Hearing loss in beta-thalassaemia: An Italian multicentre case-control study.

BACKGROUND: Despite numerous studies, the true scenario of hearing loss in beta-thalassaemia remains rather nebulous. MATERIALS AND METHODS: Pure tone audiometry, chelation therapy, demographics and laboratory data of 376 patients (mean age 38.5 ± 16.6 years, 204 females, 66 non-transfusion-dependent) and 139 healthy controls (mean age 37.6 ± 17.7 years, 81 females) were collected. RESULTS: Patient and control groups did not differ for age (p = 0.59) or sex (p = 0.44). Hypoacusis rate was higher in patients (26.6% vs. 7.2%; p < 0.00001), correlated with male sex (32.6% in males vs. 21.8% in females; p = 0.01) and it was sensorineural in 79/100. Hypoacusis rate correlated with increasing age (p = 0.0006) but not with phenotype (13/66 non-transfusion-dependent vs. 87/310 transfusion-dependent patients; p = 0.16). Sensorineural-notch prevalence rate did not differ between patients (11.4%) and controls (12.2%); it correlated with age (p = 0.01) but not with patients' sex or phenotype. Among adult patients without chelation therapy, the sensorineural hypoacusis rate was non-significantly lower compared to chelation-treated patients while it was significantly higher compared to controls (p = 0.003). CONCLUSIONS: Sensorineural hypoacusis rate is high in beta-thalassaemia (about 21%) and it increases with age and in males while disease severity or chelation treatment seems to be less relevant. The meaning of sensorineural-notch in beta-thalassaemia appears questionable.

Association of GDF15 levels with body mass index and endocrine status in ß-thalassaemia.

OBJECTIVE: GDF15 has emerged as a stress-induced hormone, acting on the brain to reduce food intake and body weight while affecting neuroendocrine function. Very high GDF15 levels are found in thalassaemia, where growth, energy balance and neuroendocrine function are impaired. We examined the relationships between GDF15 and anthropometric measures and endocrine status in ß-thalassaemia. DESIGN: Cross sectional study. PATIENTS: All ß-thalassaemia patients attending the thalassaemia unit of Colombo North Teaching Hospital for blood transfusions. MEASUREMENTS: Anthropometric data, appetite scores, circulating GDF15, IGF, thyroid and reproductive hormone levels in 103 ß-thalassaemia patients were obtained. RESULTS: GDF15 levels were markedly elevated in thalassaemia patients (24.2-fold with ß-thalassaemia major compared with healthy controls). Among patients with ß-thalassaemia major, the relationship between GDF15 and body mass index (BMI) was curvilinear with all individuals with GDF15 levels above 24,000 pg/mL having a BMI below 20 kg/m2 . After adjustment for BMI, age and Tanner stage, serum IGF1 concentrations correlated negatively with GDF15 in all thalassaemia patients (ß = -.027, p = .02). We found a significant positive relationship between GDF15 and gonadotropin (in both sexes) and testosterone (in males). CONCLUSIONS: GDF15 levels were markedly elevated in patients with ß-thalassaemia and its association with BMI is consistent with the known effect of GDF15 to reduce body weight. The inverse association between GDF15 with IGF1 levels may reflect a neuroendocrine impact of GDF15 or an indirect effect via impaired nutritional state. The positive association with testosterone in males and gonadotropins in both sexes, was surprising and should prompt further GDF15 studies on the hypothalamic pituitary gonadal axis.

The effect of ferritin levels on distal femoral cartilage thickness in patients with beta thalassaemia major.

