Patrinos, G.P.

KLF10 gene expression is associated with high fetal hemoglobin levels and with response to hydroxyurea treatment in -hemoglobinopathy patients (Article)

2012-10-01T00:00:00Z

Aim: In humans, fetal hemoglobin (HbF) production is controlled by many intricate mechanisms that, to date, remain only partly understood. Patients & methods: Pharmacogenomic analysis of the effects of hydroxyurea (HU) on HbF production was undertaken in a collection of Hellenic thalassemia and sickle cell disease (SCD) compound heterozygotes and a collection of healthy and KLF1-haploinsufficient Maltese adults, to identify genomic signatures that follow high HbF patterns. Results: KLF10 emerged as a top candidate. Moreover, genotype analysis of thalassemia major and intermedia patients and an independent cohort of thalassemia/SCD compound heterozygous patients that do or do not respond to HU treatment showed that the homozygous mutant state of a tagSNP in the KLF10 3UTR is not present in thalassemia intermedia patients and is underrepresented in thalassemia/SCD compound heterozygous patients that respond well to HU treatment. Conclusion: These data suggest that KLF10 may constitute a pharmacogenomic marker to discriminate between response and nonresponse to HU treatment. Original submitted: 2 May 2012; Revision submitted: 17 July 201.

Evidence for association of an ACCN1 gene variant with response to lithium treatment in Sardinian patients with bipolar disorder (Article)

2011-11-01T00:00:00Z

Aims: Bipolar disorder (BD) is a lifelong psychiatric illness characterized by manic and depressive episodes affecting 1-5% of the general population. Among mood-stabilizing treatments, lithium represents the mainstay in the therapeutic management of BD. However, besides the relatively high rate of excellent responders, a significant fraction of patients present patterns of partial or nonresponse to lithium. This variability might be influenced by genetic factors, even though findings have so far been inconclusive. Here, we present the results of an exploratory genome-wide scan followed by extended genotyping carried out on a sample of 204 Sardinian BD patients characterized for lithium response. Materials & methods: Phenotypic assessment of lithium response was made using the retrospective criteria of long-term treatment response scale. Using Affymetrix®6.0 SNP arrays, we genotyped a subsample of 52 BD patients evenly distributed at the extreme ends of the treatment response scale. The associated SNPs were then prioritized and selected for validation and extended genotyping in the whole sample of BD patients characterized for lithium response. Association was also tested using the scale for a quantitative trait analysis. Results: Our findings showed that several SNPs were nominally associated (p ≤ 10-5) with lithium response in the subgroup of 52 BD subjects. Some association signals were then confirmed in the extended sample. The strongest association, also supported by the quantitative trait analysis, was shown for a SNP located in intron 1 of the ACCN1 gene, encoding for a cation channel with high affinity for sodium and permeable to lithium. Conclusion: Our results indicate that ACCN1 gene is a potential candidate for response to lithium treatment that would serve as a genetic marker of lithium efficacy for BD patients. Original submitted 13 May 2011; Revision submitted 12 July 201.

Erythroid phenotypes associated with KLF1 mutations (Article)

2011-05-01T00:00:00Z

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

2011-02-01T00:00:00Z

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.

FINDbase: A worldwide database for genetic variation allele frequencies updated (Article)

2011-01-01T00:00:00Z

Frequency of INherited Disorders database (FIND base; http://www.findbase. org) records frequencies of causative genetic variations worldwide. Database records include the population and ethnic group or geographical region, the disorder name and the related gene, accompanied by links to any related external resources and the genetic variation together with its frequency in that population. In addition to the regular data content updates, we report the following significant advances: (i) the systematic collection and thorough documentation of population/ethnic group-specific pharmacogenomic markers allele frequencies for 144 markers in 14 genes of pharmacogenomic interest from different classes of drug-metabolizing enzymes and transporters, representing 150 populations and ethnic groups worldwide; (ii) the development of new data querying and visualization tools in the expanded FINDbase data collection, built around Microsoft's PivotViewer software (http:// www.getpivot.com), based on Microsoft Silverlight technology (http://www.silverlight.net) that facilitates querying of large data sets and visualizing the results; and (iii) the establishment of the first database journal, by affiliating FINDbase with Human Genomics and Proteomics, a new openaccess scientific journal, which would serve as a prime example of a non-profit model for sustainable database funding.

