Quality of analytical performance in inherited metabolic disorders: the role of ERNDIM

Summary: External quality assurance (EQA) schemes are essential for improvement of accuracy, reliability and comparability of results of biochemical genetic tests. ERNDIM (European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism), established in 1994, operates nine EQA schemes for biochemical genetic testing according to international norms and recommendations. These comprise qualitative schemes for amino acids, organic acids, purines and pyrimidines, special assays in serum and urine and white cell cystine, qualitative organic acid and acylcarnitine schemes, as well as diagnostic proficiency testing. The total number of participants has increased from 123 in 1994 to 268 in 2007. Additional activities include participation in the Eurogentest project, a laboratory directory, training, education and development of guidelines. Results from the quantitative amino acid scheme with 170 participants reveal good variation within and between laboratories of below 10% for 10 amino acids; good within-laboratory variation but intermediate inter-laboratory variation of 10-22% for 11 amino acids; and higher variation within and between laboratories for 8 amino acids. Results on samples from 51 inherited metabolic disorders from two of five centres organizing diagnostic proficiency testing indicate overall diagnostic efficiency above 80% and improved performance of individual laboratories. Comparison of results for 10 and 12 compounds in the serum and urine special assay schemes respectively for 2000 and 2007 reveal clear improvement of precision within laboratories and in inter-laboratory variation. There is considerable evidence that performance in biochemical genetic testing has improved since the introduction of ERNDIM scheme

Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation

Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc.Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease

Creatine and guanidinoacetate reference values in a French population

Creatine and guanidinoacetate are biomarkers of creatine metabolism. Their assays in body fluids may be used for detecting patients with primary creatine deficiency disorders (PCDD), a class of inherited diseases. Their laboratory values in blood and urine may vary with age, requiring that reference normal values are given within the age range. Despite the long known role of creatine for muscle physiology, muscle signs are not necessarily the major complaint expressed by PCDD patients. These disorders drastically affect brain function inducing, in patients, intellectual disability, autistic behavior and other neurological signs (delays in speech and language, epilepsy, ataxia, dystonia and choreoathetosis), being a common feature the drop in brain creatine content. For this reason, screening of PCDD patients has been repeatedly carried out in populations with neurological signs. This report is aimed at providing reference laboratory values and related age ranges found for a large scale population of patients with neurological signs (more than 6 thousand patients) previously serving as a background population for screening French patients with PCDD. These reference laboratory values and age ranges compare rather favorably with literature values for healthy populations. Some differences are also observed, and female participants are discriminated from male participants as regards to urine but not blood values including creatine on creatinine ratio and guanidinoacetate on creatinine ratio values. Such gender differences were previously observed in healthy populations; they might be explained by literature differential effects of testosterone and estrogen in adolescents and adults, and by estrogen effects in prepubertal age on SLC6A8 function. Finally, though they were acquired on a population with neurological signs, the present data might reasonably serve as reference laboratory values in any future medical study exploring abnormalities of creatine metabolism and transport

Development of a Tandem Mass Spectrometry Method for Rapid Measurement of Medium- and Very-Long-Chain Acyl-CoA Dehydrogenase Activity in Fibroblasts

International audienceMitochondrial fatty acid oxidation is a vital biochemical process for energy metabolism. Among the known fatty-acid metabolism disorders, very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency count among the most frequent. Both are potentially very serious diseases as they carry a risk of severe neurological post-crisis sequelae, and even sudden death. Diagnosis relies on plasma acylcarnitine profile analysis and urine organic acid analysis, followed by genetic testing to confirm diagnosis. However, in some cases, it is crucial to run a specific diagnostic assay for enzyme activity, which is generally performed in leukocytes or fibroblasts. The aim of this study was to address this need, first by developing a MCAD and VLCAD enzyme activity-specific diagnostic assay in fibroblasts (by measuring the reaction products, i.e. enoyl-CoA) via a rapid LC-MS/MS-based technique, and then by testing MCAD-deficient patients (n = 6), VLCAD-deficient patients (n = 10), and control patients (n = 12). MCAD activity was significantly different in the MCAD-deficiency (MCADD) group (mean = 0.07 nmol C8:1 formed/min/mg protein) compared to the control group (mean = 0.36 nmol C8:1 formed/min/mg protein). All MCADD patients showed less than 35% residual MCAD activity. VLCAD activity was significantly decreased in the VLCADD group (mean = 0.06 nmol C16:1 formed/min/mg protein) compared to the control group (mean = 0.86 nmol C16:1 formed/min/mg protein, respectively). All VLCADD patients showed less than 35% residual VLCAD activity. This technique allowed also to confirm that a novel ACADVL gene mutation (c.1400T\textgreaterC) is responsible for a defective VLCAD activity (residual activity at 10%)

Quality of analytical performance in inherited metabolic disorders : the role of ERNDIM

External quality assurance (EQA) schemes are essential for improvement of accuracy, reliability and comparability of results of biochemical genetic tests. ERNDIM (European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism), established in 1994, operates nine EQA schemes for biochemical genetic testing according to international norms and recommendations. These comprise qualitative schemes for amino acids, organic acids, purines and pyrimidines, special assays in serum and urine and white cell cystine, qualitative organic acid and acylcarnitine schemes, as well as diagnostic proficiency testing. The total number of participants has increased from 123 in 1994 to 268 in 2007. Additional activities include participation in the Eurogentest project, a laboratory directory, training, education and development of guidelines. Results from the quantitative amino acid scheme with 170 participants reveal good variation within and between laboratories of below 10% for 10 amino acids; good within-laboratory variation but intermediate inter-laboratory variation of 10-22% for 11 amino acids; and higher variation within and between laboratories for 8 amino acids. Results on samples from 51 inherited metabolic disorders from two of five centres organizing diagnostic proficiency testing indicate overall diagnostic efficiency above 80% and improved performance of individual laboratories. Comparison of results for 10 and 12 compounds in the serum and urine special assay schemes respectively for 2000 and 2007 reveal clear improvement of precision within laboratories and in inter-laboratory variation. There is considerable evidence that performance in biochemical genetic testing has improved since the introduction of ERNDIM schemes

Very long chain acyl-coenzyme A dehydrogenase deficiency in two siblings: evolution after prenatal diagnosis and prompt management

A boy had neonatal seizure, lethargy, and metabolic acidosis at presentation. He recovered completely, but the recurrence of a similar episode with associated cardiomyopathy and dicarboxylic aciduria at 10 months of age led to the recognition of a fatty acid oxidation defect. A diagnosis of very long chain acyl-coenzyme A dehydrogenase deficiency was later made by enzyme assay in culture fibroblasts from this child, as well as in cultured amniotic cells from a sibling fetus. This prenatal diagnosis forestalled neonatal injury by close clinical and metabolic monitoring of the second infant. Early diagnosis and management should potentially improve the generally poor prognosis for patients with very long chain acyl-coenzyme A dehydrogenase deficiency