Researches On The Neurotoxic Mechanism And Gene Mutation Analysis Of Methylmalonic Aciduria

Part One Effects of the mitochondrial respiratory chain complexâ…¡in rat cardiac muscle by methylmaâ…¡onic acidObjective A symmetric degeneration of the basal ganglia, particularly the globus pallidus, is usually included in the methylmalonic aciduria. Some reasearches reavealed that the neurotoxic mechanisms may be related to the mitochondrial energy metabolism and Krebs cycle affected by endogenous toxic organic acids such as methylmalonic acid, malonic acid and 2-methylcitric acid. The purpose of the present investigation was to clarify the underlying mechanisms involved in the mitochondrial respiratory chain by methylmalonic acid using mitochondria from cardiac muscle of young rats.Methods Mitochondria were extracted from cardiac muscle of 30 days old SD rats and incubated by different concentrations of methylmalonic acid (0 mM,1 mM,2.5 mM and 5 mM) and different time(30 min,2h,4h and 8h) at 37℃before determination.The mitochondrial respiratory chain complexâ…¡enzymatic activity and expression of flavoprotein subunit 70 (FP) and iron-sulfur protein subunit 30 (IP) in complexâ…¡were respectively detected using spectrophotometric method and western blot.Results 1. Enzymatic activity of mitochondrial respiratory chain complexâ…¡â‘ When exposed to methylmalonic acid for 30 min and 2h respectively, complexâ…¡activity in the groups which were incubated by different concentrations of methylmalonic acid (1 mM,2.5 mM,5 mM) had no significant difference with that in control group (all P>0.05).â‘¡hen exposed to methylmalonic acid for 4h, complexâ…¡activity in three treatment groups decreased significantly compared with the control group (all P<0.05).â‘¢When exposed to methylmalonic acid for 8h, complexâ…¡activity in low dose treatment group (1 mM) had no significant difference with that in control group (P>0.05); but in middle and high dose treatment groups (2.5 mM and 5 mM), complexâ…¡activity decreased significantly (both P<0.05);â‘£Complexâ…¡activity remained stable when exposed to 30 min,2h and 4h (all P>0.05), when the incubation time prolonged to 8h, complexâ…¡activity decreased significantly (all P<0.05).2. Expression of FP and IP in Western blotâ‘ When exposed to methylmalonic acid for 30 min, expression of FP and IP in treatment groups had no significant difference with those in control group (all P>0.05);â‘¡hen exposed for 2h, expression of IP in treatment groups had no significant difference with those in control group (all P>0.05), but expression of FP decreased significantly (all P<0.05);â‘¢When exposed for 4h, expression of IP in treatment groups had no significant difference with those in control group (all P>0.05), but expression of FP decreased significantly (all P<0.05);â‘£When the incubation time prolonged to 8h, expression of FP decreased significantly in treatment groups (all P<0.05), expression of IP in low dose treatment group (1 mM) had no significant difference with that in control group (P>0.05), but in middle and high dose treatment groups (2.5 mM and 5 mM), it decreased significantly (both P<0.05).Conclusions Time-dependent and concentration-dependent inhibition of respiratory chain complexâ…¡enzymatic activities and expression of protein subunits by methylmalonic acid found in rat heart mitochondria may suggest a direct inhibition mechanism of methymalonic acid on the respiratory chain complexâ…¡. Part Two Clinical and molecular genetic characteristics of Chinese patients with methylmalonic aciduriaObjective Methylmalonic aciduria (MMA) is a common inherited autosomal recessive disorder resulted from different causes. It is composed of mut complementation group (mut0and mut-) and Cbl complementation group (Cb1A, Cb1B, Cb1C, Cb1D, Cb1F and Cb1H) according to the defects in the enzyme methylmalonyl CoA mutase or the synthesis of the MCM cofactor adenosylcobalamin. The mut complementation group accounts for the largest number of patients with isolated MMA. More than 200 mutations in the MUT gene on chromosome 6p12 have been identified in mut patients. We aimed to investigate the clinical characteristics and genomic variations in the MUT gene of Chinese patients.Methods 18 MMA patients diagnosed by gas chromatography/mass spectrometry (GC/MS) and 10 parents were chosen. Their clinical characteristics including age onset, clinical symptoms, biochemical abnormalities, vitamin B12 responsiveness, treatment and follow up were recorded. Genomic DNA was extracted from peripheral white blood cells. PCR amplification, purification and direct sequencing of the MUT coding regions (exon 2-13) and their adjacent intronic consensus splice sites were performed in order to identify the disease causing mutations.Results 1. Major Clinical features of the patients:18 patients (13 males,5 females) presented mainly with hypotonia, feeding difficulty, vomiting and infection with onset during 2 days to 14 months. Metabolic acidosis and hyperammoniemia were the most common biochemical abnormalities. There was a pair of twin brothers in the group, patient 16 and 17. Patient 10 and 12 both had siblings died of unknown reasons may be related to this disease.12 of them were vitamin B12 unresponsive,3 responsive and 3 undone. Prognosis were disappointed,14 of them died until this research finished. 2. Mutation analysis:14 gene mutations and polymorphysims were found in all of the patients and parents, most of them were located in exon 2,3,6,9,12 and intron 9, missense mutations were the most common mutation type.â‘ Six novel mutations: c.424A>G (p.T142A), c.786T>G (p.S262R), c.808G>C (p.G270R), c.1323₁324insA, c.1445-1G>A and c.1676+77A>C were identified, all of them were heterozygous.â‘¡ther mutations and polymorphisms:c.323G>A (p.R108H), c.636A>G (p.K212K), C.682C>T (p.R228X.), c.1495G>A (p.A499T), c.1595A>G (p.H532R), c.1992G>A (p.A664A), c.2011G>A (p.V671I) and c.1677-53A>G were detected, most of them were heterozygous.â‘¢Mutations found in five families:there are seven mutations together, c.636A>G (p.K212K),(p.A499T),c.1595A>G (p.H532R),c.1992G>A (p.A664A),c.2011 G>A (p.V671I),c.1677-53A>Gå’Œc.1676+77A>C. Only one of them was novel. Mutations of the patients all came from their fathers or mothers.Conclusions In this study, we found six novel mutations previously not reported and updated the spectrum of MUT mutations. In addition, the main mutation types which caused methylmalonic aciduria in the middle part of China may be related to three polymorphisms p.K212K, p.H532R and p.V671I accompanied with other mutations.