Two new mutations in the HIF2A gene associated with erythrocytosis

Congenital or familial erythrocytosis/polycythemia can have many causes, and an emerging cause is genetic disruption of the oxygen-sensing pathway that regulates the Erythropoietin (EPO) gene. More specifically, recent studies have identified erythrocytosis-associated mutations in the HIF2A gene, which encodes for Hypoxia Inducible Factor-2a (HIF-2a), as well as in two genes that encode for proteins that regulate it, Prolyl Hydroxylase Domain protein 2 (PHD2) and the von Hippel Lindau tumor suppressor protein (VHL). We report here the identification of two new heterozygous HIF2A missense mutations, M535T, and F540L, both associated with erythrocytosis. Met-535 has previously been identified as a residue mutated in other patients with erythrocytosis; although, the mutation of this particular residue to Thr has not been reported. In contrast, Phe-540 has not been reported as a residue mutated in erythrocytosis, and we present evidence here that this mutation impairs interaction of HIF-2a with both VHL and PHD2. © 2012 Wiley Periodicals, Inc.

JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis

Background The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. Methods We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. Results We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. Conclusions JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.

Tuberous sclerosis complex: clinical features, diagnosis, and prevalence within Northern Ireland

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 and TSC2 genes on chromosomes 9 and 16 respectively. Diagnosis is based on clinical features but can be difficult as a result of variable phenotypic expression. With the advantage of mutation analysis in making a diagnosis of TSC, and improved identification of the associated clinical features, there have been few new data on its prevalence and on the proportion of cases due to new mutations. We have performed a retrospective epidemiological study on the prevalence of TSC, the clinical features attributed to it, and the availability of mutational analysis. We identified 73 known patients with TSC (5 deceased): 39 were female and 34 male. Ages ranged from 10 months to 69 years, with a mean age of 27 years 11 months (SD 16y 10mo). The point prevalence of TSC in our study was estimated at I out of 24 956 on the prevalence day (30 April 2004). The majority of patients (42.5%) were diagnosed at less than 15 months of age; 25% were not given a diagnosis on first developing symptoms. In all, 93.2% had epilepsy and 71.2% had a learning disability.* A mutation was identified in 95.8% of those tested (26% TSC1 and 74% TSC2). TSC2 mutations were correlated with a more severe phenotype. The new mutation rate was calculated at 64%. We conclude that the prevalence of TSC is higher than previously calculated. We recommend that all children with epilepsy be assessed for features of TSC. Larger studies will be required to assess the prevalence of mutations in each gene, and genotype-phenotype correlation.

Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication.

The recently discovered aging-dependent large accumulation of point mutations in the human fibroblast mtDNA control region raised the question of their occurrence in postmitotic tissues. In the present work, analysis of biopsied or autopsied human skeletal muscle revealed the absence or only minimal presence of those mutations. By contrast, surprisingly, most of 26 individuals 53 to 92 years old, without a known history of neuromuscular disease, exhibited at mtDNA replication control sites in muscle an accumulation of two new point mutations, i.e., A189G and T408A, which were absent or marginally present in 19 individuals younger than 34 years. These two mutations were not found in fibroblasts from 22 subjects 64 to 101 years of age (T408A), or were present only in three subjects in very low amounts (A189G). Furthermore, in several older individuals exhibiting an accumulation in muscle of one or both of these mutations, they were nearly absent in other tissues, whereas the most frequent fibroblast-specific mutation (T414G) was present in skin, but not in muscle. Among eight additional individuals exhibiting partial denervation of their biopsied muscle, four subjects >80 years old had accumulated the two muscle-specific point mutations, which were, conversely, present at only very low levels in four subjects <or =40 years old. The striking tissue specificity of the muscle mtDNA mutations detected here and their mapping at critical sites for mtDNA replication strongly point to the involvement of a specific mutagenic machinery and to the functional relevance of these mutations.

High-resolution organellar genome analysis of Triticum and Aegilops sheds new light on cytoplasm evolution in wheat

We have utilised polymorphic chloroplast microsatellites to analyse cytoplasmic relationships between accessions in the genera Triticum and Aegilops. Sequencing of PCR products revealed point mutations and insertions/deletions in addition to the standard repeat length expansion/contraction which most likely represent ancient synapomorphies. Phylogenetic analyses revealed three distinct groups of accessions. One of these contained all the non-Aegilops speltoides S-type cytoplasm species, another comprised almost exclusively A, C, D, M, N, T and U cytoplasm-type accessions and the third contained the polyploid Triticum species and all the Ae. speltoides accessions, further confirming that Ae. speltoides or a closely related but now extinct species was the original B-genome donor of cultivated polyploid wheat. Successive decreases in levels of genetic diversity due to domestication were also observed. Finally, we highlight the importance of elucidating longer-term evolutionary processes operating at microsatellite repeat loci.

