Abstract
Due to newborn screening for X-linked adrenoleukodystrophy (ALD), and the use of exome sequencing in clinical practice, the detection of variants of unknown significance (VUS) in the ABCD1 gene is increasing. In these cases, functional tests in fibroblasts may help to classify a variant as (likely) benign or pathogenic. We sought to establish reference ranges for these tests in ALD patients and control subjects with the aim of helping to determine the pathogenicity of VUS in ABCD1. Fibroblasts from 36 male patients with confirmed ALD, 26 healthy control subjects and 17 individuals without a family history of ALD, all with an uncertain clinical diagnosis and a VUS identified in ABCD1, were included. We performed a combination of tests: (i) a test for very-long-chain fatty acids (VLCFA) levels, (ii) a D3-C22:0 loading test to study the VLCFA metabolism and (iii) immunoblotting for ALD protein. All ALD patient fibroblasts had elevated VLCFA levels and a reduced peroxisomal ß-oxidation capacity (as measured by the D3-C16:0/D3-C22:0 ratio in the D3-C22:0 loading test) compared to the control subjects. Of the VUS cases, the VLCFA metabolism was not significantly impaired (most test results were within the reference range) in 6/17, the VLCFA metabolism was significantly impaired (most test results were within/near the ALD range) in 9/17 and a definite conclusion could not be drawn in 2/17 of the cases. Biochemical studies in fibroblasts provided clearly defined reference and disease ranges for the VLCFA metabolism. In 15/17 (88%) VUS we were able to classify the variant as being likely benign or pathogenic. This is of great clinical importance as new variants will be detected.
Highlights
The most common peroxisomal disorder, X-linked adrenoleukodystrophy (ALD), is characterized by mutations in the ABCD1 gene [1,2]
Pathological mutations cause a dysfunction of the ALD protein (ALDP) [3], resulting in the impaired peroxisomal ßoxidation of very-long-chain fatty acids (VLCFA; ≥C22:0) [4]
The fibroblasts of healthy controls subjects were collected for research purposes after the informed consent and the approval of the local Institutional Review Board
Summary
The most common peroxisomal disorder, X-linked adrenoleukodystrophy (ALD), is characterized by mutations in the ABCD1 gene [1,2]. Clinical evaluation (which should be normal in a newborn with ALD) and repeated plasma VLCFA or C26:0lysophosphatidylcholine (C26:0-lysoPC) testing can be helpful, but some uncertainty may remain This is especially common for patients without a family history of disease or plasma VLCFA levels that are elevated between the upper limit of the reference range but below the lower limit of the disease range—here referred to as the “gray zone”. In these cases, additional biochemical and functional testing in skin fibroblasts may help to determine the pathogenicity of a VUS. This study will contribute to a clearer and more uniform laboratory diagnostic process for ALD
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