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ABSTRACT Acute myeloid leukemia (AML) is one of the most common types of leukemia. With the recent advances in sequencing technology and the growing body of knowledge on the genetics of AML,

there is increasing concern about cancer predisposing germline mutations as well as somatic mutations. As familial cases sharing germline mutations are constantly reported, germline

predisposition gene mutations in patients with AML are gaining attention. We performed genomic sequencing of Korean patients diagnosed with AML to identify the prevalence and characteristics

of germline predisposition mutations. Among 180 patients, germline predisposition mutations were identified in 13 patients (13/180, 7.2%, eight adults and five children). Germline mutations

of _BLM_,_ BRCA1_,_ BRCA2_,_ CTC1_,_ DDX41_,_ ERCC4_,_ ERCC6_,_ FANCI_,_ FANCM_,_ PALB2_, and _SBDS_ were identified. Most of the mutations are in genes involved in DNA repair and genomic

stability maintenance. Patients harboring germline mutations tended to have earlier onset of AML (_p_ = 0.005), however, the presence of germline mutations did not showed significant

association with other clinical characteristics or treatment outcome. Since each mutation was rare, further study with a larger number of cases would be needed to establish the effect of the

mutations. SIMILAR CONTENT BEING VIEWED BY OTHERS AML WITH GERMLINE _DDX41_ VARIANTS IS A CLINICOPATHOLOGICALLY DISTINCT ENTITY WITH AN INDOLENT CLINICAL COURSE AND FAVORABLE OUTCOME

Article 20 October 2021 SIGNIFICANCE OF HEREDITARY GENE ALTERATIONS FOR THE PATHOGENESIS OF ADULT BONE MARROW FAILURE VERSUS MYELOID NEOPLASIA Article 20 October 2022 TARGETED GENE PANELS

IDENTIFY A HIGH FREQUENCY OF PATHOGENIC GERMLINE VARIANTS IN PATIENTS DIAGNOSED WITH A HEMATOLOGICAL MALIGNANCY AND AT LEAST ONE OTHER INDEPENDENT CANCER Article 13 April 2021 INTRODUCTION

With the recent advances in sequencing technology and the growing body of knowledge on the genetics of AML, there is increasing concern about cancer predisposing germline mutations as well

as somatic mutations. It has been widely recognized that not only somatic mutations in cancer tissue, but also germline gene mutations can affect disease characteristics, progress, and

prognosis, and In cases with solid cancers such as breast cancer, the significance of germline mutation had been already recognized and changes in treatment and genetic counseling according

to the presence of those mutations had been settled down in clinical practice. Similarly, the category myeloid neoplasm with germline predisposition mutations was included in the WHO

classification in 20161. A number of cases of familial leukemia with these mutations was reported, mainly in relatively well-recognized genes such as _DDX41_, _CEBPA_, and _RUNX1_2,3,4.

However, these studies focused on specific ethnic groups, and data regarding other ethnicities are lacking. Furthermore, for patients with AML, hematopoietic stem cell transplantation (HSCT)

is frequently performed. Germline predisposition mutations could be a significant issue in the setting of HSCT, where most donors are family members, and a higher probability of shared

mutations is expected. As in the case of _BRCA1/2_ gene mutation, there may be an increased risk of other cancers, and patients and family members having the same mutation should be involved

in a surveillance program. Therefore, there is a growing need for basic information about frequency and types of germline predisposition gene mutations. In this context, we assessed the

prevalence of germline predisposition gene mutations and identified the clinical characteristics of mutation carriers among Korean patients with AML using genomic sequencing and we

correlated the mutation status with treatment outcome, to provide a broader insight into AML biology and patient care. METHODS PATIENTS Bone marrow aspirates of 180 patients diagnosed with

AML in our center between 2013 and 2017 were obtained after receiving informed consent for genetic study from each patient. This study was approved by the Samsung Medical Center

institutional review board (2017-04-027) and was conducted in accordance with the tenets of the Declaration of Helsinki. GENOMIC SEQUENCING A total of 139 patients was evaluated by whole

exome sequencing (WES) and 41 patients were by gene panel sequencing. NGS testing was performed with initial diagnostic samples. Clinical characteristics of patients were in Supplemental

Table 1. For WES, libraries were prepared using the Twist Human Core Exome Kit (Twist Bioscience, San Francisco, CA, USA) and sequenced on a NovaSeq system (Illumina, San Diego, CA, USA).