A new polycythaemia vera-associated SOCS3 SH2 mutant (SOCS3(F136L)) cannot regulate erythropoietin responses

Recently several different JAK2 exon12 mutations have been identified in V617F negative polycythaemia vera (PV) or idiopathic erythrocytosis (IE) patients. The patients present with erythrocytosis, ligand-independent cell growth and low serum erythropoietin (EPO) levels. Within this group, a deletion of amino acids 542-543 (N542-E543del) of JAK2 is most prevalent. We have previously shown that in the presence of JAK2(V617F), suppressor of cytokine signalling 3 (SOCS3) is unable to negatively regulate EPO signalling and proliferation of V617F-expressing cells. Here we report a PV patient heterozygous for the somatic JAK2(N542-E543del) mutation and a previously unreported germline mutation within the SH2 domain of SOCS3 (F136L). Interestingly, the SOCS3(F136L) mutation was detected in a Japanese myeloproliferative disorder patient cohort at double the frequency of healthy controls. Cells expressing SOCS3(F136L) had markedly elevated EPO-induced proliferation and extended EPO-induced JAK2 phosphorylation. Additionally, compared to wild-type SOCS3, mutant SOCS3 had an extended half-life in the presence of JAK2 and JAK2(N542-E543del). Our findings suggest that this loss-of-function SOCS3 mutation may have contributed to disease onset by causing deregulated JAK2 signalling in the presence of a constitutively active JAK2(N542-E543del) mutant.

Patterns of variation at a mitochondrial sequence-tagged-site locus provides new insights into the postglacial history of European Pinus sylvestris populations

Due to their maternal mode of inheritance, mitochondrial markers can be regarded as almost 'ideal' tools in evolutionary studies of conifer populations. In the present study, polymorphism was analysed at one mitochondrial intron (nad 1, exon B/C) in 23 native European Pinus sylvestris populations. In a preliminary screening for variation using a polymerase chain reaction-restriction fragment length polymorphism approach, two length variants were identified. By fully sequencing the 2.5 kb region, the observed length polymorphism was found to result from the insertion of a 31 bp sequence, with no other mutations observed within the intron. A set of primers was designed flanking the observed mutation, which identified a novel sequence-tagged-site mitochondrial marker for P. sylvestris. Analysis of 747 trees from the 23 populations using these primers revealed the occurrence of two distinct haplotypes in Europe. Within the Iberian Peninsula, the two haplotypes exhibited extensive population differentiation (Phi(ST) = 0.59; P less than or equal to 0.001) and a marked geographical structuring. In the populations of central and northern Europe, one haplotype largely predominated, with the second being found in only one individual of one population.

Somatic point mutations in mtDNA control region are influenced by genetic background and associated with healthy aging: a GEHA stud

Tissue specific somatic mutations occurring in the mtDNA control region have been proposed to provide a survival advantage. Data on twins and on relatives of long-lived subjects suggested that the occurrence/accumulation of these mutations may be genetically influenced. To further investigate control region somatic heteroplasmy in the elderly, we analyzed the segment surrounding the nt 150 position (previously reported as specific of Leukocytes) in various types of leukocytes obtained from 195 ultra-nonagenarians sib-pairs of Italian or Finnish origin collected in the frame of the GEHA Project. We found a significant correlation of the mtDNA control region heteroplasmy between sibs, confirming a genetic influence on this phenomenon. Furthermore, many subjects showed heteroplasmy due to mutations different from the C150T transition. In these cases heteroplasmy was correlated within sibpairs in Finnish and northern Italian samples, but not in southern Italians. This suggested that the genetic contribution to control region mutations may be population specific. Finally, we observed a possible correlation between heteroplasmy and Hand Grip strength, one of the best markers of physical performance and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal functions.

Structure-Phenotype Correlations of Human CYP21A2 Mutations in Congenital Adrenal Hyperplasia

Congenital Adrenal Hyperplasia (CAH) is a family of autosomal recessive disorders involving impaired synthesis of cortisol from cholesterol by adrenal cortex. The predominant causes of the disorder are mutations in the CYP21A2 gene that encodes a Cytochrome P450 21-hydroxylase enzyme, which is central to steroidogenesis. The severity of the disease depends upon the extent of impaired enzymatic activity and can be classified under severe Classical form or the mild Non-Classical form, Molecular characterisation of CYP21A2 mutations can be used to predict clinical phenotype and disease severity based upon changes it brings in 21-hydroxylase enzyme structure. A humanized model of CYP21A2 has been used to map and investigate the structural role of all known disease-causing mutations. A structural explanation of clinical manifestation allows us to put forward criteria that might allow the prediction of clinical severity of the disease.