Locus-specific database domain and data content analysis: Evolution and content maturation toward clinical use (Article)

2010-10-01T00:00:00Z

Genetic variation databases have become indispensable in many areas of health care. In addition, more and more experts are depositing published and unpublished disease-causing variants of particular genes into locus-specific databases (LSDBs). Some of these databases contain such extensive information that they have become known as knowledge bases. Here, we analyzed 1,188 LSDBs and their content for the presence or absence of 44 content criteria related to database features (general presentation, locus-specific information, database structure) and data content (data collection, summary table of variants, database querying). Our analyses revealed that several elements have helped to advance the field and reduce data heterogeneity, such as the development of specialized database management systems and the creation of data querying tools. We also identified a number of deficiencies, namely, the lack of detailed disease and phenotypic descriptions for each genetic variant and links to relevant patient organizations, which, if addressed, would allow LSDBs to better serve the clinical genetics community. We propose a structure, based on LSDBs and closely related repositories (namely, clinical genetics databases), which would contribute to a federated genetic variation browser and also allow the maintenance of variation data.

Region-specific genetic heterogeneity of HBB mutation distribution in South-Western Greece (Article)

2010-08-01T00:00:00Z

β-Thalassemia (β-thal), is caused by reduced or absent synthesis of β-globin chains resulting in impaired erythropoiesis. It is the most common single gene defect disease in Greece, with heterozygous rates reaching, on average, 8 in the general population. Here, we performed molecular analyses on 199 unrelated β-thal and compound β-thalsickle cell disease patients, of whom 157 originated from three prefectures of South-Western Greece, namely Achaia, Ilia and Etoloakarnania. Our results indicate that the frequency of specific HBB gene mutations, namely the HBB:c.118C>T (codon 39, C>T), HBB:c.926T>C (IVS-I-6, T>C), and HBB:c.20A>T [Hb S, β6(A3)Glu→Val, GAG>GTG], present distinct distribution patterns in the Achaia and Ilia prefectures (p < 0.001, p < 0.003 and p < 0.002, respectively). This detailed analysis of the distribution of the HBB gene mutations is useful for genetic counseling in the region, and illustrates that the identification of the HBB gene mutation spectrum in this region is necessary for population carrier screening and for efficient provision of prenatal diagnosis.

Realities and expectations of pharmacogenomics and personalized medicine: Impact of translating genetic knowledge into clinical practice (Article)

2010-08-01T00:00:00Z

The implementation of genetic data for a better prediction of response to medications and adverse drug reactions is becoming a reality in some clinical fields. However, to be successful, personalized medicine should take advantage of an informational structured framework of genetic, phenotypic and environmental factors in order to provide the healthcare system with useful tools that can optimize the effectiveness of specific treatment. The impact of personalized medicine is potentially enormous, but the results that have so far been gathered are often difficult to translate into clinical practice. In this article we have summarized the most relevant applications of pharmacogenomics on diseases to which they have already been applied and fields in which they are currently emerging. The article provides an overview of the opportunities and shortcomings of the implementation of genetic information into personalized medicine and its full adoption in the clinic. In the second instance, it provides readers from different fields of expertise with an accessible interpretation to the barriers and opportunities in the use/adoption of pharmacogenomic testing between the different clinical areas.

Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin (Article)

2010-08-01T00:00:00Z

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12-13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

Allelic imbalance of expression and epigenetic regulation within the alpha-synuclein wild-type and p.Ala53Thr alleles in Parkinson disease (Article)

2010-06-01T00:00:00Z

Genetic alterations in the alpha-synuclein (SNCA) gene have been implicated in Parkinson Disease (PD), including point mutations, gene multiplications, and sequence variations within the promoter. Such alterations may be involved in pathology through structural changes or overexpression of the protein leading to protein aggregation, as well as through impaired gene expression. It is, therefore, of importance to specify the parameters that regulate SNCA expression in its normal and mutated state. We studied the expression of SNCA alleles in a lymphoblastoid cell line and in the blood cells of a patient heterozygous for p.Ala53Thr, the first mutation to be implicated in PD pathogenesis. Here, we provide evidence that: (1) SNCA shows monoallelic expression in this patient, (2) epigenetic silencing of the mutated allele involves histone modifications but not DNA methylation, and (3) steady-state mRNA levels deriving from the normal SNCA allele in this patient exceed those of the two normal SNCA alleles combined, in matching, control individuals. An imbalanced SNCA expression in this patient is thus documented, with silencing of the p.Ala53Thr allele and upregulation of the wild-type-allele. This phenomenon is demonstrated for a first time in the SNCA gene, and may have important implications for PD pathogenesis.

