George P. Patrinos

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

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 (C+G)‐ 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. Hum Mutat 30:1–10, 2009.

FINDbase: a relational database recording frequencies of genetic defects leading to inherited disorders worldwide

Frequency of INherited Disorders database (FINDbase) () 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’.

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

Abstract: 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.

AN OVERVIEW OF CURRENT MICROARRAY-BASED HUMAN GLOBIN GENE MUTATION DETECTION METHODS

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.

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

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.

Isolation and Characterization of Hematopoietic Transcription Factor Complexes by in Vivo Biotinylation Tagging and Mass Spectrometry

Abstract: We have described the application of a simple biotinylation tagging approach for the direct purification of tagged transcription factor complexes, based on the use of artificial short peptide tags that are specifically and efficiently biotinylated by the bacterial BirA biotin ligase, which is co‐expressed in cells with the tagged factor. We used this approach to initially characterize complexes formed by the hematopoietic transcription factor GATA‐1 in erythroid cells. GATA‐1 is essential for the erythroid differentiation, its functions encompassing upregulation of erythroid genes, repression of alternative transcription programs, and suppression of cell proliferation. However, it was not clear how all of these GATA‐1 functions are mediated. Our work describes, for the first time, distinct GATA‐1 interactions with the essential hematopoietic factor Gfi‐1b, the repressive MeCP1 complex, and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA‐1/FOG‐1 and GATA‐1/TAL‐1 complexes. We also provide evidence that distinct GATA‐1 complexes are associated with specific GATA‐1 functions in erythroid differentiation, for example, GATA‐1/Gfi‐1b with the suppression of cell proliferation and GATA‐1/FOG‐1/MeCP1 with the repression of other hematopoietic transcription programs. We next applied the biotinylation tag to Ldb‐1, a known partner of GATA‐1, and characterized a number of novel interaction partners that are essential in erythroid development, in particular, Eto‐2, Lmo4, and CdK9. Last, we are in the process of applying the same technology to characterize the factors that are bound to the suppressed γ‐globin promoter in vivo.

Aγ-haplotypes : A new group of genetic markers for thalassemic mutations inside the 5' regulatory region of the human Aγ-globin gene

This study illustrates the relationship between a group of nucleotide variations within the 5′ regulatory region of the Aγ‐globin gene [Aγ‐588 A→G, Aγ‐499 T→A and the 4‐bp deletion (Aγ‐225 to ‐222 AGCA)] and the spectrum of δ‐ and β‐thalassemia mutations in the Hellenic population. These sequence variations, screened by means of denaturing gradient gel electrophoresis, form four separate frameworks (Aγ‐haplotypes), each one of which was found to be linked in cis with certain δ‐ and β‐thalassemia mutations found in the Hellenic population. In addition, two novel base substitutions inside the 5′ regulatory region of the Aγ‐globin gene (Aγ‐521 C→A and Aγ‐500 C→T) were identified during this study, which together with Aγ‐haplotypes seem to be silent polymorphisms during adult life, as indicated by transient expression assays. Our data show that Aγ‐haplotypes represent genetic markers for the spectrum of thalassemic mutations, found in the Hellenic population and can constitute an important genetic repository upon which mutations leading to thalassemia and hemoglobinopathies occurred. Am. J. Hematol 66:99–104, 2001.

Pharmacogenomics and therapeutics of hemoglobinopathies

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.

A1ATVar: a relational database of human SERPINA1 gene variants leading to α1‐antitrypsin deficiency and application of the VariVis software

We have developed a relational database of human SERPINA1 gene mutations, leading to α1‐antitrypsin (AAT) deficiency, called A1ATVar, 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 A1ATVar 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 AAT allelic 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. Hum Mutat 0,1–6, 2008.

Region-Specific Genetic Heterogeneity of HBB Mutation Distribution in South-Western Greece

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-thal/sickle 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.92+6T>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.