An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia.

Essential thrombocythaemia (ET) is a chronic myeloproliferative syndrome due to sustained proliferation of megakaryocytes, which results in elevated numbers of circulating platelets, thrombotic or haemorrhagic episodes and occasional leukaemic transformation. The cause of ET is unknown. Hereditary thrombocythaemia (HT) with autosomal-dominant transmission has been described with manifestations similar to those of sporadic ET. As the thrombopoietin gene (THPO) encodes a lineage-restricted growth factor with profound stimulatory effects on megakaryopoiesis and platelet production, we tested the hypothesis that HT results from a mutation in the human THPO gene. In a Dutch family with eleven affected individuals, the thrombopoietin protein (TPO) concentrations in serum were consistently elevated in individuals with HT. We derived an intragenic CA marker for the human THPO gene and performed linkage analysis in fourteen informative meioses in this family. This resulted in a lod score of 3.5 at theta=0. A G-->C transversion was found in the splice donor site of intron 3 of the THPO gene in all affected family members. This mutation leads to THPO mRNAs with shortened 5'-untranslated regions (UTR) that are more efficiently translated than the normal THPO transcripts. We conclude that a splice donor mutation in THPO leads to systemic overproduction of TPO and causes thrombocythaemia.

Expression of c-MYC under the control of GATA-1 regulatory sequences causes erythroleukemia in transgenic mice.

To study oncogenesis in the erythroid lineage, we have generated transgenic mice carrying the human c-MYC proto-oncogene under the control of mouse GATA-1 regulatory sequences. Six transgenic lines expressed the transgene and displayed a clear oncogenic phenotype. Of these, five developed an early onset, rapidly progressive erythroleukemia that resulted in death of the founder animals 30-50 d after birth. Transgenic progeny of the sixth founder, while also expressing the transgene, remained asymptomatic for more than 8 mo, whereupon members of this line began to develop late onset erythroleukemia. The primary leukemic cells were transplantable into nude mice and syngeneic hosts. Cell lines were established from five of the six leukemic animals and these lines, designated erythroleukemia/c-MYC (EMY), displayed proerythroblast morphology and expressed markers characteristic of the erythroid lineage, including the erythropoietin receptor and beta-globin. Moreover, they also manifested a limited potential to differentiate in response to erythropoietin. Studies in the surviving transgenic line indicated that, contrary to our expectations, the transgene was not expressed in the mast cell lineage. That, coupled with the exclusive occurrence of erythroleukemia in all the transgenic lines, suggests that the GATA-1 promoter construct we have used includes regulatory sequences necessary for in vivo erythroid expression only. Additional sequences would appear to be required for expression in mast cells. Further, our results show that c-MYC can efficiently transform erythroid precursors if expressed at a vulnerable stage of their development.

The genetic polymorphism of debrisoquine/sparteine metabolism-molecular mechanisms.

The genetic polymorphism of debrisoquine/sparteine metabolism is one of the best studied examples of a genetic variability in drug response. 5-10% of individuals in Caucasian populations are 'poor metabolizers' of debrisoquine, sparteine and over 20 other drugs. The discovery and the inheritance of deficient debrisoquine/sparteine metabolism are briefly described, followed by a detailed account of the studies leading to the characterization of the deficient reaction and the purification of cytochrome P-450IID1, the target enzyme of this polymorphism. It is demonstrated by immunological methods that deficient debrisoquine hydroxylation is due to the absence of P-450IID1 protein in the livers of poor metabolizers. The cloning and sequencing of the P-450IID1 cDNA and of IID1 related genes are summarized. The P-450IID1 cDNA has subsequently led to the discovery of aberrant splicing of P-450IID1 pre-mRNA as the cause of absent P-450IID1 protein. Finally, the identification of mutant alleles of the P-450IID1 gene (CYP 2D) by restriction fragment length polymorphisms in lymphocyte DNA of poor metabolizers is presented.

The leptin receptor activates janus kinase 2 and signals for proliferation in a factor-dependent cell line.

