- Research article
- Open Access
- Open Peer Review
Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients
- Lixian Chang†1, 2,
- Weiping Yuan†1, 2,
- Huimin Zeng1, 2,
- Quanquan Zhou1, 2,
- Wei Wei1, 2,
- Jianfeng Zhou1, 2,
- Miaomiao Li3,
- Xiaomin Wang1, 2,
- Mingjiang Xu4,
- Fengchun Yang4,
- Yungui Yang3,
- Tao Cheng1, 2Email author and
- Xiaofan Zhu1, 2Email author
© Chang et al.; licensee BioMed Central Ltd. 2014
- Received: 5 January 2014
- Accepted: 29 April 2014
- Published: 15 May 2014
Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients.
We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing.
Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients’ clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents.
Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology.
- Fanconi anemia
- Exome sequencing
- DNA repair
- Concomitant mutation
Fanconi anemia (FA) is a rare inherited genetic syndrome with diverse clinical manifestations, including developmental defects, short stature, bone marrow failure, and a high risk of malignancies. The prevalence of FA is 1–5 per 1 million population, and the heterozygous carrier frequency is estimated at 1 in 300 persons . Ninety percent of the patients experience bone marrow failure by the age of 40 years old . While androgens and hematopoietic growth factors are initially effective in treating FA, the disease has a very poor prognosis that often leaves bone marrow transplantation as the only option for a cure, although gene therapy could be a potential treatment . The clinical manifestations and treatment responses of FA patients are highly variable, likely due to multiple factors that are still not quite clear. The FA diagnosis is usually confirmed by a positive chromosomal breakage test (DEB test) and by the subtyping of FA (determination of the complementation group) [3, 4].
To date, 15 genetic subtypes of FA have been identified [5–9]. An FA patient carries either homologous mutations on two of the same alleles or compound heterozygous mutations on two different alleles of one FA gene. The traditional complementation test for group studies has been used mostly in FA patients, rather than in their families, using a stepwise method [10, 11]. If homologous or compound heterozygous mutations are found in an FA gene, the subtype of FA is then determined without knowing whether other FA genes might also be mutated in the same patient. Thus, while useful, this method could lead to incomplete genetic information for FA patients. More complex changes in FA genes in the same patient are plausible, as different subtypes of FA patients can have different clinical manifestations. Moreover, the complete genetic information of FA patients and their families regarding FA genes could potentially be valuable to the patients’ prognoses and treatment options, enabling prenatal DNA testing, permitting pre-implantation genetic diagnosis (PGD) for future pregnancies and excluding FA carriers from bone marrow donation and gene therapy. To obtain a comprehensive genomic picture of the FA genes in patients, we captured and sequenced the exomes of five FA families through peripheral blood (PB) specimens.
Diagnosis of FA, patients’ sample collection and genomic DNA isolation
All of the FA patients were outpatients. The diagnosis of FA was based on the clinical manifestations of the patients, MMC testing and single-cell gel electrophoresis tests using blood lymphocytes. Subsequent studies were approved by the ethics committee of the Institute of Hematology, CAMS/PUMC (KT2010072302). A total of five FA patients (designated Fa-001 to Fa-005) and their parents were recruited for this study and signed informed consent forms. One patient was later identified as an adopted child of the family (Fa-005). Peripheral blood (PB) samples from all of the subjects were collected after they were informed of the studies and had signed an institutional consent form and we also received consent from patients to publish the images and data from these samples. Genomic DNA from the FA patients and their parents were obtained from mononuclear cells for exome sequencing.
Exome sequencing, data analysis and PCR re-sequencing
The genomic DNA samples were randomly fragmented by Covaris, and the DNA fragments with base pair peaks were approximately 150 to 200 bp long. Adapters were then ligated to both ends of the resulting fragments. The adapter-ligated templates were purified using Agencourt AMPure SPRI beads, and fragments with an insert size of approximately 250 bp were excised. The extracted DNA was amplified by ligation-mediated PCR (LM-PCR) and was purified and hybridized to the SureSelect Biotinylated RNA Library (BAITS, Agilent Inc.) for enrichment. Hybridized fragments were bound to streptavidin beads, whereas non-hybridized fragments were washed out after 24 h. Captured LM-PCR products were analyzed using an Agilent 2100 Bioanalyzer to estimate the magnitude of their enrichment. Each captured library was then loaded onto a Hiseq2000 Platform, and massive parallel sequencing was performed for each captured library independently to ensure that each sample had at least 50-fold coverage. Raw image files were processed by Illumina Pipeline software, version 1.6, for base-calling with default parameters, and the sequences of each individual were generated as 90 bp paired-end reads. The bioinformatics analyses (Additional file 1: Figure S1) were performed by BGI at Shenzhen, China. The mutations in the FANC genes identified by exome sequencing were further verified by traditional PCR. The SRA accession number for the Exome-seq data reported in this paper was SRA067806.
Diagnosis of FA patients
Clinical manifestation of FA patients
Age at diagnosis (years)
Café au lait spots
Absence of right hand thumb. Radial eversion of left hand thumb.
Right hand thumb deformity. Hypoplastic thenar eminence of right hand.
