method, originally described by Oberkanins et al29 enables us to type C282Y, H63D and S65C HFE mutations together with V53M, V59M, H63H, Q127H, E168Q, E168X, W169X and Q283P. Moreover the kit allows to evaluate Y250X mutation at TFR2 gene sequence. The test was performed according to the manufacturers instruc- tions. Briefly, multiplex PCR products (cycling was per- ° ° formed at 94 C for 2 min and 30 cycles at 94 C for 15 s, ° ° 58 C for 30 s and 72 C for 30 s, followed by a final exten- ° sion of 3 min at 72 C) obtained using biotinylated pri- mers were hybridised to aminomodified oligonucleotides immobilized on a nylon membrane able to hybridise nor- mal or mutated HFE and TFR2 sequences. Strips contain-  ing oligoprobes were hybridised with 10 l amplified DNA denatured in an equal volume of NaOH 1N for 5 min and then incubated in an adequate volume of × hybridisation solution (SSC 6 0.1% SDS) in a shaking ° bath at 45 C for 30 min. After two washes in hybridis- ation solution at room temperature, stringent wash was ° × performed at 45 C for 30 min in prewarmed SSC 6 0.1% × SDS. After two washes in SSC 1 0.1% SDS, membranes were incubated with the appropriate alkaline phospatase conjugated streptavidin dilution for 15 min at room tem- perature. After two other washes, bound PCR fragments were detected using colour substrate (NBT/BCIP) devel- opment.

Analysis of HFE gene mutations

Samples were typed for C282Y, H63D and S65C alleles using polymerase chain reaction and sequence specific primers listed in Table 4.

C282Y mutation was typed according to Takeuchi et 4s) ul al49 using a multiple PCR protocol. Briefly 20 l reaction mixtures containing 50 ng of template DNA, 0.5 U TaqGold-DNA polymerase (PE BioSystem, Milan, Italy), and a final concentration of 200 mM each deoxynucleo- × tide and 1 reaction buffer (PE Biosystem), 1 mM of F1, R1 and F2M primers and 0.2 mM of R2W primer. Cycling ° ° was performed at 96 C for 7 min and 35 cycles at 95 C ° ° for 30 s, 64 C for 30 s and 72 C for 30 s, followed by a ° final extension of 10 min at 72 C. PCR products were detected by electrophoresis on 2% agarose, obtaining a 521 bp product and a 257 and/or 136 bp bands from mutated or wild HFE Y282C sequences. H63D alleles were typed with the amplification refractory mutation system/polymerase chain reaction (ARMS/PCR) using two separated primer couples (F3/R3 and F4/R3)50 in the following conditions. 50 ng of template DNA were mixed my A) pul in a final volume of 20 l with 0.5 U TaqGold-DNA poly- merase (PE BioSystem), 200 mM each deoxynucleotide × and 1 reaction buffer and 0.5 mM of each specific ° primer. Cycling was performed at 96 C for 10 min and ° ° ° 35 cycles at 95 C for 30 s, 64 C for 30 s and 72 C for 30 s, ° followed by a final extension of 10 min at 72 C. PCR pro- ducts were evaluated by electrophoresis on 2% agarose. Detection of a 178 bp PCR product in one or both electro- phoresis lanes allows to identify homozygous or hetero- zygous subjects for H63D genotypes. S65C specificities were evaluated after H63D genotyping using the same ARMS/PCR protocol and appropriated primer couples. 63H homozygous subjects were typed using F5/R3 and F6/R3 primer mixes whereas 63D subjects were typed using F7/R3 and F8/R3. All the four primer mixes were used to type H63D heterozygous subjects.

Statistical analysis

× Allele frequencies were evaluated by gene count and 2 2 tables were constructed to determine statistical signifi- cance (Fisher exact test) of differences in allele frequency for the HFE polymorphisms between oldest old and con- trols. According to Bonferroni, obtained P values were corrected by multiplying for the number of alleles under study, ie 6.

References

To validate our results and evaluate the presence of other less common HFE mutation and of the recently described haemochromatosis related TFR2 Y250X mutation30 a commercially purchased Reverse- Hybridisation assay was used (Haemochromatosis Kit, Nuclear Laser Medicine, Settala Milanese, Italy). This

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Table 4 Details of reaction mixtures used for HFE genotyping