Page 70 - 2019_10 resto del Mondo_web

M. Mori et al.
deletions, one large duplication, and nine splicing muta- tions. All of the nine missense mutations were rated as “damaging” by both SIFT and PolyPhen-2 prediction pro- grams, including two novel variants (c.2723_2725TCT>GCC, p.LS908_909RP; c.3965T>G, p.V1322G). Three of the eight nonsense mutations, six of the 16 small indels, and four of the nine splicing mutations were novel (Online Supplementary Table S3). We consider that these 13 novel mutations are all pathogenic. The large duplication and all of the large deletions except one (c.3765+827_3814del) were detected by the MLPA assay. We did not identify the precise breakpoints of these FANCA deletions; therefore, it was unclear whether they were novel or not.
Similar to the previous reports from Western coun-
tries,20,22,23 the mutational spectrum in Japanese FA patients was broad (Figure 2B). However, some mutations were recurrently detected. The FANCA c.2546delC mutation was the most frequent (41 of 130 alleles; 31.5%), and other mutations such as c.978_c.979delGA, c.2602-2A>T, and c.2602-1G>A were detected in at least three unrelated families. c.1303C>T, c.2170A>C, c.2840C>G, c.3720_3724del, c.4168-2A>G were each detected in two unrelated families. The 45 remaining mutation variants were unique and were detected in single patients. FANCA c.2546delC existed at 0.08% frequency among 3,554 indi- viduals from 3.5KJPNv2 in the ToMMo (Table 1), but not in the ExAC database (0%). This mutation was also com- monly identified in Korean FA-A patients,24 and therefore seems to be a hotspot in the East Asian population.
B
C
Figure 1. A comprehensive analysis successfully subtyped most of the Japanese Fanconi anemia (FA) patients. (A) Schematic presentation of the diagnostic strategy for the 117 FA patients. (B) The array-compara- tive genomic hybridization (aCGH) data displayed complete loss of the FANCB gene in Case 60 and Case 61. Sanger sequencing data identi- fied the precise junctions in the two cases. (C) The whole-genome sequencing (WGS) analysis detected homozygous FANCC mutations in intron 12, resulting in a splicing defect. The Sanger sequencing data (left) identified the homozygous mutations in the patient (Case 64) and the heterozygous mutation in the patient’s mother. The real-time polymerase chain reaction (RT-PCR) analysis showed a larger product (arrowhead) than the wild-type prod- uct, and sequencing analysis of the RT-PCR product (right) revealed the 120bp intron retention (*) after exon 12, resulting in a stop codon. (D) The RNA sequence reads of exon 7 in FANCB and exon 12 in FANCN were absent for Case 62 and Case 98, respectively. Corresponding whole- exome sequencing (WES) read align- ments for Case 62 and Case 98 were diagnostic for the FANCB or FANCN mutations, as shown in Online Supplementary Figure S2A and B. N: number.
A
D
1966
haematologica | 2019; 104(10)