of the Telomerase RNA (TERC) and Telomerase Reverse Transcriptase (TERT) in Japanese Children with Aplastic Anemia.

It is well known that the incidence of aplastic anemia (AA) is much higher in Japan than in Western countries. However the reason for this finding is not known. Genetic backgrounds related to AA may be different between populations. Recent studies have shown that some patients with apparently “acquired” AA have mutations in telomerase ribonucleoprotein complex genes such as TERC and TERT. We studied 96 Japanese children with acquired AA (age range, 0–16 years; median, 7 years) and 59 healthy controls for mutations in TERC and TERT genes. Of these children, 35 were classified as having nonsevere AA, 39 as having severe AA, and 22 as having very severe AA. In 7 patients, AA was secondary to acute hepatitis. We extracted DNA samples from peripheral blood and all exons and flanking introns of TERT and TERC were amplified by PCR using 19 primer pairs (1 TERC, 18 TERT). To determine the sequence, the PCR products were analyzed by ABI/PRISM 3100 automated sequencer. Telomere lengths of leukocytes were assessed by flow-FISH. For the TERC gene, no mutation was found. One polymorphism (n514 G>A) was observed in 57/96 (59.4%) of patients. The same substitutions were detected in 31/59 (52.5%) healthy controls. For the TERT gene, two novel heterozygous, nonsynonymous mutations were identified (exon5; n2045 G>A, exon6; n2177 C>T). These base substitutions introduce an amino acid change-G682D and T726M, respectively. Neither patient had any clinical characteristics suggesting constitutional bone marrow failure syndrome. The n2177C>T substitution was identified in a 9-year-old girl with very severe AA who failed to respond to immunosuppressive therapy. She received an allogeneic bone marrow transplant (BMT) from an unrelated donor, but did not engraft. She was then treated by a second BMT from an HLA haploidentical her mother. Her blood cells had a very short telomere compared with that of age-matched controls. Another patient carrying the n2045G>A substitution had nonsevere AA and did not require any specific medication for 8 years. Six polymorphisms in exons of the TERT gene were identified in 102 unrelated patients (n915 G>A, n2097 C>T, n2520 G>A, n2946 T>C, n3039 C>T, and n3366 G>A). The allele frequencies of these silent base substitutions were 38/192 (19.8%), 3/192 (1.6%), 1/192 (0.5%), 1/192 (0.5%), 57/192 (29.7%), 2/192 (1.0%), respectively. Additionally, we identified 5 polymorphisms in introns of the TERT genes in 64 patients (IVS4+143 A>G, IVS9+11 C>T, IVS13+45 C>T, IVS15+136 G>A, and IVS16+81 C>T). The frequencies were 52/192 (27.1%), 3/192 (1.6%), 7/192 (3.6%), 1/192 (0.5%), and 1/192 (0.5%), respectively. Except for two substitutions (n915G>A and IVS4+143A>G), the other 9 were not listed in the SNP database. We found a few patients with AA carrying mutations of telomerase ribonucleoprotein complex genes. Because the incidence of these mutations is not higher than that in Western populations, this genetic difference does not explain the higher incidence of AA in Japanese children.