Improving the diagnosis of cobalamin and related defects by genomic analysis, plus functional and structural assessment of novel variants.

BACKGROUND: Cellular cobalamin defects are a locus and allelic heterogeneous disorder. The gold standard for coming to genetic diagnoses of cobalamin defects has for some time been gene-by-gene Sanger sequencing of individual DNA fragments. Enzymatic and cellular methods are employed before such sequencing to help in the selection of the gene defects to be sought, but this is time-consuming and laborious. Furthermore some cases remain undiagnosed because no biochemical methods have been available to test for cobalamin absorption and transport defects. RESULTS: This paper reports the use of massive parallel sequencing of DNA (exome analysis) for the accurate and rapid genetic diagnosis of cobalamin-related defects in a cohort of affected patients. The method was first validated in an initial cohort with different cobalamin defects. Mendelian segregation, the frequency of mutations, and the comprehensive structural and functional analysis of gene variants, identified disease-causing mutations in 12 genes involved in the absorption and synthesis of active cofactors of vitamin B12 (22 cases), and in the non-cobalamin metabolism-related genes ACSF3 (in four biochemically misdiagnosed patients) and SUCLA2 (in one patient with an unusual presentation). We have identified thirteen new variants all classified as pathogenic according to the ACGM recommendation but four were classified as variant likely pathogenic in MUT and SUCLA2. Functional and structural analysis provided evidences to classify them as pathogenic variants. CONCLUSIONS: The present findings suggest that the technology used is sufficiently sensitive and specific, and the results it provides sufficiently reproducible, to recommend its use as a second-tier test after the biochemical detection of cobalamin disorder markers in the first days of life. However, for accurate diagnoses to be made, biochemical and functional tests that allow comprehensive clinical phenotyping are also needed.

The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 2: the evolving clinical phenotype.

BACKGROUND: The disease course and long-term outcome of patients with organic acidurias (OAD) and urea cycle disorders (UCD) are incompletely understood. AIMS: To evaluate the complex clinical phenotype of OAD and UCD patients at different ages. RESULTS: Acquired microcephaly and movement disorders were common in OAD and UCD highlighting that the brain is the major organ involved in these diseases. Cardiomyopathy [methylmalonic (MMA) and propionic aciduria (PA)], prolonged QTc interval (PA), optic nerve atrophy [MMA, isovaleric aciduria (IVA)], pancytopenia (PA), and macrocephaly [glutaric aciduria type 1 (GA1)] were exclusively found in OAD patients, whereas hepatic involvement was more frequent in UCD patients, in particular in argininosuccinate lyase (ASL) deficiency. Chronic renal failure was often found in MMA, with highest frequency in mut(0) patients. Unexpectedly, chronic renal failure was also observed in adolescent and adult patients with GA1 and ASL deficiency. It had a similar frequency in patients with or without a movement disorder suggesting different pathophysiology. Thirteen patients (classic OAD: 3, UCD: 10) died during the study interval, ten of them during the initial metabolic crisis in the newborn period. Male patients with late-onset ornithine transcarbamylase deficiency were presumably overrepresented in the study population. CONCLUSIONS: Neurologic impairment is common in OAD and UCD, whereas the involvement of other organs (heart, liver, kidneys, eyes) follows a disease-specific pattern. The identification of unexpected chronic renal failure in GA1 and ASL deficiency emphasizes the importance of a systematic follow-up in patients with rare diseases.

Bioimpedance electrical spectroscopy in the first six months of life: some methodologic considerations.

OBJECTIVES: The objectives of this study were to evaluate the feasibility of using multifrequency bioelectrical impedance spectroscopy (BIS) in newborns and first-semester infants and to assess the influence of diverse methodologic and biological factors on BIS measurements. METHODS: We studied 69 infants of both sexes, from the first day after birth through age 6 mo. They were healthy term infants who had no congenital malformations and were born in a low-income, peri-urban neighborhood of Guatemala City. The design was based on serial, repeated BIS measurements. RESULTS: Overall mean values of extra- and intracellular resistance (mean+/-standard deviation) were 470.0+/-73.3 Omega and 604.6+/-179.2 Omega, respectively. We found statistical differences in both resistances in relation to advancing age and degree of movement while taking the measurements. With respect to repeat measurements by two separate observers, interobserver differences were a non-significant 7.6 Omega for both resistances. Restraining the infants and previous consumption of milk or formula had a significant effect on extracellular resistance measurements. The mean standard errors of measurement was 4.5 Omega for extracellular resistance and 73.9 Omega for intracellular resistance. CONCLUSION: The BIS technique is feasible in newborn and young children when physiologic and methodologic aspects are respected or controlled.