INTRODUCTION: To the best of our knowledge, the present study is the first in the literature to assess distal femoral cartilage thickness and its relationship with ferritin levels in adult patients with beta thalassaemia major (BTM). MATERIALS AND METHODS: 45 patients with BTM and 45 healthy controls were included in the study. Ferritin and haemoglobin levels of the patient and healthy groups were determined by blood analysis and distal femoral cartilage thicknesses were measured via ultrasound. Then, the patient group was divided into subgroups according to whether their ferritin levels were below or above 2500 µg/L. They were then compared among themselves and with the healthy control group using the available data. RESULTS: Distal femoral cartilage thickness values were statistically significantly lower in the BTM group compared to the healthy control group (p values < 0.001). Patients with a ferritin level below 2500 µg/L had statistically significantly higher right and left average distal femoral cartilage thickness values than the patients with a ferritin level above 2500 µg/L (p = 0.029 and p = 0.019, respectively). The right and left average distal femoral cartilage thickness values of the patient subgroup with low ferritin levels were statistically similar to the control group (p = 0.146 and p = 0.164, respectively). CONCLUSION: Our study showed that thalassaemia patients are more likely to develop osteoarthritis (OA) than the normal population and possible OA development can be prevented by keeping the ferritin levels of these patients in the optimum range.

Iron parameters in pregnant women with beta-thalassaemia minor combined with iron deficiency anaemia compared to pregnant women with iron deficiency anaemia alone demonstrate the safety of iron supplementation in beta-thalassaemia minor during pregnancy.

In patients with beta-thalassaemia intermedia or major, hepcidin induces iron overload by continuously promoting iron absorption. There have been no studies in pregnant women with beta-thalassaemia minor combined with iron deficiency anaemia (IDA), examining whether hepcidin is inhibited by GDF15, as may occur in patients with beta-thalassaemia intermedia or major, or whether the iron metabolism characteristics and the effect of iron supplementation are consistent with simple IDA in pregnancy. We compared and analysed routine blood parameters, iron metabolism parameters, the GDF15 levels, and the hepcidin levels among four groups, namely the beta-thalassaemia (ß) + IDA, ß, IDA, and normal groups. In addition, the ß + IDA and IDA groups received iron supplementation for four weeks. We found no statistically significant correlation between hepcidin and GDF15 in any group, but a positive correlation was observed between hepcidin and ferritin. After iron supplementation, the routine blood parameters and iron metabolism parameters in the ß + IDA group were improved, and the hepcidin content was significantly increased. These results suggest that in pregnant women with beta-thalassaemia minor, hepcidin functions normally to maintain iron homeostasis, and that iron supplementation is effective and safe.

Assessment of iron overload in a cohort of Sri Lankan patients with transfusion dependent beta thalassaemia and its correlation with pathogenic variants in HBB, HFE, SLC40A1, and TFR2 genes.

BACKGROUND: Iron overload (IO) is a complication in transfusion dependent beta thalassaemia (TDT). Pathogenic variants in genes involving iron metabolism may confer increased risk of IO. The objective of this study was to determine the magnitude of the cardiac and hepatic IO and determine whether pathogenic variants in HFE, SLC40A1 and TFR2 genes increase the risk of IO in a cohort of TDT patients in Sri Lanka. MATERIALS AND METHODS: Fifty-seven (57) patients with TDT were recruited for this study. Serum ferritin was done once in 3 months for a period of one year in all. Those who were ≥ 8 years of age (40 patients) underwent T2* MRI of the liver and heart. Fifty-two (52) patients underwent next generation sequencing (NGS) to identify pathogenic variants in HBB, HFE, SLC40A1 and TFR2 genes. RESULTS: The median age of the patients of this cohort was 10 years. It comprised of 30 (52.6%) boys and 27 (47.4%) girls. The median level of serum ferritin was 2452 ng/dl. Hepatic IO was seen in 37 (92.5%) patients and cardiac IO was seen in 17 (42.5%) patients. There was no statistically significant correlation between serum ferritin and hepatic or cardiac IO. Thirty-two (61.5%), 18 (34.6%), 2 (3.8%) of patients were homozygotes, compound heterozygotes and heterozygotes for pathogenic variants in the HBB gene. Eight (15.4%) and 1 (1.9%) patients were heterozygotes for pathogenic and likely pathogenic variants of HFE genes respectively. There were no pathogenic variants for the TfR2 and SLC40A1 genes. The heterozygotes of the pathogenic variants of the HFE were not at increased risk of IO. CONCLUSIONS: Cardiac T2* MRI helps to detect cardiac IO in asymptomatic patients. It is important to perform hepatic and cardiac T2* MRI to detect IO in patients with TDT. There was no statistically significant correlation between pathogenic variants of HBB and HFE genes with hepatic and cardiac IO in this cohort of patients.