Functional analysis of the role of the TPMT gene promoter VNTR polymorphism in TPMT gene transcription (Article)

2010-04-01T00:00:00Z

Aims: Thiopurine S-methyltransferase (TPMT) activity is polymorphic, and a trimodal distribution has been demonstrated in Caucasians (low, intermediate and high methylator groups). The TPMT gene promoter contains a variable number of three GC-rich tandem repeats, namely A, B and C, ranging from three to nine in length in a AnBmC architecture. Materials & methods: Here, we investigated the influence of number and type of TPMT gene promoter tandem repeats on human TPMT gene transcription in K562 cells transiently transfected with reporter constructs bearing various variable number of tandem repeats (VNTR) and addressed the interaction of transcription factor binding to the VNTRs by electrophoretic mobility shift assays. Results: We found that the distribution patterns of VNTR alleles do not significantly differ among acute lymphoblastic leukemia patients, acute myeloid leukemia patients and normal individuals. We also demonstrated that the A repeat has a negative effect in TPMT gene transcription and that a positive regulatory element, identified immediately upstream to the VNTR region of the TPMT gene promoter, is indispensable for TPMT gene transcription. Our electrophoretic mobility shift assay analysis indicated that the Sp1 and Sp3 transcription factors bind to the VNTR repeats. Conclusion: Overall, our data underline that both the number and type of VNTRs, as well as the upstream regulatory region of the TPMT gene promoter, determine the overall level of TPMT gene transcription. It remains to be seen whether these VNTRs can be employed as pharmacogenetic markers to individualize thiopurine therapy.

Genetic recombination as a major cause of mutagenesis in the human globin gene clusters (Article)

2009-12-01T00:00:00Z

Objectives: Homologous recombination is a frequent phenomenon in multigene families and as such it occurs several times in both the α- and β-like globin gene families. In numerous occasions, genetic recombination has been previously implicated as a major mechanism that drives mutagenesis in the human globin gene clusters, either in the form of unequal crossover or gene conversion. Unequal crossover results in the increase or decrease of the human globin gene copies, accompanied in the majority of cases with minor phenotypic consequences, while gene conversion contributes either to maintaining sequence homogeneity or generating sequence diversity. The role of genetic recombination, particularly gene conversion in the evolution of the human globin gene families has been discussed elsewhere. Conclusion: Here, we summarize our current knowledge and review existing experimental evidence outlining the role of genetic recombination in the mutagenic process in the human globin gene families.

Capturing all disease-causing mutations for clinical and research use: Toward an effortless system for the Human Variome Project (Article)

2009-12-01T00:00:00Z

The collection of genetic variants that cause inherited disease (causative mutation) has occurred for decades albeit in an ad hoc way, for research and clinical purposes. More recently, the access to collections of mutations causing specific diseases has become essential for appropriate genetic health care. Because information has accumulated, it has become apparent that there are many gaps in our ability to correctly annotate all the changes that are being identified at ever increasing rates. The Human Variome Project (www.humanvariomeproject.org) was initiated to facilitate integrated and systematic collection and access to this data. This manuscript discusses how collection of such data may be facilitated through new software and strategies in the clinical genetics and diagnostic laboratory communities.

Impact of ACE and ApoE polymorphisms on myocardial perfusion: Correlation with myocardial single photon emission computed tomographic imaging (Article)

2009-10-01T00:00:00Z

Coronary artery disease is associated with multiple genetic and environmental risk factors. In this study, we evaluated the correlation of angiotensin l-converting enzyme (ACE) (I/D) and ApoE gene polymorphisms (E2, E3, E4 and g.-219G/T) with myocardial perfusion. We examined 410 patients using exercise-rest myocardial perfusion single photon emission computed tomography (SPECT), in which the summed stress score (SSS), summed rest score (SRS) and summed difference score (SDS) indexes were calculated. Homozygotes for the ACE D allele had greater mean values of SSS (P0.001) and SDS (P0.001). In addition, E3 homozygotes, E4 heterozygotes and E4 homozygotes had significantly higher values of SSS and SDS compared with E3 heterozygotes (P0.001); E4 homozygotes had significantly higher values of SSS and SDS compared with E3 homozygotes. Furthermore, for the g.-219GT polymorphic site at the promoter region of ApoE gene, the mean values of SSS and SDS were significantly higher for T heterozygotes/homozygotes than for GG homozygotes. Adjusting for all demographic and clinical data using multiple linear regression analysis it was found that ACE D and both ApoE genotypes were independent predictors with a cumulative contribution for the prediction of SSS and SDS. Furthermore, logistic regression analysis revealed that all three genotypes had an independent predictive ability for abnormal SSS (SSS2). These data provide the first evidence of an association and significant cumulative contribution of the aforementioned genotypes in myocardial perfusion with E4 allele having the strongest association followed by ACE D and ApoE g.-219T alleles.