The antiobesity effects of leptin are mediated by the obese receptor (OB-R), a member of the cytokine receptor superfamily. Several isoforms of OB-R that differ in the length of the cytoplasmic domain have been described. An isoform with a long cytoplasmic domain of 302 amino acids, termed OB-Rb, contains the conserved box 1 and box 2 motifs and is likely to be responsible for leptin-induced signaling. A point mutation in the OB-R gene of diabetes (db) mice generates a new splice donor that interferes with the correct splicing of the OB-Rb mRNA and is predicted to cause absence of the OB-Rb protein in db/db mice. Here we examined the signaling potential of the long isoform, OB-Rb, and of a short isoform, OB-Ra, in BaF3 cells, a factor-dependent hematopoietic cell line. The long isoform was able to generate a proliferative signal and upon leptin binding, activated janus kinase 2 (Jak2). Consistently, antibodies directed against the extracellular domain of OB-R coprecipitated Jak2. The short isoform, OB-Ra, was inactive in both proliferation and Jak activation. These results provide further support for the long isoform, OB-Rb, being the principal mediator of the effects of leptin and help to explain why db/db mice are resistant to leptin, despite the presence of the short OB-R isoforms.

Stalled cerebral capillary blood flow in mouse models of essential thrombocythemia and polycythemia vera revealed by in vivo two-photon imaging.

BACKGROUND: Essential thrombocythemia (ET) and polycythemia vera (PV) are myeloproliferative neoplasms (MPNs) that share the JAK2(V617F) mutation in hematopoietic stem cells, leading to excessive production of predominantly platelets in ET, and predominantly red blood cells (RBCs) in PV. The major cause of morbidity and mortality in PV and ET is thrombosis, including cerebrovascular occlusive disease. OBJECTIVES: To identify the effect of excessive blood cells on cerebral microcirculation in ET and PV. METHODS: We used two-photon excited fluorescence microscopy to examine cerebral blood flow in transgenic mouse models that mimic MPNs. RESULTS AND CONCLUSIONS: We found that flow was 'stalled' in an elevated fraction of brain capillaries in ET (18%), PV (27%), mixed MPN (14%) and secondary (non-MPN) erythrocytosis (27%) mice, as compared with controls (3%). The fraction of capillaries with stalled flow increased when the hematocrit value exceeded 55% in PV mice, and the majority of stalled vessels contained only stationary RBCs. In contrast, the majority of stalls in ET mice were caused by platelet aggregates. Stalls had a median persistence time of 0.5 and 1 h in ET and PV mice, respectively. Our findings shed new light on potential mechanisms of neurological problems in patients with MPNs.

Specificity of signaling by hematopoietic cytokine receptors: instructive versus permissive effects.

The helical cytokines constitute a family of proteins with a common three-dimensional structure. They exert a wide variety of biological effects with a preference for the hematopoietic system. The effects of helical cytokines are mediated by cell surface receptors, which belong to the cytokine receptor superfamily and signal by activating cytoplasmic tyrosine kinases of the Janus kinase (Jak) family and other downstream signaling pathways. The relevance of each of these pathways for eliciting a specific cellular response remains to be determined. This review will focus on cytokine receptors which play a role in the regulation of hematopoiesis and summarize data the address the question how specificity of signaling is achieved.

Translational pathophysiology: a novel molecular mechanism of human disease.

In higher eukaryotes, the expression of about 1 gene in 10 is strongly regulated at the level of messenger RNA (mRNA) translation into protein. Negative regulatory effects are often mediated by the 5'-untranslated region (5'-UTR) and rely on the fact that the 40S ribosomal subunit first binds to the cap structure at the 5'-end of mRNA and then scans for the first AUG codon. Self-complementary sequences can form stable stem-loop structures that interfere with the assembly of the preinitiation complex and/or ribosomal scanning. These stem loops can be further stabilized by the interaction with RNA-binding proteins, as in the case of ferritin. The presence of AUG codons located upstream of the physiological start site can inhibit translation by causing premature initiation and thereby preventing the ribosome from reaching the physiological start codon, as in the case of thrombopoietin (TPO). Recently, mutations that cause disease through increased or decreased efficiency of mRNA translation have been discovered, defining translational pathophysiology as a novel mechanism of human disease. Hereditary hyperferritinemia/cataract syndrome arises from various point mutations or deletions within a protein-binding sequence in the 5'-UTR of the L-ferritin mRNA. Each unique mutation confers a characteristic degree of hyperferritinemia and severity of cataract in affected individuals. Hereditary thrombocythemia (sometimes called familial essential thrombocythemia or familial thrombocytosis) can be caused by mutations in upstream AUG codons in the 5'-UTR of the TPO mRNA that normally function as translational repressors. Their inactivation leads to excessive production of TPO and elevated platelet counts. Finally, predisposition to melanoma may originate from mutations that create translational repressors in the 5'-UTR of the cyclin-dependent kinase inhibitor-2A gene.

