A Familial Bleeding Disorder: Revised Diagnosis after 30 Years

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1 A Familial Bleeding Disorder: Revised Diagnosis after 30 Years Abdullah Kutlar MD-Medical College of Georgia, Augusta, GA Allison Spellman MD-Summit Cancer Care, Savannah, GA

2 Case Presentation 44y/o WF with history of excessive bleeding described as: Significant bleeding at menarche refractory to OCP Vaginal delivery at age 22 w/prolonged bleeding Bleeding w/appendectomy + wisdom teeth removal hx of menometrorrhagia (hysterectomy at 27) Treated in past w/platelets; stimate & DDAVP w/some response She & several family members were diagnosed with Bernard-Soulier. Denies any mucosal bleeding (GI; nose bleeds; gum, etc) Endorses easy bruising Labs: Plts Platelet aggregation studies: markedly decreased with col/epi & ACA (nearly absent). Normal primary response to ADP/collagen. Decreased secondary response to ADP/Collagen. Normal response to Ristocetin Suggestive of Storage pool defect; Glanzmann s; or ASA effect (not taking). Not consistent with Bernard-Soulier b/c normal aggregation with Ristocetin VWF studies normal

3 Case Presentation Family members with similar bleeding issues include: Maternal grandmother Mother Many of mother s siblings & cousins Brother Son Niece (Brother s daughter) No paternal relatives affected 5 maternal aunts - 1 died at age 3 of bleeding complications. 4 other w/milder bleeding issues 3 maternal uncles with bleeding issues Proband s mother had one cousin w/leukemia. His son died of leukemia at 9-10 yrs old as well. Pt s brother found to have a RUNX1 mutation? Sequencing revealed c159 delc in Runx1 (fraameshift)

4 RUNX1 Belongs to Runt-related transcription factor (RUNX1) family of genes Also known as AML1; CBFA2; PEBPA2B Runx1 is a transcription factor responsible for DNA binding and heterodimerization of other non-dna binding transcription factors. Encodes the DNA-binding alpha subunit of the core binding factor (CBFα) transcription complex Highly conserved runt-homology domain (RHD), located near N-terminus of RUNX1, mediates DNA binding and heterodimerization with partner CBFβ creating the CBF complex. CBFβ enhances affinity of RUNX1 to DNA protecting it from proteolytic degradation Heller 2013; Schmit 2015

5 RUNX1 Function CBF complex is expressed in hematopoietic stem cells and plays an important role in regulating hematopoiesis Critical for establishment of definitive (fetal liver) hematopoiesis as revealed in RUNX1- null mice that died during embryonic development due to bleeding Seems dispensable for HSC maintenance in adults (minor impact in self renewal). However, critical role in development of megakaryocytic & lymphoid maturation. Also in T-cell development. Deficiencies cause: Thrombocytopenia or qualitative dysfunction Reductions in CD4/CD8 T-cells; T-reg function; defective T-cell proliferative response to TCR stimulation RUNX1 deficiency also enhances myeloid cell proliferation & blocks cell differentiation resulting in leukemogenesis. Also plays a role in Regulating transcription of many tumor suppressor genes Regulating some DNA damage repair Linked to congenital neutropenia with transformation to MDS/leukemia in conjunction with CSF3R mutation Heller 2013; Ichikawa 2004; Link 2010; Yang 2005; Silva 2003; Satoh 2012; Wong 2011; Speck 2002; Skokowa 2014; Goyama 2015.

6 RUNX1 RUNX1 is located on chromosome 21 (21q22.12) Composed of 10 exons (1-6, 7A, 7B 7C & 8) Distinct promoter regions & variable splicing leads to transcription of different isoforms (1a-1c). Exons 3-5 (AA ) encode the DNA binding Runt domain in the N-terminal region Exons 7B & 8 (AA ) in the C- terminal region regulates gene transcription and encodes for the transcription activation domain DNA inhibitory domain (AA ) overlaps with C-terminal & is important in down-regulating gene expression Heller 2013; Schmit 2015

7 RUNX1 translocations RUNX1/CBFβ are common targets for chromosomal translocations in leukemia accounting for 15-20% of adult AML & 25% pediatric ALL t(8;21)(q22;q22) or RUNX1-ETO (M2 like) Inv 16 (p13q22) or CBFB-MYH11 (M4 like) t(12;21) (p13q22) or ETV-RUNX1 (ALL) t(3;21) or RUNX1-MECOM (therapy related myeloid neoplasms; CML) rarely de novo AML Inv 16 & t(8;21) are also referred to as the CBF-AML & are often favorable prognosis These fusion proteins have inability to signal transcription of RUNX1 hematopoietic target genes by loss of the transactivation domain. Also recruit corepressors further suppressing transcription Translocations can act in a dominant-negative fashion by competing for DNA or CBFβ binding sites thereby reducing wild-type Runx1 activity below 50%. Heller 2013; Speck 2002

