Pentanucleotide Repeat Insertions in RAI1 Cause Benign Adult Familial Myoclonic Epilepsy Type 8

Abstract

Background: Benign adult familial myoclonic epilepsy (BAFME) is an autosomal dominant disorder characterized by cortical tremors and seizures. Six types of BAFME, all caused by pentanucleotide repeat expansions in different genes, have been reported. However, several other BAFME cases remain with no molecular diagnosis.

Objectives: We aim to characterize clinical features and identify the mutation causing BAFME in a large Malian family with 10 affected members.

Methods: Long-read whole genome sequencing, repeat-primed polymerase chain reaction and RNA studies were performed.

Results: We identified TTTTA repeat expansions and TTTCA repeat insertions in intron 4 of the RAI1 gene that co-segregated with disease status in this family. TTTCA repeats were absent in 200 Malian controls. In the affected individuals, we found a read with only nine TTTCA repeat units and somatic instability. The RAI1 repeat expansions cause the only BAFME type in which the disease-causing repeats are in a gene associated with a monogenic disorder in the haploinsufficiency state (ie, Smith-Magenis syndrome [SMS]). Nevertheless, none of the Malian patients exhibited symptoms related to SMS. Moreover, leukocyte RNA levels of RAI1 in six Malian BAFME patients were no different from controls.

Conclusions: These findings establish a new type of BAFME, BAFME8, in an African family and suggest that haploinsufficiency is unlikely to be the main pathomechanism of BAFME. © 2023 International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Keywords: BAFME8; RAI1; TTTCA repeat insertions; haploinsufficiency; long-read sequencing.

Figure 1.

A large Malian pedigree with 10 affected and 17 unaffected family members, consistent with an autosomal dominant inheritance. Affected members are represented by black symbols. A square represents a male, a circle represents a female, and a diamond represents an individual with unspecified or unknown sex.

Figure 2.

Identification of disease-causing variant. (A) A schematic representation of exon 4, intron 4 and exon 5 of the RAI1 gene. The mutant allele has TTTTA repeat expansions and TTTCA repeat insertions. (B) RP-PCR for TTTTA repeat expansions and TTTCA repeat insertions. The primer sets P1-F, P2-TAAAA5 and P3anchor were designed to amplify the TTTTA repeats, whereas P2-TTTCA5, P1-R and P3anchor targeted the TTTCA repeats. The RP-PCR results showed that the affected individual had TTTTA repeat expansions and TTTCA repeat insertions, which were not found in unaffected individuals.

Figure 3.

RAI1 RNA studies. (A-B) Lymphoblastoid cells from six affected family members were analyzed by qRT-PCR to compare mRNA expression with that of six unaffected family members and three Malian unaffected controls. Two independent RAI1 Taqman probes were used for RNA expression (Assay 1- Hs00430773_m1; Assay 2- Hs01554690_m1). The Y-axis shows the relative fold change of RAI1 expression in affected, unaffected, and controls, normalized to Beta Actin (ACTB). Error bars indicate ±1SD. No significant difference in mRNA expression was found between the affected and unaffected family members and the Malian controls. (C) The BAM file for patient III-7 exhibited heterozygosity for the rs3818717 (C/T) variant, which was utilized for designing primers for the reverse transcription – PCR (RT-PCR). (D) Forward (RAI1-F) and reverse (RAI1-R) primers were designed between exons 3 and 6. (E) Agarose gel electrophoresis was performed on PCR products from six affected family members (Lane 3=III-7, Lane 4=III-21, Lane 5=III-23, Lane 6=IV-42, Lane 7=IV-3 and Lane 9=IV-37), six unaffected family members (Lane 8=IV-7, Lane 11=III-6, Lane 12=IV-15, Lane 13=IV-22, Lane 14=IV-17 and Lane 15=IV-20), three Malian controls (Lane 1=C1 and Lane 2=C2, Lane 10=C3) and negative control (Lane 16). No differences in band size were observed among the samples. (F) The electropherogram of the cDNA of patient III-7 showed heterozygosity for the rs3818717 (C/T). Four of six patients (III-7, IV-3, IV-37, IV-42) had the rs3818717 variant, confirming that the single band on the gel in Figure 3E represented the products of two alleles.