For gene panel sequencing, gene panel containing 215 genes (Supplemental Table 2) associated with hematologic malignancy or germline cancer predisposition was used and NextSeq 550 instrument

(Illumina) was used. Detailed sequencing methods and bioinformatics analysis were in Supplemental Methods. INTERPRETATION OF VARIANTS The following databases were used for variant

annotation: Online Mendelian Inheritance in Man (OMIM), Human Gene Mutation Database (HGMD), ClinVar, dbSNP, 1,000 Genome, he Genome Aggregation Database (gnomAD), Exome Sequencing Project

(ESP), and the Korean Reference Genome Database (KRGDB). The pathogenicity of missense variants was predicted using five in silico prediction algorithms, including Sorting Tolerant from

Intolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), MutationTaster, MutationAssessor, and Functional Analysis through Hidden Markov Models (FATHMM), implemented in dbNSFP. Effects

on splicing were predicted using SPIDEX, and dbscSNV. We first established a set of genes known to be associated with AML predisposition according to WHO classification5, and variants of

those genes were prioritized. Variants were further classified according to American College of Medical Genetics (ACMG) guideline6. For PM2 score, global population frequency cut off < 

0.00001 for dominant disease and < 0.0001 for recessive disease were applied. For PP3 score, the agreement of at least five prediction tools was applied. GERMLINE CONFIRMATION TEST

Suspected variants in germline predisposition genes were further confirmed with bone marrow specimens were collected when the patients were in complete remission. Sanger sequencing was

performed using custom primers and the BigDye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Because bone

marrow was used for germline mutation testing, possibility of confounding due to factors including residual tumor and clonal hematopoiesis cannot be ruled out. Interpretation of Sanger

sequencing results was proceeded under recognition of the limitation. STATISTICAL ANALYSIS To compare the outcome according to molecular characteristics, Fisher’s exact test and logistic

regression analysis were utilized, with single or multiple variables. Variables included in multiple logistic levels were chosen by adapting stepwise as a variable selection method.

Statistical analyses were computed using R. _p_ values < 0.05 were considered statistically significant. RESULTS/DISCUSSION For gene panel sequencing, diagnostic exome sequencing, median

sequence coverage was 1626x, with an average of genotype quality score 98. For whole exome sequencing, median sequence coverage was 182x, with an average of genotype quality score 54. GENES

INVOLVED IN DNA REPAIR OR MAINTENANCE OF GENOMIC STABILITY WERE FREQUENTLY MUTATED Among 180 patients, 13 (13/180, 7.2%) showed pathogenic mutations in germline predisposition genes (Table

1). Most of the identified germline predisposition gene mutations were in genes involved in DNA repair or maintenance of genomic stability, which were associated with inherited bone marrow

failure syndromes including Fanconi anemia (FA), dyskeratosis congenita (DC), or Shwachman–Diamond syndrome (SDS). Eight of the 13 germline mutations identified were in six FA genes:

_FANCD1_ (_BRCA2_), _FANCI_, _FANCM_,_ FANCN_ (_PALB2_), _FANCQ (ERCC4)_, and _FANCS_ (_BRCA1_). FA genes are involved in the FA signaling pathway, which is crucial in the DNA damage

response. Constant exposure to endogenous and exogenous genotoxic agents can jeopardize genomic stability when the DNA damage response is compromised7. FA is a rare genetic disorder often

accompanied by numerous other conditions, including early age of onset of symptoms, multiorgan congenital defects, bone marrow failure leading to pancytopenia, and predisposition to

hematological and non-hematological malignancies8. Monoallelic mutation carriers who had no apparent signs, were known to have increased risk for cancer9,10,11. The protein products of these

six genes are components of the DNA damage response system and participate in various cellular processes. Genomic instability resulting from defective function of those proteins can be

associated with increased cancer risk. However, elucidation of the function of each component and the consequences of deficiencies of each product have yet to be determined. In addition,

