Clinical, Pathological And Molecular Biological Study On Multiple Acyl-CoA Dehydrogenase Deficiency

BackgroundMultiple acyl-CoA dehydrogenase deficiency （MADD, OMIM#231680） also known as glutaric aciduria type II （GAII）, is an inherited autosomal recessive disease affecting fatty acid, amino acid and choline metabolism, due to mutations in one of the three genes namely, electron transfer flavoprotein alpha-subunit, ETFA （OMIM#608053）, electron transfer flavoprotein beta-subunit, ETFB （OMIM#130410） and electron transfer flavoprotein dehydrogenase, ETFDH （OMIM#231675）. Patients may present very heterogeneous clinical feature with onset from birth to adulthood. The proximal muscle weakness is the most common onset form of the condition, probably with gastrointestinal symptoms and metabolic acidosis. Some MADD patients are responsive to riboflavin treatment well with an excellent prognosis. Recently, mutations in ETFDH were found to be responsible for all riboflavin-responsive MADD patients. In this study, we present the clinical, pathological and molecular biological studies of2Chinese families with late onset riboflavin-responsive MADD.ObjectiveTo describe and verify the clinical, pathological and molecular biological features, as well as therapy and prognosis of two families of MADD, and then to analyze the relationship between phenotype and genotype.Material and methodsThe3patients of MADD in the study were from two independent family respectively, all of which were clinical cases from department of Neurology of The Affiliated Qianfoshan Hospital of Shandong University. Preliminary diagnoses were made upon clinical and pathological presentations. Then blood acylcarnitine and urine organic acid analyses were proceeded in patient1and3. Genomic DNA was extracted from patients’muscle specimen, amplified through PCR and then sequenced. After receiving the report, blood sample was gained from followed-up family members. Then related genes was sequenced for the family members and75controls. The relationship between phenotype and genotype was clarified. Enzyme linked immunosorbent assay （ELISA） was used to evaluate the quantity of the ETF:QO of the patients and controls during onsetting and remission stage respectively. The quantity of the target protein was estimated and mechanism of the riboflavin therapy was revealed preliminarily.Results1. Age of onset in patients1-3were45y,19y and17y respectively. The first symptom of the3patients is weakness of both lower extremities. Muscle weakness gradually worsened, and the involved scope gradually increased, mainly in limbgirdle muscle, paraspinal muscle, cervical muscle, cephalic muscle and proximal muscles of extremities. They presented all along with obvious amyotrophy, with or without myalgia. Interestingly, all3patients had a general aversion to meat. Changes in serum creatase were significant, with all LDH and respective CK, AST or HBDH abnormally increasing. Electromyography displayed myopathic changes or a normal pattern. Single riboflavin therapy revealed dramatic effect to the3patients.2. Light-microscopic assessment of histochemical stains showed muscular fibers had a moderate variation in size with numerous vacuoles. The O.R.O stain displayed red-stained lipid droplets varying in size and intensity. The NADH stain showed unequally deposit of dark blue granules. All the abnormalities mostly involved type I fibers.3. Urine analysis and blood acyl-carnitine profile verified the diagnoses of GAII and MADD separately.4. All the three patients were identified to carry compound heterozygous mutations in ETFDH gene.4novel mutations were found in our study. Patient1presented a missense mutation, c.1522C>A（p.P508T）, and a frameshift mutation, c.l583₁584insA（p.N528KfsX3）, which produced a premature termination codon （PTC）. Patient2&3carried a missense mutation, c.242T>C（p.L81P）, and a splice mutation, c.IVS5+7A>G. None of the mutations appeared in150control alleles. No mutations were identified in the ETFA and ETFB genes.5. ELISA results showed the patients’quantity of ETF:QO in muscle was lower than the controls’during crises （p<0.01）. Nevertheless, the quantity in peripheral blood from the patients was higher than the controls in convalescent phase （p<0.01）.ConclusionPatients from the two families in our study mainly presented as muscular weakness involving limbgirdle muscle, paraspinal muscle, cervical muscle, cephalic muscle and proximal muscles of extremities. Muscle pathology suggested lipid deposit. Biochemical studies could help to gain the initial diagnosis. Mutation analysis verified RR-MADD is mainly due to mutation in ETFDH gene. ELISA results indicated that the stability of ETF:QO could be increased to some extent. Single riboflavin therapy revealed dramatic effect to this condition.