Rare and Common Germline Variants Contribute to Occurrence of Myelodysplastic Syndrome
Authors: Hahn Christopher N , Babic Milena , Schreiber Andreas W , Kutyna Monika M , Wee L Amilia , Brown Anna L , Perugini Michelle , Singhal Deepak , Hiwase Smita , Feng Jinghua , Parker Wendy T , Moore Sarah , Bardy Peter G , Saal Russell , Marlton Paula , Moore Andrew S. , Gonda Thomas J , Leo Paul , Branford Susan , D'Andrea Richard J , Lewis Ian D , To Luen Bik , Scott Hamish S , Hiwase Devendra 
Published: October 2019

Abstract
Background: Majority of MDS cases appear to be sporadic in nature, but 10-15% have clear familial basis due to predisposing mutations in genes such as RUNX1, GATA2, CEBPA and DDX41. Contribution of germline variants in sporadic MDS is not studied. This study attempts to address the contribution of germline variants in MDS pathogenesis.
Methods: We performed amplicon-based massively parallel sequencing (AmpliSeq custom panel adapted for Illumina HiSeq2500 sequencing) on all coding regions of 29 myeloid genes for 144 MDS samples. After identifying the variants in five genes (TET2, MET, GATA2, ASXL1, NOTCH1), we tested an additional 96 MDS samples including therapy-related myeloid neoplasm (T-MN) using a Sequenom assay. We also analyzed WES data for these variants in 178 AML samples and 758 normal controls and AmpliSeq data for ASXL1 and TET2 variants in 655 CML samples.
Results: Collation of all coding variants in the 29 myeloid genes sequenced identified germline variants occurring in primary MDS at frequencies significantly higher than expected when compared to the normal population (ExAC and matched cohort were similar) (Table 1). These variants occurred in 5 genes (TET2, MET, GATA2, ASXL1 and NOTCH1) at increased frequencies of 1.5-16.6 fold. Numerous MDS samples had multiple variants (4 with 4 variants, 4 with 3 variants, 18 with 2 variants) while 70 had 1 variant.
The 3 germline MET variants have been previously investigated in solid tumorigenesis and likely generate MET variant proteins that contribute to numerous cancer types including MDS. Interestingly, 7/17 (41%) MDS cases with germline MET variants also had other cancers including pancreatic, gastric and laryngeal cancers. Of the TET2 variants, Y867H and P1723S were concurrent in 5 MDS, 5 AML and 6 CML samples indicative of them being on the same allele (i.e. a haplotype). They were seen at higher than normal frequency in MDS and AML, but were not significantly enriched in CML. We are currently confirming their coexistence on the same allele and assaying for decreased TET2 activity to determine whether one or both variants contribute to the phenotype.
Other variants identified in MDS include the rare GATA2 (P161A) variant which is present in 1% of the population and the nearby common GATA2 (A164T) allele (~20%). These were mutually exclusive in our cohort and were seen at 3.9 and 1.5-fold, respectively, above the expected population frequency. We generated the P161A variant using site-directed mutagenesis and assayed for GATA2 transactivation activity in HEK293 cells with a GATA2-responsive LYL1 promoter-Luciferase construct (Figure 1). We also included empty vector (EV), wildtype (WT) GATA2 and T354M which is the most common highly penetrant autosomal dominant mutation leading to familial MDS/AML. As expected, T354M displayed a marked decrease in transactivation ability when compared to WT. The P161A variant similarly displayed loss-of-function in this assay, but not to the same magnitude as T354M. This is consistent with the hypothesis that reduced GATA2 function predisposes to myeloid malignancy where decreasing GATA2 activity correlates with increasing risk of developing malignancy. In our study 10/36 (28%) cases harboring these variants were T-MN cases.
Apart from MET (E168D) (11.4-fold), the 2 rare variants with highest frequency in MDS versus controls were ASXL1 (N986S) (16.6-fold) and NOTCH1 (R912W) (6.5-fold). ASXL1 is an epigenetic regulator often mutated in hematopoietic malignancy and aberrant NOTCH1 function has been associated with myeloid and lymphoid malignancies.
Conclusions: We have identified common and rare germline variants in genes involved in myeloid malignancy that may contribute to MDS pathogenesis. It remains to be seen whether they contribute to initiation, maintenance and/or progression of MDS and other hematopoietic malignancies. This is the first study reporting higher frequency of germline variants in sporadic MDS cases.
Table 1. Frequency of germline variants in MDS, AML and CML in comparison to ExAC. Table 1. Frequency of germline variants in MDS, AML and CML in comparison to ExAC.

Disclosures
Hiwase: Celgene Corporation: Research Funding.