genes G C A T T A C G G C A T Article Biochemical Studies in Fibroblasts to Interpret Variants of Unknown Significance in the ABCD1 Gene Stephanie I. W. van de Stadt 1 , Petra A. W. Mooyer 2 , Inge M. E. Dijkstra 2 , Conny J. M. Dekker 2 , Divya Vats 3 , Moin Vera 3 , Maura R. Z. Ruzhnikov 4 , Keith van Haren 4 , Nelson Tang 5 , Klaas Koop 6 , Michel A. Willemsen 7 , Joannie Hui 8 , Frédéric M. Vaz 2 , Merel S. Ebberink 2 , Marc Engelen 1 , Stephan Kemp 1,2, * ,† and Sacha Ferdinandusse 2,†   Citation: van de Stadt, S.I.W.; Mooyer,P.A.W.; Dijkstra, I.M.E.; Dekker, C.J.M.; Vats, D.; Vera, M.; Ruzhnikov, M.R.Z.; van Haren, K.; Tang, N.; Koop, K.; et al. Biochemical Studies in Fibroblasts to Interpret Variants of Unknown Significance in the ABCD1 Gene. Genes 2021, 12, 1930. https://doi.org/10.3390/ genes12121930 Academic Editor: Ewa Piotrowska Received: 26 October 2021 Accepted: 28 November 2021 Published: 30 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Pediatric Neurology, Emma Children’s Hospital, Amsterdam University Medical Centers, Amsterdam Neuroscience, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; s.i.vandestadt@amsterdamumc.nl (S.I.W.v.d.S.); m.engelen@amsterdamumc.nl (M.E.) 2 Laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Amsterdam UMC, Amsterdam Gastroenterology Endocrinology Metabolism, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; p.mooyer@amsterdamumc.nl (P.A.W.M.); i.m.dijkstra@amsterdamumc.nl (I.M.E.D.); c.j.dekker@amsterdamumc.nl (C.J.M.D.); f.m.vaz@amsterdamumc.nl (F.M.V.); m.s.ebberink@amsterdamumc.nl (M.S.E.); s.ferdinandusse@amsterdamumc.nl (S.F.) 3 Regional Metabolic Clinic, Department of Medical Genetics, Southern California Permanente Medical Group, Los Angeles, CA 90027, USA; DIVYA.VATS@kp.org (D.V.); MOIN.U.VERA@kp.org (M.V.) 4 Departments of Neurology and Neurological Sciences and Pediatrics, Stanford, CA 94305, USA; ruzhnikov@stanford.edu (M.R.Z.R.); kpv@stanford.edu (K.v.H.) 5 Department of Chemical Pathology, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China; nelsontang@cuhk.edu.hk 6 Department of Metabolic Diseases, Wilhelmina Children’s Hospital, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands; K.Koop@umcutrecht.nl 7 Department of Pediatric Neurology, Radboud University Medical Centre, 6525 GA Nijmegen, The Netherlands; Michel.Willemsen@radboudumc.nl 8 Department of Pediatrics & Adolescent Medicine, Hong Kong Children’s Hospital, Hong Kong, China; joanniehui@yahoo.com.hk * Correspondence: s.kemp@amsterdamumc.nl † Co-senior authors. 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 D 3 -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 D 3 -C16:0/D 3 -C22:0 ratio in the D 3 -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. Keywords: peroxisomal disorder; fibroblasts; adrenoleukodystrophy; variants of unknown signifi- cance; newborn screening Genes 2021, 12, 1930. https://doi.org/10.3390/genes12121930 https://www.mdpi.com/journal/genes