Characterization of variants of uncertain significance in isovaleryl-CoA dehydrogenase identified through newborn screening: An approach for faster analysis

Abstract

Introduction: Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS.

Methods: An IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genome-editing strategy targeting IVD exons 2-3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity.

Results: c.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p.Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants.

Conclusions: We have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs.

Keywords: Isovaleric acidemia; Isovaleryl-CoA dehydrogenase; Newborn screening; Organic acidemia; Variants of uncertain significance.

Figure 1.. Targeted deletion of IVD exons 2-3 by using CRISPR/Cas9 genome-editing.

(A) Location of sgRNA and genotyping primers used to generate and screen for IVD null alleles. Key: Blue box, IVD exons with intervening introns; pink rectangles, sgRNA intron 1 and 3 sites; directional arrows, genotyping primers. (B) Genotyping PCR assays identifying 3 IVD null clonal HEK293T lines through presence of deletion (top) and/or inversion (second from top) breakpoint-PCR bands and lack of intact sgRNA sites (bottom). * indicates non-specific band. (C) Genomic copy number of IVD as ascribed by the ratio of IVD to RPP30 by ddPCR. Note that HEK293T is likely pseodotriploid for the RPP30 locus and that technical variability led to lower-than-expected exon 1 estimates. Bars represent absolute copy number ratio of IVD exons across the locus with Poisson distribution 95% confidence intervals.

Figure 2.. Protein and enzymatic activity of control and IVD null HEK293T lines.

(A) Western blot for IVDH confirms its absence in clonal lines 4-13, 4-16 and 4-3 and reduction in line 4-20. (B) Enzymatic activity of IVDH (isovaleryl-CoA as substrate) and (C) and MCAD (octanoyl-CoA as substrate). IVDH activity assays were done in triplicates and octanoyl-CoA assays were done in duplicates. Means and standard deviations were calculated. Data were analyzed using paired T-test. ****p<0.0001, *** p<0.001, **p<0.01, ns = no statistical difference.

Figure 3.. Protein expression and enzymatic activity assays for IVA patient-derived fibroblasts.

(A) Western blot of IVA patient and control fibroblasts for detection of IVDH and GAPDH (B) Enzymatic activity assay for IVDH (isovaleryl-CoA as substrate). (C) Enzymatic ETF assay for MCAD (octanoyl-CoA substrate). IVDH activity assays were done in triplicates and octanoyl-CoA assays were done in duplicates. Means and standard deviations were calculated. Data were analyzed using unpaired T-test. ***p<0.001, ns = no statistical difference.

Figure 4.. Expression and enzymatic activity of IVD VUS in an IVD null HEK293T cell line.

(A) Detection of IVDH protein levels by western blot to evaluate protein stability of control and variants expressed from cDNA vectors with GAPDH an internal loading standard. (B) Activity of IVDH and (C) MCAD using the ETF fluorescence reduction assay. IVDH activity assays were done in triplicates and octanoyl-CoA assays were done in duplicates. Means and standard deviations were calculated. Data were analyzed using unpaired t test showing significant difference, ****p<0.0001, ns = no statistical difference.

Figure 5.. Ribbon representation of the three dimensional structure of an IVDH monomer using published atomic coordinates, PDB: 1IVH [23].

iC5-CoA is modeled in the active site in place of the CoA-persulfide published. The A and B views, at near right angle, depict the position of the backbone carbon atom, in blue, of amino acid residues replaced due to missense mutations in the IVD gene in patients with IVA listed in Table 1. The c.1179del cause a frameshift and a Leu365Phe mutation, position marked in red, and premature termination and leading to the loss of most of the a-helix J and a-helix K and undetectable IVDH antigen band, see Figure 4.