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Am J Hum Genet
2000 May 01;665:1531-9. doi: 10.1086/302909.
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Coding and noncoding variation of the human calcium-channel beta4-subunit gene CACNB4 in patients with idiopathic generalized epilepsy and episodic ataxia.
Escayg A
,
De Waard M
,
Lee DD
,
Bichet D
,
Wolf P
,
Mayer T
,
Johnston J
,
Baloh R
,
Sander T
,
Meisler MH
.
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Inactivation of the beta4 subunit of the calcium channel in the mouse neurological mutant lethargic results in a complex neurological disorder that includes absence epilepsy and ataxia. To determine the role of the calcium-channel beta4-subunit gene CACNB4 on chromosome 2q22-23 in related human disorders, we screened for mutations in small pedigrees with familial epilepsy and ataxia. The premature-termination mutation R482X was identified in a patient with juvenile myoclonic epilepsy. The R482X protein lacks the 38 C-terminal amino acids containing part of an interaction domain for the alpha1 subunit. The missense mutation C104F was identified both in a German family with generalized epilepsy and praxis-induced seizures and in a French Canadian family with episodic ataxia. These coding mutations were not detected in 255 unaffected control individuals (510 chromosomes), and they may be considered candidate disease mutations. The results of functional tests of the truncated protein R482X in Xenopus laevis oocytes demonstrated a small decrease in the fast time constant for inactivation of the cotransfected alpha1 subunit. Further studies will be required to evaluate the in vivo consequences of these mutations. We also describe eight noncoding single-nucleotide substitutions, two of which are present at polymorphic frequency, and a previously unrecognized first intron of CACNB4 that interrupts exon 1 at codon 21.
Figure 1 Structure and interactions of CACNB4. A, Intron/exon junctions for exons 1A and 1B. The indicated 5′ end of exon 1A corresponds to the translation-initiation site. The intron sequence is from GenBank (accession number AF216867). B, Locations of mutations in CACNB4. Two domains of CACNB4 that interact with the α1 subunit of the voltage-gated calcium channel are indicated. In the mouse lethargic mutant (designated as “lh”), the protein is truncated upstream of the major binding site. In human mutation R482X, the protein is truncated in the middle of a domain that interacts with the C-terminus of the α1A subunit.
Figure 2 R482X mutation in a family with JME. A, Affected individuals (blackened symbols) are shown, with the arrow pointing to the proband. B, Results of CSGE of exon 13. A unique band seen in the proband (lane 1, arrow) is not observed in three unrelated controls (lanes 2–4). C, The sequence of exon 13 demonstrates that the proband is heterozygous for a C→T transition that generates the premature-termination codon R482X. D, Results of a restriction-site assay for R482X. A 388-bp fragment obtained by digestion of wild-type exon 13 with AluI is shown (lanes 2–4). The internal AluI site produced by R482X results in two fragments (209 bp and 179 bp; see lane 1).
Figure 3 C104F mutation cosegregating with neurological disease in two unrelated pedigrees. A, Pedigree B, which is of German origin and exhibits idiopathic generalized epilepsy. B, Pedigree C, which is of French Canadian origin and exhibits episodic ataxia. C, CSGE analysis of exon 3 from pedigree B (left) and from pedigree C (right). The variant conformer is indicated (arrow). Numbers above the lanes correspond to individuals in the pedigrees. D, Sequence of exon 3 that demonstrates a G→T transversion that changes the wild-type cysteine 104 codon to phenylalanine. CACNB4 genotypes are indicated below the symbols. G = allele 1; T = allele 2.
Figure 4 Electrophysiological analysis of mutant β4 subunits. Xenopus oocytes were injected with mRNA from either the wild-type or mutant β4 subunit, in combination with the CACNA1A α1 subunit. A, Sets of current traces at test potentials of −20, −10, 0, 10, 20, and 30 mV. The potential of half-activation was not significantly modified by the mutations, with V1/2=−12.2±0.9 mV (β4-subunit R482X, n=8), −11.3±0.6 mV (β4-subunit C104F; n=8), and -10±0.1 mV (wild-type β4 subunit; n=8). There was a small shift in the voltage dependence of inactivation of the α1A β4-subunit R482X, with half-inactivation potentials of V1/2=-41.1±0.4 mV (β4-subunit R482X; n=7) and V1/2=-37.4±0.5 (wild-type β4 subunit; n=6,). B, Average current density generated by wild-type and mutant channels at a membrane depolarization of 0 mV (peak current amplitude for each subunit combination). The average current density of the α1A wild-type β4 subunit (1.90±0.15 μA/μF; n=8) at 0 mV was increased 1.4- and 1.3-fold, to 2.72±0.25 (n=8) and 2.45±0.24 (n=8), with β4-subunit R482X and β4-subunit C104F, respectively. Asterisks (*) denote statistical significance (t test; P≤.1). C, Average time constants for the fast component of inactivation. At a membrane depolarization of 10 mV, the wild-type current inactivates along three components, a fast-inactivating component (F) with an average time constant of 89.6±5.3 ms (n=7), which constitutes 19.9±3.4% of the total current, a slower inactivating component (S) with an average time constant of 323±37.4 ms (73.9±3.1%), and a noninactivating (NI) component (6.2±0.9%). With β4-subunit R482X, there was no change in the proportion of each component, but the F component demonstrated a faster rate of decrease (50.7±4.7 ms; n=6). The time constants for β4-subunit R482X at 0 and 10 mV are statistically significantly different from those for wild type (P<.01). Values are mean ± SEM.
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