The German sickle cell disease registry reveals a surprising risk of acute splenic sequestration and an increased transfusion requirement in patients with compound heterozygous sickle cell disease HbS/ß-thalassaemia and no or low HbA expression.

Patients with sickle cell disease (SCD) in Germany exhibit a substantial genetic diversity in the ß-globin genotype. Data collected by the national German SCD registry reflect this diversity and allowed us to analyze the phenotypes associated with different SCD genotypes. Our study focused on 90 patients with HbS/ß-thalassaemia (HbS/ß-thal) and compared these to patients with HbSS and HbSC. Patients with HbS/ß-thal were classified into three groups: HbS/ß0-thal (no HbA), HbS/ß+-thal (HbA < 14%), and HbS/ß++-thal (HbA≥14%). In comparison to HbSS, patients with HbS/ß++-thal had higher Hb-levels, lower hemolytic activity and rarely required red blood cell transfusions. HbS/ß0-thal and HbS/ß+-thal closely resembled each other and are jointly referred to as HbS/ß0/+-thal. Compared to HbSS, patients with HbS/ß0/+-thal experienced a similar frequency of vasoocclusive crises and degree of hemolysis. However, the frequency of red blood cell transfusions (0.6 vs. 0.39/year, p = .0049) and splenic sequestration crises (42.4 vs. 15.5% of patients, p = 3.799e-05) was higher in HbS/ß0/+-thal than in HbSS, but close to zero in HbS/ß++-thal. In conclusion, the level of HbA expression determines the phenotype of HbS/ß+-thal. HbS/ß-thal expressing no or little HbA is hematologically similar to HbSS, but causes a previously unknown high risk of splenic sequestration.

Comprehensive in silico and functional studies for classification of EPAS1/HIF2A genetic variants identified in patients with erythrocytosis.

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

Long-term haematological response and maintained immunological function after laparoscopic subtotal splenectomy in patients with hereditary spherocytosis.

INTRODUCTION: Subtotal or total splenectomy are recommended in severe and should be considered in intermediate forms of hereditary spherocytosis (HS). Data on laparoscopic subtotal splenectomy (LSTS) in HS patients are sparse. METHODS: Thirty three patients with HS (median age 10.7 years (yrs), range 1.8-15.5) underwent LSTS. Baseline and follow-up investigation included haematological parameters, microscopic analysis of pitted erythrocytes (pitE), and B-cell subpopulations assessed by flow cytometry. Results were compared to those of non-splenectomised HS patients, HS patients after total splenectomy (TS), and healthy individuals. RESULTS: After LSTS, haemoglobin levels were normalised in all patients. During median long-term follow-up of 3.9 yrs (range 1.1-14.9), only four patients presented mild anaemia. Despite re-growing of the remnant spleen none of the patients required a second surgical intervention. As compared to TS, PitE in LSTS patients were significantly lower and indicated normal to only moderately decreased spleen function. Relative but not absolute IgM memory B-cell counts were reduced in both LSTS and TS patients. CONCLUSIONS: LSTS is effective for the treatment of patients with HS. A small remnant spleen is sufficient to provide adequate phagocytic function and to induce a pool of IgM memory B-cells.

Genetic modifiers of fetal hemoglobin affect the course of sickle cell disease in patients treated with hydroxyurea.

The course of sickle cell disease (SCD) is modified by polymorphisms boosting fetal hemoglobin (HbF) synthesis. However, it has remained an open question how these polymorphisms affect patients who are treated with the HbF-inducing drug hydroxyurea/ hydroxycarbamide. The German SCD registry offers the opportunity to answer this question, because >90% of patients are treated according to national guidelines recommending the use of hydroxyurea in all patients above 2 years of age. We analyzed the modifying effect of HbF-related genetic polymorphisms in 417 patients with homozygous SCD >2 years old who received hydroxyurea. HbF levels were correlated with higher total hemoglobin levels, lower rates of hemolysis, a lower frequency of painful crises and of red blood cell transfusions. The minor alleles of the polymorphisms in the γ-globin promoter (rs7482144), BCL11A (rs1427407) and HMIP (rs66650371) were strongly associated with increased HbF levels. However, these associations did not translate into lower frequencies of vaso-occlusive events which did not differ between patients either carrying or not carrying the HMIP and BCL11A polymorphisms. Patients on hydroxyurea carrying the γ-globin promoter polymorphism demonstrated substantially higher hemoglobin levels (P<10-4) but also higher frequencies of painful crises and hospitalizations (P<0.01) when compared to patients without this polymorphism. Taken together, these data indicate that the γ-globin, HMIP and BCL11A polymorphisms correlate with increased HbF in SCD patients on hydroxyurea. While HbF is negatively correlated with the frequency of painful crises and hospitalizations, this was not observed for the presence of known HbF-boosting alleles.