_BRCA1_/2, and _PALB2_ are not only associated with FA and predisposition for AML, but are also widely known as important risk factors for breast and ovarian cancer. Although monoallelic

mutation carriers do not develop FA, monoallelic mutations in _BRCA1_/2, or _PALB2_ are associated with increased risk of specific cancers, e.g., breast cancer. Recent National comprehensive

cancer network (NCCN) guidelines suggest that carriers of those mutations should be included in the surveillance program and should consider preventive actions

(https://www.nccn.org/professionals/physician_gls/pdf/genetics_screening.pdf). The same principle should hold for the patient with AML with germline predisposition mutations. Besides, _BLM_

is associated with Bloom syndrome, a rare chromosomal instability disorder characterized by growth retardation, immunodeficiency, and a wide spectrum of cancers12. _BLM_ mutation is known to

be associated with cancers13. _ERCC6_ plays a critical role in DNA repair, and an association between disruption of its function and increased susceptibility to cancer has been reported14.

_CTC1_ is known causative genes of the telomere biology disorder DC, characterized by accelerated telomere shortening leading to manifestations such as bone marrow failure, cancer, and

pulmonary fibrosis15. DC genes function in telomere maintenance; CTC1 functions in a telomere-associated complex to protect the telomere from lethal DNA degradation16. Although these DC

genes have autosomal recessive inheritance, an association between monoallelic deleterious germline mutations and myeloid malignancies has been reported17. Splice site mutations of the

_SBDS_,_ a causative gene of_ SDS, were identified in two patients. This mutation also had relatively high frequency among the control population (0.0048 from KRGDB and 0.004 from gnomAD

East Asians), probably reflecting high carrier frequency. _SBDS_ is characterized by exocrine pancreatic insufficiency, skeletal abnormalities, and bone marrow failure with an increased risk

of myeloid malignancy18. Approximately 90% of SDS is caused by two common mutations, c.183_184delinsCT and c.258 + 2T > G19, only the latter was detected in two of our patients. Although

monoallelic mutations are not known to cause SDS, association between monoallelic mutation and increased risk of malignancy cannot be excluded. The _DDX41_ mutations were known to have

different spectrum depending on ethnic groups. p.A500Cfs*9 mutation has been solely reported in Asian patients20. The most frequently reported mutation in Caucasians, p.D140Gfs*2, was not

found in our patients. The germline _DDX41_ mutation is one of most frequently detected germline predisposition mutations in myeloid malignancy, with around 70 families described to date21.

In these families, myeloid malignancies were associated with normal karyotypes, and about 50% were found to have a somatic second hit mutation in _DDX41_, suggesting that _DDX41_ acts as a

tumor suppressor22. _DDX41_ is associated with the dominant inheritance and donor cell derived leukemia is already reported20, screening of germline predisposition mutation should be

considered during donor selection. GERMLINE PREDISPOSITION MUTATIONS WERE ASSOCIATED WITH EARLIER AGE OF ONSET Characteristics of patients with germline predisposition mutations are given in

Table 2. There was a significant difference in age of onset between the two groups. Patients with germline predisposition mutations tended to be younger, showing an earlier age of onset

(_p_ = 0.005). Notably, while only 21 patients under age 20 were included (21/180, 11.7%), five among 13 with germline mutation (5/13, 38.5%) were children. Five of 21 children and eight of

139 adults had germline predisposition mutations (_p_ = 0.005). Although certain mutations like those in _DDX41_ are reported not to be associated with early onset malignancy2, age of AML

diagnosis was significantly lower in patients with germline mutations in other genes such as _CEBPA_3. It is understandable that inherited disorders tends to be expressed at earlier age in

childhood. < Considering better prognosis of children in patients with acute lymphoblastic leukemia, it cannot be ruled out harboring germline predisposition mutation was associated with

better outcome > The effect of germline predisposition gene mutations needs to be further investigated in this younger age group. Notably, although the association between germline

predisposition gene mutations and certain somatic mutations was not statistically clear. In addition, somatic mutations of _RUNX1_ and _ASXL1_ and complex karyotype, which are well-known

poor prognostic factors, were not identified in the germline predisposition mutation positive group. PRESENCE OF GERMLINE PREDISPOSITION GENE MUTATIONS DID NOT AFFECT THE CLINICAL OUTCOME

