Distribution of work responsibilities within the pilot 2.2 Diagrammatic Representations of Scenar... more Distribution of work responsibilities within the pilot 2.2 Diagrammatic Representations of Scenarios. 3.1 Maltese population by gender and age group in 2003 and as projected to 2015 3.2 Gross Domestic Product by Industry and Type of Income 3.3 The number of patents registered since 1994 3.4 Number of University graduates over a four year period 3.5 Standard process followed in the commercialisation of biotechnology products 3.6 Summary of the basic foundations for the successful commercialisation of biotechnology 4.1 Countries who have adopted biotechnology crops 4.2 Biotechnology Industry in Europe compared to the US 4.3 Comparison of Employment The two main vectors limiting development of the local biotechnology sector were identified to be STI education and RTDI capacity. Four main scenarios for the possible development of local biotechnology sector were developed around these two vectors. Actions to be taken to move from one scenario to another and hence reach optimal scenario are outlined. SWOT analysis of the local biotechnology sector identified that our major strengths are the well established medical and engineering faculties, good health care system, strong ICT sector and a skilled workforce. However lack of right decisions being taken at the right time might result in brain drain and low GDP. Chapter 2: The Foresight Process This chapter provides an almost chronological account of the foresight Pilot as it unfolded. Certain steps interacted with each other in synergetic, iterative loops which enhanced the process. The methodology and approaches used include stakeholder-mapping and co-nomination exercises, the setting up of expert panels, questionnaire-based surveys, SWOT analysis and scenario-building exercise.
Restriction endonuclease mapping defined a partial deletion of about 1 .35 kb in the fi-globin ge... more Restriction endonuclease mapping defined a partial deletion of about 1 .35 kb in the fi-globin gene of a black American patient with hemoglobin S-$#{176}-thalassemia and in his uncle with a ftothalassemia trait. The 5' endpoint of the deletion is about 600 bases upstream from the cap site. and T HE f.-THALASSEMIAS are inherited disorders ofhuman hemoglobin synthesis, characterized by reduced (fl-thalassemia) or absent (/3#{176}-thalassemia) production of the f3-globin of normal adult hemoglobin)3 Restriction endonuclease mapping, cloning, and DNA sequencing have revealed several types of mutations in the 3-globin gene region of such patients.
Although the precise biochemical mechanisms of globin gene switching remain elusive, considerable... more Although the precise biochemical mechanisms of globin gene switching remain elusive, considerable insight is gained by in vivo expression profiling through quantification of the hemoglobin / globin phenotype of informative heterozygosities and homozygosities / compound heterozygosities in the context of specific regulatory DNA sequence diversity such as the XMN-I or the [(AT)xTy] sequence polymorphisms. The quantification of normal and abnormal globins of Hb F Malta-I (or a2b2, 117(G19)His>Arg) heterozygotes which are in tight linkage disequilibrium with Hb Valletta (or a2b2 287(f3)Thr>Pro) i.e. Gyo, GyFMalta-I, AyI, bV and bA together with extensive haplotyping of homozygotes and heterozygotes including the XMN-I dimorphism in the Gy promoter and the (AT)xTy polymorphism (BP1 binding site) 5′ to the b globin genes had suggested that the XMN-I dimorphism was largely inactive in the normal newborn. In contrast the Hb F levels and the proportion of Gy globin in anemic adult beta-thalassemia homozygotes and compound heterozygotes…
Preimplantation Genetic Diagnosis is a fairly new form of prenatal diagnosis, which screens for g... more Preimplantation Genetic Diagnosis is a fairly new form of prenatal diagnosis, which screens for genetic disease at the embryonic stage. Its use is expanding as more knowledge is gained about genetic disorders and tests for the causative genes are developed.