Current German Guidelines on Diagnosis and Treatment of Secondary Hemochromatosis in Patients with Congenital Anemias.

Adequate diagnosis and treatment of secondary hemochromatosis in patients with congenital anemias is important for reducing long-term mortality and morbidity as for improving patients' quality of life. Due to the strong migration movements during recent years, the number of patients in Germany suffering from hemoglobinopathies as some of the most relevant disorders in the context of iron overload (IOL) has increased enormously. Many of these patients had received inadequate medical care in their countries of origin prior to migration, including diagnosis and treatment of IOL. In parallel, various medical developments and achievements took place, including the expansion of stem cell transplant as curative therapeutic option and the introduction of gene therapy for patients with hemoglobinopathies. Diagnostic tools to assess both liver and heart IOL became available at more sites in Germany. Overall experience with iron elimination therapy either as monotherapy or as combination of different chelators increased. All these aspects were considered during revision of the consensus-based guideline on the diagnosis and treatment of secondary IOL in patients with congenital anemias (AWMF Reg. No. 025/029). Here, we briefly summarize the procedure for the revision of the guideline, provide a brief overview of innovations as compared to the previous version dated from 2015, and finally present the consensus recommendations adopted in the current version.

High-resolution pediatric reference intervals for 15 biochemical analytes described using fractional polynomials.

OBJECTIVES: Assessment of children's laboratory test results requires consideration of the extensive changes that occur during physiological development and result in pronounced sex- and age-specific dynamics in many biochemical analytes. Pediatric reference intervals have to account for these dynamics, but ethical and practical challenges limit the availability of appropriate pediatric reference intervals that cover children from birth to adulthood. We have therefore initiated the multi-center data-driven PEDREF project (Next-Generation Pediatric Reference Intervals) to create pediatric reference intervals using data from laboratory information systems. METHODS: We analyzed laboratory test results from 638,683 patients (217,883-982,548 samples per analyte, a median of 603,745 test results per analyte, and 10,298,067 test results in total) performed during patient care in 13 German centers. Test results from children with repeat measurements were discarded, and we estimated the distribution of physiological test results using a validated statistical approach (kosmic). RESULTS: We report continuous pediatric reference intervals and percentile charts for alanine transaminase, aspartate transaminase, lactate dehydrogenase, alkaline phosphatase, γ-glutamyl-transferase, total protein, albumin, creatinine, urea, sodium, potassium, calcium, chloride, anorganic phosphate, and magnesium. Reference intervals are provided as tables and fractional polynomial functions (i.e., mathematical equations) that can be integrated into laboratory information systems. Additionally, Z-scores and percentiles enable the normalization of test results by age and sex to facilitate their interpretation across age groups. CONCLUSIONS: The provided reference intervals and percentile charts enable precise assessment of laboratory test results in children from birth to adulthood. Our findings highlight the pronounced dynamics in many biochemical analytes in neonates, which require particular consideration in reference intervals to support clinical decision making most effectively.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

Liver Iron Content Determination Using a Volumetric Breath-Hold Gradient-Echo Sequence With In-Line R2 * Calculation.