Vinblastine inhibits the maturation of the precursor of mitochondrial aspartate aminotransferase. Vincristine and six other cytoskeleton inhibitors do not show this effect.

Cytoskeleton inhibitors were tested in chicken embryo fibroblast cultures for possible effects on the import of the precursor of mitochondrial aspartate aminotransferase into mitochondria. Vinblastine (50 microM) increased the steady-state pool of the precursor 2.5-fold in pulse experiments with [35S]methionine. If the precursor was accumulated during a pulse in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and then chased under diluting CCCP, vinblastine (50 microM) prolonged the half-life of the precursor from 0.5 min in the control to 3 min. Other cytoskeleton inhibitors, i.e. vincristine (25 to 150 microM), colchicine (50 microM), nocodazole (50 microM), podophyllotoxin (50 microM), taxol (45 microM), cytochalasin D (20 microM) and phalloidin (25 microM) did not show this effect. The observed inhibition by vinblastine does not seem to relate to its action on microtubuli.

Thrombopoietin production is inhibited by a translational mechanism.

Thrombopoietin (TPO) is a lineage-dominant hematopoietic cytokine that regulates megakaryopoiesis and platelet production. The major site of TPO biosynthesis is the liver. Despite easily detectable levels of liver TPO mRNA, the circulating TPO serum levels are very low. We have observed that translation of TPO mRNA is inhibited by the presence of inhibitory elements in the 5'-untranslated region (5'-UTR). Alternative promoter usage and differential splicing generate at least three TPO mRNA isoforms that differ in the composition of their 5'-UTR. Using mutational analysis we show that physiologically the translation of these TPO mRNA isoforms is strongly inhibited by the presence of AUG codons, which define several short open reading frames (ORFs) in the 5'-UTR and suppress efficient initiation at the physiologic start site. The two regularly spliced isoforms, which account for 98% of TPO mRNA, were almost completely inhibited, whereas a rare splice variant that lacks exon 2 can be more efficiently translated. Thus, inhibition of translation of the TPO mRNA is an efficient mechanism to prevent overproduction of this highly potent cytokine.

Thrombopoietin in thrombocytopenic mice: evidence against regulation at the mRNA level and for a direct regulatory role of platelets.

Thrombopoietin (TPO), originally described as an activity in the serum of thrombocytopenic animals that leads to increased production of platelets, has recently been isolated and cloned. Its closest relative in the cytokine superfamily, erythropoietin (EPO), is transcriptionally regulated during anemia, and it was expected that TPO would similarly be regulated during thrombocytopenia. We induced thrombocytopenia in mice and confirmed that TPO activity was upregulated, as determined by a bioassay. Liver and kidney were found to be the major sources of TPO mRNA. Surprisingly, TPO mRNA in these tissues was not upregulated in thrombocytopenic mice. Using a sensitive RNase protection assay that can distinguish between TPO isoforms, we found no change in the profile of mRNA for these isoforms. A semiquantitative reverse transcription-polymerase chain reaction assay also did not demonstrate upregulation of TPO mRNA in the spleen. Thus, the increase of TPO activity during thrombocytopenia is not caused by regulation at the level of TPO mRNA. Furthermore, isolated mouse platelets absorbed high amounts of bioactive TPO out of TPO-conditioned medium in a dose-dependent fashion. Our results are consistent with TPO protein being regulated at a posttranscriptional level and/or directly through absorption and metabolism by platelets.

Two mutant alleles of the human cytochrome P-450db1 gene (P450C2D1) associated with genetically deficient metabolism of debrisoquine and other drugs.