The successful strategy of comprehensive pre-implantation genetic testing for beta-thalassaemia-haemoglobin E disease and chromosome balance using karyomapping.

Thalassaemia is the commonest monogenic disease and causes a health and economic burden worldwide. Karyomapping can be used for pre-implantation genetic testing of monogenic disorders (PGT-M). This study applied karyomapping in two PGT-M cycles and made a comparison to polymerase chain reaction (PCR). Two families at risk of having beta-thalassaemia-haemoglobin E disease offspring decided to join the project and informed consent was obtained. Karyomapping results of family A (beta-thalassaemia (c.41_42delTCTT)-Hb E (c.26G>A) disease) revealed four normal, two beta-thalassaemia traits, one Hb E trait and six affected. Three embryos exhibited unbalanced chromosomes. One normal male embryo was transferred. Karyomapping results of family B (beta-thalassaemia (c.17A>T)-Hb E (c.26G>A) disease) revealed six Hb E traits and three affected. Three embryos were chromosomally unbalanced. One Hb E trait embryo was transferred. Two successful karyomapping PGT-M were performed, including deletion and single-base mutations. Karyomapping provides accuracy as regards the protocol and copy number variation which is common in pre-implantation embryos. Impact StatementWhat is already known on this subject? Thalassaemia syndrome is the commonest monogenic disease and causes a health and economic burden worldwide. Modern haplotyping using SNP array (aSNP) and karyomapping algorithms can be used for pre-implantation genetic testing of monogenic disorders (PGT-M). However, few clinical karyomapping PGT-M cycles have been done and validated so far.What do the results of this study add? Two successful clinical PGT-M cycles for beta-thalassaemia (c.41_42delTCTT and c.17A>T mutations)-haemoglobin E (c.26G>A) disease were performed using karyomapping. The outcome was two healthy babies. Multiplex fluorescent polymerase chain reaction (PCR) with mini-sequencing was also used for confirmation mutation analysis results. PCR confirmed haplotyping results in all embryos. Six embryos from both PGT-M cycles exhibited unbalanced chromosomes evidenced by aSNP.What are the implications of these findings for clinical practice and/or further research? Karyomapping provides accurate information quickly and the outcomes of the study will save time as regards protocol development, provide a usable universal PGT-M protocol and add additional copy number variation (CNV) information, chromosome number variation being a common issue in pre-implantation embryos.

Development of the Thalassaemia Adult Life Index (ThALI).

BACKGROUND: Beta Thalassaemia Major (ßTM) is a chronic genetic illness whereby the challenges faced by patients exposes them to increased risk of psychosocial issues. Despite this, a disease-specific tool to measure the impact of this illness on adult patients has yet to be developed. METHODS: In collaboration with ßTM adult patients, this study aimed to develop a comprehensive, disease-specific, easy to use psychometrically sound tool to measure the impact of chelation and transfusion dependent ßTM in a cross-cultural patient group in England.The Thalassaemia Life Index (ThALI) was developed in two stages - item generation and pre-testing and item reduction - in collaboration with service users. Recruited adult patients shaped the design of the instrument including its statements and subscales. Standard item reduction techniques were used to develop the instrument. RESULTS: The final version of the ThALI encompasses 35 statements and five sub-scales - general physical health, coping, body image, appearance and confidence, social relationships and autonomy. This endorses the multidimensionality of quality of life (QoL). The factor structure of the ThALI is highly stable and its internal consistency is high (alpha = 0.87 for the overall scale; 0.83-0.94 for its subscales). The ThALI has sound scaling assumptions, acceptability and score variability. Content validity was confirmed by experts and service user interviewees. The loadings for the items retained were adequate and the item discriminant validity sound. CONCLUSIONS: The ThALI covers the impact of ßTM in adult patients. Preliminary testing shows its multidimensionality to be reliable and valid. The national authentication of the tool with patients treated in Centres of Excellence will aim to provide further evidence regarding the ThALI's psychometric properties. Once authenticated, the ThALI may be utilised in research and in clinical settings to assess the effects of new therapies and/or interventions from the patients' perspective to inform practice and/or to identify areas of concern.

Attention, response inhibition and brain event-related potential alterations in adults with beta-thalassaemia major.

We investigated neural correlates of cognitive function in adults with beta thalassaemia major (ß-TM) compared to healthy controls using scalp-recorded event-related potentials (ERPs). Event-related potential studies in the field of ß-TM are scarce and mostly limited to children. A stop-signal task was used to evaluate indices of attention and response inhibition function, considered to be the hallmark of executive control. Correlations between task performance, ERPs and haemoglobin were also examined. Results showed impaired cognitive performance in ß-TM patients, as indicated by longer response times than controls. Haemoglobin was negatively correlated with response times to Go stimuli. Electrophysiological results indicated significant ß-TM-related alterations in neuronal activity, reflected in greater peak amplitudes of several task-related ERP components. A possible interpretation of these ERP results is that ß-TM patients need to recruit additional brain resources when dealing with cognitive challenge. Significant correlations were found between levels of haemoglobin and amplitude of all ERP components; the lower the haemoglobin, the more pronounced the ERPs amplitude. The present study represents a novel investigation of cognitive function and related brain dynamics in ß-TM in adult. Integrating neuropsychological assessment and interventions into traditional disease management, may be imperative in achieving a better quality of life for these patients.

Global gene expression reveals an increase of HMGB1 and APEX1 proteins and their involvement in oxidative stress, apoptosis and inflammation pathways among beta-thalassaemia intermedia and major phenotypes.

Beta-thalassaemia (BT) is classified according to blood transfusion requirement as minor (BTMi), intermedia (BTI) and major (BTM). BTM is the most severe form, requiring regular transfusions while transfusion need is only occasional in BTI. Differential gene expression between patients has not been assessed so far. Here, we evaluated the global gene expression profiles during differentiation of human erythroid cells of two patients carrying the same mutation [CD39, (C → T)], though displaying different phenotypes (BTI and BTM). Considering the role of reactive oxygen species (ROS) in the pathophysiology of thalassaemia, we focused on differentially expressed genes involved in metabolic pathways triggered by ROS, such as inflammation and apoptosis, and, from these, we selected the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and High Mobility Group Box1 (HMGB1) genes, whose role in BT is not well established. An in-depth expression analysis of transcriptional and protein levels in patients carrying a range of mutations associated with BT phenotypes indicated that APEX1 was increased in both BTI and BTM. Furthermore, higher amounts of HMGB1 was found in the plasma of BTI patients. Our findings suggest that these proteins have important roles in BT and could represent new targets for further studies aiming to improve the management of the disease.

Brain functional impairment in beta-thalassaemia: the cognitive profile in Italian neurologically asymptomatic adult patients in comparison to the reported literature.

