Recurrent ACADVL molecular findings in individuals with a positive newborn screen for very long chain acyl-coA dehydrogenase (VLCAD) deficiency in the United States

Abstract

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.

Keywords: ACADVL; Fatty acid oxidation; Inborn error of metabolism; NBS; Newborn screening; V243A; V283A; VLCAD; VLCADD.

Fig. 1

The flowchart describes the samples sent to our laboratory for Sanger sequence analysis of ACADVL over the course of seven years. Grey boxes indicate samples analyzed in this study. pNBS= presumptive newborn screen positive.

Fig. 2. Pathogenic variants detected in patients soliciting molecular analysis after a postive NBS suggestive of VLCADD

All pathogenic variants detected in our NBS or pNBS cohort are listed. The font size is proportional to the allele frequency. Novel pathogenic variants are shown in red. Black boxes indicate exons and grey lines indicate introns of ACADVL.

Fig. 3. Structural analysis of VLCAD variants

Enriched VUSs, described in Table 1, were plotted on the crystal structure of the VLCAD homodimer (PDB# 2UXW) where the two VLCAD moieties are indicated by yellow and blue ribbon structures. Red spheres indicate the location of VUSs. Stick diagrams indicate the location of the substrate (Palmitoyl-CoA) and the cofactor, flavin adenine dinucleotide (FAD). The location of the catalytic residue, Glu462, and the p.V283A pathogenic variant are shown by green spheres.

Fig. 4. Follow-up quantitative plasma acylcarnitine data for patients with a positive NBS suggestive of VLCADD

(A) Plasma C14:1 levels from the first quantitative plasma acylcarnitine analysis following a positive NBS are plotted in relation to the patient's molecular finding. For example, red dots indicate C14.1 values for patients harboring two pathogenic variants and blue dots indicate C14.1 values for patients with no variants in ACADVL. The number of unique patients in each genotypic class is shown (n) and specific genotypes are listed for a few notable cases- indicated by numbers. The asterisk indicates findings for case PAT0165 that is further explored in Fig. 2B. (B) Plasma C14:1 levels are shown for seven different follow-up analyses completed over the course of 3 years for PAT0165. Grey dotted lines indicate the upper limit of normal (95th percentile for unaffected individuals tested in our laboratory).

Fig. 5. Quantitative plasma acylcarnitine analysis in patients homozygous for the p.V283A allele

All available plasma C14:1 test results are shown in relation to the patient's age at sampling. Presumably many of these tests were completed when the patient was receiving treatment for VLCADD. The dotted grey line is representative of the normal range of plasma C14:1 levels in unaffected individuals.