The "debrisoquine polymorphism" is a clinically important genetic defect of drug metabolism affecting 5-10% of individuals in Caucasian populations. It is inherited as an autosomal recessive trait. A full-length cDNA for human cytochrome P-450db1, the deficient enzyme (also designated P450IID1 for P450 family II subfamily D isozyme 1), has recently been cloned. Leukocyte DNA from "extensive metabolizers" (EMs) or "poor metabolizers" (PMs) of debrisoquine was examined by Southern analysis. Two polymorphic restriction fragments were associated with the PM phenotype when DNAs from 24 unrelated PM and 29 unrelated EM individuals were probed with P-450db1 cDNA after digestion with Xba I restriction endonuclease and Southern blotting: a polymorphic 44-kilobase (kb) fragment was found in 58% of PMs but only in 3.4% of EMs, and a polymorphic 11.5-kb fragment was present in 33% of PMs but in none of the EMs. Seventy-five percent of PMs had either the 44-kb or the 11.5-kb fragment or both. Segregation of these restriction fragment length polymorphisms in the families of six PM probands demonstrated that each of the two fragments is allelic with the 29-kb fragment present in all EM individuals and suggests that they identify two independent mutated allels of the P-450db1 gene (designated P450C2D1). At least a third mutated allele not detected by these restriction fragment length polymorphisms must be present in the population. The Xba I 44-kb fragment and 11.5-kb fragment were in linkage disequilibrium with restriction fragment length polymorphisms generated by four and five additional restriction endonucleases, respectively, which can be used to identify the same mutant alleles for the P-450db1 gene.

The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene.

The debrisoquine-4-hydroxylase polymorphism is a genetic variation in oxidative drug metabolism characterized by two phenotypes, the extensive metabolizer (EM) and poor metabolizer (PM). Of the Caucasian populations of Europe and North America, 5%-10% are of the PM phenotype and are unable to metabolize debrisoquine and numerous other drugs. The defect is caused by several mutant alleles of the CYP2D6 gene, two of which are detected in about 70% of PMs. We have constructed a genomic library from lymphocyte DNA of an EM positively identified by pedigree analysis to be homozygous for the normal CYP2D6 allele. The normal CYP2D6 gene was isolated; was completely sequenced, including 1,531 and 3,522 bp of 5' and 3' flanking DNA, respectively; and was found to contain nine exons within 4,378 bp. Two other genes, designated CYP2D7 and CYP2D8P, were also cloned and sequenced. CYP2D8P contains several gene-disrupting insertions, deletions, and termination codons within its exons, indicating that this is a pseudogene. CYP2D7, which is just downstream of CYP2D8P, is apparently normal, except for the presence, in the first exon, of an insertion that disrupts the reading frame. A hypothesis is presented that the presence of a pseudogene within the CYP2D subfamily transfers detrimental mutations via gene conversions into the CYP2D6 gene, thus accounting for the high frequency of mutations observed in the CYP2D6 gene in humans.

Autologous peripheral blood stem cell transplantation in a patient with chronic autoimmune thrombocytopenia.

Immunoablation by high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (PBSCT) is one approach currently under discussion for the treatment and possible cure of patients with severe autoimmune diseases. Chronic immune thrombocytopenic purpura (ITP) can be refractory to current therapies and in severe cases may constitute a life-threatening condition. We performed autologous PBSCT with T-cell depletion in a patient with severe chronic ITP but observed no effect on platelet levels. This contrasts with a recent report where complete remission was induced by a similar regimen in two patients with ITP Further studies will be necessary to identify subgroups of patients who might benefit from this form of therapy.

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization.

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).

Defective STAT signaling by the leptin receptor in diabetic mice.

Leptin and its receptor, obese receptor (OB-R), comprise an important signaling system for the regulation of body weight. Splice variants of OB-R mRNA encode proteins that differ in the length of their cytoplasmic domains. We cloned a long isoform of the wild-type leptin receptor that is preferentially expressed in the hypothalamus and show that it can activate signal transducers and activators of transcription (STAT)-3, STAT-5, and STAT-6. A point mutation within the OB-R gene of diabetic (db) mice generates a new splice donor site that dramatically reduces expression of this long isoform in homozygous db/db mice. In contrast, an OB-R protein with a shorter cytoplasmic domain is present in both db/db and wild-type mice. We show that this short isoform is unable to activate the STAT pathway. These data provide further evidence that the mutation in OB-R causes the db/db phenotype and identify three STAT proteins as potential mediators of the anti-obesity effects of leptin.

Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus.

