Congenital Thrombocytopenia and Myelofibrosis Due to Germline Mutations in G6b-B— a Megakaryocyte-Specific Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Receptor

Unlike primary myelofibrosis (PMF) in adults, which is one of the three classical BCR-ABL-negative myeloproliferative neoplasms (MPN), myelofibrosis in children is rare, occurring most commonly in patients with acute leukemia, particularly acute megakaryoblastic leukemia. Congenital or inherited forms of myelofibrosis (cMF) are exceedingly rare, and may occur in the context of patients with inherited platelet disorders. Here we describe 3 families with inherited thrombocytopenia and cMF due to germline null mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM) receptor G6b-B.

We studied 3 families with a total of 6 affected children, each of which presented with macrothrombocytopenia, mild anemia, mild leukocytosis and a distinctive pattern of bone marrow reticulin fibrosis centered around clusters of atypical megakaryocytes. The disease appeared to segregate as an autosomal recessive trait. All affected children had mild to moderate bleeding symptoms and required frequent platelet and occasional red cell transfusions. None showed evidence of extramedullary hematopoiesis, such as splenomegaly, and all were negative for MPN associated mutations in JAK2, MPL, and CALR.

To identify the disease gene, we performed Affymetrix SNP 6.0 genotyping on peripheral blood DNA from three clinically affected children and 7 first and second-degree relatives from the index family, a complex consanguineous pedigree from Kuwait. Shared regions of homozygosity by descent were assessed using a custom designed platform; multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (MHC) on chromosome 6p (LOD=2.01), were identified as potential candidate genetic intervals. We focused on this region because affected individuals in the index family and one of the two other families, which were not known to be related, shared a common homozygous human leukocyte antigen (HLA) type and had congenital adrenal hyperplasia (CAH) due to the identical 21-hydroxylase (CYP21A2) mutation; the CYP21A2 and HLA loci are located at 6p21.33 and 6p21.32-6p22.1. Whole exome sequencing was performed on all three nuclear families and revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor G6b (MPIG6B, previously G6B-B, C6orf25), encoded within the candidate linkage region. We identified 5 individuals (4 affected, one previously unaffected) with a MPIG6B frameshift mutation (c.61_61+1dup; p.20fs) in two families from Kuwait. In addition we confirmed a second MPIG6B frameshift mutation at c.147insT (p.49fs) in two individuals (one proband, one mildly affected sibling) in a family from Saudi Arabia. No other homozygous mutations that segregated with the phenotype in all three families were identified. MPIG6B is a member of the immunoglobulin superfamily and is located in the MHC class III region on chromosome 6. To validate MPIG6B as a potential disease-causing gene, we evaluated G6b-B expression in bone marrow (BM) biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody specific to G6b-B. G6b-B was strongly and selectively expressed in megakaryocytes of control BM samples, but completely absent in megakaryocytes of the clinically affected individuals.

Interestingly, a murine knockout that lacks G6b-B has a strikingly similar phenotype to these patients, the animals having macrothrombocytopenia, myelofibrosis and aberrant platelet production and function. Thus, in addition to uncovering the molecular basis of this rare disease, the clinical phenotype highlights the potential importance of MPIG6B and aberrant megakaryocytic activation in mediating other forms of BM fibrosis.