Hexadactylism of right hand
Microcephaly small eyes
Gastrointestinal tract abnormalities
Hematology 1 WBC
2.09 × 109/L
4.27 × 109/L
2.67 × 109/L
6.86 × 109/L
2.78 × 109/L
9 × 109/L
30 × 109/L
14 × 109/L
33 × 109/L
42 × 109/L
MMC (80 ng/ml)*
Abnormal cell rate (%)
Single-cell gel electrophoresis test**
Comet cell rates (%)
Traditional Chinese medicine
4 U RBC/year
6 U RBC/year
2 dose PLT/year
2 dose PLT/year
Hematology 2 WBC
4.4 × 109/L
3.55 × 109/L
3.03 × 109/L
4.7 × 109/L
3.05 × 109/L
16 × 109/L
19 × 109/L
105 × 109/L
25 × 109/L
75 × 109/L
Exome sequencing data analysis
Validated Fanconi gene mutations
-GGGCTGT deletion frameshift
G > A missense A > V
-GG deletion frameshift
-A deletion frameshift
T > C missense S > G
G > A missense R > W
G > C missense P > A
A > G intron
C > T missense V > I
A > G missense I- > V
C > G missense P > A
G > T nonsense N > N
C > T missense S > F
A > G missense I > V
G > A missense R > Q
C > G missense H > D
A > C missense N > H
A > C missense N > D
A > C 3’-UTR
C > T 3’-UTR
C > T missense P > L
G > A 3’-UTR
A > G missense I > V
FA is a recessive inherited disease with FA gene mutations that are primarily involved in DNA damage response or repair, resulting in genomic instability . Its complex clinical manifestations are associated with different FA subtypes. Interestingly, the clinical manifestations can also differ among patients with the same FA subtype. There is no clear explanation for the relationship between the clinical manifestations of FA patients and the genetic mutations in their FA genes. To this end, we performed exome sequencing on PB samples from 5 FA patients and from their parents. Although the sample size in our study was small, the high frequency of heterozygous mutations in 4 of 5 patients, compared with their biological parents, suggested that the mutational events in FA carriers might not occur randomly but rather are linked. It is probable that a core determinant for some, if not all, FA genes governs the susceptibility of FA pathway gene mutations and, thus, of FA clinical manifestations.
In clinical practice, complementation group assignment and mutational analysis have been routinely used to identify types of FA patients, with the latter becoming more prevalent in recent years. The usual strategy for mutation analysis is to sequence the FANCA, FANCC, FANCE, FANCG, FANCD2 and other FA genes sequentially until a subtype of FA is identified [10, 11]. The correct subtyping of FA patients is critical for their prognoses and treatment because of the clinical variability among subtypes [1, 15, 16]. It is conceivable that the specific mutation theory is not always reflected in FA phenotypes because siblings with identical mutations can have different FA phenotypes . Heterozygous mutations in FA genes can also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers [18–25]. This finding is also in agreement with our study, in which four patients had more than one FA mutation gene with, vastly distinct clinical manifestations and different treatment responses to androstanazol and prednisone. Furthermore, it should be noted that synonymous mutations and known SNPs can also contribute to FA, although each of these mutations must be studied individually. It will be interesting to determine whether the diverse clinical manifestations, cancer susceptibilities and biological properties in the same or different FA subtypes reflect specific combinations of multiple heterozygous FA gene mutations. Here, we suggest that all FA genes should be subjected to mutation analysis in clinically diagnosed FA patients and their parents, in addition to complementation group analysis, to ascertain the most accurate diagnosis of the FA subtype, to aid clinicians, as well as families, during genetic counseling process and to advance FA research and future gene and stem cell therapies.
Although the sample size of our study was small, the striking number of concomitant mutations in the FA genes (excluding the SNPs) lead us to believe that sequencing of large samples of FA patients and their families, as well as more detailed biological and functional studies of these samples, are needed to ascertain the relevance of heterozygous FA gene mutation(s) to the clinical manifestations and prognoses of FA patients.
To our surprise, we did not find any new mutations related to DNA repair pathway genes, especially given that FA is considered to be a genomic instability disorder. There are several possible explanations that could account for this negative finding. First, new mutations in DNA repair pathways in HSCs might occur at low frequencies such that those HSCs with new somatic mutations did not have a growth advantage at the time of the sample collection. Second, some epigenetic mechanisms other than additional genetic changes, such as mutations, could be involved in the pathogenic process [26, 27]. Third, it is also possible that some of the mutated genes detected by exome-seq might have participated in DNA repair, but they are currently unknown. Nevertheless, the sequence analysis of our current data set suggested that mutations in the FA genes were sufficient for the manifestations of FA patients. Further studies are needed to ascertain this observation with larger FA patient pool sizes, to compare the exome sequencing data of patients before and after the development of cancer.
In conclusion, although FA subtype mutations define an FA patient, we found that multiple heterozygous FA gene mutations inherited from the parents could occur concomitantly in the same FA patient. The higher mutation rate among FA genes in the same patient also hinted that a common upstream event could determine the mutational susceptibility of the FA pathway, and mutations in FA genes alone might be sufficient to initiate FA in the hematopoietic system. Therefore, our current report has important implications for the pathogenesis of FA, as well as for the clinical management (such as treatment options) of FA patients.
The authors thank the patients and their parents for their cooperation on this project. We would like also to thank the members of the State Key Laboratory of Experimental Hematology and the Department of Pediatric Hematology at the Blood Disease Hospital of CAMS/PUMC for their valuable discussion or assistance in this study. This work was supported by grants from the Ministry of Science and Technology of China (2012CB966600, 2011CB964801, 2013CB966902, 2013BAI01B09) and from the National Natural Science Foundation of China (81170470, 81330015, 81328003, 81270575, 81130074, 81300436).
The NCBI accession number for the exome sequence reported in this paper is SRA067806.
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