Objective: To search for mutations in the calcium channel gene CACNA1A and to study the genotype–phenotype correlation in a family with a severe familial hemiplegic migraine (FHM) phenotype and a slowly progressive cerebellar ataxia. Background: CACNA1A gene mutations on chromosome 19 are involved in approximately 50% of FHM families. The association of FHM and cerebellar ataxia has been reported in a small number of FHM families, all linked to chromosome 19. Methods: The proband, in addition to typical hemiplegic migraine attacks, experienced severe episodes during which hemiplegia was associated with acutely altered consciousness and fever lasting several days. She, as well as her affected sister, developed a permanent, late-onset cerebellar ataxia and cerebellar atrophy evident on MRI. Linkage analysis was performed and the whole CACNA1A gene, 47 exon–intron boundaries, was analyzed by double gradient–denaturing gradient gel electrophoresis (DG-DGGE). Results: Genetic studies suggested linkage to chromosome 19p13, and DG-DGGE analysis detected a heteroduplex fragment in exon 13 of the CACNA1A gene. By direct sequencing, a G-to-A substitution resulting in an arginine to glutamine change at codon 583 in the second putative voltage sensor domain of the channel α_1A-subunit, was identified, possibly representing the disease-causing mutation. The proband and her affected sister were treated with acetazolamide, reporting freedom from new FHM attacks but no benefit in the progression of ataxia. Conclusions: The combination of episodic dysfunction and permanent deficit could depend on the variety of functions of calcium channels and their distribution in the nervous system.

Missense mutations in the pore-forming human alpha(1A) subunit of neuronal P/Q-type Ca(2+) channels are associated with familial hemiplegic migraine. We studied the functional consequences on P/Q-type Ca(2+) channel function of three recently identified mutations, R583Q, D715E, and V1457L after introduction into rabbit alpha(1A) and expression in Xenopus laevis oocytes. The potential for half-maximal channel activation of Ba(2+) inward currents was shifted by > 9 mV to more negative potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by > 7 mV in the same direction in R583Q and D715E. Biexponential current inactivation during 3-s test pulses was significantly faster in D715E and slower in V1457L than in wild type. Mutations R583Q and V1457L delayed the time course of recovery from channel inactivation. The decrease of peak current through R583Q (30.2%) and D715E (30. 1%) but not V1457L (18.7%) was more pronounced during 1-Hz trains of 15 100-ms pulses than in wild type (18.2%). Our data demonstrate that the mutations R583Q, D715E, and V1457L, like the previously reported mutations T666M, V714A, and I1819L, affect P/Q-type Ca(2+) channel gating. We therefore propose that altered channel gating represents a common pathophysiological mechanism in familial hemiplegic migraine.

Background: Mutations in the LMNA gene, encoding human lamin A/C, have been associated with an increasing number of disorders often involving skeletal and cardiac muscle, but no clear genotype/phenotype correlation could be established to date. Methods: We analyzed the LMNA gene in a large cohort of patients mainly affected by neuromuscular or cardiac disease and clustered mutated patients in two groups to unravel possible correlations. Results: We identified 28 variants, 9 of which reported for the first time. The two groups of patients were characterized by clinical and genetic differences: 1) patients with childhood onset displayed skeletal muscle involvement with predominant scapuloperoneal and facial weakness associated with missense mutations; 2) patients with adult onset mainly showed cardiac disorders or myopathy with limb girdle distribution, often associated with frameshift mutations presumably leading to a truncated protein. Conclusions: Our findings, supported by meta-analysis of previous literature, suggest the presence of two different pathogenetic mechanisms: late onset phenotypes may arise through loss of function secondary to haploinsufficiency, while dominant negative or toxic gain of function mechanisms may explain the severity of early phenotypes. This model of patient stratification may help patient management and facilitate future studies aimed at deciphering lamin A/C pathogenesis.

N-myristoylation is a cotranslational modification involved in protein-protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. The mammalian myristoylated flavoenzyme NADH-cytochrome b5 reductase is integrated into ER and mitochondrial outer membranes via an anchor containing a stretch of 14 uncharged amino acids downstream to the NH2-terminal myristoylate glycine. Since previous studies suggested that the anchoring function could be adequately carried out by the 14 uncharged residues, we investigated a possible role for myristic acid in reductase targeting. The wild type (wt) and a nonmyristoylatable reductase mutant (gly2-->ala) were stably expressed in MDCK cells, and their localization was investigated by immunofluorescence, immuno-EM, and cell fractionation. By all three techniques, the wt protein localized to ER and mitochondria, while the nonmyristoylated mutant was found only on ER membranes. Pulse-chase experiments indicated that this altered steady state distribution was due to the mutant's inability to target to mitochondria, and not to its enhanced instability in that location. Both wt and mutant reductase were resistant to Na2CO3 extraction and partitioned into the detergent phase after treatment of a membrane fraction with Triton X-114, demonstrating that myristic acid is not required for tight anchoring of reductase to membranes. Our results indicate that myristoylated reductase localizes to ER and mitochondria by different mechanisms, and reveal a novel role for myristic acid in protein targeting.