Successful First Trimester Diagnosis in a Pregnancy at Risk for Propionic Acidaemia

Pérez‐Cerdá, Celia; Merinero, Begoña; Sanz, Pilar Perez; Jiménez, Antonio Moreno; García, Maria José; Urbón, A.; Recaséns, Joaquín Díaz; Ramos, Carmen; Ayuso, Carmen; Ugarte, Magdalena

doi:10.1007/bf03335396

Cited by 10 publications

(2 citation statements)

References 6 publications

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“…All the patients reported in this work were clinically identified after presenting a metabolic crisis and no patients came from the newborn screening metabolic program. The patients reported here were referred from physicians of clinical centers to be biochemically and/or genetically diagnosed in Madrid ( ] from leucine, used as internal metabolic control, into acid-precipitable material was assayed in intact fibroblasts grown in basal medium and in 1 mg/mL OHCbl supplemented medium, as described [Perez-Cerda et al, 1989]. To identify the defective gene, MMADHC (MMA cobalamin deficiency type D with homocystinuria) or MMACHC, genetic complementation assays were performed in 11 patients, as described previously [Merinero et al, 2008].…”

Section: Methodsmentioning

confidence: 99%

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)

Richard¹,

Jorge‐Finnigan²,

García‐Villoria³

et al. 2009

Hum. Mutat.

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Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B(12) metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC-affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B(12). The mutational spectrum includes nine previously described mutations: c.3G>A (p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1+2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real-time PCR and subsequent high-resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin-incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients.

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Section: Methodsmentioning

confidence: 99%

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)

Richard¹,

Jorge‐Finnigan²,

García‐Villoria³

et al. 2009

Hum. Mutat.

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“…18 PCC activity was assayed as described elsewhere, 22 and 14 C propionate incorporation into acid-precipitable material was determined in intact cells grown in basal medium as propionate metabolism via MCM. 23 For the detection of biotin-bound proteins, fibroblasts were harvested by trypsinization and were freeze thawed, and protein concentration in cell extracts was determined by the Bradford assay. Equal amounts of total protein (30-50 mg) from each sample were loaded onto a denaturing 6% polyacrylamide gel.…”

Section: Oligonucleotide Treatment and Analysismentioning

confidence: 99%

Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA

Rincón

Aguado

Desviat

et al. 2007

The American Journal of Human Genetics

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We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5? or 3? cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients' fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of (14)C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.

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Pseudoexon exclusion by antisense therapy in methylmalonic aciduria (MMAuria)

Pérez

Rincón²,

Jorge‐Finnigan

et al. 2009

Hum. Mutat.

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Development of pseudoexon exclusion therapies by antisense modification of pre-mRNA splicing represents a type of personalized genetic medicine. Here we present the cellular antisense therapy and the cell-based splicing assays to investigate the effect of two novel deep intronic changes c.1957-898A>G and c.1957-920C>A identified in the methylmalonyl-coenzyme A (CoA) mutase (MUT) gene. The results show that the nucleotide change c.1957-898A>G is a pathological mutation activating pseudoexon insertion and that antisense morpholino oligonucleotide (AMO) treatment in patient fibroblasts leads to recovery of MUT activity to levels 25 to 100% of control range. On the contrary, the change c.1957-920C>A, identified in two fibroblasts cell lines in cis with c.1885A>G (p.R629G) or c.458T>A (p.D153V), appears to be a rare variant of uncertain clinical significance. The functional analysis of c.1885A>G and c.458T>A indicate that they are the disease-causing mutations in these two patients. The results presented here highlight the necessity of scanning the described intronic region for mutations in MUT-affected patients, followed by functional analyses to demonstrate the pathogenicity of the identified changes, and extend previous work of the applicability of the antisense approach in methylmalonic aciduria (MMAuria) for a novel intronic mutation.

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Successful First Trimester Diagnosis in a Pregnancy at Risk for Propionic Acidaemia

Cited by 10 publications

References 6 publications

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)

Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA

Pseudoexon exclusion by antisense therapy in methylmalonic aciduria (MMAuria)

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