BACKGROUND: Liver iron overload is a serious condition occurring in patients requiring blood transfusions (eg, in thalassemia and different forms of anemia) or with dysfunctional iron resorption, since there is no physiological mechanism to excrete iron. Above a certain level of iron concentration, chelation therapy is indicated. To monitor therapy success, liver iron content should be assessed regularly. A noninvasive method is important for patient management. Existing MRI methods suffer from long acquisition times and cost. PURPOSE: To study the correlation of liver iron content (LIC) reference values to liver R2 * determined using a 3D breath-hold multigradient echo (GRE) MRI sequence, employing accelerated acquisition by parallel imaging and in-line R2 * calculation. STUDY TYPE: Prospective. POPULATION: In all, 117 patients (22.1 ± 14.1 years, 66 men) suspected of iron overload. SEQUENCE: GRE. FIELD STRENGTH: 1.5T. ASSESSMENT: For comparison, a regulatory-approved method with a considerably longer scan time was used, providing LIC reference values. Participants were divided into a calibration group (65 participants), analyzed independently by two observers, and a validation group (52 participants). STATISTICAL TESTS: Linear correlation parameters were evaluated for R2 * values with LIC reference values, and for LIC determined from R2 * for validation group participants with LIC reference values. Sensitivity/specificity for clinical relevant LIC thresholds were analyzed. Interobserver variability was determined by intraclass correlation coefficient (ICC). RESULTS: Interobserver agreement was excellent, with an ICC of 0.99, P < 0.001. Good correlation (R2 = 0.89) and congruence of LIC values obtained with our method to LIC reference values was found, and almost identical diagnostic accuracy. Sensitivity/specificity were 0.98/0.67 for the diagnostic relevant LIC threshold of 4.5 mg/g and 1.0/0.95 for the threshold of 7 mg/g. DATA CONCLUSION: MRI acquisition times for determination of LIC can be significantly reduced by the use of comprehensive in-line R2 * map generation without compromising diagnostic accuracy. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 2.

Sickle cell disease in Germany: Results from a national registry.

BACKGROUND: Limited data on the prevalence and medical care of sickle cell disease (SCD) in Germany are available. Here, we make use of a patient registry to characterize the burden of disease and the treatment modalities for patients with SCD in Germany. PROCEDURE: A nationwide German registry for patients with SCD documents basic data on diagnosis and patient history retrospectively at the time of registration. A prospective annual documentation provides more details on complications and treatment of SCD. For the current analyses, data of 439 patients were available. RESULTS: Most patients had homozygous SCD (HbSS 75.1%, HbS/ß-thalassemia 13.2%, and HbSC 11.3%). The median age at diagnosis was 1.9 years (interquartile range, 0.6-4.4 years), most patients were diagnosed when characteristic symptoms occurred. Sepsis and stroke had affected 3.2% and 4.2% of patients, respectively. During the first year of observation, 48.3% of patients were admitted to a hospital and 10.1% required intensive care. Prophylactic penicillin was prescribed to 95.6% of patients with homozygous SCD or HbS/ß thalassemia below the age of six and hydroxycarbamide to 90.4% of patients above the age of two years. At least one annual transcranial Doppler ultrasound was documented for 74.8% of patients between 2 and 18 years. CONCLUSION: With an estimated number of at least 2000, the prevalence of SCD in Germany remains low. Prospectively, we expect that the quality of care for children with SCD will be further improved by an earlier diagnosis after the anticipated introduction of a newborn screening program for SCD.

Determinants of utilization of cryopreservation of germ cells in adolescent cancer patients in four European countries.