The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the earl... more The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the early stage genetic programming of erythroid progenitors to promote physiological γ to β globin gene switching. Indeed, we showed that a truncation mutation (p.K288X) in KLF1 that deleted the DNA binding zinc finger domain resulted in marked KLF1 deficiency and a hematological condition that resembled a hereditary persistence of fetal hemoglobin (HbF) or a β thalassemia. Here, we describe five additional families with the same p.K288X mutation but varied hematological and HbF levels together with genetic and phenotypic data on a 600 data-set from the same Maltese population. The data accounted for a strong promoter embedded within a region of genetic heterogeneity of KLF1 that led to a ψβ thalassemia. Whole genome sequencing on 15 subjects of six families (FamF1 - FamF6) segregating (two) p.K288X frameworks of KLF1 had variable degrees of microcytosis (MCV; 76.1fL -77.4 fL) and HbF levels (HbF 2480 mg/dL - 802mg/dL) due to complex heterozygosity between promoter, coding and truncating mutations in KLF1. Case II-6 of FamF1 with the highest HbF (2480 mg/dL) had 2 promoter and 2 coding mutations in cis and in trans to the p.K288X truncation. Nine (9) KLF1 frameworks (A - I) were derived by transmission disequilibrium analyses of the family data, each assembled from 15 mutations and resulting in 7 genotypes among the families. The p.K288X truncation was found on a second rarer framework. Additional, rarer KLF1 frameworks were found with haploview in the population dataset. The population dataset was made up of 198 β thalassemia heterozygotes and 400 others from the clinic and the biobank and that had no β globin gene mutations, variable blood counts or hemoglobin profiles or both. They were older than 2 years of age, not pregnant and had normal iron levels. The number of KLF1 mutations differed from 0 in the wild-type framework A to 6 in one of the rare frameworks X. Six mutations were in the promoter region and 6 were in the coding region that defined a "KLF1 Variable Region" 5' genomic coordinate 12887183 - 12888273, whereas very few were found in the 3' (genomic coordinate 12884589 - 12884752) that defined the KLF1 "Constant Region" The Constant region has also been evolutionary conserved. It included the zinc finger domain and the proteasome binding site. The genotype - phenotype correlations and the family data were consistent with an autosomal recessive condition that resembled a β thalassemia, thus a ψβ thalassemia. It differed from a silent thalassemia because the β globin gene sequence was wild type. It provided a diagnosis for families with iron resistant microcytosis and borderline hemoglobin phenotypes suitable for counselling in the clinical setting. It was consistent with the observation among the families regarding the high strength of the KLF1 promoter. Multiple "hits" were necessary to suppress the biosynthesis of KLF1 for hemoglobin switching to escape perinatal suppression. The effect of the 6 promoter mutations were confirmed in vitro with native and induced K562 and Hek293T cell lines. Presumably, during normal development, the strong promoter served to rapidly drown KLF1 binding sites with KLF1 molecules to direct progenitors to erythropoiesis with the appropriate adult hemoglobin profile in the perinatal period. The diagnosis of the patients with selected genotypes due to compound and double heterozygosities in promoter and coding sequences shall further permit quantification of differential promoter function in vivo. Figure Disclosures No relevant conflicts of interest to declare.
Abstract 5162 We provide additional data on members of the family from Malta with Hereditary Pers... more Abstract 5162 We provide additional data on members of the family from Malta with Hereditary Persistence of Fetal Hemoglobin (HPFH) due to KLF1 haplo-insufficiency. The data indicated a possible role of additional loci in the pathway of globin gene control. We showed that KLF1 functions as a master regulator of erythropoiesis and developmental globin gene switching (Borg et al., Nature Genetics doi: 10. 1038/ng.630, 2010), at least partly through BCL11A. Given the phenoytpes of the HPFH heterozygotes, the truncating KLF1 p.K288X was best described as a dominant mutation with variable penetrance; most likely due to interplay with other regulatory factors that we have been seeking. Genome-wide association analysis, in the context of genome-wide expression profiles from cultured Human Erythroid Progenitors (HEPs) of critically informative family members, revealed additional loci of potential interest. The effect of the Hb F inducer Hydroxyurea on the gamma globin profiles of the KLF1- (p.K288X) HPFH HEPs was enhanced compared to the wild type, and 74 loci were differentially expressed. It is anticipated that extensive re-sequencing of these new targets may reveal the extent of the molecular pathways under control of KLF1 in erythropoiesis and globin gene switching, and in particular those that may be targeted for therapeutics in patients. Disclosures: No relevant conflicts of interest to declare.
Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type 2 Diabetes Me... more Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type 2 Diabetes Mellitus, and long-term venous insu ciency. Mesenchymal stem cells (MSCs) ability to modulate the in ammatory response represents the fundamental requisite for their applicability as a treatment of chronic wounds. Methods: This study aimed to develop a novel bioactive platelet-rich plasma (PRP)-leukocytes-depleted scaffold to reproduce typical clinical wound of patients with poor chronic skin perfusion and low leucocytes in ltration. After scratching the wound model to mimic injury three conditions were compared; an untreated condition, a condition treated with recombinant TNF to mimic an in ammatory state and a condition treated with TNF and also with MSCs to evaluate how the latter's immunomodulatory properties affect the therapeutic outcomes in an in ammatory state. Gene expression of IL8 and TGFA were analysed in biological triplicates of the three conditions. Statistical analysis was done through a paired student t-test and a p <0.05 was considered signi cant. Results: We set up a skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold, with embedded broblasts as dermal equivalent and seeded keratinocytes on it as multi-layered epidermidis. IL8 expression increased upon scratching (p=0.014) and continued to increase up to day 1 (p=0.048). IL8 expression decreased upon administration of TNF (p=0.005) but then increased again. IL8 expression decreased in the untreated condition after day 1 as the natural healing process took place and was lower than in treated conditions in day 8 (p=0.048). TGFA expression decreased upon scratching (p=0.006) and increased again in day 1, more so in the untreated than in the treated conditions (p=0.02). TGFA expression decreased again in day 4 in the study group before increasing sharply (p=0.027) in day 8 to reach pre-scratch levels. Conclusion: This study found that a leukocyte-depleted PRP-based skin equivalent can be useful in the study of treatments of chronic wounds. This study also indicates that MSCs appear to modulate the expression of IL8 by switching from an immunosuppressive phenotype to a pro-in ammatory phenotype. These results indicate that the administration of MSCs could offer a potential therapeutic approach for the treatment of leg ulcers in patients with poor skin perfusion. Background Leg ulcers are common, and their incidence rises with age, leading to a negative impact on the quality of life and considerable cost for the health service [1]. Haemolytic disorders such as Sickle Cell Disease and β-thalassaemia are common in Malta and other Mediterranean countries due to a number of β globin gene mutations [2]. Haemoglobin disorders are associated with chronic cutaneous wounds due to peripheral hypoxia [3, 4], lower bioavailability of nitric oxide (NO), iron overload, and an impaired endothelial function [5]. Chronic leg ulceration has also been seen in Type 2 Diabetes Mellitus (T2DM)
Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually charac... more Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually characterized by high levels of HbF since this compensates for the lack of, or abnormal HbA that is expressed in vivo. Further insight on how the hemoglobin switch is controlled and regulated can become crucial in the treatment of hemoglobin disorders. The present treatment options currently include routine blood transfusions with the occasional complications attributed to iron overload in major organs. Human stem cell therapy and bone marrow transplantation are some other options that are very risky and not without complications. Previous studies have shown that HbF levels differ in adults and that at least three loci independent of the β globin locus are responsible including XMN1, the HBS1L-MYB intergenic interval, and BCL11A. In another study conducted on an extended Maltese family, revealed that KLF1 also controls HbF levels in vivo. This study also suggested that other genes in conjuncti...
GMS Interdisciplinary Plastic and Reconstructive Surgery DGPW, 2022
Objectives: Platelet-derived products have been shown as promising novel therapeutic agents for c... more Objectives: Platelet-derived products have been shown as promising novel therapeutic agents for chronic wounds. However, their clinical use requires a greater degree of method standardisation, part of which involved more extensive cataloguing of their biochemical composition. This study aimed to quantify and compare total protein and 6 angiogenically-active growth factors in three distinct platelet products. Methods: Platelet Lysate (PL, n=5), Calcium-activated Platelet Rich Plasma (Ca-PRP, n=5) and Platelet-Rich Fibrin (PRF, n=5) were prepared from pooled platelet apheresis products (n=10). Ca-PRP and PRF were prepared from the same units (n=5) by activation with 20 mmolL-1 calcium chloride. PL was prepared from the remaining (n=5) units using an established lysate. Total protein was quantified with the Bradford Assay. Sandwich enzyme-linked immunosorbent assay was used to quantify six growth factors: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), stromal cell-derived growth factor-1α (SDF-1α), endostatin, and transforming growth factor-β1 (TGF-β1). Results: Protein retrieval differed significantly (p&lt;0.05) between the three products: PL (11.35±0.80 mg/mL) &lt; Ca-PRP (20.44±8.17 mg/mL) &lt; PRF (40.67±3.13 mg/mL). Growth factor yield was considerable in all three products and differed significantly for: VEGF (PL&lt;PRF); EGF (Ca-PRP&lt;PRF); HFG (PL&lt;Ca-PRP); Endostatin (PL&lt;Ca-PRP, PRF&lt;Ca-PRP, PL&lt;PRF) and TGF-β1 (Ca-PRP&lt;PL, Ca-PRP&lt;PRF). Conclusions: Platelet apheresis products contain a substantial quantity of the investigated pro- and anti-angiogenic growth factors. Their release varies depending on the manufacturing protocol used. Clinically, alternate products could thus be combined to provide a therapeutically optimal mix of growth factors.
