These “mixed” families were assigned a “family diagnosis” using the following criteria: “MO” when the proband suffered from MO and less than 20% of affected members suffered from a different type of migraine and “MA” when at least 20% of the affected family members suffered from headaches preceded by an aura. According to these criteria, 30 (49%) families were “MO,” 27(44%) were “MA,” and 4 (7%) were FHM. Known migraine loci already had mTOR inhibitor been excluded in this sample by SSCP screening and linkage (specifically, CACNA1A, chromosome 19 and 1, and “Finnish Locus” on chromosome 4). Two hundred and seventeen of all included persons were affected;
126 had MO, 70 had MA, and 15 had migraine with R428 hemiparetic aura. One was
diagnosed with “acephalgic” migraine, and 5 with probable migraine. We assigned the status “unknown” to all patients with insufficient information available and also to all not meeting all criteria for migraine but also not meeting criteria for any other type of headache; 42 patients were recorded in this manner. We had 163 blood samples from unaffected relatives. On 32 family members we had full information but no blood samples. Details of family structure are shown in the Table. Genotyping.— Venous blood samples were collected from 380 subjects, and genomic DNA was extracted by standard procedure.30 In a first round, we screened 18 fluorescent-labeled markers spaced on an average of 10 cM apart and spanning the entire X-chromosome (Linkage Mapping Panel 28, Applied Biosystems, Foster City, CA, USA). A positive logarithm of the odds (LOD) score at Xp22 and allele sharing at Xq24-q28 then prompted further evaluation of additional markers in both regions (Xp22: DXS7100, DXS996, DXS1223, DXS1053; Xq24-q28: DXS8028, DXS1200,
DXS1193, DXS1123, DXS8069, DXS8011). The order and distances between the markers were determined on the basis of their physical and genetic location, respectively, and according to the combined published data (http://www.sanger.ac.uk; http://genome.cse.ucsc.edu). The amplification reactions were run in microtiter 96-well MCE公司 plates with standard conditions on a MJ Research thermocycler. Depending on marker performance, there was some minor variation of annealing temperature and MgCl concentration. The PCR products subsequently were pooled for electrophoresis and supplemented with an internal size standard. A high through-put capillary electrophoresis system was used (ABI DNA Analyzer 3700, Applied Biosystems), and GENESCAN® and GENOTYPER® software was used for allele scoring (Applied Biosystems). All genotypes were verified by human inspection. Pedcheck 1.1 was used to detect genotyping errors.31 If the mistyping could not be resolved by review of the data, the suspected genotypes were set to unknown. Linkage Analysis.
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