Michele Rees

SCN9A Mutations in Paroxysmal Extreme Pain Disorder: Allelic Variants Underlie Distinct Channel Defects and Phenotypes

Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, or OMIM 167400), is an inherited condition characterized by paroxysms of rectal, ocular, or submandibular pain with flushing. A genome-wide linkage search followed by mutational analysis of the candidate gene SCN9A, which encodes hNa(v)1.7, identified eight missense mutations in 11 families and 2 sporadic cases. Functional analysis in vitro of three of these mutant Na(v)1.7 channels revealed a reduction in fast inactivation, leading to persistent sodium current. Other mutations in SCN9A associated with more negative activation thresholds are known to cause primary erythermalgia (PE). Carbamazepine, a drug that is effective in PEPD, but not PE, showed selective block of persistent current associated with PEPD mutants, but did not affect the negative activation threshold of a PE mutant. PEPD and PE are allelic variants with distinct underlying biophysical mechanisms and represent a separate class of peripheral neuronal sodium channelopathy.

The ducky2J Mutation in Cacna2d2 Results in Reduced Spontaneous Purkinje Cell Activity and Altered Gene Expression

The mouse mutant ducky and its allele ducky2J represent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the α2δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2du2J mutation results in a 2 bp deletion in the coding region and a complete loss of α2δ-2 protein. Here we show that du2J/du2J mice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C, du2J/du2J PCs show no spontaneous intrinsic activity. Du2J/du2J mice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du2J/+ mice have a marked reduction in α2δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However, du2J/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α2δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α2δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma.

Evaluation of the positional candidate gene CHRNA7 at the juvenile myoclonic epilepsy locus (EJM2) on chromosome 15q13-14

A previous study of 34 nuclear pedigrees segregating juvenile myoclonic epilepsy (JME) gave significant evidence of linkage with heterogeneity to marker loci on chromosome 15q13-14 close to the candidate gene CHRNA7 (Hum. Mol. Genet. 6 (1997) 1329). The aim of this work was to further evaluate the putative aetiological role of CHRNA7 in JME within the 34 families originally described, and to assess the contribution of this locus to a broader phenotype of idiopathic generalised epilepsy (IGE). Multipoint linkage analysis and intrafamilial association studies were performed with microsatellite markers that encompass both CHRNA7 and its partial duplication (CHRFAM7A). A maximum HLOD of 3.45 [alpha=0.58; (Zall=2.88, P=0.0008)] was observed 8 cM distal to D15S1360, a CHRNA7 intragenic marker. Significant exclusion lod scores were obtained across the region in 12 mixed phenotype JME/IGE families. Mutation screening of the CHRNA7 gene (and consequently exons 5-10 of CHRFAM7A) and its putative promoter sequence identified a total of 13 sequence variants across 23 of 34 JME-affected families. Two variants (c.1354G>A and c.1466C>T) are predicted to result in amino acid changes and one (IVS9+5G>A) is predicted to result in aberrant transcript splicing. However, none of the variants alone appeared either necessary or sufficient to cause JME in the families in which they occurred. In conclusion, linkage analyses continue to support the existence of a locus on chromosome 15q13-14 that confers susceptibility to JME but not to a broader IGE phenotype. Causal sequence variants in the positional candidate CHRNA7 have not been identified but the presence of multiple segmental duplications in this region raises the possibility of undetected disease-causing genomic rearrangements.

Evaluation of CACNA1H in European patients with childhood absence epilepsy

CACNA1H was evaluated in a resource of Caucasian European patients with childhood absence epilepsy by linkage analysis and typing of sequence variants previously identified in Chinese patients. Linkage analysis of 44 pedigrees provided no evidence for a locus in the CACNA1H region and none of the Chinese variants were found in 220 unrelated patients.

Linkage and association analysis of CACNG3 in childhood absence epilepsy.