Notwithstanding our increase in knowledge on the events that lead to atherosclerosis and myocardi... more Notwithstanding our increase in knowledge on the events that lead to atherosclerosis and myocardial infarction, the literature on the genetic determinants of these related diseases is ridden with conflicting results. In this study a novel RNA profiling technique was applied in a case-control setting including 524 men with a history of myocardial infarction and 628 control subjects. The relationship between a selection of polymorphisms, RNA expression and other intermediate phenotypes, and disease outcome was investigated. Patients had higher levels of inflammatory molecules and Toll-like receptors (TLRs) than controls. Macrophage migration inhibitory factor (MlF) and the intracellular regulator proteinase inhibitor 9 (PI9) gave the highest odds ratios for myocardial infarction. Analysis of genetic data with the RNA data revealed that DNA changes in inflammation-related genes can influence several disease-related intermediate phenotypes. The underlying levels of expression of genes of related function were shown to have considerable impact on the effect of a particular gene on disease outcome. The overall effect of polymorphisms on risk outcome tended to be small, but additive, and was frequently modified by smoking. Aberrant RNA profiles acted as sentinels for particularly deleterious or protective outcomes. Bioinformatics tools were applied to detect a new MlF splice variant and to determine different roles of alternative transcripts of TLR4. To date, this is the largest RNA profiling study on myocardial infarction. This innovative approach highlights a degree of complexity in the expression and regulation of inflammatory molecules that needs to be accounted for to improve our understanding of the mechanism of genetic risk in atherosclerosis and myocardial infarction.
Hb F-Malta-I [Ggamma117(19)His--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;... more Hb F-Malta-I [Ggamma117(19)His--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;Arg, CAT--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;CGT] is a stable and benign variant of Hb F found in 1.8% of Maltese newborn. We studied 120 Hb F-Malta-I heterozygotes and four Hb F-Malta-I homozygotes. The mean proportion of Ggamma-F-Malta-I in Hb F was 0.26 +/- 0.03 for the Hb F-Malta-I heterozygotes and 0.58 +/- 0.06 for the Hb F-Malta-I homozygotes. The Hb F-Malta-I allele was shown to occur on a background of the common Mediterranean haplotype Va [+ + - - - - - + + -]. Furthermore, the common Mediterranean haplotypes Va, IIIb [- + + + - + + + + -], I [+ + - - - - - + + +] and II [- + - + + - + + + +] accounted for most (66.2%) of the wild-type alleles among the tested Hb F-Malta-I heterozygotes. Different genotypes at the 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; epsilon HincII, Ggamma and Agamma HindIII, and 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;psibeta HincII sites (but not at the 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; Ggamma XmnI site) were found to be linked to significant variations in the proportion of Ggamma-F-Malta-I and Ggamma-globins in the Hb F of newborn Hb F-Malta-I heterozygotes. Moreover, the 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; Ggamma XmnI site was found to be associated with variations in Hb F and Ggamma-globin levels in a population of adult Maltese beta-thalassemia (thal) homozygotes. This implies that a determinant linked to the XmnI site which effects Ggamma-globin gene expression is active in anemic adults but not in normal infants.
ABSTRACT Muscarinic receptors are important in the development of airway hyperresponsiveness, and... more ABSTRACT Muscarinic receptors are important in the development of airway hyperresponsiveness, and dysfunction of these receptors has been suggested to be present in asthma. The human muscarinic M2 and M3 receptor genes were screened for polymorphic variation using single-stranded conformation polymorphism (SSCP) analysis, complemented by direct fluorescent sequencing. Forty-six random DNA samples and 46 respiratory physician diagnosed asthmatic samples were used as a template for analysis. Within the muscarinic M2 receptor gene, we identified two degenerate single base substitutions (1197T→C, Thr→Thr and 976A→C, Arg→Arg) in one random and one asthmatic sample respectively. Analysis of the 3′ UTR region revealed an additional ‘A’ at bp 1793 (c.f. ATG). This was present in all of 49 samples analysed by sequencing or BsmI digest, suggesting that the published sequence (GenBank Accession No. M16404) is incorrect. A common 3′ UTR polymorphism (T→A) was found at bp 1696 (c.f. ATG) (allelic frequency=65%, n=60), but this does not alter transcription factor recognition sites. We were unable to identify any polymorphic variation within the muscarinic M3 coding region or the flanking regions investigated, using the methods described. The coding regions for the human muscarinic M2 and M3 receptor genes are both highly conserved. These data suggest that polymorphic variation within these coding sequences is unlikely to account for inter-individual variability in response to methacholine or anticholinergic therapy. The potential functional significance of the muscarinic M2 receptor 3′ UTR polymorphism (bp 1696) remains to be determined. British Journal of Pharmacology (2001) 133, 43–48; doi:10.1038/sj.bjp.0704039
The percentages of the a-chain variant Hb G-Philadelphia (Hb G) or a2 68 Asn?Lysß2 were evaluated... more The percentages of the a-chain variant Hb G-Philadelphia (Hb G) or a2 68 Asn?Lysß2 were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another
Distribution of work responsibilities within the pilot 2.2 Diagrammatic Representations of Scenar... more Distribution of work responsibilities within the pilot 2.2 Diagrammatic Representations of Scenarios. 3.1 Maltese population by gender and age group in 2003 and as projected to 2015 3.2 Gross Domestic Product by Industry and Type of Income 3.3 The number of patents registered since 1994 3.4 Number of University graduates over a four year period 3.5 Standard process followed in the commercialisation of biotechnology products 3.6 Summary of the basic foundations for the successful commercialisation of biotechnology 4.1 Countries who have adopted biotechnology crops 4.2 Biotechnology Industry in Europe compared to the US 4.3 Comparison of Employment The two main vectors limiting development of the local biotechnology sector were identified to be STI education and RTDI capacity. Four main scenarios for the possible development of local biotechnology sector were developed around these two vectors. Actions to be taken to move from one scenario to another and hence reach optimal scenario are outlined. SWOT analysis of the local biotechnology sector identified that our major strengths are the well established medical and engineering faculties, good health care system, strong ICT sector and a skilled workforce. However lack of right decisions being taken at the right time might result in brain drain and low GDP. Chapter 2: The Foresight Process This chapter provides an almost chronological account of the foresight Pilot as it unfolded. Certain steps interacted with each other in synergetic, iterative loops which enhanced the process. The methodology and approaches used include stakeholder-mapping and co-nomination exercises, the setting up of expert panels, questionnaire-based surveys, SWOT analysis and scenario-building exercise.
