Dietary therapy in two patients with vitamin B12-unresponsive methylmalonic acidemia

The biochemical and therapeutic responses to dietary therapy were studied in a 25-month-old girl and a 1-month-old girl with methylmalonic acidemia (MMA-emia), which was unresponsive to vitamin B₁₂.The minimum daily intake of protein which patients could tolerate and display a good development was between 1.0 and 1.2 g per kg body weight. Supplementation with amino acid mixture devoid of toxic amino acids was required to prevent protein malnutrition when daily protein intake was restricted to 0.6 g per kg body weight. Caloric intake should be sufficient, not only to promote growth but also to prevent a rise in MMA level, especially when a patient has ketoacidosis.It was found that MMA excretion per mg creatinine in random urine specimens correlated significantly with serum MMA and twenty four-hour output of MMA per kg body weight. Therefore measurement of MMA in a single urine specimen is useful for evaluating the in vivo accumulation of MMA.     

PRENATAL DIAGNOSIS OF METHYLMALONIC ACIDURIA

Abstract. Mahoney, M. J., Rosenberg, L. E., Lindblad, B., Waldenström, J. and Zetterström, R. (Departments of Human Genetics and Pediatrics, Yale University, New Haven, USA, the Department of Clinical Chemistry, University of Gothenburg, Gothenburg, Sweden and the Department of Pediatrics, Karolinska Institutet, St. Göran's Hospital for Children, Stockholm, Sweden). Prenatal diagnosis of methylmalonic aciduria. Acta Paediatr Scand, 64: 44, 1975.–Prenatal diagnosis using amniocentesis was sought in two midtrimester pregnancies, each at risk for a different type of inherited methylmalonic aciduria. In one pregnancy a normal fetus was diagnosed from studies of cultured amniotic fluid cells and the diagnosis confirmed after the baby was born. In the second pregnancy a fetus with a methylmalonyl-CoA mutase apoenzyme defect was found. The diagnosis was based on Cultured cell studies and supported by an elevation of methyl-malonate in both amniotic fluid and maternal urine. Confirmatory studies were obtained using cultured cells from the aborted fetus. At the present time, assays of cultured amniotic fluid cells are imperative for firm diagnosis. With more experience, quantities of amniotic fluid and maternal urine methylmalonate may prove sufficient if differentiation among the various types of methylmalonic aciduria is not required.     

Immediate newborn outcome and mode of delivery: Use of standardized fetal heart rate pattern management

Objective: To determine whether a rule-based system for fetal heart rate interpretation can result in reduced metabolic acidemia without increasing obstetrical intervention. Methods: Rates of vacuum-assisted delivery and Cesarean sections, and umbilical artery pH and base excess values were determined over a 5-year period in a single hospital with 3907 deliveries in Japan. Results were compared for 2 years before and 2 years after a 6-month training period in rule-based fetal heart rate interpretation. Results: The pre- and post-training rates of unscheduled Cesarean deliveries (4.8% vs. 6.0%) and vacuum deliveries (21.2% vs. 18.1%) did not differ significantly. The rates of umbilical arterial pH <7.15 (1.51% vs. 0.18%, p < 0.05) and base excess <–12 mEq/L (1.76% vs. 0.25%, p < 0.05) were significantly lower after training. Conclusion: A standardized fetal heart rate pattern management system was associated with a 7-fold reduction of newborn metabolic acidemia with no change in operative intervention.     

Methylmalonic and propionic acids increase the in vitro incorporation of P into cytoskeletal proteins from cerebral cortex of young rats through NMDA glutamate receptors

In this study we investigated the effects of methylmalonic acid (MMA) and propionic acid (PA) on the phosphorylation of cytoskeletal proteins of cerebral cortex of rats. Slices of tissue were incubated with ³²P-orthophosphate in the presence or absence of glutamate, MMA, PA and ionotropic or metabotropic glutamate receptor agonists. The cytoskeletal fraction was isolated and the radioactivity incorporated into the cytoskeletal proteins was measured. Results demonstrated that the acids, glutamate and NMDA increased the phosphorylation of the proteins studied. However, this effect was not observed for non-NMDA ionotropic agonists or metabotropic agonists. Experiments using glutamate receptor antagonists confirmed that MMA and PA at the same concentrations as found in tissues from propionic or methylmalonic acidemic children increase the phosphorylation of cytoskeletal proteins, possibly via NMDA glutamate receptors. Therefore, it is feasible that these findings may be related to the neurological dysfunction characteristic of these disorders.     

A male case with CDKL5-associated encephalopathy manifesting transient methylmalonic acidemia

Mutations in the X-linked gene CDKL5 cause early-onset epileptic encephalopathy and severe developmental delay. Because this disorder predominantly affects females, the full clinical spectrum of male patients remains elusive. We herein report a 16-year-old boy, who suffered from intractable seizures 20 days after birth. Serial electroencephalograms detected recurrent focal epileptiform discharges from age 4 months, which evolved to hypsarrhythmia later in infancy. Mass-spectrometric analyses revealed increase in urinary excretion of methylmalonic acid without perturbed concentrations of propionic acid, homocystein and methionine. Whole-exome sequencing identified a de novo, truncating mutation in CDKL5 (NM_003159.2:c.419dupA, p.Asn140Lysfs*8). Targeted sequencing excluded concomitant mutations in methylmalonic academia-associated genes. No methylmalonic acidemia has been reported in children with CDKL5 disorder. Extensive analyses on organic acid metabolism for males with CDKL5 mutations will gain more insight into their biochemical profiles in infancy.     

