400 - Mosaic X-Linked Adrenoleukodystrophy in Males Identified by Newborn Screening and Next-Generation Sequencing.

Publication Number: 400.5579

Alexandra C. Keefe, Seattle Children's, Seattle, WA, United States; Dana Jensen, Seattle Children's, Seattle, WA, United States; Meranda M.. Pham, Seattle Children's Research Institute Center for Developmental Biology and Regenerative Medicine, Seattle, WA, United States; Natalie YT. Au, Seattle Children's Research Institute, Seattle, WA, United States; Monica Penon-Portmann, University of Washington School of Medicine, Seattle, WA, United States; Emily Shelkowitz, Seattle Children's, Seattle, WA, United States; Renee C. Bend, PreventionGenetics, Salt Lake City, UT, United States; Michelle M. Morrow, GeneDX LLC, Gaithersburg, MD, United States; Paul Kruszka, GeneDx, Alexandria, VA, United States; Divya Vats, Southern California Permanente Medical Group, Los Angeles, CA, United States; Bianca E. Russell, UCLA, Los Angeles, CA, United States; Ahna M. Rabani, University of California, Los Angeles David Geffen School of Medicine, Los Angeles, CA, United States; Lauren O'Grady, MassGeneral Hospital for Children, Boston, MA, United States; Ethan D. Sperry, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States; Rebecca Tryon, University of Minnesota Masonic Children's Hospital, Saint Paul, MN, United States; Troy C. Lund, University of Minnesota Medical School, MINNEAPOLIS, MN, United States; Angela Sun, Seattle Children's, Coos Bay, OR, United States; James T. Bennett, Sea, Seattle, WA, United States

Background: Somatic mosaicism produces genetic differences between cells and is an underrecognized contributor to phenotypic variability. X-linked adrenoleukodystrophy (XALD) is a neurometabolic disorder caused by pathogenic variants in ABCD1. Factors contributing to XALD phenotypic variability are unclear, and, although peroxisomal dysfunction is central, the mechanisms contributing to disease severity remain an area of active inquiry. Because of advances in treatment, 44 states now include XALD on newborn screening (NBS), yielding a large, pre-symptomatic cohort of male children diagnosed with XALD.

Objective: We hypothesized that somatic mosaicism for pathogenic ABCD1 variants might be present in pre-symptomatic children with elevated very long chain fatty acids (VLCFA) identified on NBS, and set out to estimate the incidence of this potential phenotypic modifier.

Design/Methods: A proband (mXALD-1) was identified through elevated NBS, and follow up DNA sequencing demonstrated the presence of a likely pathogenic variant (c.1988T>C) in ABCD1 in ~82% of reads (Table 1), suggesting somatic mosaicism. To determine the tissue distribution of this mosaic variant, 6 tissues were obtained, and genomic and cell-free DNA were isolated to determine the variant allele fraction (VAF) across multiple tissues. We additionally contacted several commercial sequencing laboratories and state NBS programs to find other males with mosaic ABCD1 variants.

Results: Our proband (mXALD-1) and 5 other males were identified to have mosaic XALD. In mXALD-1, we found that the variant allele fraction (VAF) ranged from 66%-82%, and was lowest in the skin (Figure 1). These results are consistent with an asymptomatic male individual with a post-zygotic mosaic variant in ABCD1 manifesting as biochemically and molecularly confirmed XALD. In 4 additional male individuals (mXALD-2, 3, 4, 5) clinical information was available (Table 1). All 5 males are reported as pre/asymptomatic with normal adrenal screening labs and stable MRIs. Cumulative data from two large labs found that in total 1.8% (4/227) of males with P/LP variants in ABCD1 were reported to have VAFs that less than 100%, therefore we estimate the frequency of mosaic XALD within a biochemically and molecularly confirmed XALD cohort to be 1.8% or higher (Table 2).

Conclusion(s): X-linked mosaicism is rare and has only been published in two other X-linked conditions, to our knowledge. Our study suggests that mosaicism in XALD, and likely other X-linked genetic disorders is underrecognized, and adds to the growing body of literature detecting mosaicism in non-neoplastic, developmental disease.