The presence or absence of germline predisposition mutations, however, did not affect clinical outcome. Factors confirmed as significant in this study were well-recognized good or poor

prognostic factors of AML. In multivariate Cox proportional hazards regression analysis for overall survival (OS), complex karyotype, older age, absence of gene fusion, poor outcome of

induction chemotherapy, and _FLT3_ ITD mutation were factors for unfavorable outcome (Supplemental Table 3). In the same analysis for relapse-free survival (RFS), poor outcome factors were

_RUNX1_ somatic mutation and _FLT3_ ITD (Supplemental Table 4). On the other hand, complete remission after induction chemotherapy, the presence of gene fusions, and CD34-negative

immunophenotype mutation were identified as favorable factors. Achievement of complete remission after induction chemotherapy and carrying well-known poor prognostic features like _FLT3_ ITD

mutation were important factors for both OS and RFS. The presence or absence of germline predisposition mutations did not affect clinical outcome. As reason for the negative finding, the

number of identified mutations was possibly insufficient to determine the effects of those mutations. More patients and germline mutation carriers would be needed to establish whether

germline predisposition mutations are beneficial or harmful. Clinical and therapeutic heterogeneity of patients might also play the role. Comparison of patients between groups with otherwise

identical condition would be desirable. Although younger age could be linked to higher durable intensity of and better response to chemotherapy, the association of germline predisposition

mutation and treatment outcome was not definite. Because statistical significance could not be achieved partially due to insufficient sample size, further study with more patients is needed.

In conclusion, we identified 13 patients with germline predisposition mutation among 180 patients with AML. Most of the mutated genes are involved in the DNA repair system, contributing

genomic stability. Although the effect of these mutations on clinical outcome, including OS and RFS, was not significant, we confirmed that this group of patients tends to develop AML at a

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This study was supported by a grant from the National Research Foundation of Korea (NRF-2018R1C1B6005185). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Laboratory Medicine,

Korea University College of Medicine, Seoul, Korea Borahm Kim * Department of Laboratory Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea

Borahm Kim, Seung-Tae Lee & Jong Rok Choi * Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea Woobin Yun * Department of Pediatrics, Samsung Medical

Center, Sungkyunkwan University School of Medicine, Seoul, Korea Keon Hee Yoo & Hong Hoe Koo * Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of

Medicine, Seoul, Korea Chul Won Jung * Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Ilwon-ro, Gangnam-gu, Seoul,

06351, Korea Sun Hee Kim Authors * Borahm Kim View author publications You can also search for this author inPubMed Google Scholar * Woobin Yun View author publications You can also search

for this author inPubMed Google Scholar * Seung-Tae Lee View author publications You can also search for this author inPubMed Google Scholar * Jong Rok Choi View author publications You can

also search for this author inPubMed Google Scholar * Keon Hee Yoo View author publications You can also search for this author inPubMed Google Scholar * Hong Hoe Koo View author

publications You can also search for this author inPubMed Google Scholar * Chul Won Jung View author publications You can also search for this author inPubMed Google Scholar * Sun Hee Kim

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS B.K. performed the research, analyzed the data, and wrote the paper. W.Y. performed the

experiment and analyzed the data. S.L., J.C. and S.K. designed the study and review the paper. K.Y., H.K., and C.J. obtained and analyzed the patient data. CORRESPONDING AUTHORS

Correspondence to Seung-Tae Lee or Sun Hee Kim. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE Springer

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Yun, W., Lee, ST. _et al._ Prevalence and clinical implications of germline predisposition gene mutations in patients with acute myeloid leukemia. _Sci Rep_ 10, 14297 (2020).

https://doi.org/10.1038/s41598-020-71386-z Download citation * Received: 16 December 2019 * Accepted: 14 August 2020 * Published: 31 August 2020 * DOI:

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