The Significance of the Next Generation Targeted Sequencing in the Precise Diagnosis of Pediatric Acquire Aplastic Anemia and Inherited Bone Marrow Failure Syndromes

Background

Diagnosis of inherited bone marrow failure syndromes (IBMFs) depend on classic clinical manifestation including early onset, physical anomalies, family history of cancer and/or bone marrow failure and chromosome breakage testing (MMC and/or DEB), mutation analyses and bone marrow chromosome analyses. At present, more than 70 pathogenic gene mutations had been identified. However, in some patients, physical anomalies is absent or delayed, and were misdiagnosed as acquired aplastic anemia(AA). Genetic analysis is very important to establish a precise diagnosis, predict cancer risk, direct treatment and genetic counseling. In this study, we focus on the application of next generation targeted sequencing in precise diagnosis of pediatric acquired AA/IBMFs, and the association between genetic abnormalities and clinical and laboratory characteristics.

Methods

We designed a targeted sequencing assay to test a panel of 417 genes. The panel contain reported gene associated with IBMFs and other diseases need be differentiated. Pediatic patients (≤14 year old) with suspected diagnosis of AA/IBMFs were enrolled. Peripheral blood (PB) DNA was used to genetic analysis and oral epithelia cells or PB DNA from their parents were used to identify somatic mutations and unreported polymorphism. All the results were validated by Sanger sequencing.

Results

The average coverage of targeted region was 98.15%, and the average sequencing depth was 315.9×. Totally, 283 patients were enrolled, including 176 clinically diagnosed acquired AA, 51 Fanconi anemia (FA), 8 dyskeratosis congenital(DKC), 30 Diamond-Blanckfan anemia(DBA), 15 congenital neutropenia(CN), and 3 congenital thrombocytopenia. Totally, 19% subjects had IBMFs related genetic mutations. In the patients who were clinically diagnosed as acquired AA patients, about 7% had IBMFs related disease-causing genetic mutations. Finally, 7 patients were genetically diagnosed as FA, 2 were DKC, 1 was WAS and 1 was SDS. In patients who were clinically diagnosed as FA, 33.4% had FANC related gene mutations. Telomere associated gene mutations were detected in 75% of clinical diagnosed DKC. For patients clinically diagnosed as DBA and CN, 36.7% and 20% were detected disease-causing mutations. After genetic screening, 2 patients who had been diagnosed as FA were modified as WAS and 1 DBA was modified as SDS.

Only 26% genetic diagnosed IBMFs patients had family history of bone marrow failure, leukemia, tumor or physical anomalies. Compared with acquired AA, patients with genetic diagnosed FA were more likely to have physical anomalies of short stature and development retardation, Cafe au lait spots and finger or toe malformation(P<0.001).However, 46% patients with IBMFs did not have any type of physical anomalies. Moreover, there were only 24% patients with genetic diagnosed IBMFs had positive results of MMC induced chromosome breakage test or SCGE, and both the examinations could not differentiate subtype of IBMFs. FANCD2 mono-ubiquitination test were performed recently. However, even in the genetically confirmed FA, the positive rate was only 18% (2/11). And, there were positive results in some acquired AA patients.

For FA patients with definitely genetic mutations, 62.5%(15/24) were compound heterozygous mutations,37.5%(9/24) were homozygous mutations.

Mutational frequencies of FANC were: FANCA 65%, FANCD2 23%, FANCG 9%, FANCI 9% and FANCB 4%. For the mutated type, the frequencies of missense, frameshift, nonsense, splicing mutation were 42%, 26%, 16%, 16%. In our study, there were 4 undetermined patients met the clinical diagnostic criteria of FA, and having heterozygous damaged mutations in FANC genes.

Conclusion

In conclusion, our IBMFs associated genes targeted sequencing assay is an effective strategy for precise diagnosis of bone marrow failure diseases, especially for those without family history or physical anomalies. However, nearly half of the clinically diagnosed IBMFs patients in our study were not detected the disease-causing mutations. This may be due to the mutations in the intron area, or large fragment deletion, which cannot be detected by targeted sequencing. And the novel gene involved in IBMFs need further study.