Loss of function of the retinoid-related nuclear receptor (RORB) gene and epilepsy

Abstract

Genetic generalized epilepsy (GGE), formerly known as idiopathic generalized epilepsy, is the most common form of epilepsy and is thought to have predominant genetic etiology. GGE are clinically characterized by absence, myoclonic, or generalized tonic-clonic seizures with electroencephalographic pattern of bilateral, synchronous, and symmetrical spike-and-wave discharges. Despite their strong heritability, the genetic basis of generalized epilepsies remains largely elusive. Nevertheless, recent advances in genetic technology have led to the identification of numerous genes and genomic defects in various types of epilepsies in the past few years. In the present study, we performed whole-exome sequencing in a family with GGE consistent with the diagnosis of eyelid myoclonia with absences. We found a nonsense variant (c.196C>T/p.(Arg66*)) in RORB, which encodes the beta retinoid-related orphan nuclear receptor (RORβ), in four affected family members. In addition, two de novo variants (c.218T>C/p.(Leu73Pro); c.1249_1251delACG/p.(Thr417del)) were identified in sporadic patients by trio-based exome sequencing. We also found two de novo deletions in patients with behavioral and cognitive impairment and epilepsy: a 52-kb microdeletion involving exons 5-10 of RORB and a larger 9q21-microdeletion. Furthermore, we identified a patient with intellectual disability and a balanced translocation where one breakpoint truncates RORB and refined the phenotype of a recently reported patient with RORB deletion. Our data support the role of RORB gene variants/CNVs in neurodevelopmental disorders including epilepsy, and especially in generalized epilepsies with predominant absence seizures.

Figure 1

Inherited nonsense RORB variant segregating in a family with GGE with eyelid myoclonia (family 1). (a) Pedigree with GGE. DNA numbers are indicated under each corresponding family member. Empty symbols represent unaffected individuals. ILS, intermittent light stimulation; PPR, photoparoxysmal response. Patients 19–25 have been phenotyped and collected after the initial report on the family. Note that the variant was not found in neither individual 1 nor 2, which – together with the genetic exclusion of non-paternity (data not shown) – suggest germline mosaicism. (b) DNA sequence traces (both strands) corresponding to the heterozygous c.196C>T variant (arrow) in exon 3 of the RORB gene (patient no. 4, top) and to a wild-type unaffected individual (family member no. 16, bottom). (c) Predicted mutant p.Arg66* RORβ protein. The different domains of the wild-type RORβ protein (Genbank NP_008845) (top) and the corresponding truncated mutant protein (bottom) are schematically represented. Numbers at the top of each protein indicate amino-acid positions from the N-terminus (left) to the C-terminus (right). DBD, DNA-binding domain; LBD, ligand-binding domain; AF2, activation function domain. (d) cDNA sequence traces showing the presence of the heterozygous c.196C>T variant (arrow) in RORB transcripts extracted from lymphoblastoid cell lines of patients 4 (top) and 13 (bottom) and of control individual no. 16 (middle). No difference was observed upon treatment with puromycin (data not shown).

Figure 2

The de novo 9q21 microdeletions found in epileptic patients converge on RORB. (a) aCGH profile of the 8.5-Mb microdeletion event (from 70 984 481 to 79 549 501 nt, hg19 coordinates) detected in patient 9A1117. (b) aCGH profile of the 52-kb de novo partial microdeletion event (from 77 261 322 to 77 313 598 nt, hg19 coordinates) detected in patient GE0705. (c) Map of all microdeletions comprising RORB reported to date in epileptic patients. Note the existence of two de novo truncating events on either 5′ and 3′ sides of RORB, in addition to the partial microdeletion of exons 5–10 reported here (patient GE0705).

Figure 3

Conventional (RHG) karyotype and fluorescent in situ hybridization (FISH) showing the complex chromosomal rearrangement in patient 9A1117. (Top) Conventional karyotype. (a) Small supernumerary ring chromosome (arrow) (b) Derivative chromosome 9 from a t(9;21) translocation replacing a normal chromosome 21 (arrow). (Bottom) FISH analysis. (c–e) FISH with RP11-404E6 (9q21.3) probe in TRITC (red) and 9pter probe (Cytocell) in FITC (green) showing the normal chromosome 9 (chr9), the 9q13q21.13 deletion (del9q) lacking the RP11-404E6 signal. Note that RP11-404E6 probe cross-hybridizes on chromosome 14. Additional rearrangements included (d) isochromosome 9p i(9p) showing two 9pter green signals and (e) a derivative 9 chromosome from a t(9;21) translocation der(9) showing a single 9pter green signal. (f) FISH with centromeric chromosome 9 probe in Texas Red and 9pter probe in FITC (green) (Cytocell) showing the supernumerary small ring chromosome 9r(9) with a centromeric red signal but no telomeric signal. A full color version of this figure is available at the European Journal of Human Genetics journal online.

Figure 4

Mapping of seq[GRch37]t(9;19)(q21;q12)dn breakpoint in case DK8393. (a) Mate-pair mapping place the chromosome 9 breakpoint (vertical arrow) within the first intron of RORB, between two oppositely oriented clusters of 19 mate-pair reads (b). (c) Sanger sequencing with forward (F) and reverse primer (R) pairs reveal two small duplications (blue bases in italics) associated with the chromosome 9 breakpoint at nt 77174984 (arrow). The black bars illustrate the BLAT results of the forward and reverse sequences in the UCSC Genome Browser. The amino acids (AA) encoded by the first three exons are shown on the top. If the gene could be transcribed despite the loss of the highly conserved and complex promoter (unlikely, see Supplementary Figure S3), the use of the next start codon (green arrow) distal to the translocation breakpoint (in exon 3) will result in the deletion of the first 74 AA of RORB (blue box). A full color version of this figure is available at the European Journal of Human Genetics journal online.