Infertility is a relevant late-effect following cancer treatment; yet, a large proportion of survivors cannot recall having been informed of this risk. In an intervention study, we examined if and how supportive patient information material on fertility/fertility-preserving measures influences utilization of cryopreservation in adolescent cancer patients. The control group, recruited 03/2014-01/2016, received the usual patient education at initial diagnosis. The intervention group, recruited 04/2016-10/2017, received patient education supported by a fertility flyer and brochure. Patients and parents were each asked questions on utilization of cryopreservation in a questionnaire 3 and 6 months after initial diagnosis. Patient core and therapy data were obtained from medical records. Overall, cryopreservation rates showed no significant difference between the control (32.7%, n = 37/113) and intervention group (36.6%, n = 37/101). In the control group, cryopreservation was associated with gender (OR 0.100, CI 0.023-0.427), age (OR 1.559, CI 1.077-2.258) and recalling information on fertility protection (OR 33.663, CI 2.100-539.574); in the intervention group, cryopreservation was related to gender (OR 0.093, CI 0.026-0.330) and the estimated infertility risk (OR 43.665, CI 2.157-883.974).Conclusion: Cryopreservation rates did not overall increase following the intervention; however, the individual risk seemed to be brought into attention more: Those at risk, including younger patients, cryopreserved at higher rates.What is Known:•Infertility is a relevant late-effect following adolescent cancer.•Guidelines recommend to offer fertility protection before cancer treatment.•A relevant proportion of adolescents with cancer are not aware of this risk.•Fertility protection seems under-used in cancer patients at risk for infertility.What is New:•Information material on fertility and protection in adolescents did not increase overall rates of cryopreservation.•Cryopreservation rates were improved according to individual risk for infertility.•Our flyers and brochures on fertility in cancer patients are available in various languages.

Bone marrow failure unresponsive to bone marrow transplant is caused by mutations in thrombopoietin.

We report 5 individuals in 3 unrelated families with severe thrombocytopenia progressing to trilineage bone marrow failure (BMF). Four of the children received hematopoietic stem cell transplants and all showed poor graft function with persistent severe cytopenias even after repeated transplants with different donors. Exome and targeted sequencing identified mutations in the gene encoding thrombopoietin (THPO): THPO R99W, homozygous in affected children in 2 families, and THPO R157X, homozygous in the affected child in the third family. Both mutations result in a lack of THPO in the patients' serum. For the 2 surviving patients, improvement in trilineage hematopoiesis was achieved following treatment with a THPO receptor agonist. These studies demonstrate that biallelic loss-of-function mutations in THPO cause BMF, which is unresponsive to transplant due to a hematopoietic cell-extrinsic mechanism. These studies provide further support for the critical role of the MPL-THPO pathway in hematopoiesis and highlight the importance of accurate genetic diagnosis to inform treatment decisions for BMF.

Fertility knowledge and associated empowerment following an educational intervention for adolescent cancer patients.

INTRODUCTION/OBJECTIVES: Fertility preservation is a major concern for adolescent cancer patients; yet, educational gaps remain. Our intervention study examined whether specially designed educational materials regarding fertility preservation increase knowledge and empowerment of patients and parents. METHODS: Eleven paediatric-oncological centres in four European countries agreed to enrol all eligible patients and parents in a questionnaire survey at 3 and 6 months after diagnosis. Treating physicians were surveyed on their medical consultation regarding fertility. RESULTS: Educational intervention increased knowledge in both patients (n = 113 and n = 101 in the control and intervention groups, respectively) and parents (n = 111 and n = 99 in the control and intervention groups, respectively), but the difference did not achieve statistical significance (knowledge difference patients: 5.6% (t0)/13.1% (t1); parents: 6.4% (t0)/3.8% (t1)). Parents of older patients (OR = 1.3, 95%CI = 1.1-1.7) and higher educational groups (OR = 6.2, 95%CI = 2.1-18.3) in the intervention group (OR = 1.9, 95%CI = 1.03-3.7) achieved higher knowledge levels. Empowerment was significantly improved in both patients (p = 0.046, d = 0.27) and parents (p = 0.046, d = 0.48) in the intervention group. DISCUSSION/CONCLUSIONS: In our study, the use of specifically prepared flyers and brochures successfully raised the level of fertility preservation knowledge in parents of older patients as well as parents with higher educational levels. Overall, the intervention improved patient and parent empowerment. Subsequent projects will include simpler information and digital material to particularly reach out to younger and less educated individuals.

Next-generation reference intervals for pediatric hematology.