Cognitive involvement in beta-thalassaemia is strikingly controversial and poorly studied in adulthood. This multicentre prospective study investigated 74 adult neurologically-asymptomatic beta-thalassaemia patients (mean-age 34·5 ± 10·3 years; 53 transfusion-dependent [TDT], 21 non-transfusion dependent [NTDT]) and 45 healthy volunteers (mean-age 33·9 ± 10·7 years). Participants underwent testing with Wechsler Adult Intelligence Scale-Fourth Edition (WAIS-IV), Brief Psychiatric Rating Scale (BPRS) and multiparametric brain 3T-magnetic resonance imaging (MRI) for parenchymal, vascular and iron content evaluation. Patients had lower Full-Scale Intelligence Quotient (FSIQ) than controls (75·5 ± 17·9 vs. 97·4 ± 18·1, P < 0·0001) even after correction for education level. Compared to TDT, NTDT showed a trend of higher FSIQ (P = 0·08) but a similar cognitive profile at WAIS-subtests. FSIQ correlated with total and indirect bilirubin (P < 0·0001 and P = 0·002, respectively); no correlation was found with splenectomy, intracranial MRI/magnetic resonance-angiography findings, brain tissue iron content or other disease-related clinical/laboratory/treatment data. FSIQ did not correlate with BPRS scores, although the latter were higher among patients (28·74 ± 3·1 vs. 27·29 ± 4·8, P = 0·01) mainly because of increased depression and anxiety levels. Occupation rate was higher among controls (84·4% vs. 64·9%, P = 0·004) and correlated with higher FSIQ (P = 0·001) and education level (P = 0·001). In conclusion, Italian adult beta-thalassaemia patients seem to present a characteristic cognitive profile impairment and an increased rate of psychological disorders with possible profound long-term socio-economic consequences.

Consanguineous unions and endogamy in families of beta-thalassaemia patients from two Mediterranean populations: Tunisia and Italy.

Background: Consanguinity increases the incidence of recessive diseases such as beta-thalassaemia major (ßTM), one of the most prevalent lethal inherited diseases in the world.Aim: This study aims to identify the frequency of endogamy and consanguinity in two Mediterranean ßTM populations and to study the implication of socio-economic factors.Subjects and methods: A trans-sectional study was conducted in 203 Tunisian families and 75 Italian families. Data were collected using a questionnaire completed by patients and parents.Results: Complete endogamy and consanguinity were observed in 82.75% and 62.56% of Tunisian families, respectively. Complete endogamy was found in 90.67% of Italian families, no consanguinity was noted. The low occupation status of Tunisian mothers was associated with an increasing frequency of consanguinity (p = .01) and endogamy (p = .0003). Consanguinity was associated with low education level (p = .012) and low occupation status (p=.047) of fathers. No significant association was found between endogamy and socio-economic factors in the Italian sample.Conclusions: High consanguinity and endogamy rates in Tunisian families may explain the frequency of ßTM in Tunisia. The high endogamy rate in Italian families could also increase the frequency of ßTM. Identification of geographical distribution and socio-economic factors leading to endogamy and consanguinity in these populations might help to improve ßTM prevention.

Co-existing iron deficiency/overload in beta-thalassemia trait.

OBJECTIVE: To identify the co-existence of iron deficiency and iron overload in individuals with beta thalassaemia trait. METHODS: The cross-sectional study was conducted at Rehman Medical Institute and Khyber Medical University, Peshawar, Pakistan, September 1, 2015, to December 31, 2017, and comprised individuals with hypochromic microcytic blood picture. Haemoglobin electrophoresis was performed on their blood samples. Serum ferritin levels of subjects with Haemoglobin Subunit Alpha 2 levels between 3.5% and 7% were checked. Data were analysed using analysed using GraphPad Prism v6. RESULTS: Of the 292 subjects, 159(54.5%) were males and 133(45.5%) were females. Of these, 240 (82.2%) were anaemic and 52 (17.8%) had haemoglobin within the normal range. Serum ferritin level of 55(18.8%) subjects was low and 207(70.9%) were iron-replete. Notably, 30(10.3%) subjects had serum ferritin levels higher than the reference range, and this was more common among adults (p<0.001). CONCLUSIONS: Ferritin levels in beta thalassaemia trait can be low, normal or higher than the normal values..

Clinical application of a protocol based on universal next-generation sequencing for the diagnosis of beta-thalassaemia and sickle cell anaemia in preimplantation embryos.

