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Huntington disease (HD) is associated with abnormal expansions of a CAG repeat close to the 5[prime] end of the IT15 gene. We have assembled a set of 293 HD subjects whose ages of onset were known and sized their HD CAG repeats. These repeats accounted for 69% of the variance of age of onset when we used the most parsimonious model, which relates the logarithm of age of onset to a function of CAG repeat number. Since other familial factors have been proposed to influence the age of onset of HD, we have examined a number of candidate loci. The CAG repeat number on normal chromosomes, the [Delta]2642 polymorphism in the HD gene, and apolipoprotein E genotypes did not affect the age of onset of HD. Although mitochondrial energy production defects in HD have led to suggestions that variants in the mitochondrial genome may be associated with clinical variability in HD, this suggestion was not supported by our preliminary experiments that examined the DdeI mitochondrial restriction fragment length polymorphism at position 10,394. Excitotoxicity has been a favored mechanism to explain the cell death in HD, particularly since intrastriatal injection of excitatory amino acids in animals creates HD-like pathology. Accordingly, we investigated the GluR6 kainate receptor. Of the variance in the age of onset of HD that was not accounted for by the CAG repeats, 13% could be attributed to GluR6 genotype variation. These data implicate GluR6-mediated excitotoxicity in the pathogenesis of HD and highlight the potential importance of this process in other polyglutamine repeat expansion diseases.

A reference genotyping laboratory was established in 2000 at Queen's University, Kingston, to provide genetic testing for Hemophilia A (HA) and B (HB) and create a Canadian mutation database. Canadian hemophilia treatment centers and genetics clinics provided DNA and clinical information from November 2000 to March 2011. The factor VIII (F8) gene was analyzed in 1,177 patients (47% of HA population) and 787 female family members and the factor IX (F9) gene in 267 patients (47% of HB population) and 123 female family members, using Southern Blot, PCR, conformation sensitive gel electrophoresis, and/or direct sequencing. The mutation detection rates for HA and HB were 91% and 94%, respectively. 380 different F8 mutations were identified: inversions of intron 22 and intron 1, 229 missense, 45 nonsense, eight deletions, 70 frameshifts, 25 splice site, and one compound mutation with a splice site and intron 1 inversion. Of these mutations, 228 were novel to the Hemophilia A Database (HADB, http://hadb.org.uk/). A total 125 different F9 mutations were identified: 80 missense, 12 frameshift, 12 splice site, nine nonsense and seven promoter mutations, three large deletions, and two compound mutations with both missense and nonsense changes. Of these mutations, 36 were novel to the International Haemophilia B Mutation database (http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html). The Canadian F8 and F9 mutation database reflects the allelic heterogeneity of HA and HB, and is similar to previously described populations. This report represents the largest and longest duration experience of a national hemophilia genotyping program documented, to date., (Copyright © 2013 Wiley Periodicals, Inc.)

Objective: To establish a simple, rapid and easy method for screening the gene mutation in hemophilia A, which was further applied to a direct diagnosis and carrier detection at gene level., Methods: Twenty-four clinically diagnosed hemophilia pedigrees, including all the hemophilia patients and female members, were tested for the introns 22 and 1 in factor VIII gene by using inversion polymerase chain reaction (PCR) and regular PCR techniques. All the 26 exons of factor VIII gene were consecutively screened in the 17 patients manifesting non-inverted sequences in intron 22 by using PCR, subsequently all the 37 amplicons resulted from 26 exons were analyzed by conformation sensitive gel electrophoresis (CSGE), finally the mutated exons were subjected to sequencing verification. According to the mutation results, mothers and twin sisters of the hemophilia probands were tested by CSGE or subjected to nucleotide sequencing directly, to ascertain if those individuals had the same mutation or were the carriers of disease-causing gene., Results: Intron 22 inversion was detected in 7 hemophilia probands out of 24 hemophilia pedigrees, intron 1 inversion was not detected in these pedigrees. Single-base mutations distributed in different exons of factor VIII gene were detected in 13 pedigrees with family history and 3 sporadic pedigrees, diagnosed as non-inverted 22 intron patients. By comprehensive usage of PCR-CSGE and nucleotide sequencing, the positive rate and the diagnosable rate of gene diagnosis or carrier detection in the 24 hemophilia pedigrees was 94.12% and 100% respectively., Conclusion: PCR-CSGE is a highly sensitive and special assay for detecting single base mutation. By integrated utilization of introns 22 and 1 of factor VIII gene detection and PCR-CSGE genotyping, combining with nucleotide sequencing, a direct diagnosis of all hemophilia pedigrees be could nearly make at gene level, including the sporadic families. This method might be used to screen new mutation theoretically and ascertain the mutation type. It is a simple, rapid and low-cost method, possessing unique advantages in direct diagnosis of hemophilia A and carrier screening. It should have important application value in hemophilia diagnosis.