A novel RUNX1 exon 3 - 7 deletion causing a familial platelet disorder

Abstract Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare inherited disorder confirmed with the presence of a pathogenic germline RUNX1 variant and is thought to be heavily underdiagnosed. RUNX1 has also been found to be mutated in up to 10% of adult AML cases and other cell malignancies. We performed targeted next-generation sequencing and subsequent MLPA analysis in a kindred with multiple affected individuals with low platelet counts and a bleeding history. We detected a novel heterozygous exon 3–7 large deletion in the RUNX1 gene in all affected family members which is predicted to remove all of the Runt-homology DNA-binding domain and a portion of the Activation domain. Our results show that the combination of targeted NGS and MLPA analysis is an effective way to detect copy number variants (CNVs) which would be missed by conventional sequencing methods. This precise diagnosis offers the possibility of accurate counseling and clinical management in such patients who could go onto develop other cell malignancies.


Introduction
Inherited thrombocytopenias (ITs) comprise a heterogeneous group of disorders with a sustained reduction in platelet count which often manifest as a bleeding diathesis. To date, 26 forms of IT have been reported with known disease associations in 30 genes which makes genetic diagnosis challenging [1][2][3]. A precise genetic diagnosis provides clinical benefits for the patients where some patients with IT have unnecessary procedures and treatments such as splenectomies or mistaken for idiopathic thrombocytopenic purpura (ITP), which may be treated with steroids or immunosuppressive drugs with many side effects. Some of the gene variants in IT patients, e.g., RUNX1 result in patients having a predisposition to hematological malignancies and once a genetic defect is proven the information can be used to monitor the patients' hematological parameters more closely. Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) (OMIM# 601399) is confirmed with the presence of a pathogenic germline mutation in RUNX1. RUNX1 variants have been reported in approximately 10% of adult AML, which are mainly acquired but can also be germline and thought to be underdiagnosed [4]. This highlights the need for a definitive genetic diagnosis to aid clinical management of the condition. Current Next-Generation Sequencing (NGS) technologies allow the detection of both point mutations and CNVs with a single platform and workflow [5][6][7]. In this study, we describe and elucidate the genetic basis of a single family with three affected individuals who suffer from a bleeding diathesis and low platelet counts with previously unknown genetic diagnosis.

Cases
The family presented here has three affected individuals, all with a strong family history of clinical bleeding including epistaxis and easy bruising associated with low platelet counts and recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study [8] ( Figure 1A). The index case (P1) was a 67 year old male when initially recruited to the GAPP study with a 10 year history of a 'probable' hereditary platelet disorder. He had a history of bleeding episodes which included epistaxis, cutaneous bleeding, bleeding from minor wounds, GI bleeding, oral cavity bleeding, bleeding after tooth extraction and muscle hematomas. He bled excessively after a hip replacement. He had a platelet count of 129 x 10 9 /L and mean platelet volume (MPV) of 10.2 fL. He had Coronary artery bypass graft surgery aged 69 following which he suffered a thromboembolic stroke.
His relatives P2 and P3 (son and grandson, respectively) were also referred for genetic testing to the GAPP study. P2 is a 44 year old man with a clinical history of life-long muco-cutaneous bleeding. He had a platelet count of 83 x 10 9 /L and MPV of 9.3 fL. P3 is 22 years old with clinical features of platelet dysfunction. His platelet count at testing was 96 x 10 9 /L and MPV of 12.6 fL. (Table I). To date, no evidence of AML or other associated hematological malignancies have been reported in any of these patients. All other hematological parameters were within normal ranges.
The index case P1 was subjected to the ThromboGenomics targeted sequencing platform (TGv3.0) [5] where CNVs are called using an experimental pipeline based on ExomeDepth [9] version 1.1.10. This detected a novel heterozygous CNV Deletion of RUNX1 from exons 3-7 ( Figure 1B). The genetic variant was scored   with strong evidence of pathogenicity based on the current ACMG guidelines [10] (PM2-Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium, PVS1 -Null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where LOF is a known mechanism of disease). To confirm if the RUNX1 CNV deletion was present in all affected patients, Multiplex ligationdependent probe amplification (MLPA) analysis was performed which includes all exons of the RUNX1/CEBPA/GATA2/TERC/ TERT genes (MRC Holland kit P437-B1), and can detect both deletions and duplications. The index patient P1 and relatives P2 and P3 were all confirmed to have the heterozygous RUNX1 exon 3-7 deletion by MLPA analysis ( Figure 1C). The deletion removes 870 bp of the mRNA sequence of RUNX1 which is in frame, but completely removes the DNA/Runt DNA-binding domain and part of the activation domain ( Figure 1D).

Discussion
This report describes a family with an inherited thrombocytopenia and history of excessive bleeding. FPDMM is confirmed in this family studied with the presence of a pathogenic germline RUNX1 mutation [11][12][13]. To date, more than 70 FPDMM families have been reported with a 40% lifetime risk of developing MDS and/or AML [14]. While the majority of familial cases reported in the literature with RUNX1 mutations are point mutations, different extended deletions have also been described [5,11,15,16] ( Figure 1D). Here using targeted NGS and subsequent CNV/MLPA analysis, we have detected a novel heterozygous deletion covering exons 3-7 of the RUNX1 gene which is predicted to remove the entire DNA Runt/DNA binding domain. RUNX1/AML1 encodes the DNA-binding unit of the heterodimeric core-binding factor (CBF) that is a critical regulator of definitive hematopoiesis [17]. RUNX1 is also a recurrently mutated gene in sporadic myelodysplastic syndrome and leukemia. In sporadic AML, mutations in RUNX1 are usually secondary events, whereas in FPDMM they are initiating events. None of the patients included in this study have developed AML as a result of a secondary somatic event occurring within RUNX1 to date. It is thought that RUNX1 variants causing haploinsufficiency are thought to be associated with a lower incidence of myeloid malignancies when compared to those patients with dominant negative RUNX1 defects. The detection of a RUNX1 variant in such patients with low platelet counts allows the possibility of accurate counseling and clinical management for both them and extended family members at 50% risk of inheriting the pathogenic variant, whom could go onto develop other cell malignancies.