RESEARCH QUESTION: Mutations of the beta-globin gene (HBB) cause beta-thalassaemia and sickle cell anaemia. These are the most common cause of severe inherited disease in humans. Traditional preimplantation genetic testing protocols for detecting HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. We, therefore, asked the question whether a universally applicable preimplantation genetic testing method can be developed to test for HBB gene mutations. DESIGN: A multiplex polymerase chain reaction protocol was designed, allowing simultaneous amplification of multiple overlapping DNA fragments encompassing the entire HBB gene sequence in addition to 17 characterized, closely linked single nucleotide polymorphisms (SNP). Amplicons were then analysed using a next-generation sequencing method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified DNA derived from embryos of three couples carrying different combinations of beta-thalassaemia mutations. RESULTS: The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Analysis of 141 heterozygous sites showed no instances of allele dropout and the test displayed 100% concordance compared with the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive next-generation sequencing may provide superior accuracy than typically achieved using traditional preimplantation genetic testing methods. Importantly, no patient-specific test design or optimization was needed. CONCLUSIONS: It is hoped that protocols that deliver almost universally applicable low-cost tests, without compromising diagnostic accuracy, will improve patient access to preimplantation genetic testing, especially in less affluent parts of the world.

The performance of MALBAC and MDA methods in the identification of concurrent mutations and aneuploidy screening to diagnose beta-thalassaemia disorders at the single- and multiple-cell levels.

AIM: To select an optimal whole-genome amplification (WGA) method to improve the efficiency of the preimplantation genetic diagnosis and screening (PGD/PGS) of beta-thalassaemia disorders. METHODS: Fifty-seven fibroblast samples with defined beta-thalassaemia variations and forty-eight single-blastomere samples were amplified from single-, two-, and five-cell samples by multiple annealing and looping-based amplification cycles (MALBAC) and the multiple displacement amplification (MDA) method. Low-depth, high-throughput sequencing was performed to evaluate and compare the coefficiencies of the chromosomal copy number variation (CNV) detection rate and the allele dropout (ADO) rate between these two methods. RESULTS: At the single-cell level, the success rates of the CNV detection in the fibroblast samples were 100% in the MALBAC group and 91.67% in the MDA group; the coefficient of variation in the CNV detection in the MALBAC group was significantly superior to that in the MDA group (0.15 vs 0.37). The total ADO rate in the HBB allele detection was 4.55% in the MALBAC group, which was significantly lower than the 22.5% rate observed in the MDA group. However, when five or more cells were used as the starting template, the ADO rate significantly decreased, and these two methods did not differ significantly. CONCLUSIONS: For the genetic diagnosis of HBB gene variation at the single-cell level, MALBAC is a more suitable method due to its higher level of uniformity and specificity. When five or more cells are used as the starting template, both methods exhibit similar efficiency, increased accuracy, and a similar success rate in PGD/PGS.

Hepatitis E in transfusion-dependent thalassaemia patients, in Greece: a single centre experience.

Hepatitis E is considered an emerging disease that may be a threat in both developing and industrialized countries all over the world. The risk of chronic hepatitis E virus infection is higher among immunocompromised patients. This study aimed to assess the status of hepatitis E infection in patients with transfusion-dependent thalassaemia from a single centre, in Greece. Our results suggest that the prevalence of hepatitis E infection in this group of patients is low.

Prenatal screening for genetic disorders: Suggested guidelines for the Indian Scenario.

