-850条带阶段无异常。3、gDNA的MLYCD基因测序,患者存在c.920T>G(p. Leu307Arg)杂合错义突变,父亲存在杂合错义突变c.920T>G(p. Leu307Arg)和杂合同义SNP(c.732G>A;p. ser266ser),母亲存在杂合错义SNP(c.776G> C;p. Gly259A1a);4、cDNA的MLYCD基因测序,患者存在纯合父源错义突变c.920T>G(p. Leu307Arg),母亲存在纯合错义SNP(c.776G>C;p. Gly259Ala),父亲存在杂合错义突变c.920T>G(p. Leu307Arg)和杂合同义SNP(c.732G>A;p. ser266ser);5、基因芯片显示患者没有微缺失微重复异常；6、MLPA技术示患者及母亲gDNA的MLYCD基因皆存在Exonl杂合缺失,父亲正常。7、新错义突变在50个无关个体中未有发现。且通过与其他物种行序列同源比对(包括人,猩猩,牛,大鼠,小鼠),发现该错义突变位点皆为保守T。结论：1.患者父源MLYCD基因存在c.920T>G(p. Leu307Arg),为文献未报导的新突变,该新突变位点在不同物种中皆保守。2.患者母源MLYCD基因存在Exon 1缺失,且该链MLYCD基因不表达。3.确诊患者为MLYCD基因复合杂合突变,导致丙二酰辅酶A脱羧酶缺乏症。其父母确诊为MCD杂合子,再生育时必须行产前诊断。更多还原

【Abstract】 Background:Malonyl Coenzyme A decarboxylase(MCD) deficiency (OMIM 248360) is an extremely rare metabolic disorder decided by autosomal recessive inheritance, which is also named malonic aciduria.The first report of the disease was by Brown in 1984. K.A.Sacksteder, D.R.FitzPatrick and Jim Gao cloned MCD-related MLYCD gene respectively in 1999.32 unrelated MCD deficient patients have been reported all over the world to date, and 23 different MLYCD gene mutations are discovered (see Table 1). Clinical features change frequently. It leads to central nervous system abnormalities such as early childhood mental retardation, developmental delay, epilepsy, hypotonia, organic brain disease, diarrhea, vomiting, hypertrophic cardiomyopathy and so on. Its more serious form is neonatal death. Other rare symptoms include short stature, lethargy, anorexia, gastroenteritis, hearing impairment, facial malformations. Laboratory examination usually demonstrate irregular urinary compounds (such as urinary malonyl, methyl malonyl urine), hypoglycemia, metabolic acidosis, ketoacidosis, lactic acidosis, etc. No case has been reported in China,so far.Objective:We use cytogenetic and a variety of molecular genetic methods to study an MCD deficiency patient, to provide accurate diagnosis and genetic counseling for the patient and his family, and to provid gene diagnosis guidance for the other MCD deficiency patients.Methods:①Adopt GC-MS instrument screened patient’s urinary metabolites.②Using conventional cytogenetic analyzed patient’s peripheral blood chromosome.③Using PCR amplified and sequeced the five exons of MLYCD gene and its flanking sequences from patient’s and his parents’peripheral blood cells and patient’s skin cells.④Investigate their corresponding cDNA sequences by RT-PCR.⑤Meanwhile, we use Illumina Human 1M-Duo V3 gene chip to confirm/exclude the whole genome DNA micro-deletions and micro-duplication CNV abnomaly by patient’s peripheral blood gDNA.⑥Using MLPA technology to explore MLYCD gene copy munber of patient and his parents’peripheral blood and patient’s skin cells.⑦Using PCR amplified and sequeced the novel mutation site of MLYCD gene in 50 unrelated normal individuals.Results:1、Screening of urine organic acid metabolism:August 24,2009 found the patient had increased urinary malonate; Re-examination on September 24,2009 showed more increased urinary malonic acid, methylmalonic acid increased slightly.2、The patient’s peripheral blood chromosome G-band and High solution demonstrate the karyotyoe is normal.3、Sequencing of the MLYCD gene revealed a heterozygous missense mutation (c.920T> G; p. Leu307Arg) in the patient and his father. A synonymous SNP (c.732G> A; p. serine 266 serine) is also found exisitng in his father’s MLYCD gene. The patient inherited a "gDNA-normal strand" from his mother, of which the other strand has a missense SNP (c.776G> C; p. Gly 259 Ala).4、Further cDNA sequencing of MLYCD gene showed existence of homozygous missense mutation (c.920T> G; p. Leu307Arg) in the patient, homozygous missense SNP(c.776G>C;p. Gly259Ala) in the patient’s mother, heterozygous missense mutation c.920T>G(p. Leu307Arg) and heterozygous SNP(c.732G>A;p. ser266ser) in the father.5、Genechip assay excluded the possibility of CNV abnormality in the patient.6、MLPA confirmed "gDNA-normal strand" actually was Exonl deletion in proband DNA strand which was also found in maternal MLPA, the patient’s father’s MLPA result is normal.7、Paternal novel missense mutation is not found in 50 unrelated normal individuals.And we compared conservation of this site from chimpanzee,human, cow,mouse and rat and comfirmed that this missense mutation is a conservative T.Conclusion:1.The patient’s missense mutation derived from his father is novel (c.920T> G; p. Leu307Arg), which has not been reported in literature before. The mutation site is conserved in different species,suggesting that it may be the pathogenicity of the disease.2.MLYCD gene in the patient’s maternal chain was not expressed. MLPA proved that this maternal srtand is Exonl missing, indicating that Exonl deletion may lead to gene silence,therefore cause MCD deficiency.3.We found that the proband’s MLYCD gene was compound heterozygous mutation at the level of molecular genetics. Thus, the patient can be diagnosed as malonyl-CoA decarboxylase A deficiency correctly. This research suggests that parents of this patient who are demonstrated apparently as heterozygous mutation of MCD should carry out prenatal diagnosis when they attempt to conceive a baby.更多还原