Argyro Sgourou

Genotypic heterogeneity and correlation to intergenic haplotype within high HbF β-thalassemia intermedia

Abstract:  Objectives: A molecular study was carried out of β‐thalassemia intermedia patients, compound heterozygotes for mutations usually found in β‐thalassemia major, with high levels of HbF in the absence of hereditary persistence of fetal hemoglobin (HPFH) syndrome. Our objective was to locate cis‐DNA structures, DNA haplotypes, motifs, or polymorphisms that may correlate with the presence of high HbF. Methods: Allele‐specific oligonucleotide (ASO) hybridization was used for the detection of mutations and restriction fragment length polymorphism (RFLP) analysis and automated sequencing for motifs, haplotypes, and polymorphisms. Southern blot was used for investigating α‐thalassemia and/or α‐ or γ‐globin genes triplications. RNA extracted from burst forming unit‐erythroid (BFU‐e) colonies of peripheral blood mononuclear cell cultures was used in reverse transcriptase‐polymerase chain reaction (RT‐PCR) to investigate intergenic transcription. Results: We established that (i) the combination: T haplotype of the Aγ‐δ‐globin intergenic region, the motif (TA)9N10(TA)10 in the HS2 site of locus control region (LCR), and TAG pre‐Gγ haplotype is sufficient but not necessary for high HbF, (ii) the genetic determinant(s) for high HbF involves an element associated with this combination and must be present in the specific R haplotype occurring in β‐thalassemia intermedia and (iii) the genetic determinant(s) for high HbF does not involve the abolition of intergenic transcription in the Aγ‐δ‐globin intergenic region. Conclusions: The genetic determinant(s) of high HbF in the absence of HPFH is linked to intergenic haplotype T and does not disrupt intergenic transcription.

Functional significance of the thyrotropin receptor germline polymorphism D727E.

In a toxic thyroid adenoma we identified a novel somatic mutation that constitutively activates the thyrotropin receptor (TSHR). Two heterozygous point mutations at adjacent nucleotides led to a substitution of alanine with asparagine at codon 593 (A593N) in the fifth transmembrane helix of TSHR. This somatic mutation resided on the same TSHR allele with the germline polymorphism D727E. The functional characteristics of the single TSHR mutants A593N and D727E and of the double mutant A593N/D727E were studied in transiently transfected COS-7 cells. The TSHR mutants A593N and A593N/D727E constitutively activated the cAMP cascade, whereas the D727E mutant did not differ from the wild-type TSHR. Surprisingly, the double mutants specific constitutive activity was 2.3-fold lower than the A593N mutant. Thus, the polymorphism significantly ameliorates G(alphas) protein activation in the presence of the gain-of-function mutation A593N, although it is functionally inert in the context of the wild-type TSHR.

Thalassaemia mutations within the 5 ' UTR of the human beta-globin gene disrupt transcription

The mechanisms by which mutations within the 5′ untranslated region (UTR) of the human β‐globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four ‘silent’ mutations in the human β‐globin 5′UTR, namely: +10(−T), +22(G → A), +33(C → G) and +(40–43)(−AAAC), which are present in patients with β‐thalassaemia intermedia. Expression of these genes under the control of the β‐globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61·6%, 68%, 85·2% and 70·6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3′ end processing or mRNA stability. Nuclear run‐on experiments demonstrated that mutations +10(−T) and +33(C → G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G → A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post‐transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment.

Kallmann's syndrome and schizophrenia.

Objective: Kallmanns Syndrome is a heritable disorder characterized by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. A common pathogenesis for KS and schizophrenia had been proposed based on shared pathologies of these two disorders, although no such clinical associations have ever been reported. Method: We report a 35 year old man with schizophrenia and Kallmanns Syndrome. The patient presented with signs and symptoms of hypogonadism, severe hyposmia and normal endocrine functions of the anterior pituitary. Hyposmia has been attributed to the absence of the olfactory bulbs and tracts and atrophy of the olfactory gyri, but normal olfactory mucosa. The patient presented with paranoid schizophrenia with persecutory delusions, auditory hallucinations, thought disorder, depersonalization, and gradual but marked global deterioration. Results: Psychiatric evaluation revealed an entirely different psychopathological and personality profile between the patient and the six other Kallmann patients studied. Cycle sequencing analysis revealed a normal sequence of all 14 exons of the KAL gene. In conclusion, based on the presented case, Kallmanns Syndrome and schizophrenia represent a rare clinical association rather than a syndrome with a common pathogenesis, which if present should be confined to the olfactory dysfunction.

Individualizing fetal hemoglobin augmenting therapy for β-type hemoglobinopathies patients.

Individual genetic composition is an important cause of variations in the response and tolerance to drug treatment. Pharmacogenomics is a modern discipline aiming to delineate individual genomic profiles and drug response. To date, there are several medical disciplines where pharmacogenomics is readily applicable, while in others its usefulness is yet to be demonstrated. Recent experimental evidence suggest that besides genomic variation within the human β-globin gene cluster, other variants in modifier genes residing outside the human β-globin gene cluster are significantly associated with response to hydroxyurea treatment in β-type hemoglobinopathies patients, deducted from the increase in fetal hemoglobin levels. This article aims to provide an update and to discuss future challenges on the application of pharmacogenomics for β-type hemoglobinopathies therapeutics in relation to the current pharmacological treatment modalities for those disorders.

