Novel Therapy Approaches in β-Thalassemia Syndromes — A Role of Genetic Modifiers

The β-thalassemia syndromes are heterogeneous autosomal recessive hereditary disorders, caused by alterations in the HBB gene and characterized by absent or reduced β-globin chain synthesis. The β-thalassemia phenotypes are variable, ranging from severe, transfusion-dependent thalassemia major to mild, asymptomatic thalassemia trait. This interpatient clinical variability has swayed researchers toward identifying genetic modifiers for these disorders. Primary modifiers refer to type of alterations affecting β-globin gene. Secondary modifiers include variations in genes affecting α/β-globin chain equilibrium, such as genes involved in the γ-globin gene expression and genes affecting the amount and stability of α-globin chains. Tertiary modifiers are gene variations affecting the phenotype with regard to the complications caused by β-thalassemia syndromes. A role of secondary genetic modifiers in ameliorating the clinical phenotype has been observed. Secondary genetic modifiers are the most common targets for modern therapy and could be located within α- and γ-globin genes or outside globin gene cluster. The most potent secondary modifier genes are γ-globin genes. Production of fetal hemoglobin (HbF) trough adulthood ameliorates the severity of β-thalassemia phenotype. Large family and genome-wide association studies have shown that regions outside of the β-globin gene cluster are also implicated in γ-globin gene expression regulation. HBS1-MYB intragenic region and BCL11A gene have been particularly studied. Variants within these loci, along with γ-globin gene variants, account for approximately 50% of the HbF level variation, suggesting that additional factors are involved (transcription regulators (KLF1), regulators of α-globin chain stability (AHSP), epigenetic regulators (FoP)). Until recently a definitive cure for β-thalassemia could be achieved with bone marrow transplantation. However, it is available for less than 30% of the patients and bears a significant risk of morbidity and mortality. Alternative strategies, such as gene therapy and development of induced pluripotent stem cells (iPSCs) have been explored. The targets for gene therapy are hematopoietic stem cells, which are harvested from patient bone marrow or peripheral blood, purified by immunoselection, transduced by “therapeutic gene” aimed at correcting the effect of defective β-globin gene, and returned to the patient. Various types of vectors have been considered for gene transfer, including non viral (tRNK and ribozymes) and viral (retroviral and lentiviral vectors). In the past few years, iPSCs emerged as an interesting candidate for gene transfer. The feature that makes these cells appealing in the field of gene therapy is their susceptibility to gene correction by homologous recombination. Therapy protocols based on molecular basis of β-thalassemia are the best example of novel approaches in disease treatment

Untreated PKU patients without intellectual disability: what do they teach us?

Phenylketonuria (PKU) management is aimed at preventing neurocognitive and psychosocial dysfunction by keeping plasma phenylalanine concentrations within the recommended target range. It can be questioned, however, whether universal plasma phenylalanine target levels would result in optimal neurocognitive outcomes for all patients, as similar plasma phenylalanine concentrations do not seem to have the same consequences to the brain for each PKU individual. To better understand the inter-individual differences in brain vulnerability to high plasma phenylalanine concentrations, we aimed to identify untreated and/or late-diagnosed PKU patients with near-normal outcome, despite high plasma phenylalanine concentrations, who are still alive. In total, we identified 16 such cases. While intellectual functioning in these patients was relatively unaffected, they often did present other neurological, psychological, and behavioral problems. Thereby, these "unusual" PKU patients show that the classical symptomatology of untreated or late-treated PKU may have to be rewritten. Moreover, these cases show that a lack of intellectual dysfunction despite high plasma phenylalanine concentrations does not necessarily imply that these high phenylalanine concentrations have not been toxic to the brain. Also, these cases may suggest that different mechanisms are involved in PKU pathophysiology, of which the relative importance seems to differ between patients and possibly also with increasing age. Further research should aim to better distinguish PKU patients with respect to their cerebral effects to high plasma phenylalanine concentrations

Expanded national database collection and data coverage in the FINDbase worldwide database for clinically relevant genomic variation allele frequencies

