Intronic ATTTC repeat expansions in STARD7 in familial adult myoclonic epilepsy linked to chromosome 2

Abstract

Familial Adult Myoclonic Epilepsy (FAME) is characterised by cortical myoclonic tremor usually from the second decade of life and overt myoclonic or generalised tonic-clonic seizures. Four independent loci have been implicated in FAME on chromosomes (chr) 2, 3, 5 and 8. Using whole genome sequencing and repeat primed PCR, we provide evidence that chr2-linked FAME (FAME2) is caused by an expansion of an ATTTC pentamer within the first intron of STARD7. The ATTTC expansions segregate in 158/158 individuals typically affected by FAME from 22 pedigrees including 16 previously reported families recruited worldwide. RNA sequencing from patient derived fibroblasts shows no accumulation of the AUUUU or AUUUC repeat sequences and STARD7 gene expression is not affected. These data, in combination with other genes bearing similar mutations that have been implicated in FAME, suggest ATTTC expansions may cause this disorder, irrespective of the genomic locus involved.

Fig. 1

Identification of an expanded pentameric ATTTC repeat causing FAME2. a Estimated sizes of the AAATG repeats in two affected individuals from Family 1 (red, orange), one from Family 3 (brown) and three affected individuals from Family 19 (blue, green, purple), compared to 69 individuals without FAME using TruSeq Nano (grey) or KAPA Hyper (tan) library preparation. Left panel shows empirical cumulative distribution functions from exSTRa panel while the right panel shows the estimated repeat size by Expansion Hunter (the sum of both alleles suggests repeat sizes of 0.75–2.3 kb). Data underlying this part of the figure are available in Source Data. b WGS data from two individuals in Family 1 and one from Family 3 show reads suggesting expansion of AAAAT and insertion of AAATG repeats in the chr2 linkage interval. c Upper section shows the location of the repeat in the context of chr2. The approximate location of the FAME2 minimal linkage interval is shown above the ideogram with two blue arrow heads. The STARD7 gene is on the reverse chromosome strand and the endogenous AAAAT repeat is found in the first intron of the gene. Schema in the lower section shows the primers used in the RP-PCR to detect the ATTTT “3′ assay” and ATTTC “5′ assay” expanded repeats, respectively. d Example results of the RP-PCR 5′ assay obtained in an individual negative for the ATTTC insert (top panel) and in an individual affected by FAME, positive for the ATTTC repeat insertion (bottom panel). Full screening results are provided in Supplementary Data 1. e Summary of 184 individuals from 22 families tested with the RP-PCR assay. Individuals under category (+) tested positive for the ATTTC repeat and individuals under category (−) tested negative for the repeat

Fig. 2

Long-read sequencing identifies the structure of the AAATG/AAAAT repeat expansion in intron one of STARD7. a Upper panel shows CCS reads from one member of Family 1 (IV-98) mapped to GRCh38. A read with a 3261 bp insert (blue arrow) which contains both AAATG and AAAAT sequences and flanking sequences that map to either side of the endogenous AAAAT repeat is present. Lower panel shows the component subreads mapped to the same region. b Top panel shows combined PacBio and nanopore reads mapped to hg38, following correction with Canu v1.7, with base pair mismatches in the reads masked for clarity. For Family 1 IV-98 (upper panel), a 2154 bp insert is shown (black arrow) on IGV; however, the read sequence contains a 3672 bp combined AAATG/AAAAT repeat insertion. Lower panel shows a nanopore read in one individual from Family 5 (II-37) with a 1705 bp insert on IGV (blue arrow), however the read contains a 4645 bp combined AAATG/AAAAT repeat insertion. Complete sequences for all reads that span the repeat expansion are included in Supplementary Data

Fig. 3

Expression of STARD7 is unaltered in patient-derived skin fibroblasts. a Graph shows average STARD7 expression by relative standard curve quantitative PCR (qPCR) normalised to HPRT1 expression in fibroblast cell lines from four control donors (white bars) and four affected male individuals from Family 1 (IV-52, V-118, V-124 and V-161; black bars). Individual data points overlay the each bar. Tests for significance were performed using Student’s two-tailed t-test assuming unequal variances (p = 0.50 Exon 1–2; p = 0.85 Exon 3–4). b Western blot of STARD7 protein compared to β-actin on the same blot of fibroblasts from the same four individuals from Family 1 as assayed by qPCR and two male control donors (C1 and C2). Data underlying this figure are available in Source Data

Fig. 4

Expression patterns of ATTTC repeat genes in brain, skin fibroblast and lymphoblastoid cell lines. The heatmap shows relative gene expression expressed as transcripts per kilobase per million mapped reads (TPM) based on the colour scale as shown. Data and image downloaded from the GTEx Portal https://www.gtexportal.org