In silico prediction of the effects of mutations in the human UDP-galactose 4'-epimerase gene:Towards a predictive framework for type III galactosemia

The enzyme UDP-galactose 4'-epimerase (GALE) catalyses the reversible epimerisation of both UDP-galactose and UDP-N-acetyl-galactosamine. Deficiency of the human enzyme (hGALE) is associated with type III galactosemia. The majority of known mutations in hGALE are missense and private thus making clinical guidance difficult. In this study a bioinformatics approach was employed to analyse the structural effects due to each mutation using both the UDP-glucose and UDP-N-acetylglucosamine bound structures of the wild-type protein. Changes to the enzyme's overall stability, substrate/cofactor binding and propensity to aggregate were also predicted. These predictions were found to be in good agreement with previous in vitro and in vivo studies when data was available and allowed for the differentiation of those mutants that severely impair the enzyme's activity against UDP-galactose. Next this combination of techniques were applied to another twenty-six reported variants from the NCBI dbSNP database that have yet to be studied to predict their effects. This identified p.I14T, p.R184H and p.G302R as likely severely impairing mutations. Although severely impaired mutants were predicted to decrease the protein's stability, overall predicted stability changes only weakly correlated with residual activity against UDP-galactose. This suggests other protein functions such as changes in cofactor and substrate binding may also contribute to the mechanism of impairment. Finally this investigation shows that this combination of different in silico approaches is useful in predicting the effects of mutations and that it could be the basis of an initial prediction of likely clinical severity when new hGALE mutants are discovered.

Evaluating Many Treatments and Biomarkers in Oncology: A New Design

There is a pressing need for more-efficient trial designs for biomarker-stratified clinical trials. We suggest a new approach to trial design that links novel treatment evaluation with the concurrent evaluation of a biomarker within a confirmatory phase II/III trial setting. We describe a new protocol using this approach in advanced colorectal cancer called FOCUS4. The protocol will ultimately answer three research questions for a number of treatments and biomarkers: (1) After a period of first-line chemotherapy, do targeted novel therapies provide signals of activity in different biomarker-defined populations? (2) If so, do these definitively improve outcomes? (3) Is evidence of activity restricted to the biomarker-defined groups? The protocol randomizes novel agents against placebo concurrently across a number of different biomarker-defined population-enriched cohorts: BRAF mutation; activated AKT pathway: PI3K mutation/absolute PTEN loss tumors; KRAS and NRAS mutations; and wild type at all the mentioned genes. Within each biomarker-defined population, the trial uses a multistaged approach with flexibility to adapt in response to planned interim analyses for lack of activity. FOCUS4 is the first test of a protocol that assigns all patients with metastatic colorectal cancer to one of a number of parallel population-enriched, biomarker-stratified randomized trials. Using this approach allows questions regarding efficacy and safety of multiple novel therapies to be answered in a relatively quick and efficient manner, while also allowing for the assessment of biomarkers to help target treatment.

Polycythaemia-inducing mutations in the erythropoietin receptor (EPOR):mechanism and function as elucidated by epidermal growth factor receptor-EPOR chimeras

Primary familial and congenital polycythaemia (PFCP) is a disease characterized by increased red blood cell mass, and can be associated with mutations in the intracellular region of the erythropoietin (EPO) receptor (EPOR). Here we explore the mechanisms by which EPOR mutations induce PFCP, using an experimental system based on chimeric receptors between epidermal growth factor receptor (EGFR) and EPOR. The design of the chimeras enabled EPOR signalling to be triggered by EGF binding. Using this system we analysed three novel EPOR mutations discovered in PFCP patients: a deletion mutation (Del1377-1411), a nonsense mutation (C1370A) and a missense mutation (G1445A). Three different chimeras, bearing these mutations in the cytosolic, EPOR region were generated; Hence, the differences in the chimera-related effects are specifically attributed to the mutations. The results show that the different mutations affect various aspects related to the signalling and metabolism of the chimeric receptors. These include slower degradation rate, higher levels of glycan-mature chimeric receptors, increased sensitivity to low levels of EGF (replacing EPO in this system) and extended signalling cascades. This study provides a novel experimental system to study polycythaemia-inducing mutations in the EPOR, and sheds new light on underlying mechanisms of EPOR over-activation in PFCP patients.

Molecular pathology of prostate cancer:the key to identifying new biomarkers of disease

Microarray technology has recently accelerated the study of the molecular events involved in prostate cancer, offering the prospect of more precise prognosis and new therapeutic strategies. This review summarises current knowledge of the molecular pathology of prostate cancer. The expression and function of numerous genes have been shown to be altered in prostate cancer. Many of these genes are involved in cell cycle regulation, steroid hormone metabolism or regulation of gene expression. The mechanisms by which androgen independence arises are discussed, including cross-activation, gene amplification and point mutations of the androgen receptor. Analysis of changes in the levels of expression of large numbers of genes during prostate cancer progression have provided a better understanding of the basis of the disease, yielding new molecular markers, such as hepsin, with potential use in diagnosis and prognosis.