A total of 200 random DNA samples from healthy individuals (100 Macedonians, 50 Albanians and 50 Gypsies), provided by the Macedonian human DNA bank (hDNAMKD), Institute of Immunobiology and Human Genetics at Skopje, Republic of Macedonia, were genotyped for HFE mutations (C282Y, H63D and S65C). Written consent was obtained from each individual enrolled in this study. DNA was isolated from peripheral blood leukocytes by the phenol-chloroform extraction method [12]. The mutations were detected by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method [1,2,12]. Exon 4 was amplified in a 50 µL PCR reaction (MgCl2 1.5 µM, 2.5 U Taq Gold; Perkin Elmer, Roche Molecular Systems, Inc., Branchburg, NJ, USA; Tm 62EC, 40 cycles) and exon 2 in a 100 mL PCR reaction (MgCl2 2.0 µM, 2.5U Taq Gold; Perkin Elmer; Tm 62EC, 40 cycles), on a 96-well thermal cycler (PTC-100; MJ Research, Inc., Waltham, MA, USA) [14,15]. Twenty mL of the PCR products and 5 U of the corresponding restriction enzyme were used for the digestion reaction (buffer, temperature and time according to the manufacturer's recommendations; New England Biolabs (UK), Ltd., Hitchin, Hertfordshire, UK). The restriction fragments were visualized after electrophoresis in 3% agarose gel stained with ethidium bromide. The primers and restriction enzymes used are shown in Table 1. Quality control was ascertained by participation in the UK NEQAS HFE quality control scheme 5, 2001 (an international system of external quality control provided by UK NEQAS; 20 external quality control samples analyzed; accuracy result 100%).

Mutation	Forward Primer (5'÷3')	Reverse Primer (5'÷3')	Restriction Enzyme
C282Y  exon 4	TGGCAAGGGTAAACAGATCC	CTCAGGCACTCCTCTCAACC	RsaI; SnaBI
H63D  exon 2	ACATGGTTAAGGCCTGTTGC	CTTGCTGTGGTTGTGATTTTCC	MboI, BclI
S65C  exon 2	ACATGGTTAAGGCCTGTTGC	CTTGCTGTGGTTGTGATTTTCC	HinfI