Clinical Exome Sequencing Unravels New Disease Causing Mutations in Myeloproliferative Neoplasms (MPNs): A Pilot Study in Patients from the State of Qatar

In Qatar, a cluster of ET familial cases (25 families from 5 major tribes) has been documented. This observation represents a unique interest for the scientific community worldwide, as the cultural traditions of tribal marriages between 1^stcousins is expected to lead to the preservation of a limited genetic pool (giving rise to founder effects).

CES was employed to analyze a 3 probands from three unrelated families (S1, S2, S3). All 6 patients were Qatari belonging to consanguineous families with a positive family history of MPNs.

CES confirmed the diagnosis in the 3 probands: two patients (S1 and S2) were classified as Essential Thrombocythemia (ET), one patient (S3) as unclassified MPNs. In the other 3 subjects (S4, S4, S6), several shared deleterious mutations and polymorphisms were uncovered.

CES identified 10 novel mutations in known genes as well as 7 novel candidate genes and 2 (6%) previously reported mutations in the three probands (S1, S2, S3) who's diagnosis was confirmed .

S1: a confirmed diagnosis and phenotype of ET (JAK2 and THPO), S2: a confirmed diagnosis and phenotype of ET (JAK2 and MPL) and S3: a confirmed diagnosis of U-MPNs (TERT, ATM, NUMA1 and ASXL)

The mutations/variants were classified according to ACMG recommendations into 5 classes. The number of mutations/variants detected in each class is as follows:

Class 1 - Previously reported as disease-causing: 6

Class 2 - Previously unreported, but of the type which can cause the disorder: 3

Class 3 - Previously unreported, may or may not be the cause of the disorder (Variant of uncertain clinical significance): 48

Class 4 - Previously unreported but likely neutral: 1

Class 5 - Previously reported as neutral: 3

CES identified a total of 61 interesting variants which occurred in 50 genes, 13 were recurrently. The vast majority of the prioritized variants were missense 50(82%); the remaining were frameshift (small insertions and deletions) 10(16%) or affecting splicing sites 1(2%).

The most interesting finding is the presence of 10 novel mutations in known genes related to MPNs as well as 7 variants in novel candidate genes in the three probands who's diagnosis was confirmed via CES. This study presents novel data that contributes to understanding the high level of genetic complexity in MPNs Qatari patients. It has identified new variants in cancer-related genes potentially involved in the pathogenesis of the disease

Our study of familial MPNs led to the identification of new disease-causing mutations and variants, some previously described as either germline or somatic. A clinical cohort of 3 Qatari probands suspected to have MPNs as well as three family members with a positive family history of MPNs presented at National Center for Cancer Care (NCCCR) S1) is a female who presented with high hemoglobin (Hb) level, red blood cells (RBCs) count, leukocytosis with neutrophilia, very high platelets and normal erythropoietin (EPO); subject 2 (S2) is a female who presented with normal Hb, RBCs count, slightly high WBCs and high platelets; subject 3 (S3) is a male who presented with high Hb, normal WBCs and EPO

3 family members, subject 4 -a mother presented with normal Hb level, sustained high RBCs count and slightly high WBCs count, subject 5 a daughter presented with unexplained sustained erythrocytosis, leukocytosis (absolute neutrophilia, for more than 2 years Chronic headache, normal MRI brain and no progression to cancer no Ischemic events, subject 6- healthy daughter with normal CBC indicesThe pattern of mutations identified was unique. In this study 10 novel mutations in known genes already implicated in the pathogenesis of MPNs were detected including THPO, CBL, ASXL1, MPL (2x), BCL2, MYO18B, MTOR, PDGFRB, TERT. Mutations/variants were detected in 9 genes not previously associated with MPNs suggesting potential novel gene associations. Pathogenic/likely pathogenic mutations, uncertain variants and neutral/likely neutral variants were identified using the CES approach. A complex combination of mutations was observed in our cohort of subjects. For example in subject 1 (S1), an ET patient, the following mutations were observed: THPO S184R, ITGA2 R76Q, JAK2 V617F, CBL E196G, CBFB G180S, FANCA S858R, ASXL1 T600P fs103. The following detailed analysis of each mutation reveals that all these mutations could potentially play a role in the pathogenesis of MPNs