Gene conversion causing human inherited disease: Evidence for involvement of non-B-DNA-forming sequences and recombination-promoting motifs in DNA breakage and repair (Article)

2009-08-01T00:00:00Z

A variety of DNA sequence motifs including inverted repeats, minisatellites, and the χ recombination hotspot, have been reported in association with gene conversion in human genes causing inherited disease. However, no methodical statistically based analysis has been performed to formalize these observations. We have performed an in silico analysis of the DNA sequence tracts involved in 27 nonoverlapping gene conversion events in 19 different genes reported in the context of inherited disease. We found that gene conversion events tend to occur within (C1G)- and CpG-rich regions and that sequences with the potential to form non-B-DNA structures, and which may be involved in the generation of double-strand breaks that could, in turn, serve to promote gene conversion, occur disproportionately within maximal converted tracts and/or short flanking regions. Maximal converted tracts were also found to be enriched (P<0.01) in a truncated version of the χ-element (a TGGTGG motif), immunoglobulin heavy chain class switch repeats, translin target sites and several novel motifs including (or overlapping) the classical meiotic recombination hotspot, CCTCCCCT. Finally, gene conversions tend to occur in genomic regions that have the potential to fold into stable hairpin conformations. These findings support the concept that recombination-inducing motifs, in association with alternative DNA conformations, can promote recombination in the human genome.

An electronic infrastructure for research and treatment of the thalassemias and other hemoglobinopathies: The Euro-Mediterranean ITHANET project (Article)

2009-06-01T00:00:00Z

Hemoglobin (Hb) disorders are common, potentially lethal monogenic diseases, posing a global health challenge. With worldwide migration and intermixing of carriers, demanding flexible health planning and patient care, hemoglobinopathies may serve as a paradigm for the use of electronic infrastructure tools in the collection of data, the dissemination of knowledge, the harmonization of treatment, and the coordination of research and preventive programs. ITHANET, a network covering thalassemias and other hemoglobinopathies, comprises 26 organizations from 16 countries, including non-European countries of origin for these diseases (Egypt, Israel, Lebanon, Tunisia and Turkey). Using electronic infrastructure tools, ITHANET aims to strengthen cross-border communication and data transfer, cooperative research and treatment of thalassemia, and to improve support and information of those affected by hemoglobinopathies. Moreover, the consortium has established the ITHANET Portal, a novel web-based instrument for the dissemination of information on hemoglobinopathies to researchers, clinicians and patients. The ITHANET Portal is a growing public resource, providing forums for discussion and research coordination, and giving access to courses and databases organized by ITHANET partners. Already a popular repository for diagnostic protocols and news related to hemoglobinopathies, the ITHANET Portal also provides a searchable, extendable database of thalassemia mutations and associated background information. The experience of ITHANET is exemplary for a consortium bringing together disparate organizations from heterogeneous partner countries to face a common health challenge. The ITHANET Portal as a web-based tool born out of this experience amends some of the problems encountered and facilitates education and international exchange of data and expertise for hemoglobinopathies.

The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter (Article)

2009-06-01T00:00:00Z

Nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH), a rare hereditary condition resulting in elevated levels of fetal hemoglobin (Hb F) in adults, is associated with promoter mutations in the human fetal globin (HBG1 and HBG2) genes. In this paper, we report a novel type of nd-HPFH due to a HBG2 gene promoter mutation (HBG2:g.-109G>T). This mutation, located at the 3′ end of the HBG2 distal CCAAT box, was initially identified in an adult female subject of Central Greek origin and results in elevated Hb F levels (4.1%) and significantly increased Gγ-globin chain production (79.2%). Family studies and DNA analysis revealed that the HBG2:g.- 109G>T mutation is also found in the family members in compound heterozygosity with the HBG2:g.-158C>T single nucleotide polymorphism or the silent HBB:g.-101C>T β-thalassemia mutation, resulting in the latter case in significantly elevated Hb F levels (14.3%). Electrophoretic mobility shift analysis revealed that the HBG2:g.-109G>T mutation abolishes a transcription factor binding site, consistent with previous observations using DNA footprinting analysis, suggesting that guanine at position HBG2/1:g.-109 is critical for NF-E3 binding. These data suggest that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in our index cases.