Background Interpreting hematology analytes in children is challenging due to the extensive changes in hematopoiesis that accompany physiological development and lead to pronounced sex- and age-specific dynamics. Continuous percentile charts from birth to adulthood allow accurate consideration of these dynamics. However, the ethical and practical challenges unique to pediatric reference intervals have restricted the creation of such percentile charts, and limitations in current approaches to laboratory test result displays restrict their use when guiding clinical decisions. Methods We employed an improved data-driven approach to create percentile charts from laboratory data collected during patient care in 10 German centers (9,576,910 samples from 358,292 patients, 412,905-1,278,987 samples per analyte). We demonstrate visualization of hematology test results using percentile charts and z-scores (www.pedref.org/hematology) and assess the potential of percentiles and z-scores to support diagnosis of different hematological diseases. Results We created percentile charts for hemoglobin, hematocrit, red cell indices, red cell count, red cell distribution width, white cell count and platelet count in girls and boys from birth to 18 years of age. Comparison of pediatricians evaluating complex clinical scenarios using percentile charts versus conventional/tabular representations shows that percentile charts can enhance physician assessment in selected example cases. Age-specific percentiles and z-scores, compared with absolute test results, improve the identification of children with blood count abnormalities and the discrimination between different hematological diseases. Conclusions The provided reference intervals enable precise assessment of pediatric hematology test results. Representation of test results using percentiles and z-scores facilitates their interpretation and demonstrates the potential of digital approaches to improve clinical decision-making.

Serum ferritin is not a reliable predictor to determine iron overload in thalassemia major patients post-hematopoietic stem cell transplantation.

OBJECTIVE: Iron overload (IO) in transfusion-dependent anemia persists after hematopoietic stem cell transplantation (HSCT) and can cause long-term organ damage. In many studies, the diagnosis of IO before and after HSCT is based on serum ferritin (SF) levels rather than on assessment of liver iron concentration (LIC) by MRI or SQUID. METHOD: In a retrospective multicenter study, we analyzed the concordance for indication of iron depletion therapy and correlation between LIC and SF of 36 thalassemia patients after HSCT. LIC was determined either by MRI-R2 (FerriScan®) or SQUID. RESULTS: The concordance between LIC and SF varies over time after transplant (P = 0.011). The correlation between SF and LIC was strong in the first year (Spearman's rho 0.75; P < 0.001). In agreement, the concordance between SF and LIC concerning indication for treatment was close to 1 with an overall error rate ca. of 10%. In particular in the first year after HSCT, SF underestimates the degree of iron overload. However, in the longitudinal analysis since the second year post-HSCT onward no association was found between LIC and SF (P = 0.217). Furthermore, in the second year after HSCT, the overall error rate was 35%, whereas in the 3rd, 4th, and >4th year, it was 58%, 60%, and 25%, respectively. CONCLUSIONS: Our data suggest serum ferritin is not a reliable predictor to determine iron overload in thalassemia patients after HSCT.

The epidemiology of sickle cell disease in Germany following recent large-scale immigration.

BACKGROUND: The epidemiology of sickle cell disease (SCD) in Germany is currently changing fundamentally with ongoing immigration. Here, we address the challenges resulting from the increased frequency, that is, the morbidity, and mortality of SCD in this population. PROCEDURE: The number of immigrants with SCD was estimated based on the data of the German central registry of migrants (2007-2015) and published epidemiologic data. Additional data analysis was based on nationwide aggregated data from the diagnosis-related groups' (DRG) statistics of the German Federal Statistical Office. RESULTS: The total number of patients with SCD among migrants was estimated at 2,016 in 2007 and 3,216 in 2015, thus showing a 60% increase, which was particularly remarkable during 2014 and 2015. The countries of origin included those of West sub-Saharan Africa, followed by Syria, and other countries of the Middle East. In parallel, the number of SCD inpatient treatments increased from 780 in 2002 to 1,340 in 2015. Between 2012 and 2014, 42 patients with SCD died in hospital, mostly at an age of less than 5 years (n = 7) or over 30 years (n = 29). CONCLUSION: More than 3,000 patients with SCD are estimated to live among the immigrant population in Germany. In addition, the number of SCD patients of German nationality is not known. The increasing number of inpatient treatments and the death of young children from SCD indicate the need for a general newborn screening program and an increased awareness of this disease among medical practitioners in a country in which SCD used to be rare.

