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. 2019 Jan 3;104(1):35-44.
doi: 10.1016/j.ajhg.2018.11.005. Epub 2018 Dec 13.

GGC Repeat Expansion and Exon 1 Methylation of XYLT1 Is a Common Pathogenic Variant in Baratela-Scott Syndrome

Amy J LaCroix  1 , Deborah Stabley  2 , Rebecca Sahraoui  3 , Margaret P Adam  4 , Michele Mehaffey  1 , Kelly Kernan  1 , Candace T Myers  5 , Carrie Fagerstrom  6 , George Anadiotis  6 , Yassmine M Akkari  6 , Katherine M Robbins  2 , Karen W Gripp  2 , Wagner A R Baratela  7 , Michael B Bober  8 , Angela L Duker  8 , Dan Doherty  4 , Jennifer C Dempsey  1 , Daniel G Miller  1 , Martin Kircher  9 , Michael J Bamshad  4 , Deborah A Nickerson  10 ; University of Washington Center for Mendelian Genomics; Heather C Mefford  11 , Katia Sol-Church  12
Affiliations

GGC Repeat Expansion and Exon 1 Methylation of XYLT1 Is a Common Pathogenic Variant in Baratela-Scott Syndrome

Amy J LaCroix et al. Am J Hum Genet. .

Abstract

Baratela-Scott syndrome (BSS) is a rare, autosomal-recessive disorder characterized by short stature, facial dysmorphisms, developmental delay, and skeletal dysplasia caused by pathogenic variants in XYLT1. We report clinical and molecular investigation of 10 families (12 individuals) with BSS. Standard sequencing methods identified biallelic pathogenic variants in XYLT1 in only two families. Of the remaining cohort, two probands had no variants and six probands had only a single variant, including four with a heterozygous 3.1 Mb 16p13 deletion encompassing XYLT1 and two with a heterozygous truncating variant. Bisulfite sequencing revealed aberrant hypermethylation in exon 1 of XYLT1, always in trans with the sequence variant or deletion when present; both alleles were methylated in those with no identified variant. Expression of the methylated XYLT1 allele was severely reduced in fibroblasts from two probands. Southern blot studies combined with repeat expansion analysis of genome sequence data showed that the hypermethylation is associated with expansion of a GGC repeat in the XYLT1 promoter region that is not present in the reference genome, confirming that BSS is a trinucleotide repeat expansion disorder. The hypermethylated allele accounts for 50% of disease alleles in our cohort and is not present in 130 control subjects. Our study highlights the importance of investigating non-sequence-based alterations, including epigenetic changes, to identify the missing heritability in genetic disorders.

Keywords: 16p13 deletion; Desbuquois dysplasia; XYLT1; epigenetic; fragile site; methylation; repeat expansion; skeletal dysplasia; trinucleotide repeat.

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Figures

Figure 1
Figure 1
XYLT1 Exon 1 and Surrounding Region Schematic of XYLT1 (GRCh37/hg19, GenBank: NM_022166) exon 1 and surrounding region, noting the region upstream previously described as XYLT1-238, which contains the GGC repeat region; genomic coordinates notated. Methylation-specific PCR was performed on the bracketed segment of exon 1, which includes 48 CpG sites.
Figure 2
Figure 2
BSS Cohort (A) Pedigrees of 10 families with 12 affected individuals included in this study. XYLT1 variants are noted for those individuals tested. CH3, methylated allele; del, 16p13 deletion; ∗, individual underwent exome sequencing; hmz, homozygous. (B) Proband 06-1 as an infant. (C) Proband 04-1 at 9 years of age. Dysmorphic features include flat midface, depressed nasal bridge, epicanthal folds.
Figure 3
Figure 3
Methylated Alleles Are Not Transcribed (A) Sequence analysis of MS-PCR product from bisulfite-treated genomic DNA (top) of individual 02-1 confirms that the XYLT1 point mutation (c.319G>T) is located on the unmethylated allele in genomic DNA. CpG sites are notated (∗). Fibroblast cDNA sequence (bottom) confirms that only the unmethylated allele is transcribed. (B) RT-PCR of fibroblast cDNA from 04-1 shows that transcription of exon 1-3 is absent and levels of shortened transcripts that begin downstream are reduced compared to the control. (C) Sashimi Plot of RNA-seq data for 04-1 shows exon 1 is not transcribed and the exon 1-2 junction is absent. Overall transcription levels are severely reduced compared to the control.
Figure 4
Figure 4
Southern Blot of Four Affected Individuals and Available Family Members The expected wild-type fragment (2,800 bp) encompasses exon 1 and surrounding DNA including XYLT1-238. For each individual with one or two methylated alleles, a fragment 300–2,500 bp larger than expected was detected. Individual 07-4 has two methylated alleles; 04-1 and 06-1 each have one methylated and one deleted allele, and 02-1 is heterozygous for methylation. In families 07 and 04, there is further expansion when transmitted from unaffected mother to affected child. Fragments that are >400 bp larger than wild-type exhibit instability in somatic cells as indicated by the non-distinct bands seen; particularly in 02-1 and 04-1. Each panel is from the same Southern blot but with different exposure times (panel 1, 4 day; panel 2, 24 hr; panel 3, 4 day).
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