Planning the human variome project: The Spain report (Article)

2009-04-01T00:00:00Z

The remarkable progress in characterizing the human genome sequence, exemplified by the Human Genome Project and the HapMap Consortium, has led to the perception that knowledge and the tools (e.g., micro-arrays) are sufficient for many if not most biomedical research efforts. A large amount of data from diverse studies proves this perception inaccurate at best, and at worst, an impediment for further efforts to characterize the variation in the human genome. Because variation in genotype and environment are the fundamental basis to understand phenotypic variability and heritability at the population level, identifying the range of human genetic variation is crucial to the development of personalized nutrition and medicine. The Human Variome Project (HVP; http://www.humanvariomeproject.org/) was proposed initially to systematically collect mutations that cause human disease and create a cyber infrastructure to link locus specific databases (LSDB). We report here the discussions and recommendations from the 2008 HVP planning meeting held in San Feliu de Guixols, Spain, in May 2008.

A1ATVar: A relational database of human SERPINA1 gene variants leading to α1-antitrypsin deficiency and application of the VariVis software (Article)

2009-03-01T00:00:00Z

We have developed a relational database of human SERPINA1 gene mutations, leading to α1-antitrypsin (AAT) deficiency, called A 1ATVar, which can be accessed over the World Wide Web at www.goldenhelix.org/A1ATVar. Extensive information has been extracted from the literature and converted into a searchable database, including genotype information, clinical phenotype, allelic frequencies for the commonest AAT variant alleles, methods of detection, and references. Mutation summaries are automatically displayed and user-generated queries can be formulated based on fields in the database. A separate module, linked to the FINDbase database for frequencies of inherited disorders allows the user to access allele frequency information for the three most frequent AAT alleles, namely PiM, PiS, and PiZ. The available experimental protocols to detect AAT variant alleles at the protein and DNA levels have been archived in a searchable format. A visualization tool, called VariVis, has been implemented to combine A 1ATVar variant information with SERPINA1 sequence and annotation data. A direct data submission tool allows registered users to submit data on novel AAT variant alleles as well as experimental protocols to explore SERPINA1 genetic heterogeneity, via a password-protected interface. Database access is free of charge and there are no registration requirements for querying the data. The A1ATVar database is the only integrated database on the Internet offering summarized information on AATallelic variants and could be useful not only for clinical diagnosis and research on AAT deficiency and the SERPINA1 gene, but could also serve as an example for an all-in-one solution for locus-specific database (LSDB) development and curation. © 2008 Wiley-Liss, Inc.

Copy number variation and genomic alterations in health and disease (Article)

2009-02-20T00:00:00Z

A report of the 1st GOLDEN HELIX Symposium Copy number variation and genomic alterations in health and disease, Athens, Greece, 28-29 November 2008.

The Israeli national genetic database (Article)

2009-01-01T00:00:00Z

The Israeli National Genetic Database http//www.goldenhelix.org/israeli is a continuously updated depository on monogenic genetic disorders that are present in the various Israeli populations. It provides the means of obtaining information for clinical purposes, genetic counseling and research.

The 5′ regulatory region of the human fetal globin genes is a gene conversion hotspot (Article)

2008-11-01T00:00:00Z

The human fetal globin genes consist of the first mammalian genomic loci for which gene conversion was reported. To date, 14 gene conversions have been described in the human Gγ- and Aγ-globin genes, the vast majority of which are restricted to the coding sequences. Here, we provide evidence for three new gene conversion events in the 5′ regulatory region of the human fetal globin genes, identified during a large genetic screening effort in adult individuals with high fetal hemoglobin (Hb) levels. The sequence variations, resulting from these conversion events, are transcriptionally silent polymorphisms that do not contribute to increased fetal Hb levels. Our results suggest that the 5′ regulatory region of the human fetal globin genes is a gene conversion hotspot that prevent globin gene promoter sequence diversification, further underlining the need for two functional fetal globin genes in fetal erythropoiesis. Copyright

Towards a hybrid model for the cryopreservation of umbilical cord blood stem cells (Article)

2008-10-01T00:00:00Z

Compound heterozygosity of non-deletional hereditary persistence of fetal hemoglobin and δβ-thalassemia (Article)

2008-09-01T00:00:00Z

Compound heterozygosity for the Cretan type of non-deletional hereditary persistence of fetal hemoglobin and β-thalassemia or Hb Sabine confirms the functional role of the Aγ - 158 C > T mutation in γ-globin gene transcription (Article)

2008-08-20T00:00:00Z

A T-to-G transversion at nucleotide -567 upstream of HBG2 in a GATA-1 binding motif is associated with elevated hemoglobin F (Article)

