Diagnosis Of A Patient With MCD Deficiency By Cytogenetic And Multiple Molecular Genetic Techniques

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.