Pathogenic Germline Variants in Acquired Aplastic Anemia (AA) and Paroxysmal Nocturnal Hemoglobinuria (PNH)
Authors: Shen Wenyi , Hirsch Cassandra M. , Przychodzen Bartlomiej P. , Makishima Hideki , Williams Louis , Mukherjee Sudipto , Carraway Hetty E. , Sekeres Mikkael A. , Maciejewski Jaroslaw P. 
Published: October 2019

Abstract
Next generation sequencing (NGS) methods, including whole exome sequencing (WES) and sequencing of large panels of genes have become standard tools facilitating the discovery of the genetic etiology of complex disorders, including the inherited bone marrow failure syndromes (IBMFS). This method has also revealed pathogenic variants in genes not previously associated with IBMFS. Our understanding of disease prevalence/incidence has also changed as exomes and genomes from healthy individuals or those with atypical presentations are being sequenced.
We hypothesized that in a proportion of otherwise typical AA/PNH, known or new germ line variants (GLVs) may be present. These variants may also contribute to timing of onset and disease evolution of AA/PNH. We screened a cohort of 258 AA/PNH pts (median age 44 years, range 6-89, male vs female 115:143). In aggregate, 564 exonic variants with an average 2.18 variants per sample were found. We then sub-classified these candidate alterations into Tier-1 and Tier-2 lesions: Tier-1 were defined as known disease-prone sequence alterations/mutations, new nonsense/frameshift mutations and highly recurrent missense mutations with low frequency in the general population. Tier-2 were defined as missense mutations with a high general population frequency of ˃=0.01% and were not studied further. In the final analysis, we identified 80 GLVs of Tier-1 and 484 GLVs of Tier-2.
In analyses focused on Tier-1 lesions, 61/258 pts (24%) with AA/PNH had pathogenic GLVs and 19 pts carried co-occurrent GLVs. High frequencies of mutations in DNA repair genes including BRCA2 (8 pts), ATM (6 pts), BRCA1 (5 pts), FANCC (3 pts), MSH2 (3pts), MLH1 (2 pts) were found in our cohort, all heterozygous, as well as telomere-associated genes including POT1 (5 pts), CTC1 (4 pts), DKC1 (3 pts), WRAP53 (2 pts) and NOP10 (1 pt) together with several other genes including NF1 (8 pts), CFTR (7 pts), APC (3 pts), PTEN (2 pts) and single carried GLVs in FH, GAR1, SBDS, SETBP1 and TP53. These GLVS are enriched in our cohort compared with the ExAC database controls (p<.01). As we can see TELO-associated genes including POT1, CTC1, DKC1, WRAP53 and NOP10 were all overrepresented in our cohort, FANCC was the only FA gene overrepresented in the same cohort, a completely opposite result when compared with previous study focusing on MDS cohort, in which FANCA, FANCE and FANCG were present at higher rates.
Further exploration focused on the relationship between GLVs and disease evolution. 10/61 Tier-1 GLVs carriers (16%) and 15/197 without Tier-1 GLVs (7.6%) evolved to MDS or AML (p<0.05). Further focusing on Tier-1 GLVs carriers, pts with co-occurring GLVs had a higher tendency to develop advanced disease compared to pts with single GLVs (p<.01). In particular, DKC1 (3 cases) and PTEN (2 cases) mutations always existed in a co-occurring pattern, although we cannot draw definitive conclusions from this apparent relationship given the relative paucity of cases. Compared to non-carriers (3/197, 1.5%), Tier-1 GLVs carriers (7/61, 11.4%) were significantly more likely to undergo PNH clone expansion (p<.01). Among 8 AA/PNH cases carrying NF1 GLVs, 4/8 (50%) underwent disease evolution, and 3 of these 4 cases were co-occurring GLV carriers. When we subgroup total cohort into typical AA, AA with small PNH clone and typical PNH separately, we can see that GLVs carriers in typical AA still had a higher tendency to develop advanced disease compared to non-carriers in this subgroup(p<.05).
In our cohort, none of the carriers of Tier-1 GLVs showed any physical signs of inherited congenital BMF syndromes. We identified 6 cases with a family history (FH) of hematologic disease and none of them were Tier-1 GLVs carriers. We also did not see any difference in FH of other cancer in these two groups.
In conclusion, our results indicate that important GL alterations in cancer predisposition genes can be found in acquired AA/PNH patients. Some of the genes seems to contribute to disease persistence and disease evolution/progression. Inherited factors likely play an important role in the pathogenesis of acquired AA/PNH and a clinical family history appears to be insufficient alone to determine/identify these predisposition states.

Disclosures
Carraway: Agios: Consultancy, Speakers Bureau; FibroGen: Consultancy; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy.