The beta thalassaemia trait in Jamaica.

The objective of this study was to review the prevalence and features of the beta thalassaemia trait in Jamaican populations. Screening of 221,306 newborns over the last 46 years has given an indication of the distribution and prevalence of beta thalassaemia genes, and screening of 16,612 senior school students in Manchester parish, central Jamaica, has provided their haematological features. The prevalence of the beta thalassaemia trait predicted from double heterozygotes was 0.8% of 100,000 babies in Kingston, 0.9% of 121,306 newborns in southwest Jamaica, and 0.9% of school students in Manchester. Mild beta+ thalassaemia variants (-88 C>T, -29 A>G, -90 C>T, polyA T>C) accounted for 75% of Kingston newborns, 76% of newborns in southwest Jamaica, and 89% of Manchester students. Severe beta+ thalassaemia variants were uncommon. Betao thalassaemia variants occurred in 43 patients and resulted from 11 different variants of which the IVSII-849 A>G accounted for 25 (58%) subjects. Red cell indices in IVSII-781 C>G did not differ significantly from HbAA, and this is probably a harmless polymorphism rather than a form of beta+ thalassaemia; the removal of 6 cases in school screening had a minimal effect on the frequency of the beta thalassaemia trait. Red cell indices in the beta+ and betao thalassaemia traits followed established patterns, although both were associated with increased HbF levels. The benign nature of beta+ thalassaemia genes in Jamaica means that cases of sickle cell-beta+ thalassaemia are likely to be overlooked, and important clinical questions such as the role of pneumococcal prophylaxis remain to be answered.

Hb Santa Juana (ß 108(G10) Asn > Ser): a low oxygen affinity hemoglobin variant in a family of Bosnian background.

We present a family that carries the ß-hemoglobin variant Hb Santa Juana (HBB:c.326A>G, ß 108(G10) Asn>Ser), also known as Hb Serres, in three generations. All affected family members had an anomal hemoglobin fraction as detected by HPLC but normal blood count without evidence of anemia or hemolysis. Oxygen affinity (p50 (O2) = 31.9-40.4 mmHg) was decreased in all probands, compared to 24.9-28.1 mmHg in unaffected individuals. Clinical symptoms likely related to the hemoglobin variant were cyanosis during anaesthesia, while other complaints such as shortness of breath or dizziness were less clearly linked with the hemoglobin variant.

The haematology of Jamaicans: red cell indices in HbAA, HbAS, HbAC, and HbA-HPFH genotypes.

Based in the parish of Manchester in central Jamaica, the Manchester Project offered free detection of haemoglobin genotype to senior classes in 15 secondary schools between 2008 and 2013. Restricting the database to 15,103 students aged 15.0-19.9 years provided an opportunity to examine the red cell characteristics of the different haemoglobin genotypes, including normal (HbAA) in 85.0%, the sickle cell trait (HbAS) in 9.7%, HbC trait (HbAC) in 3.5% and hereditary persistence of foetal haemoglobin (HbA-HPFH) in 0.4%. Compared to the normal HbAA phenotype, HbAS had significantly increased mean cell haemoglobin concentration (MCHC), red cell count (RBC), and red cell distribution width (RDW) and decreased mean cell volume (MCV) and mean cell haemoglobin (MCH), these differences being even more marked in HbAC. Compared to HbAA, the HbA-HPFH had significantly increased RDW, but there were no consistent differences in other red cell indices, and there were no significant differences in haematological indices between the two common deletion HPFH variants, HPFH-1 and HPFH-2. Although these changes are unlikely to be clinically significant, they contribute to an understanding of the haematological spectrum of the common haemoglobin genotypes in peoples of African origin.

Thrombocytosis in children and adolescents-classification, diagnostic approach, and clinical management.

Secondary thrombocytosis is a frequent secondary finding in childhood infection and inflammation. Primary hereditary thrombocytosis may be caused by germline mutations within the genes encoding key regulators of thrombopoiesis, i.e., thrombopoietin (THPO) and its receptor c-MPL (MPL) or the receptor's effector kinase Januskinase2 (JAK2). Furthermore, somatic mutations in JAK2, MPL, and in the gene-encoding calreticulin (CALR) have been described to act as driver mutations within the so-called Philadelphia-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Increasing knowledge on the molecular mechanisms and on the clinical complications of these diseases is reflected by the WHO diagnostic criteria and European LeukemiaNet (ELN) recommendations on the management of adult MPN. However, data on childhood thrombocytosis are rare, and no consensus guidelines for pediatric thrombocytosis exist. Current literature has highlighted differences in the epidemiology and molecular pathogenesis of childhood thrombocytosis as compared to adults. Furthermore, age-dependent complications and pharmacological specificities suggest that recommendations tailored to the pediatric population are necessary in clinical practice. Here we summarize literature on classification, diagnostics, and clinical management of childhood thrombocytosis.

Genomic profiling of Acute lymphoblastic leukemia in ataxia telangiectasia patients reveals tight link between ATM mutations and chromothripsis.

Recent developments in sequencing technologies led to the discovery of a novel form of genomic instability, termed chromothripsis. This catastrophic genomic event, involved in tumorigenesis, is characterized by tens to hundreds of simultaneously acquired locally clustered rearrangements on one chromosome. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, an essential guardian of genome stability, would show a higher prevalence of chromothripsis due to the associated defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) arising in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared with tumors from individuals with other types of DNA repair syndromes (27 cases total, 10 with Ataxia Telangiectasia). Our data suggest that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients may be linked with frequent chromothripsis. Furthermore, we show that ATM loss is associated with increased chromothripsis prevalence in additional tumor entities.

Identification of a genetically defined ultra-high-risk group in relapsed pediatric T-lymphoblastic leukemia.

In the search for genes that define critical steps of relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) and can serve as prognostic markers, we performed targeted sequencing of 313 leukemia-related genes in 214 patients: 67 samples collected at the time of relapse and 147 at initial diagnosis. As relapse-specific genetic events, we identified activating mutations in NT5C2 (P=0.0001, Fisher's exact test), inactivation of TP53 (P=0.0007, Fisher's exact test) and duplication of chr17:q11.2-24.3 (P=0.0068, Fisher's exact test) in 32/67 of T-ALL relapse samples. Alterations of TP53 were frequently homozygous events, which significantly correlated with higher rates of copy number alterations in other genes compared with wild-type TP53 (P=0.0004, Mann-Whitney's test). We subsequently focused on mutations with prognostic impact and identified genes governing DNA integrity (TP53, n=8; USP7, n=4; MSH6, n=4), having key roles in the RAS signaling pathway (KRAS, NRAS, n=8), as well as IL7R (n=4) and CNOT3 (n=4) to be exclusively mutated in fatal relapses. These markers recognize 24/49 patients with a second event. In 17 of these patients with mostly refractory relapse and dire need for efficient treatment, we identified candidate targets for personalized therapy with p53 reactivating compounds, MEK inhibitors or JAK/STAT-inhibitors that may be incorporated in future treatment strategies.

Newborn Screening for Sickle Cell Disease: Jamaican Experience.

OBJECTIVES: To review the history of newborn screening for sickle cell disease with especial reference to Jamaica. METHODS: A summary was done of the history, the development of associated laboratory technology and the implementation of newborn screening for sickle cell disease in Jamaica. RESULTS: Screening was initiated at Victoria Jubilee Hospital, Kingston from 1973-1981, reactivated in 1995 and extended to the University Hospital of the West Indies in 1997 and to Spanish Town Hospital in 1998. From August 2008, there was a progressive recruitment of 12 hospitals in the south and west of Jamaica which has raised the frequency of islandwide newborn coverage from 25% in 1973 to 81%. The results of this extended programme in southwest Jamaica are presented. Dried blood spots collected from the umbilical cord proved stable, cheap and efficient; mean sample collection rates were 98%, maternal contamination occurred in < 1% and caused diagnostic confusion in < 0.1%. By March 31, 2015, a total of 54 566 births have been screened, detecting 161 with homozygous sickle cell (SS) disease, 125 with sickle cell-haemoglobin C (SC) disease and 36 with sickle cell-beta thalassaemia. Of the 327 babies with clinically significant sickle cell syndromes, all except five who died within seven days of birth were confirmed by four to six weeks and recruited to local sickle cell clinics. CONCLUSION: Early detection of sickle cell disease and recruitment to clinics is known to reduce its morbidity and mortality. The methods currently detailed provide an effective and economic model of newborn screening which may be of value elsewhere.

Radiosensitization by histone deacetylase inhibition in an osteosarcoma mouse model.

BACKGROUND: Osteosarcomas (OS) are highly malignant and radioresistant tumors. Histone deacetylase inhibitors (HDACi) constitute a novel class of anticancer agents. We sought to investigate the effect of combined treatment with suberoylanilide hydroxamic acid (SAHA) and radiotherapy in OS in vivo. METHODS: Clonogenic survival of human OS cell lines as well as tumor growth delay of OS xenografts were tested after treatment with either vehicle, radiotherapy (XRT), SAHA, or XRT and SAHA. Tumor proliferation, necrosis, microvascular density, apoptosis, and p53/p21 were monitored by immunohistochemistry. The CD95 pathway was performed by flow cytometry, caspase (3/7/8) activity measurements, and functional inhibition of CD95 death signaling. RESULTS: Combined treatment with SAHA and XRT markedly reduced the surviving fraction of OS cells as compared to XRT alone. Likewise, dual therapy significantly inhibited OS tumor growth in vivo as compared to XRT alone, reflected by reduced tumor proliferation, impaired angiogenesis, and increased apoptosis. Addition of HDACi to XRT led to elevated p53, p21, CD95, and CD95L expression. Inhibition of CD95 signaling reduced HDACi- and XRT-induced apoptosis. CONCLUSION: Our data show that HDACi increases the radiosensitivity of osteosarcoma cells at least in part via ligand-induced apoptosis. HDACi thus emerge as potentially useful treatment components of OS.

Evaluation of the prognostic meaning of C-reactive protein (CRP) in children and adolescents with classical Hodgkin's lymphoma (HL).

BACKGROUND: In adult cancer patients the negative predictive value of elevated CRP levels has been described for several malignancies. Only few studies have analyzed the prognostic role of CRP in children and adolescents with classical HL. In these studies elevated CRP levels correlate with the presence of classical risk factors and adverse outcome. PATIENTS AND METHODS: The prognostic role of CRP for patients with classical HL admitted to the GPOH-HD-2002 study was analyzed retrospectively. RESULTS: CRP levels were documented for 369 of 573 patients. Significant (p<0.05) increased median CRP levels were found in the presence of B-Symptoms (25.7 vs. 5.1 mg/l), extranodal involvement (21.5 vs. 7.5 mg/l), elevated erythrocyte sedimentation rate (ESR, 13.0 vs. 1.0 mg/l) and stage III/IV disease (15.5 vs. 5.3 mg/l). 83.9% of patients with elevated and 45.8% of patients with normal CRP had an ESR >30 mm/h. CONCLUSION: Elevated CRP levels were associated with classical risk factors of HL. CRP and ESR may reflect different biological processes. CRP was prognostic within early stage TG-1 patients treated with reduced treatment, but not within advanced stage TG-2+3.

Phase I/II intra-patient dose escalation study of vorinostat in children with relapsed solid tumor, lymphoma or leukemia.

Members of the histone deacetylase (HDAC) family exhibit great promise as potential drug targets in pediatric tumors including neuroblastoma, medulloblastoma, ependymoma and Ewing's sarcoma. HDAC inhibitors of various structural classes have shown anti-tumoral effects in pre-clinical pediatric tumor models as single agents or in combination treatments. Suberoylanilidehydroxamic acid (SAHA=vorinostat) is the most clinical advanced compound of the class and was approved by the US FDA in October 2006 for the treatment of refractory cutaneous T-cell lymphoma. In this phase I/II trial, pediatric patients with relapsed solid tumors, lymphoma or leukemias are treated according to an individualized dose escalation concept ensuring each individual patient to receive his optimal dose with respect to toxicity and efficacy. The study is accompanied by an extensive pharmacokinetic, pharmacodynamic and biomarker program.

Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.

PURPOSE: Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. METHODS: Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. RESULTS: SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. CONCLUSION: SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.

The favorable effect of activating NOTCH1 receptor mutations on long-term outcome in T-ALL patients treated on the ALL-BFM 2000 protocol can be separated from FBXW7 loss of function.

Precursor T-cell acute lymphoblastic leukemia (T-ALL) remains an important challenge in pediatric oncology. Because of the particularly poor prognosis of relapses, it is vital to identify molecular risk factors allowing early and effective treatment stratification. Activating NOTCH1 mutations signify a favorable prognosis in patients treated on ALL-BFM protocols. We have now tested if NOTCH pathway activation at different steps has similar clinical effects and if multiple mutations in this pathway function synergistically. Analysis of a validation set of 151 T-ALL patients and of the total cohort of 301 patients confirms the low relapse rate generally and the overall favorable effect of activating NOTCH1 mutations. Subgroup analysis shows that the NOTCH1 effect in ALL-BFM is restricted to patients with rapid early treatment response. Inactivation of the ubiquitin ligase FBXW7 is associated with rapid early treatment response and synergizes with NOTCH1 receptor activation. However, the effect of FBXW7 inactivation is separable from NOTCH1 activation by not synergizing with NOTCH1 mutations in predicting favorable long-term outcome, which can probably be explained by the interaction of FBXW7 with other clients. Finally, the comparison with other European protocols suggests that the NOTCH effect is treatment dependent generally and may depend on the intensity of central nervous system-directed therapy specifically.

Novel homozygous mutation (c.175delG) in platelet glycoprotein ITGA2B gene as cause of Glanzmann's thrombasthenia type I.

BACKGROUND: Glanzmann's thrombasthenia (GT), is a rare autosomal recessive bleeding disorder. Platelets from patients with GT show quantitative or qualitative defects of the platelet membrane glycoprotein (GP) IIb/IIIa complex. A variety of genetic defects in ITGA2B and ITGB3 (genes for GPIIb and GPIIIa) has been described causing the clinical entity of GT. PATIENTS: A newborn with bleeding symptoms (petechiae) platelet analyses revealed an inherited primary hemostasis disorder. METHODS/RESULTS: Analyses of patient's platelets using flow cytometry and immunoblotting showed absence of GPIIb protein and reduced amount of GPIIIa. Using restriction fragment length polymorphism heterozygosity for the deletion could be identified in the parents and in two siblings. Expression studies in mammalian cells revealed that the mutant GPIIb is missing and additionally affects the expression of wildtype GPIIIa. This deletion leads to a truncation at the very N-terminal region of the GPIIb protein. CONCLUSION: The present study describes a patient with GT associated with a novel homozygous deletion (c.175delG) in exon 1 of ITGA2B. This deletion led to a reading frameshift and caused a severely truncated form of GPIIb.

Pyoderma gangraenosum, a rare, but potentially fatal complication in paediatric oncology patients.

Pyoderma gangraenosum (PG) is a serious chronic, ulcerative skin disorder afflicting both adults and children. As PG is often associated with systemic diseases (>50%) such as inflammatory bowel disease, rheumatoid arthritis or haematological disorders, it requires a multidisciplinary approach. This disorder is not commonly reported in paediatrics; therefore children with PG represent a particularly difficult diagnostic challenge. Clinical diagnosis is often delayed and PG is only considered after eliminating other causes of cutaneous ulcers. We report a 4-year-old boy with secondary myelodysplastic syndrome following treatment for acute lymphoblastic leukaemia who presented with a massive inflammatory, ulcerative proliferation of the lower lip which was diagnosed as PG. We have reviewed the literature with reference to diagnostic criteria and treatment options of this disorder that is particularly rare in childhood.

[Genetic modifiers of homozygous sickle cell disease]. / Genetische modulierende Faktoren der Klinik der homozygoten Sichelzellkrankheit.

Homozygous sickle cell (HbSS) disease is paradigmatic for the complex influence of genetic modifiers of a monogenic disease. The genetically determined variability of HbF concentration has a strong impact on the clinical phenotype. The pharmacologically induced increase of HbF leads to a reduced morbidity which demonstrates that the knowledge of genetic modifiers enables the development of new therapeutic strategies. The presence of alpha-thalassemia also ameliorates the disease phenotype albeit not to the same extent as HbF does. Both factors, HbF and alpha-thalassemia are insufficient to explain the clinical variability of HbSS disease. The introduction of genome analysis has now provided the tools to identify relevant gene loci that will likely be helpful in estimating the probability of severe complications such as the occurrence of stroke. Following the validation in prospective studies, the subtle analysis of genetic modifiers will therefore influence the management of patients with sickle cell disease.

The prothrombin 20209 C-->T mutation in Jewish-Moroccan Caucasians: molecular analysis of gain-of-function of 3' end processing.

BACKGROUND: Mutations of the 3' end mRNA-processing signal of the prothrombin (F2) gene have been reported to cause elevated F2 plasma concentrations, thrombosis, and complications of pregnancy. Whereas the common F2 20210*A mutation is almost exclusively found in Caucasians, the F2 20209*T mutation has been reported in Afro-Americans and Afro-Caribbeans only. PATIENTS AND METHODS: Using LightCycler technology, three unrelated Jewish-Moroccan patients tested for obstetric complications were found to be carriers of the F2 20209*T allele. A detailed molecular analysis was performed to identify the functional impact of this mutation. RESULTS: We report three unrelated women of Jewish-Moroccan origin with a F2 20209*T mutation and fetal loss or infertility. The functional analysis revealed that the F2 20209*T mutation stimulates 3' end processing and up-regulates prothrombin protein expression as assessed by a highly sensitive luminescence-based reporter system. CONCLUSIONS: This is the first report of 20209*T in Caucasians, and functional analysis demonstrates that F2 20209*T falls into a general category of mutations of the F2 gene, which may possibly contribute to thrombophilia and complications of pregnancy by interfering with a tightly balanced architecture of non-canonical F2 3' end formation signals.

mRNA Metabolism and hereditary disorders: a tale of surveillance and escape.

The exploration of the molecular origin of hereditary diseases focused on genes and proteins for many years. Recently, mRNA has gained increasing attention. Most human genes contain introns and a considerable proportion of transcripts are not only alternatively spliced but also regulated posttranscriptionally in manifold ways. mRNA processing as well as a complex network of interactions between the steps of gene expression and associated quality control mechanisms are guided by a multitude of regulative mRNA sequence elements. Therefore it is not surprising that mutations of such elements can cause or modify human disease. The purpose of this review is the illustration of principles of physiological and dysregulated mRNA metabolism as well as a survey of new analytical tools and therapeutic approaches.

Chronic haemolytic anaemia and glucose-6 phosphate dehydrogenase deficiency. Case report and review of the literature.

Deficiency in glucose-6-phosphate dehydrogenase (G6PD) is the most common enzymopathy, and more than 125 different mutations causing G6PD deficiency have been identified. Chronic haemolytic anaemia (CHA) associated with G6PD deficiency is rare, but there is a cluster of mutations causing CHA between amino acids 361-428 which are encoded by exon 10 of the G6PD gene. This region is involved in the dimer formation of the active G6PD enzyme and therefore plays an important role for enzyme stability and activity. Here, we report a 17-year-old patient with CHA, who carries a rare G --> A mutation at nucleotide 1160 which causes an R387H amino acid substitution. We review the reports of the seven previously described patients with this mutation, concluding that G6PD deficiency should be considered as a rare differential diagnosis of chronic haemolytic, non-spherocytic anaemia.

Increased efficiency of mRNA 3' end formation: a new genetic mechanism contributing to hereditary thrombophilia.

The G-->A mutation at position 20210 of the prothrombin or coagulation factor II gene (F2) represents a common genetic risk factor for the occurrence of thromboembolic events. This mutation affects the 3'-terminal nucleotide of the 3' untranslated region (UTR) of the mRNA and causes elevated prothrombin plasma concentrations by an unknown mechanism. Here, we show that the mutation does not affect the amount of pre-mRNA, the site of 3' end cleavage or the length of the poly(A) tail of the mature mRNA. Rather, we demonstrate that the physiological F2 3' end cleavage signal is inefficient and that F2 20210 G-->A represents a gain-of-function mutation, causing increased cleavage site recognition, increased 3' end processing and increased mRNA accumulation and protein synthesis. Enhanced mRNA 3' end formation efficiency emerges as a novel principle causing a genetic disorder and explains the role of the F2 20210 G-->A mutation in the pathogenesis of thrombophilia. This work also illustrates the pathophysiologic importance of quantitatively minor aberrations of RNA metabolism.