Familial hemiplegic migraine: a ion channel disorder

At present, little information is available on the genetics of common migraines, most likely to be considered a multifactorial disease. Recently, the CACNA1A gene encoding the brain-specific P/Q type calcium channel alpha(1) subunit, has been cloned and mutations in this gene, located on chromosome 19p13, have been shown to be involved in familial hemiplegic migraine (FHM), a rare autosomal dominantly inherited subtype of migraine with aura. Being part of the migraine spectrum, FHM represents a good model to study the genetics of more common forms of migraine. Different classes of mutations within the CACNA1A gene have been associated with different diseases, thus identifying a new member among 'channelopathies'. Variable clinical expression and genetic heterogeneity of FHM will be discussed

Denaturing HPLC analysis of DNA deletions and insertions

Denaturing HPLC (DHPLC) is a useful technique for the fast screening of known and unknown heterozygous gene mutations. Most DNA mutations causing genetic disorders consist of nucleotide substitutions, but insertions and deletions occur, albeit less frequently. The heteroduplexes with insertions/deletions have gaps that may affect molecular stability differently from the mismatches caused by substitutions. Therefore, gaps and mismatches may be distinguished by DHPLC analysis, which is based on the differential thermal stability of amplicons with different characteristics. To verify this hypothesis, we examined 12 DNA samples containing insertions and deletions of different sizes (one to 29 residues) from four different genes (ABCA4, CFTR, FTL, and SLC11A3). We found that all of them were detected by DHPLC runs at 50\ub0C, which is considered a non-denaturing temperature, as well as by runs at the temperature optimized for mismatch recognition. The finding confirms that gaps reduce heteroduplex stability more than mismatches, and indicates that DHPLC analysis at low temperature may be applied to distinguish DNA deletions/insertions from substitutions

Denaturing HPLC analysis of DNA deletions and insertions

Denaturing HPLC (DHPLC) is a useful technique for the fast screening of known and unknown heterozygous gene mutations. Most DNA mutations causing genetic disorders consist of nucleotide substitutions, but insertions and deletions occur, albeit less frequently. The heteroduplexes with insertions/deletions have gaps that may affect molecular stability differently from the mismatches caused by substitutions. Therefore, gaps and mismatches may be distinguished by DHPLC analysis, which is based on the differential thermal stability of amplicons with different characteristics. To verify this hypothesis, we examined 12 DNA samples containing insertions and deletions of different sizes (one to 29 residues) from four different genes (ABCA4, CFTR, FTL, and SLC11A3). We found that all of them were detected by DHPLC runs at 50 degrees C, which is considered a non-denaturing temperature, as well as by runs at the temperature optimized for mismatch recognition. The finding confirms that gaps reduce heteroduplex stability more than mismatches, and indicates that DHPLC analysis at low temperature may be applied to distinguish DNA deletions/insertions from substitution

Dual-color microchip electrophoresis with single-photon avalanche diodes: Application to mutation detection

A novel microchip electrophoresis instrument based on single-photon avalanche diodes was used for the molecular characterization of mutations in disease genes. The identification of the main mutation causing cystic fibrosis, named ΔF508, by the Amplification Refractory Mutation System was used to validate the technology. In our implemented protocol the wild-type and mutant allele-specific primers are labeled with Cy5 and Cy5.5, respectively. The protocol enables the amplification of the DNA sample in a single PCR. The genotype was deduced from the fluorescence of the amplicons run in the CE microchip. Validation on 15 DNA samples from either homozygous wild-type or heterozygous and homozygous mutated control subjects proved the complete reliability of the system, thus confirming its high diagnostic potential

Denaturing HPLC Profiling of the ABCA4 Gene for Reliable Detection of Allelic Variations

Background: Mutations in the retina-specific ABC transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes scanning of the ABCA4 gene cumbersome, we describe here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations. Methods: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions. Results: DHPLC profiling not only identified all 132 sequence alterations previously detected by doublegradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before. Conclusions: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene

A new CACNA1A gene mutation in acetazolamide-responsive familial hemiplegic migraine and ataxia.

11OBJECTIVE: 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 alpha1A-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.reservedmixedBATTISTINI S; STENIRRI S; PIATTI M; GELFI C; RIGHETTI PG; ROCCHI R; F. GIANNINI; BATTISTINI N; GUAZZI GC; FERRARI M; CARRERA P.Battistini, Stefania; Stenirri, S; Piatti, M; Gelfi, C; Righetti, Pg; Rocchi, Raffaele; Giannini, Fabio; Battistini, Noe'; Guazzi, Gc; Ferrari, M; Carrera, P

Deciphering Variability of PKD1 and PKD2 in an Italian Cohort of 643 Patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common hereditary kidney disease. We analysed PKD1 and PKD2, in a large cohort of 440 unrelated Italian patients with ADPKD and 203 relatives by direct sequencing and MLPA. Molecular and detailed phenotypic data have been collected and submitted to the PKD1/PKD2 LOVD database. This is the first large retrospective study in Italian patients, describing 701 variants, 249 (35.5%) already associated with ADPKD and 452 (64.5%) novel. According to the criteria adopted, the overall detection rate was 80% (352/440). Novel variants with uncertain significance were found in 14% of patients. Among patients with pathogenic variants, in 301 (85.5%) the disease is associated with PKD1, 196 (55.7%) truncating, 81 (23%) non truncating, 24 (6.8%) IF indels, and in 51 (14.5%) with PKD2. Our results outline the high allelic heterogeneity of variants, complicated by the presence of variants of uncertain significance as well as of multiple variants in the same subject. Classification of novel variants may be particularly cumbersome having an important impact on the genetic counselling. Our study confirms the importance to improve the assessment of variant pathogenicity for ADPKD; to this point databasing of both clinical and molecular data is crucial