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy characterised by absence seizures manifested by transitory loss of awareness with 2.5–4 Hz spike–wave complexes on ictal EEG. A genetic component to aetiology is established but the mechanism of inheritance and the genes involved are not fully defined. Available evidence suggests that genes encoding brain expressed voltage-gated calcium channels, including CACNG3 on chromosome 16p12–p13.1, may represent susceptibility loci for CAE. The aim of this work was to further evaluate CACNG3 as a susceptibility locus by linkage and association analysis. Assuming locus heterogeneity, a significant HLOD score (HLOD=3.54, α=0.62) was obtained for markers encompassing CACNG3 in 65 nuclear families with a proband with CAE. The maximum non-parametric linkage score was 2.87 (P<0.002). Re-sequencing of the coding exons in 59 patients did not identify any putative causal variants. A linkage disequilibrium (LD) map of CACNG3 was constructed using 23 single nucleotide polymorphisms (SNPs). Transmission disequilibrium was sought using individual SNPs and SNP-based haplotypes with the pedigree disequilibrium test in 217 CAE trios and the 65 nuclear pedigrees. Evidence for transmission disequilibrium (P≤0.01) was found for SNPs within a ∼35 kb region of high LD encompassing the 5’UTR, exon 1 and part of intron 1 of CACNG3. Re-sequencing of this interval was undertaken in 24 affected individuals. Seventy-two variants were identified: 45 upstream; two 5’UTR; and 25 intronic SNPs. No coding sequence variants were identified, although four variants are predicted to affect exonic splicing. This evidence supports CACNG3 as a susceptibility locus in a subset of CAE patients.

Linkage analysis between childhood absence epilepsy and genes encoding GABAA and GABAB receptors, voltage-dependent calcium channels, and the ECA1 region on chromosome 8q

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy (IGE) characterised by onset of typical absence seizures in otherwise normal children of school age. A genetic component to aetiology is well established but the mechanism of inheritance and the genes involved are unknown. Available evidence suggests that mutations in genes encoding GABA receptors or brain expressed voltage-dependent calcium channels (VDCCs) may underlie CAE. The aim of this work was to test this hypothesis by linkage analysis using microsatellite loci spanning theses genes in 33 nuclear families each with two or more individuals with CAE. Seventeen VDCC subunit genes, ten GABA(A)R subunit genes, two GABA(B) receptor genes and the ECA1 locus on 8q24 were investigated using 35 microsatellite loci. Assuming locus homogeneity, all loci gave statistically significant negative LOD scores, excluding these genes as major loci in the majority of these families. Positive HLOD scores assuming locus heterogeneity were observed for CACNG3 on chromosome 16p12-p13.1 and the GABRA5, GABRB3, GABRG3 cluster on chromosome 15q11-q13. Association studies are required to determine whether these loci are the site of susceptibility alleles in a subset of patients with CAE.

Mouse models of spike-wave epilepsy

The mouse is a well‐established model for human genetic disorders. An increasing number of single‐gene human diseases are being elucidated through the use of mouse models. Recently genes for three of the six well‐characterised single‐locus models for human spike‐wave epilepsy have been isolated and published. The tottering mouse has been shown to be due to mutations in the gene encoding the high voltage‐activated α1A calcium channel subunit. The lethargic mouse has been shown to be due to mutations in the gene encoding another calcium channel subunit, β4. The slow‐wave epilepsy mouse phenotype is the result of loss of function of the ubiquitous sodium hydrogen exchanger NHEI. These genes and the pathways they are involved in are now candidates for human spike‐wave epilepsy. The six mouse models and those genes underlying the spike‐wave phenotype are discussed in conjunction with how these mutations were discovered and how they may give rise to the seizure phenotypes. Several nonepilepsy human neurologic disorders have been shown to be allelic with the tottering mouse. The question this raises as to the validity of these models for human spike‐wave epilepsy is considered. Finally, the effect these discoveries will have on the understanding and treatment of human spike‐wave epilepsy are discussed.

Genomic organization of the mouse and human alpha2delta2 voltage-dependent calcium channel subunit genes.

