Mutational Analysis Of MMACHC Gene In Cohorts Of Patients With CblC Type Methylmalonicacidemia

Research backgroundMethylmalonic acidemia?methylmalonicacidemia,MMA?is also known as methylmalonic aciduria?methylmalonicaciduria?,is one of the most common diseases of amino acid and organic acid metabolism abnormal,is a branched chain amino acid metabolism pathway,is an autosomal recessive inherited disease of organic acidemias.MMA is mainly due to two methyl coenzyme A?methylmalonyl coenzyme A,MMCoA?mutase?methylmalonyl-CoA mutase,MCM?defect and coenzyme-5 'deoxyadenosyl cobalamin?vitamin B12??Ado-Cbl?synthesis lead to metabolic disorders,methylmalonic acid,propionic acid and methylcitrate abnormal accumulation,causing multiple organ function by injury,especially the nervous system damage.Ado-Cbl metabolic abnormalities include cblA,cblB,cblC,cblD,cblE,cblF,cblG,and cblH 8 subtypes,among which cblC is the most common.Patients with cblC disease can exhibit a wide range of clinical manifestations,spanning the prenatal stage to the late adulthood.Increased homocysteine concentration and methyl metabolism may contribute to disease related complications,characteristics of macular and retinal degeneration,and different clinical manifestations at the onset of each individual is different: lethargy,repeated vomiting,feeding difficulties,hypotonia,low body temperature,respiratory distress,recurrent seizures or status epilepticus severe ketoacidosis,lactic acidosis,hyperammonemia,liver function,kidney damage loss,eye abnormalities,skin abnormalities,megaloblastic anemia,neutropenia and thrombocytopenia,cerebral edema,cerebral hemorrhage,severe cases can lead to death.The pathogenic gene of cblC disease MMACHC gene,the gene is located on chromosome 1 p34 encoding region,encoding cblC protein,function of catalytic reduction reaction and cyanide removal of deoxyadenosylcobalamin synthesis reaction in the cytoplasm.When a gene mutation occurs,the protein that encodes cblC fails to function properly and leads to disease.In this paper,3 cases of methylmalonic acidemia were summarized according to its typical clinical symptoms,biochemical characteristics,the disease can be found early,early diagnosis and effective treatment,help to improve the prognosis of patients.At the same time,the first positive permit and parental genetic testing,genetic methods of understanding disease?genetic mutation or genetic parents?,and provide information for genetic counseling,reduce the genetic diseases,gene mutation of related information,improve the MMACHC gene mutation type.Objective1.The clinical symptoms,biochemical features and treatment effects of 3 patients with MMA from 3 families were summarized;2.to study the pathogenesis of MMA in patients with molecular genetics,a clear diagnosis,understanding of the occurrence of MMACHC mutations,to infer the relevance of genetic counseling and prenatal diagnosis.Method1.A total of 3 patients with MMA from 3 families were included in the study,including clinical symptoms,biochemical characteristics,and treatment outcomes;2.sample collection: the research object for consent signed in the case of collecting the research object and the parents,50 healthy patients with fasting blood 5ml;3.Genomic DNA extraction: genomic DNA extraction kit was used to extract genomic DNA from blood samples;4.Primer design: according to the human genome database design MMACHC gene sequence primers,polymerase chain reaction?PCR,Polymerase Chain Reaction?amplification of the target gene;5.Product recovery: PCR product recycling kit for product recovery;6.sequencing: the PCR products were directly sequenced by DNA,and the original gene sequences in the human genome database GenBank were compared and analyzed.Result1.the product of PCR amplification was approximately the same as the length of the amplified fragment,and it was no longer the specific amplified fragment;2.Among the 1 probands of pedigrees,the MMACHC gene found a heterozygous heterozygous nucleotide mutation: third heterozygous heterozygous mutations in exon c.394C>T,and a heterozygous mutation in deletion of the nucleotide sequence of exon fourth of c.656₆58del.Heterozygous mutations in the exon third of the father c.394C>T heterozygous nonsense mutation and deletion of the c.656₆58del nucleotide sequence of the mother's fourth exon;3.In pedigree 2,proband MMACHC gene found: fourth exon c.609G>A homozygous null mutation.The parents of the subjects were fourth exon c.609G>A heterozygous null mutations;4.Among the 3 probands of pedigrees,the MMACHC gene found a heterozygous heterozygous nucleotide variant: first heterozygous missense mutations in exon c.80A>G,and heterozygous nonsense mutations in exon fourth of c.609G>A.Heterozygous missense mutation in father first exon c.80A>G;heterozygous mutation in mother fourth exon c.609G>A.Conclusion1.Among the 3 families,the proband was consistent with the clinical and biochemical characteristics of MMACHC.The diagnosis was clear and the prognosis was improved by early clinical intervention;2.The molecular genetic basis for 1 families in the proband is a combination of c.394C>T and c.656₆58del compound heterozygous mutations,the query gene pool,pathogenic c.656₆58del mutation has not been reported,1 new genetic mutations suspected.Parents can have genetic counseling when they are pregnant again,and gene sequencing of the fetus is used to make pre production diagnosis based on the results;3.The molecular genetic basis of pedigree 2 and pedigree 3 probands,c.609G>A homozygous mutations,and c.80A>G and c.609G>A heterozygous heterozygous mutation loci have been reported,and their pathogenicity is consistent with previous reports.