Molecular study of electron transfer flavoprotein alpha-subunit deficiency in two Japanese children with different phenotypes of glutaric acidemia type II

BACKGROUND: Electron transfer flavoprotein is a mitochondrial matrix protein composed of alpha- and beta-subunits (ETF alpha and ETF beta, respectively). This protein transfers electrons between several mitochondrial dehydrogenases and the main respiratory chain via ETF dehydrogenase (ETF-DH). Defects in ETF or ETF-DH cause glutaric acidemias type II (GAII). MATERIALS AND METHODS: We investigated the molecular basis of ETF alpha deficiency in two Japanese children with different clinical phenotypes using expression study. RESULTS: Patient 1 had the severe form of GAII, a compound heterozygote of two mutations: 799G to A (alpha G267R) and nonsense 7C to T (alpha R3X). Patient 2 had the mild form and carried two heterozygous mutations: 764G to T (alpha G255V) and 478delG (frameshift). Both patients had one each of missense mutations in one allele; the others were either nonsense or truncated. Restriction enzyme digestion assay using genomic DNAs from 100 healthy Japanese revealed that these mutations were all novel. No signal for ETF alpha was detected by immunoblotting in cases of missense mutants, while wild-type cDNA resulted in expression of ETF alpha protein. Transfection with wild-type ETF alpha cDNA into cultured cells from both patients elevated incorporation of radioisotope-labelled fatty acids. CONCLUSION: These four mutations were pathogenic for GAII and missense mutations, alpha G255V and alpha G267R were considered anecdotal for mild and severe forms, respectively.     

Stop codon read-through of a Methylmalonic aciduria mutation

A stop codon defect in methylmalonyl-CoA mutase (resulting in a truncated unstable protein) accounts for up to 14% of mutations identified as causes of Methylmalonic aciduria. There are currently limited treatment regimes for patients with this inherited condition. We aimed to investigate the use of stop codon read-through drugs in a genomic reporter assay cell line with a defect in the mutase gene. A single C–T base change was introduced into exon 6 of the human MUT sequence in the BAC clone RP11-463L20 resulting in an arginine residue being replaced with a TGA stop codon. An enhanced green fluorescent protein reporter gene was introduced in-frame with exon 13 of the MUT gene. The construct was transfected into HeLa cells to produce the genomic reporter assay cell line. To test the suppression of nonsense mutations, cells were incubated in the presence of different compounds for a period of 72 h then analysed by flow cytometry. Treatment of the cells with gentamicin resulted in a 1.6-fold increase in reporter protein, whilst G418 treatment resulted in no change, however the two drugs together acted synergistically to increase the production of methylmalonyl-CoA mutase 2.0-fold (confirmed by mRNA, flow cytometry and enzyme activity). Zidovudine, adefovir and cisplatin were also found to have some activity in the stop codon read-through genomic reporter assay. These results encourage further testing of compounds as well as follow up animal studies. This is the first study to demonstrate the use of stop codon read-through drugs for the potential treatment of Methylmalonic aciduria.     

High frequency of large genomic deletions in the PCCA gene causing propionic acidemia

Mutations in either the PCCA or PCCB genes are responsible for propionic acidemia (PA), one of the most frequent organic acidemias inherited in autosomal recessive fashion. Most of the mutations detected to date in both genes are missense. In the case of PCCA deficient patients, a high number of alleles remain uncharacterized, some of them suspected to carry an exonic deletion. We have now employed multiplex ligation probe amplification (MLPA) and long-PCR in some cases to screen for genomic rearrangements in the PCCA gene in 20 patients in whom standard mutation detection techniques had failed to complete genotype analysis. Eight different deletions were found, corresponding to a frequency of 21.3% of the total PCCA alleles genotyped at our center. Two of the exonic deletions were frequent, one involving exons 3–4 and another exon 23 although in the first case two different chromosomal breakpoints were identified. Absence of exons 3 and 4 which is also the consequence of the novel splicing mutation c.231 + 1g > c present in two patients, presumably results in an in-frame deletion covering 39 aminoacids, which was expressed in a eukaryotic system confirming its pathogenicity. This work describes for the first time the high frequency of large genomic deletions in the PCCA gene, which could be due to the characteristics of the PCCA gene structure and its abundance in intronic repetitive elements. Our data underscore the need of using gene dosage analysis to complement routine genetic analysis in PCCA patients.     

新生儿有机酸血症临床分析

目的探讨新生儿有机酸血症的临床特点和治疗方法。方法回顾性分析24例新生儿有机酸血症的临床资料,采用气相色谱-质谱法（gas chromatography-mass spectrometry,GC/MS）进行尿或血有机酸分析。结果本组患儿症状主要表现为喂养困难（20例）,反应减弱（18例）,呕吐（7例）,呼吸急促（4例）,惊厥（7例）,肌张力减低（17例）等。血小板减少13例,血气分析提示失代偿性酸中毒7例。10例确诊甲基丙二酸血症患儿接受维生素B12治疗,同时予低蛋白饮食及左旋肉碱治疗。2例死亡。随访8例,时间2个月～3年5个月,4例临床症状完全消失,4例明显好转。结论新生儿有机酸血症的临床表现是非特异性的,可依据GC/MS尿有机酸分析确诊,早期发现和治疗是改善预后的关键。