2008-07-01T00:00:00Z

Increased fetal hemoglobin (Hb F; α2γ2) production in adults can ameliorate the clinical severity of sickle cell disease and β-thalassemia major. Thus, understanding the regulation of γ-globin gene expression and its silencing in adults has potential therapeutic implications. We studied a father and son in an Iranian-American family who had elevated Hb F levels and found a novel T-to-G transversion at nucleotide (nt) -567 of the HBG2 promoter. This mutation alters a GATA-1 binding motif to a GAGA sequence located within a previously identified silencing element. DNA-protein binding assays showed that the GATA motif of interest is capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of the HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between nt -675 and -526. In addition, the T-to-G mutation at the GATA motif increased the promoter activity by two- to threefold in transiently transfected erythroid cell lines. The binding motif is uniquely conserved in simian primates with a fetal pattern of γ-globin gene expression. These results suggest that the GATA motif under study has a functional role in silencing γ-globin gene expression in adults. The T-to-G mutation in this motif disrupts GATA-1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up-regulation of γ-globin gene expression in adults. Copyright

Mutations in the paralogous human α-globin genes yielding identical hemoglobin variants (Article)

2008-01-01T00:00:00Z

The human α-globin genes are paralogues, sharing a high degree of DNA sequence similarity and producing an identical α-globin chain. Over half of the α-globin structural variants reported to date are only characterized at the amino acid level. It is likely that a fraction of these variants, with phenotypes differing from one observation to another, may be due to the same mutation but on a different α-globin gene. There have been very few previous examples of hemoglobin variants that can be found at both HBA1 and HBA2 genes. Here, we report the results of a systematic multicenter study in a large multiethnic population to identify such variants and to analyze their differences from a functional and evolutionary perspective. We identified 14 different Hb variants resulting from identical mutations on either one of the two human α-globin paralogue genes. We also showed that the average percentage of hemoglobin variants due to a HBA2 gene mutation (α2) is higher than the percentage of hemoglobin variants due to the same HBA1 gene mutation (α1) and that the α2/α1 ratio varied between variants. These α-globin chain variants have most likely occurred via recurrent mutations, gene conversion events, or both. Based on these data, we propose a nomenclature for hemoglobin variants that fall into this category.

Pharmacogenomics and therapeutics of hemoglobinopathies (Article)

2008-01-01T00:00:00Z

Individual genetic constitution is an important cause of variations in the response and tolerance to drug treatment. Single nucleotide polymorphisms (SNPs) in genes located within as well as outside the human β-globin cluster have recently been shown to be significantly associated with Hb F increase in relation to hydroxyurea (HU) treatment in hemoglobinopathies patients. This article provides an update and discusses future challenges on the application of pharmacogenetic testing and pharmacogenomics for hemoglobinopathies therapeutics in relation to the current pharmacological treatment modalities for those disorders. Copyright

Increased γ-globin gene expression in β-thalassemia intermedia patients correlates with a mutation in 3′HS1 (Article)

2007-11-01T00:00:00Z

We report a novel set of genetic markers in the DNasel hypersensitive sites comprising the human β-globin locus chromatin hub (CH), namely HS-111 and 3′HS1. The HS-111 (-21 G>A) and 3HS1 (+179 C>T) transitions form CH haplotypes, which occur at different frequencies in β-thalassemia intermedia and major patients and normal (nonthalassemic) individuals. We also show that the 3′HS1 (+179 C>T) variation results in a GATA-1 binding site and correlates with increased fetal hemoglobin production in β-thalassemia intermedia patients. In contrast, the HS-111 (+126 G>A) transition, found in three normal chromosomes, is simply a rare polymorphism. We conclude that the CH haplotypes are useful genetic determinants for β-thalassemia major and intermedia patients, while the 3′HS1 (+179 C>T) mutation may have functional consequences in γ-globin genes expression.

Documentation of inherited disorders and mutation frequencies in the different religious communities in Israel in the Israeli national genetic database (Article)

2007-10-01T00:00:00Z

The National and Ethnic Mutation Databases (NEMDBs) are continuously updated mutation depositories that contain extensive information on the described genetic heterogeneity of an ethnic group or population. Here, we report the construction of the Israeli National Genetic database (Available at: www.goldenhelix.org/israeli; Last accessed: 20 April 2007) to document the sheer genetic heterogeneity found in the Jewish and non-Jewish populations in Israel. The database is built and maintained online using a newly developed customized version of the ETHNOS platform. The Israeli NEMDB is the richest in information among individual NEMDB, containing summaries of 347 genetic disorders studied for the Israeli populations with numerous relevant references and links to the respective Online Mendelian Inheritance in Man (OMIM) entries. Summaries can be selected from an alphabetical summary index or queried using a keyword-based search functionality. An easy-to-use query interface provides access to the over 600 entries on allelic and carrier frequencies of the different mutations responsible for certain inherited disorders in the Jewish and non-Jewish populations, although such documentation is not as extensive as in the other ETHNOS-based NEMDBs. Also, the Israeli NEMDB provides a comprehensive listing of all laboratories providing molecular genetic testing services in Israel with a separate query interface for the user to select which genetic service is provided to a certain laboratory. The Israeli NEMDB is a useful user-friendly and extendable online resource for genetic services in Israel, while the modified version of the ETHNOS software can be particularly useful for similar projects in other populations.

