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Molecular diagnosis and clinical features of 117 Japanese FA patients
Mutation screening for FANCA and FANCG, and ALDH2 genotyping
Mutation analyses by PCR of FANCA or FANCG genes, Multiplex Ligation-mediated Probe Amplification (MLPA) tests for FANCA (Falco Biosystems), and ALDH2 genotyping were per- formed as previously described.11,12
Targeted-sequencing and whole-exome sequencing
Ten and 67 patients were examined by targeted-seq and WES, respectively, as previously described.8 In targeted-seq, 184 genes, including 15 FA genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O and P), were covered. All the mutation variants identified by targeted-seq or WES were verified by PCR and Sanger sequencing.
Array-comparative genomic hybridization analysis
For 10 patients, aCGH was performed as previously described.6 The probes covered 19 FA genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, Q, S, T, U) as well as FA-related genes, including NBS1, three RAD51 paralogs (XRCC3, RAD51B, and RAD51D), FAAP20, FAAP24, and FAAP100.
RNA-sequencing
We performed RNA-seq for three patients (Cases 62, 98, and 104). Libraries for RNA-seq were prepared using the TruSeq RNA Sample Prep Kit (Illumina) at Macrogen, and sequenced using the Illumina HiSeq 2500 platform with a standard 126-bp paired-end read protocol. Exon skipping events were identified using Genomon-fusion13 in which patient-specific spliced junc- tions were identified compared with those identified in a control sample.
Whole-genome sequencing
We performed WGS of DNA samples from one patient (Case 64) and his parents. The TruSeq DNA PCR-Free Library Preparation Kit (Illumina, San Diego, CA, USA) was used for library preparation. The prepared libraries were subjected to next- generation sequencing using a HiSeq X platform. We detected mutation variants as previously described.8
Estimating allele frequencies of the Fanconi anemia-associated deleterious genetic variations in the general Japanese population
We analyzed the 3.5KJPNv2 database, which was created with data generated by WGS of 3,554 individuals of the resident cohort of the ToMMo Project. The ToMMo project was estab- lished to develop a biobank that combines medical and genome information in the Tohoku area.14 As of 5th November 2018, the allele frequencies, including indel variations, were released in the publicly accessible 3.5KJPNv2 database (https://jmorp.mega- bank.tohoku.ac.jp/201811/). Our analysis focused on nonsense mutations, frameshift mutations (indels) and splicing donor or acceptor site mutations with less than 1% allele frequencies.
Results
Genetic subtyping of 117 Japanese Fanconi anemia patients through a comprehensive mutation screening
We started mutation analysis of FA patients by direct sequencing of FANCA and FANCG, and MLPA analysis for FANCA in 2009. WES and targeted-seq analyses were initiated in 2012, and molecular diagnosis was successful- ly achieved in 107 (91.5%) of the 117 patients (Figure 1A). We also examined the ALDH2 genotype which has been
Introduction
Fanconi anemia (FA) is a rare recessive disease character- ized by multiple congenital abnormalities, progressive bone marrow failure, and predisposition to malignancies. It results from mutations in one of the 22 known FANC genes.1 These genes are summarized in Online Supplementary Table S1. The proteins encoded by these genes participate in a DNA interstrand cross-link repair pathway that deals with DNA damage due to endogenous aldehydes, which are particularly deleterious to hematopoietic stem cells.2 However, more recent studies have shown that biallelic mutations in FANCM cause infertility and early onset cancer but not a typical FA phe- notype, and some of the FA genes are actually 'FA-like' since the patients with mutations in these genes do not display hematologic defects (Online Supplementary Table S1). Molecular subtyping is critical for the accurate diag- nosis and clinical management of the FA patients. However, finding causative mutations for a FA patient is not an easy task.3,4
In this study, we successfully subtyped 113 of the 117 Japanese FA patients and identified 215 mutant alleles through a comprehensive strategy starting from a simple genome polymerase chain reaction (PCR)-direct sequenc- ing approach, then progressing to next generation sequencing. The co-ordinated strategies included whole- exome sequencing (WES) and targeted exome sequencing (targeted-seq). In some cases in which we could not reach a conclusive diagnosis, additional methods, such as array- comparative genomic hybridization (aCGH) or RNA- sequencing (RNA-seq) and whole-genome sequencing (WGS) analysis, were extremely useful in detecting dele- tions or splicing abnormalities, respectively. Similar to other ethnic groups, we found that the FA-A and FA-G groups are the most prevalent in Japan. The FANCC muta- tion is rare and, a little surprisingly, FA-B is the third most prevalent subtype in Japan. The patients with the rare complementation groups, such as FA-D1, E, F, I, N, P, and T, were detected in less than 5% of the cases. We noted striking genotype-phenotype correlation in Japanese FA-B, D1, I, and N cases. In addition, we report the allele fre- quency of FA-associated deleterious genetic variations in the general Japanese population using the 3.5KJPNv2 data- base from the Tohoku Medical Megabank Organization (ToMMo).
Methods
Patients and samples
We studied 117 Japanese FA patients from 104 families in total. They overlap with previously reported cases (Online Supplementary Table S2)5-10 and an additional 13 new FA patients were recruited. The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Informed consent was obtained from the family for all subjects involved in this study, and the study was approved by the Research Ethics Committees of all participating hospitals and universities, including Tokai University, Kyoto University, and Nagoya University. Genomic DNA or total RNA was isolated from peripheral blood or cultured fibroblasts using Puregene (Qiagen) or RNAeasy (Qiagen) kit, respectively. cDNA was synthesized with a PrimeScript RT reagent kit (Takara).
haematologica | 2019; 104(10)
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