Hereditary thrombocythemia (HT) is an autosomal dominant disorder with clinical
features resembling sporadic essential thrombocythemia. HT families share
similar clinical symptoms caused by heterogeneous genetic alterations. Inherited
germ-line mutations in the thrombopoietin (TPO) gene and its receptor MPL have
been found causing thrombocytosis in a number of HT families. Five reported
mutations in the thrombopoietin gene are all located in the 5 prime untranslated
region (5’UTR) and cause overproduction of Tpo protein by the same
mechanism: increased translation efficiency for the mutant mRNAs. One
mutation identified in the MPL gene is located at the transmembrane domain and
results in a hyperactive receptor, thereby leading to thrombocytosis. All these
germ-line mutations have not been found in sporadic patients and are only
responsible for the etiology of some HT families, indicating that the occurrence of
these germ-line mutations is a rare event. The disease-causing genes for many
HT families remain unknown. Identifying genetic lesions in these families will
increase our knowledge of the physiology of thrombopoiesis and some of these
unknown genetic components may contribute to the pathogenesis in sporadic
MPD patients.
In the first part of the project for genetic studies of HT families, the TPO and MPL
genes were analyzed by genomic DNA exon sequencing and linkage analysis. A
splice donor mutation in the TPO gene was identified in a Polish family. This
mutation was previously identified in a Dutch family and the reoccurrence of this
rare mutation has not been reported to date. In order to determine whether the
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mutation mutation in these two families arose de novo or from a founder effect, haplotype
analysis was performed to examine polymorphic DNA sequences in the vicinity of
the mutation using microsatellites and single nucleotide polymorphism (SNP) in
these two families. Six microsatellite markers on the affected allele showed
different sizes in PCR products and 3 SNPs close to the mutation differed in their
sequences between the two families. We therefore concluded that the mutation
in these two families occurred de novo. The previously reported MPL mutation at
the transmembrane domain of MPL protein was identified in one of the HT
families studied here. Recently, 5 additional HT families were found carrying this
mutation. We conducted haplotype analysis using microsatellite markers in the
MPL gene locus for the 6 HT families. Four microsatellite markers surrounding
the MPL mutation showed identical sizes in the PCR products on the affected
allele, suggesting that the MPL mutation occurred from a single founder event.
This may explain the high frequency of this mutation in HT families.
In a large US family with HT, where the TPO and MPL genes were excluded as
disease causes, genome-wide linkage analysis was performed aiming to identify
novel genetic component for the thrombocytosis phenotype. Two genetic regions
with significant logarithm of odds (LOD) score values have been located using
microsatellites and SNP chip arrays. Candidate gene sequencing revealed one
novel polymorphism in the gelsolin gene, which encodes an actin-binding protein
abundant in platelets. Gelsolin has multiple biological functions in addition to
cytoskeletal actin modulation. Functional studies in cell proliferation assays and
mouse bone marrow transplantation did not validate this polymorphism as an
active disease causing mutation. Further studies on this polymorphism in platelet
biogenesis are planned for the future. In addition, sequencing of all the candidate
genes in the segregating regions is in progress.
In a second project, genome-wide linkage analyses were performed using
microsatellites and SNP chip arrays in a family with secondary polycythemia
inherited in an autosomal recessive mode. Both parametric and nonparametric linkage analysis were conducted for this family. Five genetic regions were found
linked to the disease phenotype. A few candidate genes were sequenced and
studied, however no genetic variation was found so far. Additionally, no
mutations were found in several genes involved in erythropoiesis and oxygen
sensing pathway. Burst forming units-Erythroid cultures in hypoxia condition
showed high expression of the EPO gene in 3 out of 4 affected family members,
suggesting a potential unknown defect in the oxygen-sensing pathway
