Fasting Vs Nonfasting Plasma Homocysteine Concentra- Tions for Diagnosis of Hyperhomocysteinemia, M. Re

X-linked diseases, this requires the equimolar mixing of PCR products between a wild-type DNA sample and the sample to be tested. (c) The type of mutations searched for must be detectable by D-HPLC. D-HPLC is more sensitive in the screening of point mutations, which is the case of most FIX mutations (1 ). In any case, once the D-HPLC conditions have been defined, a prospective study of a novel population sample should be performed to confirm the detection rate of the D-HPLC procedure. The D-HPLC scanning procedure described here is fast because all of the DNA fragments can be amplified under the same conditions, the post-PCR phase is automated, and each D-HPLC run requires only 6 min. The protocol for D-HPLC scanning of the entire FIX gene is completed in 5 h. Finally, the D-HPLC method is cost-effective: we calculated that it costs approximately US $25 to scan the whole FIX gene, excluding instrument and personnel costs. Direct sequencing has a 100% sensitivity, and rapid protocols have been set up to analyze all FIX gene fragments under the same PCR conditions (6 ) or in single multiplex PCR amplifications (5 ). Direct FIX gene sequencing has been shown to be efficient in various ethnic-geographic groups (5–8). However, direct sequencing is more expensive than D-HPLC. On the other hand, scanning procedures have a sensitivity of 75–90% (10 ), and in most studies D-HPLC was more sensitive than other scanning procedures, as recently demonstrated for the factor VIII gene (11 ) and for several other disease genes (12, 13). Furthermore, denaturing gradient gel electrophoresis and single-strand conformation polymorphism analysis are more difficult to automate (9 ). In conclusion, D-HPLC scanning of the FIX gene with the procedure described here is suitable for the routine diagnosis of HB and for carrier diagnosis if the proband is not available. Using this procedure, we have confirmed the heterogeneity of FIX gene mutations in HB patients from Southern Italy.