Peer reviewe

The Genetic Landscape and Epidemiology of Phenylketonuria

Phenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]–1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A>G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C>T (p.Arg408Trp) (22.2%), c.1066−11G>A (IVS10−11G>A) (6.4%), and c.782G>A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066−11G>A];[1066−11G>A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcome.Fil: Hillert, Alicia. No especifíca;Fil: Anikster, Yair. No especifíca;Fil: Belanger Quintana, Amaya. No especifíca;Fil: Burlina, Alberto. No especifíca;Fil: Burton, Barbara K.. No especifíca;Fil: Carducci, Carla. No especifíca;Fil: Chiesa, Ana Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Christodoulou, John. No especifíca;Fil: Dordevic, Maja. No especifíca;Fil: Desviat, Lourdes R.. No especifíca;Fil: Eliyahu, Aviva. No especifíca;Fil: Evers, Roeland A.F.. No especifíca;Fil: Fajkusova, Lena. No especifíca;Fil: Feillet, Francois. No especifíca;Fil: Bonfim Freitas, Pedro E.. No especifíca;Fil: Gizewska, María. No especifíca;Fil: Gundorova, Polina. No especifíca;Fil: Karall, Daniela. No especifíca;Fil: Kneller, Katya. No especifíca;Fil: Kutsev, Sergey I.. No especifíca;Fil: Leuzzi, Vincenzo. No especifíca;Fil: Levy, Harvey L.. No especifíca;Fil: Lichter Koneck, Uta. No especifíca;Fil: Muntau, Ania C.. No especifíca;Fil: Namour, Fares. No especifíca;Fil: Oltarzewsk, Mariusz. No especifíca;Fil: Paras, Andrea. No especifíca;Fil: Perez, Belén. No especifíca;Fil: Polak, Emil. No especifíca;Fil: Polyakov, Alexander V.. No especifíca;Fil: Porta, Francesco. No especifíca;Fil: Rohrbach, Marianne. No especifíca;Fil: Scholl Bürgi, Sabine. No especifíca;Fil: Spécola, Norma. No especifíca;Fil: Stojiljkovic, Maja. No especifíca;Fil: Shen, Nan. No especifíca;Fil: Santana da Silva, Luiz C.. No especifíca;Fil: Skouma, Anastasia. No especifíca;Fil: van Spronsen, Francjan. No especifíca;Fil: Stoppioni, Vera. No especifíca;Fil: Thöny, Beat. No especifíca;Fil: Trefz, Friedrich K.. No especifíca;Fil: Vockley, Jerry. No especifíca;Fil: Yu, Youngguo. No especifíca;Fil: Zschocke, Johannes. No especifíca;Fil: Hoffmann, Georg F.. No especifíca;Fil: Garbade, Sven F.. No especifíca;Fil: Blau, Nenad. No especifíca

A European spectrum of pharmacogenomic biomarkers: Implications for clinical pharmacogenomics

Pharmacogenomics aims to correlate inter-individual differences of drug efficacy and/or toxicity with the underlying genetic composition, particularly in genes encoding for protein factors and enzymes involved in drug metabolism and transport. In several European populations, particularly in countries with lower income, information related to the prevalence of pharmacogenomic biomarkers is incomplete or lacking. Here, we have implemented the microattribution approach to assess the pharmacogenomic biomarkers allelic spectrum in 18 European populations, mostly from developing European countries, by analyzing 1,931 pharmacogenomics biomarkers in 231 genes. Our data show significant interpopulation pharmacogenomic biomarker allele frequency differences, particularly in 7 clinically actionable pharmacogenomic biomarkers in 7 European populations, affecting drug efficacy and/or toxicity of 51 medication treatment modalities. These data also reflect on the differences observed in the prevalence of high-risk genotypes in these populations, as far as common markers in the CYP2C9, CYP2C19, CYP3A5, VKORC1, SLCO1B1 and TPMT pharmacogenes are concerned. Also, our data demonstrate notable differences in predicted genotype-based warfarin dosing among these populations. Our findings can be exploited not only to develop guidelines for medical prioritization, but most importantly to facilitate integration of pharmacogenomics and to support pre-emptive pharmacogenomic testing. This may subsequently contribute towards significant cost-savings in the overall healthcare expenditure in the participating countries, where pharmacogenomics implementation proves to be cost-effective

