An alanine-to-threonine substitution in protein 4.2 cDNA is associated with a Japanese form of hereditary hemolytic anemia (protein 4.2NIPPON)

Abstract

Erythrocyte (RBC) protein 4.2 (P4.2)-deficiency observed in Japanese individuals results in a hemolytic anemia associated with abnormally shaped (spherocytic, ovalocytic, and elliptocytic), osmotically fragile RBCs, the clinical presentation of which resembles hereditary spherocytosis (HS). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, P4.2-deficient individuals contain less than 1% of the normal membrane content of P4.2 and immunologic analysis shows that the P4.2 present exists as an equimolar doublet of 74-Kd and 72-Kd bands, in contrast to normal RBC membranes where a discrete 74-Kd band is not observed. RBC membranes from both of the biologic parents of a P4.2- deficient individual contained both the 74-Kd and the 72-Kd bands, demonstrating their heterozygosity for the P4.2 defect. The molecular basis of Japanese P4.2-deficiency was investigated by reverse transcription of total reticulocyte RNA, followed by polymerase chain reaction (PCR) amplification, subcloning, and sequencing. The complete cDNA sequence of a P4.2-deficient patient showed a single point mutation that changes codon 142 from GCT (alanine) to ACT (threonine) (Protein 4.2NIPPON). The mutation also eliminated an HgaI restriction site, therefore allowing rapid screening for the presence of the mutation. Screening of PCR-amplified genomic DNA showed that the mutation was present in the homozygous state in four (eight chromosomes) unrelated Japanese P4.2-deficient individuals and absent in 35 (70 chromosomes) P4.2-normal controls (including 15 Japanese [30 chromosomes]). The presence of the mutation was confirmed by allele- specific hybridization. The mutation occurred in an alternatively spliced exon that is present in two of four P4.2 mRNA splicing isoforms. These results demonstrate that Japanese P4.2-deficiency is closely associated with the P4.2 gene and does not arise secondarily to a defect in another membrane protein, and further suggest that the P4.2- deficiency is related to the pathogenesis of the hemolytic anemia in this variant form of recessively inherited spherocytosis.

Disorders of red cell membrane

“…Mushroom-shaped or ''pincered'' red cells in addition to spherocytes may be seen on peripheral blood smear. Recessive HS due to homozygous mutations in EPB42 is common in Japan but is rare in other populations accounting for less than 5% of HS cases (Bouhassira et al, 1992;Yawata et al, 2000). In these cases, an almost complete deficiency of protein 4.2 is noted.…”

Genotypic and phenotypic expressions of protein 4.2 in human erythroid cells

“…Seven kinds of total protein 4.2 deficiency due to mutations of the protein 4.2 gene have been identified [32−39]: four missense mutations [32,35,36,38], one frameshift mutation [37], one nonsense mutation [34], and one donor site mutation due to intronic substitution [39], as summarized in Table 3. Therefore, missense mutations are predominant, especially allele protein 4.2 Nippon (142 GCTǞACT: Al-aǞThr) [32][33][34]36,39], which has been observed in 17 patients of 13 kindreds among 28 patients of 19 kindreds with complete protein 4.2 deficiency [90], as shown in Table 2. These protein 4.2 gene mutations have been found mostly or nearly exclusively in the Japanese population, i.e., protein 4.2 Nippon [32,33] Table 2).…”

“…Therefore, missense mutations are predominant, especially allele protein 4.2 Nippon (142 GCTǞACT: Al-aǞThr) [32][33][34]36,39], which has been observed in 17 patients of 13 kindreds among 28 patients of 19 kindreds with complete protein 4.2 deficiency [90], as shown in Table 2. These protein 4.2 gene mutations have been found mostly or nearly exclusively in the Japanese population, i.e., protein 4.2 Nippon [32,33] Table 2). Only two mutations have been observed in the non-Japanese population, i.e., protein 4.2 Tozeur in Tunisia (310 CGAǞCAA) [38] and protein 4.2 Lisboa in Portugal (88 AAG GTGǞAAG TG) [37], in addition to one Caucasian patient who was a homozygous protein 4.2 Nippon.…”

“…Only two mutations have been observed in the non-Japanese population, i.e., protein 4.2 Tozeur in Tunisia (310 CGAǞCAA) [38] and protein 4.2 Lisboa in Portugal (88 AAG GTGǞAAG TG) [37], in addition to one Caucasian patient who was a homozygous protein 4.2 Nippon. Among these protein 4.2 gene mutations, the mutation of the Nippon type is most important [5,6,90], because it is involved in homozygotes of protein 4.2 Nippon [32,33] and also in compound heterozygotes of protein 4.2 Shiga with 317 (CGCǞTGC) [35], protein 4.2 Fukuoka with 119 (TGGǞTGA) [34], and protein 4.2 Notame with GǞA at the intron 6 donor site [39] (Table 3).…”

Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis

“…In addition, the primary and almost total absence of protein 4.2 is observed due to the occurrence in the homozygous (or the compound heterozygous) state of mutations in the EPB42 gene encoding protein 4.2 (Bouhassira et al, 1992;Yawata, 1994;Takaoka et al, 1994;Hayette et al, 1995a, b;Kanzaki et al, 1995a, b;Matsuda et al, 1995). The inheritance pattern is also recessive.…”