Large-scale population genetic analysis for hemoglobinopathies reveals different mutation spectra in central Greece compared to the rest of the country (Article)

2007-07-01T00:00:00Z

We have undertaken a large population screening study to identify the molecular basis of hemoglobinopathies in the central Greece region. A total of 845 unrelated β-thalassemia patients and α-, β-, and δβ-thalassemia carriers have been recruited and screened for mutations in the α- and β-globin gene clusters. The α-MEDdeletion and the Turkish inversion/deletion are the most frequent genetic rearrangements leading to α- and δβ- thalassemia respectively, contrary to the situation in the rest of the country, while the β-101 (C>T) promoter mutation is surprisingly frequent in the central part of Greece. Our data indicate that determination of mutation frequencies in different regions is vital for accurate provision of genetic services and counseling and for precise estimation of genetic diversity.

An overview of current microarray-based human globin gene mutation detection methods (Article)

2007-07-01T00:00:00Z

□ The panoply of human globin gene mutation detection methods could become significantly enriched with the advent of microarray-based genotyping platforms. The aim of this article is to provide an overview of the current medium and high-throughput microarray-based globin gene mutation detection platforms, namely the microelectronic array, the "thalassochip" arrayed primer extension (APEX) technology and the single base extension methods. This article also outlines an emerging method based on multiple ligation probe amplification (MLPA) and discusses the implications of customized solutions for resequencing of genomic loci in relation to molecular genetic testing of hemoglobinopathies. Copyright

Association study of human VN1R1 pheromone receptor gene alleles and gender (Article)

2007-06-01T00:00:00Z

Pheromones are water-soluble chemicals that elicit neuroendocrine and physiological changes, while they also provide information about gender within individuals of the same species. VN1R1 is the only functional pheromone receptor in humans. We have undertaken a large mutation screening approach in 425 adult individuals from the Hellenic population to investigate whether the allelic differences, namely alleles 1a and 1b present in the human VN1R1 gene, are gender specific. Here we show that both VN1R1 1a and 1b alleles are found in chromosomes of both male and female subjects at frequency of 26.35% and 73.65%, respectively. Given the fact that those allelic differences potentially cause minor changes in the protein conformation and its transmembrane domains, as simulated by the TMHMM software, our data suggest that the allelic differences in the human VN1R1 gene are unlikely to be associated with gender and hence to contribute to distinct gender-specific behavior.

PhenCode: Connecting ENCODE data with mutations and phenotype (Article)

2007-06-01T00:00:00Z

PhenCode (Phenotypes for ENCODE; www.bx.psu.edu/phencode) is a collaborative, exploratory project to help understand phenotypes of human mutations in the context of sequence and functional data from genome projects. Currently, it connects human phenotype and clinical data in various locus-specific databases (LSDBs) with data on genome sequences, evolutionary history, and function from the ENCODE project and other resources in the UCSC Genome Browser. Initially, we focused on a few selected LSDBs covering genes encoding alpha- and beta-globins (HBA, HBB), phenylalanine hydroxylase (PAH), blood group antigens (various genes), androgen receptor (AR), cystic fibrosis transmembrane conductance regulator (CFTR), and Bruton's tyrosine kinase (BTK), but we plan to include additional loci of clinical importance, ultimately genomewide. We have also imported variant data and associated OMIM links from Swiss-Prot. Users can find interesting mutations in the UCSC Genome Browser (in a new Locus Variants track) and follow links back to the LSDBs for more detailed information. Alternatively, they can start with queries on mutations or phenotypes at an LSDB and then display the results at the Genome Browser to view complementary information such as functional data (e.g., chromatin modifications and protein binding from the ENCODE consortium), evolutionary constraint, regulatory potential, and/or any other tracks they choose. We present several examples illustrating the power of these connections for exploring phenotypes associated with functional elements, and for identifying genomic data that could help to explain clinical phenotypes.