Pediatric reference intervals for alkaline phosphatase.

BACKGROUND: Interpretation of alkaline phosphatase activity in children is challenging due to extensive changes with growth and puberty leading to distinct sex- and age-specific dynamics. Continuous percentile charts from birth to adulthood allow accurate consideration of these dynamics and seem reasonable for an analyte as closely linked to growth as alkaline phosphatase. However, the ethical and practical challenges unique to pediatric reference intervals have restricted the creation of such percentile charts, resulting in limitations when clinical decisions are based on alkaline phosphatase activity. METHODS: We applied an indirect method to generate percentile charts for alkaline phosphatase activity using clinical laboratory data collected during the clinical care of patients. A total of 361,405 samples from 124,440 patients from six German tertiary care centers and one German laboratory service provider measured between January 2004 and June 2015 were analyzed. Measurement of alkaline phosphatase activity was performed on Roche Cobas analyzers using the IFCC's photometric method. RESULTS: We created percentile charts for alkaline phosphatase activity in girls and boys from birth to 18 years which can be used as reference intervals. Additionally, data tables of age- and sex-specific percentile values allow the incorporation of these results into laboratory information systems. CONCLUSIONS: The percentile charts provided enable the appropriate differential diagnosis of changes in alkaline phosphatase activity due to disease and changes due to physiological development. After local validation, integration of the provided percentile charts into result reporting facilitates precise assessment of alkaline phosphatase dynamics in pediatrics.

Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations.

Erythrocytosis is a rare disorder characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit. Several genetic variants have been identified as causes for erythrocytosis in genes belonging to different pathways including oxygen sensing, erythropoiesis and oxygen transport. However, despite clinical investigation and screening for these mutations, the cause of disease cannot be found in a considerable number of patients, who are classified as having idiopathic erythrocytosis. In this study, we developed a targeted next-generation sequencing panel encompassing the exonic regions of 21 genes from relevant pathways (~79 Kb) and sequenced 125 patients with idiopathic erythrocytosis. The panel effectively screened 97% of coding regions of these genes, with an average coverage of 450×. It identified 51 different rare variants, all leading to alterations of protein sequence, with 57 out of 125 cases (45.6%) having at least one of these variants. Ten of these were known erythrocytosis-causing variants, which had been missed following existing diagnostic algorithms. Twenty-two were novel variants in erythrocytosis-associated genes (EGLN1, EPAS1, VHL, BPGM, JAK2, SH2B3) and in novel genes included in the panel (e.g. EPO, EGLN2, HIF3A, OS9), some with a high likelihood of functionality, for which future segregation, functional and replication studies will be useful to provide further evidence for causality. The rest were classified as polymorphisms. Overall, these results demonstrate the benefits of using a gene panel rather than existing methods in which focused genetic screening is performed depending on biochemical measurements: the gene panel improves diagnostic accuracy and provides the opportunity for discovery of novel variants.

LNK mutations and myeloproliferative disorders.

The lymphocyte adaptor protein (LNK) is one of a family of adaptor proteins involved cell signaling and control of B cell populations. It has a critical role in regulation of signaling in hematopoiesis. Lnk negatively regulates cytokine initiated cell signaling and it functions as a negative regulator of the mutant protein in myeloproliferative neoplasms JAK2V617F. A number of mutations in LNK have been described in a variety of myeloproliferative neoplasms some of which have been demonstrated to cause increased cellular proliferation. The majority of mutations occur in exon 2. In a small number of cases idiopathic erythrocytosis with subnormal erythropoietin levels LNK mutations have been found which may account for the clinical phenotype. Thus investigation for LNK mutations should be considered in the investigation of idiopathic erythrocytosis and perhaps other myeloproliferative neoplasms.

Genetic basis of congenital erythrocytosis: mutation update and online databases.

Congenital erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3-bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1, and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive Internet-based database focusing on the registration of clinical history, hematological, biochemical, and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database.