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases

Mutations in the PAH gene: A Tool for population genetics study

Fenilketonurija je urođena metabolička bolest prouzrokovana mutacijama u genu za fenilalanin hidroksilazu (PAH). U srpskoj populaciji je identifikovano 19 različitih PAH mutacija. PAH mutacije korišćene su kao molekularni markeri za populaciono-genetičko istraživanje. Niska vrednost homozigotnosti PAH gena (0,10) ukazuje na heterogenost fenilketonurije u Srbiji i odražava brojne migracije u regionu jugoistočne Evrope. U skladu sa tim, osmišljena je strategija molekularne dijagnostike fenilketonurije za Srbiju. U cilju rasvetljavanja porekla najčešće mutacije koja uzrokuje fenilketonuriju u Srbiji, L48S, urađena je haplotipska analiza PCR-RFLP metodom. Naši rezultati sugerišu da je L48S mutacija poreklom iz više populacija sa različitim genetičkim karakteristikama. .Phenylketonuria (PKU), an inborn error of metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. In the Serbian population, 19 different PAH mutations have been identified. We used PAH mutations as molecular markers for population genetics study. The low homozygosity value of the PAH gene (0.10) indicates that PKU in Serbia is heterogeneous, reflecting numerous migrations throughout Southeast Europe. The strategy for molecular diagnostics of PKU was designed accordingly. To elucidate the origin of the most common (L48S) PKU mutation in Serbia, we performed haplotype analysis by PCR-RFLP. Our results suggest that the L48S mutation was imported into Serbia from populations with different genetic backgrounds

The role of FasR/FasL system in pathogenesis of myeloprolyferative neoplasms

Mijeloproliferativne neoplazije (MPN) su hematološki maligniteti koji se karakterišu nekontrolisanom ćelijskom proliferacijom i poremećajem u procesu apoptoze. Sistem FasR/FasL je uključen u kontrolu apoptoze u različitim tipovima ćelija. U ovom radu je izučavana uloga sistema FasR/FasL u patogenezi mijeloproliferativnih neoplazija. Upoređena je ekspresija FasR i FasL između pacijenata sa MPN (24) i zdravih kontrola korišćenjem metode 'real-time' PCR. Detektovana je povećana ekspresija FasR kod pacijenata sa MPN. Nije utvrđena razlika u ekspresiji FasL. Mutacija B617F u JAK2 genu, karakteristična za MPN, je nađena kod 13 od 24 pacijenta. Pokazano je da ekspresija FasR i FasL nije povezana sa prisustvom B617F JAK2 mutacije.Myeloproliferative neoplasms (MPN) are hematological malignancies characterized by uncontrolled cell proliferation and impaired apoptosis. The FasR/FasL system is involved in the control of apoptosis in different cell types. Here we have investigated the role of FasR/FasL in the pathogenesis of MPNs. We compared FasR/FasL expression between MPN patients (24) and healthy individuals using the real-time PCR assay. We found an increase of FasR expression in MPN patients. No difference was detected in FasL expression. Mutation V617F in the JAK2 gene, a hallmark of MPN, was detected in 13/24 patients. We found that neither FasR nor FasL expression were related to the presence of JAK2 V617F mutation

Molecular genetic strategy for diagnosis of congenital adrenal hyperplasia in Serbia

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common endocrine diseases, yet genetic diagnosis is among the most complicated of all monogenic disorders. It has an overall incidence of 1: 10000-1: 20000, it is inherited in autosomal recessive pattern and caused by mutations affecting CYP21A2 gene. Based on the phenotypic expression, this disease is categorized into severe, classical form revealed at birth and mild, non-classical form. Although diagnosis could be established based on biochemical tests and distinctive clinical features, molecular genetic testing is crucial for diagnosis confirmation, detection of carriers and asymptomatic patients, disease prognosis, as well as for providing proper genetic counselling and prenatal diagnosis. Based on CYP21A2 mutational spectrum and frequencies in Serbia, in this paper we propose an optimal molecular genetic diagnostic algorithm for CAH and discuss genetic mechanisms underlying the disease. The complete diagnostic procedure combines multiplex minisequencing technique (SNaPshot PCR) as a method for rapid detection of common point mutations, direct sequencing of whole CYP21A2 gene and PCR with sequence specific primers (PCR-SSP) for large gene rearrangements detection (CYP21A1P/CYP21A2 chimeras). While SNaPshot PCR assay analyses ten common mutations (c. 290-13A/C gt G, p.P30L, p.R356W, p.G110fs, p.V281L, p.Q318X, p.L307fs, p.I172N, Cluster p.[I236N;V237E;M239K] and p.P453S) which account for over 80% of all CYP21A2 mutations in Serbian population, direct sequencing of CYP21A2 gene is needed to identify potential rare or novel mutations present in Serbian population with frequency of 1.8%. Additionally, large gene rearrangements which are present with frequency of 16.7% make PCR-SSP analysis an unavoidable part of molecular characterization of CAH in Serbia. Described molecular genetic strategy is intended to facilitate correct diagnosis assessment in CAH affected individuals and their families in Serbia but it will also contribute to molecular genetic testing of CAH patients across Europe