Restriction endonuclease mapping defined a partial deletion of about 1 .35 kb in the fi-globin ge... more Restriction endonuclease mapping defined a partial deletion of about 1 .35 kb in the fi-globin gene of a black American patient with hemoglobin S-$#{176}-thalassemia and in his uncle with a ftothalassemia trait. The 5' endpoint of the deletion is about 600 bases upstream from the cap site. and T HE f.-THALASSEMIAS are inherited disorders ofhuman hemoglobin synthesis, characterized by reduced (fl-thalassemia) or absent (/3#{176}-thalassemia) production of the f3-globin of normal adult hemoglobin)3 Restriction endonuclease mapping, cloning, and DNA sequencing have revealed several types of mutations in the 3-globin gene region of such patients.
Although the precise biochemical mechanisms of globin gene switching remain elusive, considerable... more Although the precise biochemical mechanisms of globin gene switching remain elusive, considerable insight is gained by in vivo expression profiling through quantification of the hemoglobin / globin phenotype of informative heterozygosities and homozygosities / compound heterozygosities in the context of specific regulatory DNA sequence diversity such as the XMN-I or the [(AT)xTy] sequence polymorphisms. The quantification of normal and abnormal globins of Hb F Malta-I (or a2b2, 117(G19)His&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;Arg) heterozygotes which are in tight linkage disequilibrium with Hb Valletta (or a2b2 287(f3)Thr&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;Pro) i.e. Gyo, GyFMalta-I, AyI, bV and bA together with extensive haplotyping of homozygotes and heterozygotes including the XMN-I dimorphism in the Gy promoter and the (AT)xTy polymorphism (BP1 binding site) 5′ to the b globin genes had suggested that the XMN-I dimorphism was largely inactive in the normal newborn. In contrast the Hb F levels and the proportion of Gy globin in anemic adult beta-thalassemia homozygotes and compound heterozygotes…
Preimplantation Genetic Diagnosis is a fairly new form of prenatal diagnosis, which screens for g... more Preimplantation Genetic Diagnosis is a fairly new form of prenatal diagnosis, which screens for genetic disease at the embryonic stage. Its use is expanding as more knowledge is gained about genetic disorders and tests for the causative genes are developed.
