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

Abstract

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.

Keywords: Cobalamin disorders; Homocystinuria; Massive parallel sequencing; Methylmalonic aciduria.

Fig. 1

Functional analysis of c.1084-10G > A identified in the MUT gene. Splicing pattern observed in the minigene construct bearing the empty vector (pSPL3), normal minigene (c.1084-10G), and mutant vector c.1084-10G. The figure shows the splicing scores obtained using the Human Splicing Finder (www.umd.be/HSF3/). The ESE sequences were obtained using RESCUE-ESE software (www.genes.mit.edu/burgelab/rescue-ese)

Fig. 2

Functional rescue of mitochondrial dysfunction. Bioenergetic profiles of SUCLA2 in patient P23 fibroblasts transduced with a lentiviral plasmid bearing wild-type SUCLA2 cDNA (Lv Wt) or a null construct (Lv Co), and control-derived fibroblasts. Experiments were performed using a Seahorse XF device. A Oxygen consumption rates of control and patient fibroblasts were measured in DMEM with galactose (1 g/L) instead of glucose, and upon the subsequent addition of a 6 μM oligomycin, b 50 μM FCCP, c 1 μM rotenone or, d 1 μM antimycin. B Basal and maximum respiration (Rmax) were calculated for each situation. C Oligomycin-sensitive respiration (OSR) was calculated as the difference between the basal respiration and the oxygen consumption rate measured as described in (C), after the addition of 6 μM oligomycin. The results reflect the mean of three biological repetitions. Control values are the mean for two different control cell lines. (*p < 0.05; **p < 0.01; ***p < 0.001 [Student t test])

Fig. 3

Structure and conservation analysis of SUCLA2 variants (p.G326R, p.I312T). For each variant, p.G326R (a) or p.I312T (b), the location of the wild-type amino acid (red) and its network of interactions (light orange) are shown. Close residue-residue contacts are drawn with dashed lines. The “phosphate shuttle” loop is marked in dark orange [44]. The conservation pattern for each variant is represented below the structural models. The size of the letters reflects the degree of conservation