The β‐globin C→G mutation at 6 bp 3′ to the termination codon causes β‐thalassaemia by decreasing the mRNA level

Summary. We have studied the expression of the silent β‐thalassaemia term+6 (C→G) mutation, at nucleotide 6 after the stop codon within the human β‐globin 3′ untranslated regions (3′UTR), by stable transfection in murine erythroleukaemia (MEL) cells. Steady state mRNA levels from transfected MEL cells containing the term+6 mutant allele were reduced by 52–60%, compared with those obtained from the normal β‐globin gene, in both total and cytoplasmic RNA fractions, showing that the mutation itself is responsible for the similar data obtained from patients. Upon analysis of nuclear RNA, the term+6 mutation was found to also lower the ratio of cleaved/uncleaved transcripts by 22–30%, thus revealing that it interferes with correct 3′‐end formation of β‐globin mRNA. The term+6 mutation lies within a polypyrimidine track, similar to that in the β‐intervening sequence II (β‐IVSII), which is known to be an important contributor to the promotion of premRNA 3′‐end formation. We propose that the two polypyrimidine tracks flanking the translated region of exon III of the human β‐globin gene may co‐operate during β‐globin mRNA biogenesis.

Correlation of SIN3A genomic variants with β-hemoglobinopathies disease severity and hydroxyurea treatment efficacy

AIMS Hemoglobinopathies, particularly β-thalassemia and sickle cell disease, are characterized by great phenotypic variability in terms of disease severity, while notable differences have been observed in hydroxyurea treatment efficacy. In both cases, the observed phenotypic diversity is mostly dependent on the elevated fetal hemoglobin levels, resulting from the persistent fetal globin gene expression in the adult erythroid stage orchestrated by intricate mechanisms that still remain only partly understood. We have previously shown that several protein factors act as modifiers of fetal hemoglobin production, exerting their effect via different pathways. MATERIALS & METHODS Here, we explored whether SIN3A could act as a modifier of fetal globin gene expression, as it interacts with KLF10, a known modifier of fetal hemoglobin production. RESULTS We show that SIN3A genomic variants are associated both with β-thalassemia disease severity (rs11072544) as well as hydroxyurea treatment response (rs7166737) in β-hemoglobinopathies patients. CONCLUSION Our findings further underline that fetal hemoglobin production is the result of a complex interplay in which several human globin gene cluster variants interact with protein factors encoded by modifier genes to produce the observed clinical outcome.

Enhanced Central Nervous System Transduction with Lentiviral Vectors Pseudotyped with RVG/HIV-1gp41 Chimeric Envelope Glycoproteins

ABSTRACT To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the “Kennedy” sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease.

Genomic variants in the ASS1 gene, involved in the nitric oxide biosynthesis and signaling pathway, predict hydroxyurea treatment efficacy in compound sickle cell disease/β-thalassemia patients

AIM Hemoglobinopathies exhibit a remarkable phenotypic diversity that restricts any safe association between molecular pathology and clinical outcomes. PATIENTS & METHODS Herein, we explored the role of genes involved in the nitric oxide biosynthesis and signaling pathway, implicated in the increase of fetal hemoglobin levels and response to hydroxyurea treatment, in 119 Hellenic patients with β-type hemoglobinopathies. RESULTS We show that two ASS1 genomic variants (namely, rs10901080 and rs10793902) can serve as pharmacogenomic biomarkers to predict hydroxyurea treatment efficacy in sickle cell disease/β-thalassemia compound heterozygous patients. CONCLUSION These markers may exert their effect by inducing nitric oxide biosynthesis, either via altering splicing and/or miRNA binding, as predicted by in silico analysis, and ultimately, increase γ-globin levels, via guanylyl cyclase targeting.

Association of genome variations in the renin- angiotensin system with physical performance

BackgroundThe aim of this study was to determine the genotype distribution and allelic frequencies of ACE (I/D), AGTR1 (A +1166 C), BDKRB2 (+9/−9) and LEP (G–2548A) genomic variations in 175 Greek athletes who excelled at a national and/or international level and 169 healthy Greek adults to identify whether some particular combinations of these loci might serve as predictive markers for superior physical condition.ResultsThe D/D genotype of the ACE gene (p = 0.034) combined with the simultaneous existence of BDKRB2 (+9/−9) (p = 0.001) or LEP (G/A) (p = 0.021) genotypes was the most prevalent among female athletes compared to female controls. A statistical trend was also observed in BDKRB2 (+9/−9) and LEP (G–2548A) heterozygous genotypes among male and female Greek athletes, and in ACE (I/D) only in male athletes. Finally, both male and female athletes showed the highest rates in the AGTR1 (A/A) genotype.ConclusionsOur results suggest that the co-existence of ACE (D/D), BDKRB2 (+9/−9) or LEP (G/A) genotypes in female athletes might be correlated with a superior level of physical performance.