Pathogenic mutations identified by a multimodality approach in 117 Japanese Fanconi anemia patients

Abstract

Fanconi anemia is a rare recessive disease characterized by multiple congenital abnormalities, progressive bone marrow failure, and a predisposition to malignancies. It results from mutations in one of the 22 known FANC genes. The number of Japanese Fanconi anemia patients with a defined genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FANC genes in Japan and clarify the genotype-phenotype correlations. We studied 117 Japanese patients and successfully subtyped 97% of the cases. FANCA and FANCG pathogenic variants accounted for the disease in 58% and 25% of Fanconi anemia patients, respectively. We identified one FANCA and two FANCG hot spot mutations, which are found at low percentages (0.04-0.1%) in the whole-genome reference panel of 3,554 Japanese individuals (Tohoku Medical Megabank). FANCB was the third most common complementation group and only one FANCC case was identified in our series. Based on the data from the Tohoku Medical Megabank, we estimate that approximately 2.6% of Japanese are carriers of disease-causing FANC gene variants, excluding missense mutations. This is the largest series of subtyped Japanese Fanconi anemia patients to date and the results will be useful for future clinical management.

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-comparative genomic hybridization (aCGH) data displayed complete loss of the FANCB gene in Case 60 and Case 61. Sanger sequencing data identified 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 product, 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 alignments 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.

Figure 2.

Frequency distribution of total (A) versus unique (B) Fanconi anemia (FA) gene mutations in the 117 Japanese FA patients. The frequency of the total FA gene mutation was based on subtyping of 117 FA cases, while frequency of unique FA gene mutations was derived from 84 genetic variants detected in the 117 FA patients.

Figure 3.

The two FANCI variants in Case 96 caused two types of splicing defects. Real-time quantitative polymerase chain reaction (RT-PCR) analysis was carried out using a forward flanking primer on exon 3 and a reverse flanking primer on exon 5 as indicated. Two types of products were obtained, and the sequencing analyses revealed a single nucleotide insertion (top) and exon 4 skipping (bottom).