Table 1. Comparison of ABCD1 mosaicism in 5 male individuals identified through newborn screening.
Note: NBS = newborn screening. C26, C24, C22 = very long-chain fatty acids. RR = reference range. NGS = next-generation sequencing. “Sanger” = Sanger sequencing. Clinical “cutoff” for abnormal values and/or RR provided, as reported by clinical sequencing laboratories. Labs 1 and 2 indicate samples were sent for verification at a second lab but are not necessarily obtained from identical labs or the labs listed in Table 2. *Areas of non-enhancing T2 hyperintensity, T1 hypointensity in the periventricular white matter along the frontal horns. **mild hyperintensity involving the bilateral parietal occipital periventricular white matter on axial FLAIR images, score 1. Tiny foci of FLAIR hyperintensities noted involving the central part of the bilateral parietal occipital white matter, score 1. ***Suspected minor T2/FLAIR hyperintensities. Did not initially identify mosaic ABCD1 variant, whole genome sequencing was sent at 1 day old for non-ALD indications, and resulted prior to knowledge of abnormal NBS. The lab later able to identify presence of variant, but only after Sanger sequencing of ABCD1 performed through a second lab. ****non-ALD related clinical features include arthrogryposis.

Figure 1. Comparison of levels of ABCD1 1988T>C mosaicism in multiple tissues of proband (mXALD-1).
A) Various tissue samples obtained, and proposed germ layer of origin. Graph shows the comparison of the variant allele fraction (VAF) from genomic DNA in 6 different tissues including blood, CSF (pellet), urine (pellet), tonsil, skin, and buccal sample and cell free DNA derived from plasma and urine. Representative data presented as individual values of n=4 replicates. Error bars represent the min/max “Total Error” of the mutant reads based on 4 merged replicates as provided by Quantasoft software. VAF calculated by mutant concentration divided by total concentration.

Table. 2. Frequency of mosaicism detected in male individuals with Pathogenic or Likely Pathogenic ABCD1 variants identified through Next-Generation Sequencing.
Mosaicism was defined as a male who presented with elevations of VLCFA and was found to have mosaic pathogenic or likely pathogenic (P/LP) ABCD1 variants. The number of males with mosaic P/LP ABCD1 variants was divided by the total number of males with identified P/LP ABCD1 variants found in large NGS-sequencing databases (includes de novo and known familial variants). Variants of uncertain significance were excluded. Variants were verified to confirm 4 independent variants. Three of the 4 variants are included in Table 1. For the remaining variant, we obtained the level of mosaicism but the ordering provider was not reachable to provide clinical information.

Table 1. Comparison of ABCD1 mosaicism in 5 male individuals identified through newborn screening.
Note: NBS = newborn screening. C26, C24, C22 = very long-chain fatty acids. RR = reference range. NGS = next-generation sequencing. “Sanger” = Sanger sequencing. Clinical “cutoff” for abnormal values and/or RR provided, as reported by clinical sequencing laboratories. Labs 1 and 2 indicate samples were sent for verification at a second lab but are not necessarily obtained from identical labs or the labs listed in Table 2. *Areas of non-enhancing T2 hyperintensity, T1 hypointensity in the periventricular white matter along the frontal horns. **mild hyperintensity involving the bilateral parietal occipital periventricular white matter on axial FLAIR images, score 1. Tiny foci of FLAIR hyperintensities noted involving the central part of the bilateral parietal occipital white matter, score 1. ***Suspected minor T2/FLAIR hyperintensities. Did not initially identify mosaic ABCD1 variant, whole genome sequencing was sent at 1 day old for non-ALD indications, and resulted prior to knowledge of abnormal NBS. The lab later able to identify presence of variant, but only after Sanger sequencing of ABCD1 performed through a second lab. ****non-ALD related clinical features include arthrogryposis.

Figure 1. Comparison of levels of ABCD1 1988T>C mosaicism in multiple tissues of proband (mXALD-1).
A) Various tissue samples obtained, and proposed germ layer of origin. Graph shows the comparison of the variant allele fraction (VAF) from genomic DNA in 6 different tissues including blood, CSF (pellet), urine (pellet), tonsil, skin, and buccal sample and cell free DNA derived from plasma and urine. Representative data presented as individual values of n=4 replicates. Error bars represent the min/max “Total Error” of the mutant reads based on 4 merged replicates as provided by Quantasoft software. VAF calculated by mutant concentration divided by total concentration.

Table. 2. Frequency of mosaicism detected in male individuals with Pathogenic or Likely Pathogenic ABCD1 variants identified through Next-Generation Sequencing.
Mosaicism was defined as a male who presented with elevations of VLCFA and was found to have mosaic pathogenic or likely pathogenic (P/LP) ABCD1 variants. The number of males with mosaic P/LP ABCD1 variants was divided by the total number of males with identified P/LP ABCD1 variants found in large NGS-sequencing databases (includes de novo and known familial variants). Variants of uncertain significance were excluded. Variants were verified to confirm 4 independent variants. Three of the 4 variants are included in Table 1. For the remaining variant, we obtained the level of mosaicism but the ordering provider was not reachable to provide clinical information.