Remotely acting SMCHD1 gene regulatory elements: in silico prediction and identification of potential regulatory variants in patients with FSHD

Mayes et al. Human Genomics Remotely acting SMCHD1 gene regulatory elements: in silico prediction and identification of potential regulatory variants in patients with FSHD Mary B. Mayes 0 Taniesha Morgan 2 Jincy Winston 2 Daniel S. Buxton 0 Mihir Anant Kamat 0 3 Debbie Smith 1 Maggie Williams 1 Rebecca L. Martin 0 Dirk A. Kleinjan 4 David N. Cooper 2 Meena Upadhyaya 2 Nadia Chuzhanova 0 0 School of Science and Technology, Nottingham Trent University , Clifton Lane, Nottingham NG11 8NS , UK 1 Blood Sciences Department and Bristol Genetics Laboratory, Southmead Hospital , Westbury-on-Trym, Bristol BS10 5NB , UK 2 Institute of Medical Genetics, School of Medicine, Cardiff University , Heath Park, Cardiff CF14 4XN , UK 3 Present address: Cardiovascular Epidemiology Unit, Department of Public Health and Primary Care, University of Cambridge , Cambridge CB1 8RN , UK 4 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh EH4 2XU , UK Background: Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of the D4Z4 macro-satellite repeat on chromosome 4q35 (FSHD1) or mutations in the SMCHD1 gene (FSHD2). Recent studies have shown that the clinical manifestation of FSHD1 can be modified by mutations in the SMCHD1 gene within a given family. The absence of either D4Z4 contraction or SMCHD1 mutations in a small cohort of patients suggests that the disease could also be due to disruption of gene regulation. In this study, we postulated that mutations responsible for exerting a modifier effect on FSHD might reside within remotely acting regulatory elements that have the potential to interact at a distance with their cognate gene promoter via chromatin looping. To explore this postulate, genome-wide Hi-C data were used to identify genomic fragments displaying the strongest interaction with the SMCHD1 gene. These fragments were then narrowed down to shorter regions using ENCODE and FANTOM data on transcription factor binding sites and epigenetic marks characteristic of promoters, enhancers and silencers. Results: We identified two regions, located respectively ~14 and ~85 kb upstream of the SMCHD1 gene, which were then sequenced in 229 FSHD/FSHD-like patients (200 with D4Z4 repeat units <11). Three heterozygous sequence variants were found ~14 kb upstream of the SMCHD1 gene. One of these variants was found to be of potential functional significance based on DNA methylation analysis. Further functional ascertainment will be required in order to establish the clinical/functional significance of the variants found. Conclusions: In this study, we propose an improved approach to predict the possible locations of remotely acting regulatory elements that might influence the transcriptional regulation of their associated gene(s). It represents a new way to screen for disease-relevant mutations beyond the immediate vicinity of the specific disease gene. It promises to be useful for investigating disorders in which mutations could occur in remotely acting regulatory elements. Regulatory mutations; Regulatory elements; Hi-C data; SMCHD1; FSHD - Background Facioscapulohumeral muscular dystrophy (FSHD) is the most prevalent of the nine primary types of muscular dystrophy affecting adults and children; it is characterized by the weakness and atrophy of the facial and shoulder girdle muscle extending to the abdominal and lower limb muscle [1]. Two genetic loci are associated with the disease. The FSHD1 locus maps to 4q35 [2] and accounts for 95 % of clinical diseases in an FSHD context [1, 3]. A second FSHD locus, FSHD2, exists and is phenotypically indistinguishable from FSHD1 [2]. FSHD1 patients harbour a large deletion in the polymorphic D4Z4 macro-satellite repeat array at 4q35 and invariably present with 1–10 repeats whereas nonaffected individuals possess 11–150 repeats. Each 3.3-kb D4Z4 unit contains a double homeobox 4 (DUX4) gene that, among others, is transcriptionally activated on contraction of the 4q35 repeat array as a consequence of the induction of chromatin remodelling of the 4qter region. A number of 4q subtelomeric sequence variants are now recognized, although FSHD1 only occurs in association with ‘permissive’ haplotypes, each of which is associated with a polyadenylation signal located immediately distal of the last D4Z4 unit [4]. Approximately 5 % of FSHD patients lack a contraction of the D4Z4 array, and the disease aetiology has been ascribed to a putative FSHD2 locus. Whole-exome sequencing identified the SMCHD1 gene as the causative agent at the FSHD2 locus [5]. In FSHD2 families, the disease exhibits a more complex digenic inheritance because mutations in the chromosome-18-located SMCHD1 gene segregate independently from the FSHD-permissive 4q haplotype [5]. SMCHD1 is a member of a condensing/ cohesion family of chromatin compact complexes that bind to the D4Z4 array [6]. However, not all FSHD2 patients can be explained by the lack (...truncated)