8 Common Translocations Fusion proteins have dominant inhibitory activity repressing RUNX1 mediated transcription of hematopoietic target genes Inv 16 & RUNX1-ETO exhibit dominant negative inhibition of residual RUNX1 allele Led to same phenotype as homozygous deficient mice Arrest cells at immature stages switch of cell-fate decisions from differentiation to self-renewal Speck 2002

9 RUNX1 mutations RUNX1 somatic, point mutations have also been identified in 5-15% of sporadic leukemia predominantly M0 subtype Often normal cytogenetics Can be biallelic (no documented biallelic germline mutations in FPD/AML) - RUNX1 point mutations in 6-11% of MDS; 10% of CMML; 25% of secondary AML; 20% of systemic mastoycytosis; 10-15% of T-ALL Mutants lose ability to promote early hematopoietic development & induce myeloid maturation. However, low levels of RUNX1 are needed to maintain the leukemogenic cell phenotype also serve as oncogene? Cell cycle arrest & apoptosis ensue without any RUNX1 activity Usually associated with worse prognosis with less response to therapy & shortened OS. Heller 2013; Tang 2009; Mendler 2012; Osato 2004; Goyama 2015

10 Familial platelet dysfunction with propensity to myeloid malignancies Heterozygous germline mutations in RUNX1 cause autosomal dominant familial platelet disorder (FPD) with propensity to transform into myeloid malignancies (MDS; CMML; AML) Originally characterized in 1978 Some FPD/AML pedigrees with risk of T-ALL Quantitative & qualitative platelet defects with >35% risk of developing myeloid malignancies. Varying age of onset of malignancy. >35 reported pedigrees identified more common? 5/10 families with more than one first degree relative with MDS/AML had inherited RUNX1 mutations Disease with complete penetrance, but platelet count may be normal or only mild bleeding manifestations. Individual mutations result in different degrees of functional loss of RUNX1 protein, accounting for variable phenotypes of FPD/AML between families Ludy 1978; Liew 2011; Owen 2005; Nishimoto 2010

11 Identified Germline Mutations Figure 2. Schematic structure of RUNX1 protein and position of germline mutations identified in thirty-six FPD/AML pedigrees. Missense mutations are shown in green dotted lines, frameshift mutations are represented by blue dashed lines, while nonsense mutations are shown in red. Intragenic deletion and duplication were identified in three and one pedigree each, respectively. Numbers in parentheses indicate mutations identified in more than one pedigree. Heller 2013

12 FPD/AML Thrombocytopenia is usually mild-moderate w/normal plt size. Some with normal plt counts. Platelet aggregation is abnormal in response to several agonists & ASA like defects have been described PFA with abnormal response to collagen/epi with varying response to other agonists Also platelet storage pool deficiency & impaired GPIIb-IIIa activation Platelet production defects: Decreased megakaryocyte number Decreased maturation Decreased polyploidization & impaired pro-platelet formation Runx1 plays a role in the transcription of many genes involved in normal platelet production and function Heller 2013; Gerrard 1991; Sun 2004; Ickawa 2004; Bluteau 2012

13 Our Family: Proband Normal platelet count on all occasions except for once PFA & platelet aggregation studies suggestive of dysfunction but does not fit into a specific category. Platelet aggregation studies: markedly decreased with col/epi & ACA (nearly absent). Normal primary response to ADP/collagen. Decreased secondary response to ADP/Collagen. Normal response to Ristocetin RUNX1 testing revealed c.159del (S53Rfs*19) identical to her brother s mutational analysis To our knowledge, novel FPD/AML mutation Located on exon 3: Runt Domain mutation Flow cytometry analysis of Platelet Glycoprotein Expression (PGE) revealed normal MAB binding to GP1b & GPIIb/IIIa. R/o Bernard Soulier & Glanzman s Heller 2013

14 Our Family

15 Platelet Aggregation ASA: absent aggregation to ACA; dec or absent to collagen; only primary wave of ADP aggregation Plavix: absent ADP aggregation Storage pool: no secondary wave to ADP/epi. Glanzmanns: no aggregation w/any agonist only ristocetin response BSS/VWD: absent ristocetin Sharathkumar 2008; Practical-Haemostasis; UpToDate

16 Future Directions Changes in Classification: AML with recurrent genetic abnormalities Already have AML with t(8:21) & inv 16 Different subgroup for AML with RUNX1 mutation Revised criteria for section on familial myeloid neoplasms Associated not only with RUNX1 but also GATA2; CEPBA; ANKRD26 inherited mutations Who to screen? Mild thrombocytopenia & vague bleeding history without family history? All myeloid malignancies? Who will progress to myeloid neoplasms? Are FPD/AML family s also at risk of developing solid tumors?

17 The 51 genes underlying IPDs. The cartoon depicts the process of megakaryopoiesis and platelet formation. Claire Lentaigne et al. Blood 2016;127: by American Society of Hematology