Sex-specific newborn screening for X-linked adrenoleukodystrophy

Abstract

Males with X-linked adrenoleukodystrophy (ALD) are at high risk for developing adrenal insufficiency and/or progressive leukodystrophy (cerebral ALD) at an early age. Pathogenic variants in ABCD1 result in elevated levels of very long-chain fatty acids (VLCFA), including C26:0-lysophosphatidylcholine (C26:0-LPC). Newborn screening for ALD enables prospective monitoring and timely therapeutic intervention, thereby preventing irreversible damage and saving lives. The Dutch Health Council recommended to screen only male newborns for ALD without identifying untreatable conditions associated with elevated C26:0-LPC, like Zellweger spectrum disorders and single peroxisomal enzyme defects. Here, we present the results of the SCAN (Screening for ALD in the Netherlands) study which is the first sex-specific newborn screening program worldwide. Males with ALD are identified based on elevated C26:0-LPC levels, the presence of one X-chromosome and a variant in ABCD1, in heel prick dried bloodspots. Screening of 71 208 newborns resulted in the identification of four boys with ALD who, following referral to the pediatric neurologist and confirmation of the diagnosis, enrolled in a long-term follow-up program. The results of this pilot show the feasibility of employing a boys-only screening algorithm that identifies males with ALD without identifying untreatable conditions. This approach will be of interest to countries that are considering ALD newborn screening but are reluctant to identify girls with ALD because for girls there is no direct health benefit. We also analyzed whether gestational age, sex, birth weight and age at heel prick blood sampling affect C26:0-LPC concentrations and demonstrate that these covariates have a minimal effect.

Keywords: ABCD1; C26:0-LPC; X-chromosome; adrenoleukodystrophy; dried bloodspots; heel prick; newborn screening; sex-specific.

FIGURE 1

The Dutch 4‐tier ALD newborn screening algorithm. In the Dutch 4‐tier ALD newborn screening algorithm, males with ALD are identified based on elevated C26:0‐LPC concentrations, the presence of one X‐chromosome and a variant in ABCD1, in heel prick dried bloodspots. The first tier consists of quantification of C26:0‐LPC using a high‐throughput flow injection analysis/tandem mass spectrometric (FIA–MS/MS) method. Samples that are above the cut‐off are screened in Tier 2 for the presence of one X‐chromosome. Next, C26:0‐LPC is re‐analyzed in Tier 3 using the more sensitive and specific high‐performance liquid chromatography (HPLC)‐MS/MS method. If C26:0‐LPC is above the cut‐off the ABCD1 gene is sequenced in Tier 4. Numbers in black and in parentheses indicate the number of samples analyzed in each tier. Numbers and percentages indicate the number and percentage of screen positive (green) and screen negative (red) samples in each tier.

FIGURE 2

Correlation of C26:0‐LPC with FIA–MS/MS and HPLC–MS/MS. Correlation plot showing the correlation between the “putative” C26:0‐LPC (+isobaric interferent(s)) concentration measured in Tier 1 with FIA–MS/MS (X‐axis) and the (accurate) C26:0‐LPC concentration measured in Tier 3 with HPLC–MS/MS (Y‐axis). Blue circles are Tier 1 positive, but Tier 3 negative samples and orange triangles are both Tier 1 and Tier 3 positive samples. The dashed lines represent the cut‐offs for Tier 1 (≥0.32 mol/L) and Tier 3 (≥0.150 mol/L), respectively.

FIGURE 3

Effect of age at blood sampling, gestational age and birth weight on C26:0‐LPC. (A–C) Correlation plots showing the correlation between age of the newborn at heel prick blood sampling (A), gestational age at birth (B), birth weight (C) and C26:0‐LPC concentrations by FIA–MS/MS. Blue dots indicate males and pink dots indicate females. (D) Correlation plot showing the correlation between gestational age at birth and birth weight for all boys in Tier 1 (blue), 249 boys in Tier 2 (green), 20 screen‐positive boys in Tier 3 (pink) and the four ALD newborns (yellow).