Prenatal testing is the best strategy for reducing the burden of genetic disorders and congenital disabilities that cause significant postnatal functional impairment. Universal prenatal screening is advisable for common genetic disorders and congenital anomalies such as Down syndrome, beta-thalassaemia and neural tube defects. Several prenatal-screening tests are now available for Down syndrome, but knowledge about the appropriate timing of the test and the need for pre- and post-test counselling may not be updated among the primary care physicians. There is also a considerable degree of confusion regarding the prenatal screening test to be chosen in each case, due to the availability of a number of new and advanced screening techniques. At present, there is no nation-wide consensus regarding the nature and timing of these prenatal-screening protocols. Due to the absence of any definite guidelines and the additional lacunae in the awareness regarding the appropriate prenatal screening in the country, the optimum benefits of these screening protocols are not reaching the population. This review focuses on the various prenatal screening and diagnostic tests that are available for common genetic conditions and congenital disabilities and attempts to outline the most cost-effective and gestational age-appropriate strategies for prenatal screening for the Indian healthcare set-up. The recommendations suggested would serve as a source guide for formulating prenatal-screening guidelines for reducing the incidence of common genetic disorders and congenital disabilities in India.

Comparing the Efficacy of Tadalafil Versus Placebo on Pulmonary Artery Systolic Pressure and Right Ventricular Function in Patients with Beta-Thalassaemia Intermedia.

BACKGROUND: Conventional oral therapies in the management of pulmonary hypertension in people without haemoglobinopathies are of limited value in thalassaemia patients because of toxicity and poor effectiveness. This study was conducted to assess the effect of tadalafil on pulmonary artery pressure and right ventricular systolic function in patients with beta-thalassaemia intermedia. METHODS: Forty-four patients with beta-thalassaemia intermedia with pulmonary hypertension based on transthoracic echocardiography (TTE) were entered in the study. Patients with hepatic or renal insufficiency and also patients who were treated with organic nitrates or alpha-blockers were excluded. The patients were randomly divided into two groups (n=22) and they were treated for six weeks with tadalafil (40mg daily) or placebo. The pulmonary artery systolic pressure (PASP), tricuspid regurgitation velocity (TRV) and parameters related to systolic function of the right ventricle were measured by the TTE before and after treatment. RESULTS: Significant improvement in TRV (3.02±0.02 m/s-2.52±0.06 m/s), PASP (45.31±0.66 mmHg-34.26±1.15mmHg) and parameters related to systolic function of the right ventricle were observed in the group who received tadalafil compared to placebo (p< 0.05). CONCLUSIONS: Tadalafil significantly decreased PASP and TRV in patients with beta-thalassaemia intermedia. Likewise, tadalafil improved right ventricular systolic function in the patients.

ASH1L (a histone methyltransferase protein) is a novel candidate globin gene regulator revealed by genetic study of an English family with beta-thalassaemia unlinked to the beta-globin locus.

In 1993, we described an English family with beta-thalassaemia that was not linked to the beta-globin locus. Whole genome sequence analyses revealed potential causative mutations in 15 different genes, of which 4 were consistently and uniquely associated with the phenotype in all 7 affected family members, also confirmed by genetic linkage analysis. Of the 4 genes, which are present in a centromeric region of chromosome 1, ASH1L was proposed as causative through functional mRNA knock-down and chromatin-immunoprecipitation studies in human erythroid progenitor cells. Our data suggest a putative role for ASH1L (Trithorax protein) in the regulation of globin genes.

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

OBJECTIVES: Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. METHODS: The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. RESULTS: A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. CONCLUSION: We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Evaluation of the validity of Hb A2 and mean corpuscular haemoglobin action values in antenatal screening for beta thalassaemia carriers in England.

National antenatal screening of all pregnant women in England is carried out using standards and guidelines produced by the National Health Service Sickle Cell and Thalassaemia Screening Programme. The algorithms for detection of beta thalassaemia carrier status rely on action criteria, which are set using the percentage Hb A2 and mean corpuscular haemoglobin (MCH) values. Three groups of samples: MCH <27 pg and Hb A2 3·5-3·9%, MCH ≥27 pg and Hb A2 4-4·3% and MCH ≥27 pg and Hb A2 3·5-3·9% were selected from a sample population of 59 500 to assess the validity and predictive value of the action criteria - 25 false positives (0·042% of total) and nine false negatives (0·015% of total) were detected. These findings support the continuation of the current action values.