A versatile denaturing HPLC approach for human β-globin gene mutation screening (Article)

2007-02-01T00:00:00Z

Hemoglobinopathies represent the most common genetic disorder worldwide, with a higher prevalence among populations with a history of malaria endemicity. More than 690 mutations in the human β-globin gene are usually the cause of β-type hemoglobinopathies. Here, we report a rapid and highly sensitive β-globin gene mutation screening approach based on denaturing high-performance liquid chromatography (DHPLC), which contrary to the previously described ones can be used in every HPLC apparatus. The sensitivity and specificity of the method were tested in 120 healthy Greek subjects and 25 β-thalassemia heterozygotes and homozygotes, in which 11 different β-globin sequence variations had been previously characterized by denaturing gradient gel electrophoresis. Using this method, we were able to rapidly identify the commonest β-globin gene mutations, accounting for more than 90% of the mutant β-globin alleles reported for the Hellenic population. Compared to classical mutation screening approaches, our DHPLC approach provides the means for rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples.

FINDbase: A relational database recording frequencies of genetic defects leading to inherited disorders worldwide (Article)

2007-01-01T00:00:00Z

Frequency of INherited Disorders database (FINDbase) (http://www.findbase.org) is a relational database, derived from the ETHNOS software, recording frequencies of causative mutations leading to inherited disorders worldwide. Database records include the population and ethnic group, the disorder name and the related gene, accompanied by links to any corresponding locus-specific mutation database, to the respective Online Mendelian Inheritance in Man entries and the mutation together with its frequency in that population. The initial information is derived from the published literature, locus-specific databases and genetic disease consortia. FINDbase offers a user-friendly query interface, providing instant access to the list and frequencies of the different mutations. Query outputs can be either in a table or graphical format, accompanied by reference(s) on the data source. Registered users from three different groups, namely administrator, national coordinator and curator, are responsible for database curation and/or data entry/ correction online via a password-protected interface. Databaseaccess is free of charge and there are no registration requirements for data querying. FINDbase provides a simple, web-based system for population-based mutation data collection and retrieval and can serve not only as a valuable online tool for molecular genetic testing of inherited disorders but also as a non-profit model for sustainable database funding, in the form of a 'database-journal'.

Multiple interactions between regulatory regions are required to stabilize an active chromatin hub. (Article)

2004-06-15T00:00:00Z

The human beta-globin locus control region (LCR) is required for the maintenance of an open chromatin configuration of the locus. It interacts with the genes and the hypersensitive regions flanking the locus to form an active chromatin hub (ACH) transcribing the genes. Proper developmental control of globin genes is largely determined by gene proximal regulatory sequences. Here, we provide the first functional evidence of the role of the most active sites of the LCR and the promoter of the beta-globin gene in the maintenance of the ACH. When the human beta-globin gene promoter is deleted in the context of a full LCR, the ACH is maintained with the beta-globin gene remaining in proximity. Additional deletion of hypersensitive site HS3 or HS2 of the LCR shows that HS3, but not HS2, in combination with the beta-globin promoter is crucial for the maintenance of the ACH at the definitive stage. We conclude that multiple interactions between the LCR and the beta-globin gene are required to maintain the appropriate spatial configuration in vivo.

Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. (Article)

2004-01-01T00:00:00Z

HbVar (http://globin.cse.psu.edu/globin/hbvar/) is a relational database developed by a multi-center academic effort to provide up-to-date and high quality information on the genomic sequence changes leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Extensive information is recorded for each variant and mutation, including sequence alterations, biochemical and hematological effects, associated pathology, ethnic occurrence and references. In addition to the regular updates to entries, we report two significant advances: (i) The frequencies for a large number of mutations causing beta-thalassemia in at-risk populations have been extracted from the published literature and made available for the user to query upon. (ii) HbVar has been linked with the GALA (Genome Alignment and Annotation database, available at http://globin.cse.psu.edu/gala/) so that users can combine information on hemoglobin variants and thalassemia mutations with a wide spectrum of genomic data. It also expands the capacity to view and analyze the data, using tools within GALA and the University of California at Santa Cruz (UCSC) Genome Browser.

Modification of human beta-globin locus PAC clones by homologous recombination in Escherichia coli (Article)

2000-01-01T00:00:00Z

We report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for plasmid loss in this host. High frequencies of recombination are observed even with very small lengths of homology and the method has general utility for introducing insertions, deletions and point mutations. No rearrangements were detected with the exception of one highly repetitive genomic sequence when either the E.COLI: RecA- or the lambdoid phage encoded RecT and RecE-dependent recombination systems were used.