The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the earl... more The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the early stage genetic programming of erythroid progenitors to promote physiological γ to β globin gene switching. Indeed, we showed that a truncation mutation (p.K288X) in KLF1 that deleted the DNA binding zinc finger domain resulted in marked KLF1 deficiency and a hematological condition that resembled a hereditary persistence of fetal hemoglobin (HbF) or a β thalassemia. Here, we describe five additional families with the same p.K288X mutation but varied hematological and HbF levels together with genetic and phenotypic data on a 600 data-set from the same Maltese population. The data accounted for a strong promoter embedded within a region of genetic heterogeneity of KLF1 that led to a ψβ thalassemia. Whole genome sequencing on 15 subjects of six families (FamF1 - FamF6) segregating (two) p.K288X frameworks of KLF1 had variable degrees of microcytosis (MCV; 76.1fL -77.4 fL) and HbF levels (HbF 2480 mg/dL - 802mg/dL) due to complex heterozygosity between promoter, coding and truncating mutations in KLF1. Case II-6 of FamF1 with the highest HbF (2480 mg/dL) had 2 promoter and 2 coding mutations in cis and in trans to the p.K288X truncation. Nine (9) KLF1 frameworks (A - I) were derived by transmission disequilibrium analyses of the family data, each assembled from 15 mutations and resulting in 7 genotypes among the families. The p.K288X truncation was found on a second rarer framework. Additional, rarer KLF1 frameworks were found with haploview in the population dataset. The population dataset was made up of 198 β thalassemia heterozygotes and 400 others from the clinic and the biobank and that had no β globin gene mutations, variable blood counts or hemoglobin profiles or both. They were older than 2 years of age, not pregnant and had normal iron levels. The number of KLF1 mutations differed from 0 in the wild-type framework A to 6 in one of the rare frameworks X. Six mutations were in the promoter region and 6 were in the coding region that defined a &amp;amp;amp;quot;KLF1 Variable Region&amp;amp;amp;quot; 5&amp;amp;amp;#39; genomic coordinate 12887183 - 12888273, whereas very few were found in the 3&amp;amp;amp;#39; (genomic coordinate 12884589 - 12884752) that defined the KLF1 &amp;amp;amp;quot;Constant Region&amp;amp;amp;quot; The Constant region has also been evolutionary conserved. It included the zinc finger domain and the proteasome binding site. The genotype - phenotype correlations and the family data were consistent with an autosomal recessive condition that resembled a β thalassemia, thus a ψβ thalassemia. It differed from a silent thalassemia because the β globin gene sequence was wild type. It provided a diagnosis for families with iron resistant microcytosis and borderline hemoglobin phenotypes suitable for counselling in the clinical setting. It was consistent with the observation among the families regarding the high strength of the KLF1 promoter. Multiple &amp;amp;amp;quot;hits&amp;amp;amp;quot; were necessary to suppress the biosynthesis of KLF1 for hemoglobin switching to escape perinatal suppression. The effect of the 6 promoter mutations were confirmed in vitro with native and induced K562 and Hek293T cell lines. Presumably, during normal development, the strong promoter served to rapidly drown KLF1 binding sites with KLF1 molecules to direct progenitors to erythropoiesis with the appropriate adult hemoglobin profile in the perinatal period. The diagnosis of the patients with selected genotypes due to compound and double heterozygosities in promoter and coding sequences shall further permit quantification of differential promoter function in vivo. Figure Disclosures No relevant conflicts of interest to declare.
Abstract 5162 We provide additional data on members of the family from Malta with Hereditary Pers... more Abstract 5162 We provide additional data on members of the family from Malta with Hereditary Persistence of Fetal Hemoglobin (HPFH) due to KLF1 haplo-insufficiency. The data indicated a possible role of additional loci in the pathway of globin gene control. We showed that KLF1 functions as a master regulator of erythropoiesis and developmental globin gene switching (Borg et al., Nature Genetics doi: 10. 1038/ng.630, 2010), at least partly through BCL11A. Given the phenoytpes of the HPFH heterozygotes, the truncating KLF1 p.K288X was best described as a dominant mutation with variable penetrance; most likely due to interplay with other regulatory factors that we have been seeking. Genome-wide association analysis, in the context of genome-wide expression profiles from cultured Human Erythroid Progenitors (HEPs) of critically informative family members, revealed additional loci of potential interest. The effect of the Hb F inducer Hydroxyurea on the gamma globin profiles of the KLF1- (p.K288X) HPFH HEPs was enhanced compared to the wild type, and 74 loci were differentially expressed. It is anticipated that extensive re-sequencing of these new targets may reveal the extent of the molecular pathways under control of KLF1 in erythropoiesis and globin gene switching, and in particular those that may be targeted for therapeutics in patients. Disclosures: No relevant conflicts of interest to declare.
Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type 2 Diabetes Me... more Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type 2 Diabetes Mellitus, and long-term venous insu ciency. Mesenchymal stem cells (MSCs) ability to modulate the in ammatory response represents the fundamental requisite for their applicability as a treatment of chronic wounds. Methods: This study aimed to develop a novel bioactive platelet-rich plasma (PRP)-leukocytes-depleted scaffold to reproduce typical clinical wound of patients with poor chronic skin perfusion and low leucocytes in ltration. After scratching the wound model to mimic injury three conditions were compared; an untreated condition, a condition treated with recombinant TNF to mimic an in ammatory state and a condition treated with TNF and also with MSCs to evaluate how the latter's immunomodulatory properties affect the therapeutic outcomes in an in ammatory state. Gene expression of IL8 and TGFA were analysed in biological triplicates of the three conditions. Statistical analysis was done through a paired student t-test and a p <0.05 was considered signi cant. Results: We set up a skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold, with embedded broblasts as dermal equivalent and seeded keratinocytes on it as multi-layered epidermidis. IL8 expression increased upon scratching (p=0.014) and continued to increase up to day 1 (p=0.048). IL8 expression decreased upon administration of TNF (p=0.005) but then increased again. IL8 expression decreased in the untreated condition after day 1 as the natural healing process took place and was lower than in treated conditions in day 8 (p=0.048). TGFA expression decreased upon scratching (p=0.006) and increased again in day 1, more so in the untreated than in the treated conditions (p=0.02). TGFA expression decreased again in day 4 in the study group before increasing sharply (p=0.027) in day 8 to reach pre-scratch levels. Conclusion: This study found that a leukocyte-depleted PRP-based skin equivalent can be useful in the study of treatments of chronic wounds. This study also indicates that MSCs appear to modulate the expression of IL8 by switching from an immunosuppressive phenotype to a pro-in ammatory phenotype. These results indicate that the administration of MSCs could offer a potential therapeutic approach for the treatment of leg ulcers in patients with poor skin perfusion. Background Leg ulcers are common, and their incidence rises with age, leading to a negative impact on the quality of life and considerable cost for the health service [1]. Haemolytic disorders such as Sickle Cell Disease and β-thalassaemia are common in Malta and other Mediterranean countries due to a number of β globin gene mutations [2]. Haemoglobin disorders are associated with chronic cutaneous wounds due to peripheral hypoxia [3, 4], lower bioavailability of nitric oxide (NO), iron overload, and an impaired endothelial function [5]. Chronic leg ulceration has also been seen in Type 2 Diabetes Mellitus (T2DM)
Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually charac... more Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually characterized by high levels of HbF since this compensates for the lack of, or abnormal HbA that is expressed in vivo. Further insight on how the hemoglobin switch is controlled and regulated can become crucial in the treatment of hemoglobin disorders. The present treatment options currently include routine blood transfusions with the occasional complications attributed to iron overload in major organs. Human stem cell therapy and bone marrow transplantation are some other options that are very risky and not without complications. Previous studies have shown that HbF levels differ in adults and that at least three loci independent of the β globin locus are responsible including XMN1, the HBS1L-MYB intergenic interval, and BCL11A. In another study conducted on an extended Maltese family, revealed that KLF1 also controls HbF levels in vivo. This study also suggested that other genes in conjuncti...
GMS Interdisciplinary Plastic and Reconstructive Surgery DGPW, 2022
Objectives: Platelet-derived products have been shown as promising novel therapeutic agents for c... more Objectives: Platelet-derived products have been shown as promising novel therapeutic agents for chronic wounds. However, their clinical use requires a greater degree of method standardisation, part of which involved more extensive cataloguing of their biochemical composition. This study aimed to quantify and compare total protein and 6 angiogenically-active growth factors in three distinct platelet products. Methods: Platelet Lysate (PL, n=5), Calcium-activated Platelet Rich Plasma (Ca-PRP, n=5) and Platelet-Rich Fibrin (PRF, n=5) were prepared from pooled platelet apheresis products (n=10). Ca-PRP and PRF were prepared from the same units (n=5) by activation with 20 mmolL-1 calcium chloride. PL was prepared from the remaining (n=5) units using an established lysate. Total protein was quantified with the Bradford Assay. Sandwich enzyme-linked immunosorbent assay was used to quantify six growth factors: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), stromal cell-derived growth factor-1α (SDF-1α), endostatin, and transforming growth factor-β1 (TGF-β1). Results: Protein retrieval differed significantly (p&lt;0.05) between the three products: PL (11.35±0.80 mg/mL) &lt; Ca-PRP (20.44±8.17 mg/mL) &lt; PRF (40.67±3.13 mg/mL). Growth factor yield was considerable in all three products and differed significantly for: VEGF (PL&lt;PRF); EGF (Ca-PRP&lt;PRF); HFG (PL&lt;Ca-PRP); Endostatin (PL&lt;Ca-PRP, PRF&lt;Ca-PRP, PL&lt;PRF) and TGF-β1 (Ca-PRP&lt;PL, Ca-PRP&lt;PRF). Conclusions: Platelet apheresis products contain a substantial quantity of the investigated pro- and anti-angiogenic growth factors. Their release varies depending on the manufacturing protocol used. Clinically, alternate products could thus be combined to provide a therapeutically optimal mix of growth factors.
