Comprehensive Genomic Evaluation For Inherited Bone Marrow Failure/Myelodysplastic Syndromes

Accurate and timely diagnosis of inherited bone marrow failure syndromes and inherited myelodysplastic syndromes (iBMFS/iMDS) is essential to guide optimal medical management and treatment. Their early diagnosis allows appropriate clinical monitoring to initiate hematopoietic stem cell transplantation (HSCT) prior to the development of leukemia, which carries a poor prognosis in these patients. Additionally these inherited syndromes typically do not respond to standard medical therapies for aplastic anemia (i.e. ATG and cyclosporine) and are associated with excessive toxicity with standard HSCT conditioning regimens. The recognition of an inherited disorder also informs donor selection as it allows unambiguous identification of affected siblings. Currently, testing of individual genes for iBMFS/iMDS is guided by clinical suspicion. Therefore, diagnosis is easily missed in patients with cryptic presentations. Moreover, sequential genetic testing is often cost-prohibitive and may not be completed within a clinically useful timeframe.

To address these clinical challenges, we developed a targeted gene capture pool coupled with next generation sequencing for the exons and flanking intronic sequences of 108 genes implicated in inherited and acquired marrow failure and MDS. Median base coverage was 192X, with 94.5% of targeted bases having >50x coverage and 99.6% of targeted bases having >10X coverage. This level of coverage together with our bioinformatic pipeline enabled identification of mutations spanning all classes (point mutations, small insertions and deletions, copy number variants, and genomic rearrangements). Rigorous criteria were applied to identify functionally deleterious mutations. Mutations were validated by conventional Sanger sequencing. Both germline and acquired mutations could be discerned, depending on DNA source (fibroblasts, peripheral blood or marrow mononuclear cells). The assay was validated with a blinded analysis of 12 patients harboring known mutations spanning different mutation classes. All mutations were successfully identified, including discerning mutations in genes from those in cognate pseudogenes.

We next tested for cryptic presentations of iBMFS/iMDS in 71 patients with idiopathic marrow failure. Fifty-nine subjects were drawn from the pediatric clinic and 12 from the adult clinic. Patients previously diagnosed with a known iBMFS/iMDS were excluded from this analysis. Twenty seven of the 71 subjects (38%) had a family history suggestive of an inherited marrow failure syndrome. Diagnoses of iBMFS/iMDS were made in 7 patients (10%), of whom only 2 patients had a history suggestive of familial marrow failure. An additional 4 patients had possible mutations in iBMFS/iMDS genes (TINF2 and SRP72) with functional validation pending. Deleterious mutations in GATA2 were identified in 4 patients (6%). Four additional patients had deleterious mutations in RUNX1 of which 2 were constitutional and 2 were somatically acquired. The diagnosis had previously been missed by clinical laboratory testing of these genes in 2 patients, possibly due to technical limitations of standard approaches. One additional patient with marrow failure had pathogenic mutations in a DNA repair gene not previously reported to cause iBMFS/iMDS. These data demonstrate the utility of this unbiased approach to diagnose patients irrespective of prior clinical preconceptions. The diagnosis of iBMFS/iMDS in 2 of the 12 adult patients highlights the need to consider inherited syndromes in adults with idiopathic marrow failure. More broadly, our results draw attention to the large number of patients (85-90%) remaining genetically undefined despite comprehensive screening for mutations in known iBMFS/iMDS genes.

In summary, we have developed and applied a comprehensive genomic approach to diagnose iBMFS/iMDS in 10% of patients presenting with idiopathic marrow failure. Mutations in GATA2 or RUNX1 were the most common causes of idiopathic marrow failure. Testing should be considered in both pediatric and adult patients even in the absence of prior family history. This unbiased diagnostic approach has revealed previously unexpected phenotypic features of these disorders. This efficient and cost-effective (<$400 reagents/sample) clinical diagnostic assay allows comprehensive unbiased screening for cryptic presentations of iBMFS/iMDS.