Evaluation of multiple putative risk alleles within the 15q13.3 region for genetic generalized epilepsy

Highlights

• Genetic analysis of patients with generalized epilepsy was performed.

• Three missense variants in CHRNA7 and CHRFAM7A were found.

• The common 2-bp deletion in CHRFAM7A was not enriched in patients.

• Missense variants contribute to complex inheritance in generalized epilepsy.

Abstract

The chromosome 15q13.3 region has been implicated in epilepsy, intellectual disability and neuropsychiatric disorders, especially schizophrenia. Deficiency of the acetylcholine receptor gene CHRNA7 and the partial duplication, CHRFAM7A, may contribute to these phenotypes and we sought to comprehensively analyze these genes in genetic generalized epilepsy. We analyzed using DHPLC, Sanger sequencing and long range PCR, 174 probands with genetic generalized epilepsy with or without intellectual disability or psychosis, including 8 with the recurrent 15q13.3 microdeletion. We searched CHRNA7 and CHRFAM7A for single sequence variants, small copy number variants, and the common 2-bp deletion in CHRFAM7A. We identified two novel and one reported missense variants. The common 2-bp deletion was not enriched in patients compared to controls. Our data suggest that missense mutations in CHRNA7 contribute to complex inheritance in genetic generalized epilepsy in a similar fashion to the 15q13.3 microdeletion. They do not support a pathogenic role for the common 2-bp CHRFAM7A deletion.

Introduction

CHRNA7, at chromosome 15q13.3, encodes the alpha7 subunit of the neuronal nicotinic acetylcholine receptor and has long been a candidate gene for neuropsychiatric disorders. Linkage analyses and neurophysiological studies initially associated the CHRNA7 region with schizophrenia (Freedman et al., 1997). Definitive evidence for involvement of this region in epilepsy and neuropsychiatric disorders emerged with robust demonstration of association between the 15q13.3 microdeletion (del15q13.3) and intellectual disability (ID) in 2008 (Sharp et al., 2008); then subsequently with autism spectrum disorder (ASD), schizophrenia and genetic generalized epilepsy (GGE) (Helbig et al., 2009, Miller et al., 2009, Shinawi et al., 2009, Stefansson et al., 2009). Among the associated heterogeneous phenotypes, GGE is most frequent with over 1% of patients carrying del15q13.3 (Dibbens et al., 2009, Helbig et al., 2009). In those with both intellectual disability and epilepsy consistent with GGE (ID-GGE) over 5% carry del15q13.3 (Mullen et al., 2013). Despite these remarkable carriage rates, particularly in “dual-phenotype” cases, del15q13.3 acts as a risk allele rather than a Mendelian cause of disease (Dibbens et al., 2009).

CHRNA7 and its partial duplication CHRFAM7A, lie within the common 15q13.3 microdeletion. As other neuronal nicotinic acetylcholine receptor subunits are known monogenic causes of epilepsy (CHRNA4, CHRNA2 and CHRNA2), interest focused on CHRNA7 as the likely critical gene in the 15q13.3 region (Taske et al., 2002). This was reinforced by the description of small 15q13.3 deletions encompassing CHRNA7 and CHRFAM7A associated with ID (Liao et al., 2011). This is a complex region, however. The partially duplicated gene alongside CHRNA7, CHRFAM7A, comprises exons 5–10 of CHRNA7 fused to the FAM7A gene. Although previously thought non-functional, more recent evidence suggests CHRFAM7A may affect CHRNA7 currents by regulating surface expression of the channel (de Lucas-Cerrillo et al., 2011).

A number of studies have examined sequence variation in this region. One study in 2002, seeking molecular variation responsible for a putative linkage signal at 15q13-14 in GGE, reported three rare variants, although no significant association was found (Flomen et al., 2006). Variation in CHRNA7 and CHRFAM7A in schizophrenia has also been reported but its significance remains unclear (Flomen et al., 2006, Gault et al., 2003). These studies have been limited by the sequence homology between CHRNA7 and CHRFAM7A. With conventional PCR it is not possible to determine, for the duplicated exons at least, the gene in which variants are located.

In addition, the contribution of a common 2-base pair (bp) deletion in CHRFAM7A to psychiatric disease has been suggested. This deletion is a marker of inversion of CHRFAM7A. An association has been claimed in some cohorts (Gault et al., 2003) but not in others (Flomen et al., 2006, Raux et al., 2002). An association with GGE has also been reported (Rozycka et al., 2013).

Here we examine sequence variation in CHRNA7 and CHRFAM7A in the phenotypes most strongly associated with del15q13.3. We selected probands with either GGE alone or a “dual phenotype” including GGE and a second del15q13.3 phenotype such as ASD, schizophrenia or ID. We hypothesized that sequence variation would act either as a risk allele in a similar manner to del15q13.3 or modify the expression of a co-existing deletion of the other chromosome 15 allele. We utilized long-range PCR to reliably define in which gene variants reside. In addition, we sought to confirm the association of the common 2 bp deletion in CHRFAM7A with GGE.