Notwithstanding our increase in knowledge on the events that lead to atherosclerosis and myocardi... more Notwithstanding our increase in knowledge on the events that lead to atherosclerosis and myocardial infarction, the literature on the genetic determinants of these related diseases is ridden with conflicting results. In this study a novel RNA profiling technique was applied in a case-control setting including 524 men with a history of myocardial infarction and 628 control subjects. The relationship between a selection of polymorphisms, RNA expression and other intermediate phenotypes, and disease outcome was investigated. Patients had higher levels of inflammatory molecules and Toll-like receptors (TLRs) than controls. Macrophage migration inhibitory factor (MlF) and the intracellular regulator proteinase inhibitor 9 (PI9) gave the highest odds ratios for myocardial infarction. Analysis of genetic data with the RNA data revealed that DNA changes in inflammation-related genes can influence several disease-related intermediate phenotypes. The underlying levels of expression of genes of related function were shown to have considerable impact on the effect of a particular gene on disease outcome. The overall effect of polymorphisms on risk outcome tended to be small, but additive, and was frequently modified by smoking. Aberrant RNA profiles acted as sentinels for particularly deleterious or protective outcomes. Bioinformatics tools were applied to detect a new MlF splice variant and to determine different roles of alternative transcripts of TLR4. To date, this is the largest RNA profiling study on myocardial infarction. This innovative approach highlights a degree of complexity in the expression and regulation of inflammatory molecules that needs to be accounted for to improve our understanding of the mechanism of genetic risk in atherosclerosis and myocardial infarction.
Hb F-Malta-I [Ggamma117(19)His--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;... more Hb F-Malta-I [Ggamma117(19)His--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;Arg, CAT--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;CGT] is a stable and benign variant of Hb F found in 1.8% of Maltese newborn. We studied 120 Hb F-Malta-I heterozygotes and four Hb F-Malta-I homozygotes. The mean proportion of Ggamma-F-Malta-I in Hb F was 0.26 +/- 0.03 for the Hb F-Malta-I heterozygotes and 0.58 +/- 0.06 for the Hb F-Malta-I homozygotes. The Hb F-Malta-I allele was shown to occur on a background of the common Mediterranean haplotype Va [+ + - - - - - + + -]. Furthermore, the common Mediterranean haplotypes Va, IIIb [- + + + - + + + + -], I [+ + - - - - - + + +] and II [- + - + + - + + + +] accounted for most (66.2%) of the wild-type alleles among the tested Hb F-Malta-I heterozygotes. Different genotypes at the 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; epsilon HincII, Ggamma and Agamma HindIII, and 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;psibeta HincII sites (but not at the 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; Ggamma XmnI site) were found to be linked to significant variations in the proportion of Ggamma-F-Malta-I and Ggamma-globins in the Hb F of newborn Hb F-Malta-I heterozygotes. Moreover, the 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; Ggamma XmnI site was found to be associated with variations in Hb F and Ggamma-globin levels in a population of adult Maltese beta-thalassemia (thal) homozygotes. This implies that a determinant linked to the XmnI site which effects Ggamma-globin gene expression is active in anemic adults but not in normal infants.
ABSTRACT Muscarinic receptors are important in the development of airway hyperresponsiveness, and... more ABSTRACT Muscarinic receptors are important in the development of airway hyperresponsiveness, and dysfunction of these receptors has been suggested to be present in asthma. The human muscarinic M2 and M3 receptor genes were screened for polymorphic variation using single-stranded conformation polymorphism (SSCP) analysis, complemented by direct fluorescent sequencing. Forty-six random DNA samples and 46 respiratory physician diagnosed asthmatic samples were used as a template for analysis. Within the muscarinic M2 receptor gene, we identified two degenerate single base substitutions (1197T→C, Thr→Thr and 976A→C, Arg→Arg) in one random and one asthmatic sample respectively. Analysis of the 3′ UTR region revealed an additional ‘A’ at bp 1793 (c.f. ATG). This was present in all of 49 samples analysed by sequencing or BsmI digest, suggesting that the published sequence (GenBank Accession No. M16404) is incorrect. A common 3′ UTR polymorphism (T→A) was found at bp 1696 (c.f. ATG) (allelic frequency=65%, n=60), but this does not alter transcription factor recognition sites. We were unable to identify any polymorphic variation within the muscarinic M3 coding region or the flanking regions investigated, using the methods described. The coding regions for the human muscarinic M2 and M3 receptor genes are both highly conserved. These data suggest that polymorphic variation within these coding sequences is unlikely to account for inter-individual variability in response to methacholine or anticholinergic therapy. The potential functional significance of the muscarinic M2 receptor 3′ UTR polymorphism (bp 1696) remains to be determined. British Journal of Pharmacology (2001) 133, 43–48; doi:10.1038/sj.bjp.0704039
The percentages of the a-chain variant Hb G-Philadelphia (Hb G) or a2 68 Asn?Lysß2 were evaluated... more The percentages of the a-chain variant Hb G-Philadelphia (Hb G) or a2 68 Asn?Lysß2 were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another
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