channels (VDCCs) are found in the plasma membranes of all excitable cells and are hetero-oligomeric complexes composed of up to five subunits:a1, a2, d, b and g. The a1 subunit acts as a channel pore, voltage sensor, and receptor for many drugs. The other accessory subunits modify channel properties such as current magnitude and kinetics (Walker and De Waard 1998). Thea2 andd chains are derived by proteolytic cleavage of the same gene product, the d subunit acting as a membrane anchor for the extracellulara2 subunit. Three genes encoding a2d subunits have been described: human, mouse, rat, and rabbit CACNA2D1 (a2d1), human CACNA2D2 ( a2d2), and mouseCacna2d3(a2d3) (Klugbauer et al. 1999). Genes encoding VDCCs have been implicated in the aetiology of a wide range of mammalian phenotypes (Fletcher et al. 1998). The genes encoding the a1A, b4, and g2 subunits have been shown to underlie the mouse epilepsy phenotypes tottering, lethargic, and stargazer (Fletcher and Frankel 1999). Recently, we have demonstrated that the Cacna2d2gene is also associated with a phenotype of ataxia and epilepsy in ducky mice (unpublished). TheCacna2d2gene is located on mouse Chromosome (Chr) 9 approximately 59–60 cM from the centromere, and the human ortholog CACNA2D2 is located on human Chr 3p21.3 (Gao et al. 2000). Here, the genomic structures of the mouse Cacna2d2and human CACNA2D2 genes are described and compared for the first time. The two regions of alternative splicing identified in mouse brain RNA are discussed together with a consideration of the associated non-consensus splice sites. The 5.5-kb human CACNA2D2 cDNA (GenBank AF042792) sequence has been previously described, and we have isolated the mouseCacna2d2cDNA sequence (GenBank AF247139). Genomic clones containing the Cacna2d2gene were identified from the WI/MIT YAC library (y203E7, y257D12, y465F1) and the RPCI21 mouse PAC library (p428C5, p432G2, p524O8, and p524G24). Most of the intron/exon boundaries were determined by using the ExpandTM Long Template PCR system (Roche Diagnostics, UK) to amplify mouse genomic and YAC DNA with primers contained within the cDNA sequence. PCR products were sequenced to determine the positions of the intron/exon boundaries. Smaller introns were sequenced in their entirety and exact sizes determined. Sizes of larger introns (1–8 kb) were estimated by comparison of PCR products to size standards. Introns 1 and 2 could not be amplified by PCR; therefore, intron/exon boundaries were determined by direct sequencing of the PAC clones by using Big-Dye technology (Applied Biosystems, UK). These intron sizes were estimated by Southern blot hybridization of digested PAC clones.Cacna2d2is organized into 39 exons (Table 1 and Fig. 1A) that are distributed over 85 kb of genomic DNA. Exon 1 contains the start codon, and exon 39 contains the stop signal. The mouseCacna2d2cDNA sequence was aligned with the genomic sequence derived from a cosmid contig of human Chr 3p21.3 (GenBank Z84493, Z84494, Z84495, Z75743, Z75742, and Z84492). The positions of the mouse intron/exon boundaries were compared with regions of divergence between the mouse cDNA sequence and the human genomic DNA sequence. The positions of all the intron/exon boundaries and exon sizes are conserved between human and mouse, so CACNA2D2 is also organized into 39 exons over a genomic distance of at least 118 kb (Table 1 and Fig. 1B). Overall, the genomic organizations of the two genes are highly similar. Two regions of alternative splicing were identified in mouse brain RNA (Fig. 2). Exon 23 (Fig. 2A) is present in 13% of subclones of an RTPCR product spanning exons 22–24. At the protein level, it results in the sequence change KYF to KLPISKLKDF. A splice variant at the same position has been described in human cDNA (Hobom et al. 2000). The 5 8 splice donor does not conform to the consensus gt, but contains a gc in both the mouse and human sequences (Table 1). Such a variation at the 58 splice site has been previously described (Shapiro and Senapathy 1987) and is not expected to significantly impair splicing. Presumably, this represents a mechanism whereby the recognition of the splice site is slightly reduced, consistent with the relatively low abundance of RNA species containing exon 23. The second region of alternative splicing involves the 3 8 splice acceptor of intron 38. The 6 bp indicated (Fig. 2B) was identified in 25% of subclones from an RTPCR product spanning exons 37–39 and results in the insertion of two amino acids (CP). Thus, two 38 splice donor sites are associated with intron 37. The more favored more 3 8 splice site contains the consensus ag motif. The second splice site contains a non-consensus at motif, again suggesting that divergence from the consensus produces alternatively spliced products of relatively lower abundance. This nonconsensus splice site is conserved in humans, and the same splice variant has been described in human medulla thyroid carcinoma cells (Hobom et al. 2000). Sequence analysis of full-length Cacna2d2cDNA clones (n4 9) and in silico analysis of cDNA sequences have not identified transcripts that contain exon 23 and the 6-bp variant of exon 38. Tissue-specific splicing of human CACNA2D2 has been described, although functional expression of splice variants did not reveal differences at the electrophysiological level (Hobom et al. 2000). Analysis at the protein level is required to provide definitive proof that the alternatively spliced transcripts represent native proCorrespondence to: J. Barclay; E-mail: jane.barclay@pharma.novartis.com