Representative blood smears and GPC genotyping results.

(A) North American control. Arrows indicate examples of ovalocytes: (B) Melanesian, Southeast Asian ovalocytosis (heterozygous for AE1Δ27); (C) Melanesian homozygous for GPCΔex3; and (D) Melanesian heterozygous for GPCΔex3. GPC genotyping; lane 1: homozygous wt (wt/wt); lane 2: GPCΔex3 heterozygote (wt/Δ); lane 3: GPCΔex3 homozygote (Δ/Δ); lane 4: North American, wt/wt genotype. Because exon 2 and exon 3 of the GPC gene are highly homologous, 2 reactions are needed to delineate all 3 genotypes. All PCR amplifications were performed as previously described.12(E) To assess the presence of exons 2 and 3, primers (GPCup 5′-CAGATTCTTGTCCTCTGTTCACAG-3′ and GPCdn 5′-TCAAAACCACCTCTGAGGGAGAG-3′) annealing to conserved sequence around exons 2 and 3 were used (thermocycling program: 94°C for 30 seconds, 68°C for 30 seconds, and 72°C for 30 seconds [× 40]). PCR products were subjected to electrophoresis on a 4% 5:1 GTG NuSieve:LE agarose (FMC Bioproducts, Rockland, ME) gel. A 264-bp band (exon 2) is detected in wt/wt, wt/Δ, and Δ/Δ. A 240 bp band (exon 3) is detected in only wt/wt and wt/Δ. The heteroduplex product is created by hybridization between complementary strands of exon 2 and 3 amplicons. (F) To identify individuals with the wt allele, primers annealing to a 51-bp intron 2 insert (GenBank AF342984) and downstream intron 2 polymorphism (GenBank M24627)5 (GPC349up 5′-GGAAACTGCCGTGACTTCAGA-3′ and 1679dn 5′-CATGGTCCTTGCAACAGTTGC-3′) were used to amplify a 1376-bp product (thermocycling program: 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds [× 40]). PCR products were subjected to electrophoresis on a 1.5% 5:1 GTG NuSieve:LE agarose gel. (G) To identify the GPCΔex3 allele and differentiate between wt/wt and wt/Δ, primers annealing to a 51-bp intron 2 insert and downstream intron 3 polymorphism (GenBank M24628)5 (GPC349up and GPC1680dn 5′-AGGTTAGAATCATACCCCAGG-3′) were used. Because the 3.4-kb deletion of the GPC gene includes exon 3, this PCR produces a 1377-bp band. To ensure that absence of the 1376-bp (D) and 1377-bp (E) bands was not caused by heterogeneity in PCR master mix, an additional primer, GPCdn, along with GPC349up, amplified conserved regions downstream of exon 2. The 155-bp product produced in all genotypes by GPC349up and GPCdn is not shown. All gels were stained with a 1:10 000 dilution of SYBR Gold (Molecular Probes, Eugene, OR), and products were visualized using a Storm 860 (Molecular Dynamics, Sunnyvale, CA).