Identification Of Pathogenic Mechanism For Two Families With Neurogenetic Diseases

Duchenne muscular dystrophy（DMD）and Becker muscular dystrophy（BMD）are both caused by mutations in the DMD gene effecting the expression of dystrophin.Generally female carriers are asymptomatic;however,it has been suggested that carriers may exhibit symptoms if skewed X-chromosome inactivation（XCI）patterns are present.We investigated a 6-year-old Chinese girl exhibiting a BMD-like phenotype,including persistently elevated creatine kinase（CK）and creatine kinase isoenzyme（CK-MB）levels,with a maximum of 6050 u/L and 123 u/L.The proband harbored a novel heterozygous mutation,c.3458₃459insAA,in the DMD gene from her mother who had completely normal phenotype upon examination.However,XCI patterns in the peripheral blood of the child were slightly skewed:proband with 62%（mutant allele）/38%（normal allele）when compared to her mother with 32/68%.Amplification of regions of the cDNA revealed different ratios for the expression of these alleles:proband with 50%（mutant allele）/50%（normal allele）and her mother with 20/80%.Real-time PCR showed that mRNA expression was significantly decreased in both.It is the first reported point mutation,within exon 26 of DMD to date,which has resulted in a manifestation of symptoms.We proposed that a frameshift or nonsense mutation,resulting in a loss-of-function truncated protein with premature termination,may contribute to the development of symptoms in carriers.These phenotypes correlate with nonrandom XCI patterns and are compounded by the locus of the mutation.For incompletely skewed XCI patterns,although the mutant allele could suppress the expression of a normal allele,resulting in significant reduction in expression,carriers would remain asymptomatic as long as there was adequate compensation from the normal allele.We further hypothesis that the normal allele could in turn suppress the expression of a mutant allele,to some extent,described as a "resistance behavior".Developmental delay（DD）/Intellectual disability（ID）is considered one of the most genetically heterogeneous human diseases.Herein,we investigated the mutation of HCFC1 in a Chinese boy affected by DD with 3-methylcrotonyl-CoA carboxylase deficiency（MCCD）and 3-hydroxy-3-methyl glutaric aciduria（3-HMG）,and its potential molecular mechanism.The blood of all family members was analyzed by Sanger sequencing to identify the novel mutation.The pathogenesis and function of the mutation was studied by bioinformatics analysis,subcellular localization,real-time PCR and western blotting,in addition to its effect on cell proliferation,apoptosis and cell cycle progression by flow cytometer.A novel missense hemizygous mutation,c.4442C>T,was found within the acidic domain of HCFC1.The mutation had not only affected the subcellular localization,but also increased the protein expression in the whole cell.Different from mutations of the Kelch domain,this mutation had no effect on MMACHC responsible for the metabolism of cobalamin,but decreased the expressions of MCCC1,MCCC2,HMGCL,relating to MCCD and 3-HMG.Over-expression of mutant HCFC1 would probably inhibit apoptosis without affecting the proliferation and cell cycle process.This novel mutation,related to DD/ID with MCCD and 3-HMG,was the first reported genetic mutation occurring within the acidic domain of HCFC1 to date.This X-linked disease revealed a new insight into a distinct disease mechanism that transcriptional dysregulation can lead to metabolic disorders with a much more serious and complex clinical phenotype.