The adipose tissue-derived hormone leptin regulates body weight homeostasis by decreasing food intake and increasing energy expenditure. The weight-reducing action of leptin is thought to be mediated primarily by signal transduction through the leptin receptor (LR) in the hypothalamus. We have used immunohistochemistry to localize LR-immunoreactive (LR-IR) cells in the rat brain using an antiserum against a portion of the intracellular domain of LR that is common to all LR isoforms. The antiserum recognized the short and long isoforms of LR in transfected hematopoietic BaF3 cells. To examine the chemical nature of target cells for leptin, direct double-labeling immunofluorescence histochemistry was applied. The results show extensive distribution of LR-like immunoreactivity (LR-LI) in the brain with positively stained cells present, e.g., in the choroid plexus, cerebral cortex, hippocampus, thalamus, and hypothalamus. In the hypothalamus, strongly LR-IR neurons were present in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), periventricular nucleus, arcuate nucleus, and lateral hypothalamus. Weaker LR-IR neurons were also demonstrated in the lateral and medial preoptic nuclei, suprachiasmatic nucleus, ventromedial and dorsomedial nuclei, and tuberomammillary nucleus. Confocal laser scanning microscopy showed LR-LI in the periphery of individual cells. In magnocellular neurons of the SON and PVN, LR-LI was demonstrated in vasopressin- and oxytocin-containing neurons. In parvocellular neurons of the PVN, LR-LI was demonstrated in many corticotropin-releasing hormone-containing neurons. LR-IR neurons were mainly seen in the ventromedial aspect of the arcuate nucleus, where LR-LI co-localized with neuropeptide Y. In the ventrolateral part of the arcuate nucleus, LR-LI was present in many large adrenocorticotropic hormone-IR proopiomelanocortin-containing neurons and in a few galanin-, neurotensin-, and growth hormone-releasing hormone-containing neurons. In the dorsomedial arcuate nucleus, few tyrosine hydroxylase (dopamine)-containing neurons were seen to have LR-LI. Melanin-concentrating hormone-containing neurons in the lateral hypothalamus had LR-LI. Based on the immunohistochemical results, possible interactions of leptin with brain mechanisms are discussed.

Myeloproliferative disorders: complications, survival and causes of death.

This retrospective single-center study compared thromboembolic and hemorrhagic complications, survival and causes of death in a cohort of 102 consecutive patients with myeloproliferative disorders (MPD). We included 17 patients with essential thrombocythemia (ET), 59 with polycythemia vera (PV), and 26 with osteomyelofibrosis (OMF). The median follow-up was 3.7 years. Estimated 8-year probability of complications for the entire cohort was 80 +/- 11% (95% confidence interval), without significant differences among MPD subgroups. The rate of thromboembolic complications, expressed as the number of events per 100 patient years, was 16.7 for patients with PV, 13.8 for OMF, and 7.5 for ET. Fifty-four percent of thromboembolic events in PV involved cerebral or limb arteries. The rate of bleeding complications was highest in patients with OMF (31.8 per 100 patient years), followed by ET and PV (11.8). Ninety percent of bleeding episodes affected the skin. mucosal membranes, and the gastrointestinal tract. Eight-year survival was highest in ET with 91 +/- 17%, followed by PV (66 +/- 18%) and OMF (40 +/- 31%) (P< 0.01). Twenty-four patients died during the observation period, and fatal thrombosis (in five patients) represented the leading cause of death. Only two patients with MPD died from fatal hemorrhage and one from acute leukemia. We conclude that survival is highest in ET and lowest in OMF. Both thromboembolic and hemorrhagic complications are frequent. However, thrombosis appears to be more often fatal than bleeding complications. Prophylaxis of thromboembolic events remains a key issue in the management of MPD.

Hereditary thrombocythaemia in a Japanese family is caused by a novel point mutation in the thrombopoietin gene.

Hereditary thrombocythaemia (HT) with clinical features very similar to essential thrombocythaemia (ET) has been found to be transmitted as an autosomal dominant trait in several families. Here we studied the pathogenesis of HT in a previously described Japanese kindred. We found markedly elevated thrombopoietin (TPO) serum levels in all affected individuals and identified a novel point mutation in the TPO gene, a G --> T transversion at position 516 of the TPO mRNA (G516T) that co-segregated with the HT phenotype in all affected family members. This mutation is located in the 5'-untranslated region (5'-UTR) of the TPO mRNA and when assayed in reticulocyte lysates, improved translational efficiency of in vitro transcribed TPO mRNA. Cell lines transfected with the mutant TPO cDNA secreted up to 8-fold more TPO protein than cells transfected with the normal cDNA. We provide a molecular model of how the mutation partially disables the physiologic repression of TPO translation and thereby causes thrombocytosis. This is the third family in which HT has been caused by the loss of translational inhibition of TPO mRNA.