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Informativeness of a novel multiallelic marker-set comprising an F8 intron 21 and three tightly linked loci for haemophilia A carriership analysis

2010; Wiley; Volume: 17; Issue: 2 Linguagem: Inglês

10.1111/j.1365-2516.2010.02404.x

1365-2516

Filipe Brum Machado, Antônio Francisco Alves da Silva, Liliana Carmen Rossetti, Carlos Daniel De Brasi, Enrique Medina‐Acosta,

Blood Coagulation and Thrombosis Mechanisms

HaemophiliaVolume 17, Issue 2 p. 257-266 Informativeness of a novel multiallelic marker-set comprising an F8 intron 21 and three tightly linked loci for haemophilia A carriership analysis F. B. MACHADO, F. B. MACHADO Núcleo de Diagnóstico e Investigação Molecular, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil Hospital Escola Álvaro Alvim, Campos dos Goytacazes, RJ, Brazil Present address: Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, Monte Alegre, CEP 14049-900, Ribeirão Preto, SP, Brazil.Search for more papers by this authorA. F. ALVES DA SILVA, A. F. ALVES DA SILVA Núcleo de Diagnóstico e Investigação Molecular, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil Hospital Escola Álvaro Alvim, Campos dos Goytacazes, RJ, BrazilSearch for more papers by this authorL. C. ROSSETTI, L. C. ROSSETTI Sección Genética Molecular de la Hemofilia, Departamento de Genética, Academia Nacional de Medicina de Buenos Aires, Buenos Aires, ArgentinaSearch for more papers by this authorC. D. DE BRASI, C. D. DE BRASI Sección Genética Molecular de la Hemofilia, Departamento de Genética, Academia Nacional de Medicina de Buenos Aires, Buenos Aires, ArgentinaSearch for more papers by this authorE. MEDINA-ACOSTA, E. MEDINA-ACOSTA Núcleo de Diagnóstico e Investigação Molecular, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, BrazilSearch for more papers by this author F. B. MACHADO, F. B. MACHADO Núcleo de Diagnóstico e Investigação Molecular, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil Hospital Escola Álvaro Alvim, Campos dos Goytacazes, RJ, Brazil Present address: Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, Monte Alegre, CEP 14049-900, Ribeirão Preto, SP, Brazil.Search for more papers by this authorA. F. ALVES DA SILVA, A. F. ALVES DA SILVA Núcleo de Diagnóstico e Investigação Molecular, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil Hospital Escola Álvaro Alvim, Campos dos Goytacazes, RJ, BrazilSearch for more papers by this authorL. C. ROSSETTI, L. C. ROSSETTI Sección Genética Molecular de la Hemofilia, Departamento de Genética, Academia Nacional de Medicina de Buenos Aires, Buenos Aires, ArgentinaSearch for more papers by this authorC. D. DE BRASI, C. D. DE BRASI Sección Genética Molecular de la Hemofilia, Departamento de Genética, Academia Nacional de Medicina de Buenos Aires, Buenos Aires, ArgentinaSearch for more papers by this authorE. MEDINA-ACOSTA, E. MEDINA-ACOSTA Núcleo de Diagnóstico e Investigação Molecular, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, BrazilSearch for more papers by this author First published: 11 November 2010 https://doi.org/10.1111/j.1365-2516.2010.02404.xCitations: 4 Enrique Medina-Acosta, Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego 2000, Parque Califórnia, Campos dos Goytacazes, RJ, CEP 28013-602, Brazil.Tel./fax: +55 22 273 97086; e-mail: quique@uenf.br Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Summary. The extraordinary heterogeneous nature of the deleterious mutations in the F8 gene that lead to functional deficiency of clotting factor VIII in haemophilia A makes routine direct mutation profiling difficult. When direct mutation analysis cannot be performed or a causative/candidate mutation is not found, a second-line approach to track the defective F8 gene within at-risk families is linkage genetic analysis with, tried-and-tested, F8-intragenic and/or extragenic non-recombining multiallelic short tandem repeats (STR). Although several typing STR loci within and around F8 have been described, there is need for improving assessment, because the combined informativeness of available assays rarely reaches 100%. Here, we characterized a newly identified 0.28 cM-resolution marker-set, consisting of a dinucleotide STR located on F8 intron 21 (F8Int21; [AC]n) and three extragenic tetranucleotide STR located on GAB3 intron 1 (GAB3Int1; [TAAA]n) and TMLHE intron 1 (TMLHEInt1.1; [GAAA]n and TMLHEInt1.3; [ATTC]n). Heterozygosity rates determined in 100 unrelated females ranged from 0.25 (GAB3Int1) to 0.63 (F8Int21). The set rendered a combined informativeness of 0.91 for at least one marker and 0.60 for a minimum of two loci, with at least one F8-intragenic. Multiallelic interlocus non-random association analysis revealed that GAB3Int1 is not in significant gametic disequilibrium (GD) with F8Int21, F8Int9.2, TMLHEInt1.3 or TMLHEInt1.1. Gametic disequilibrium breakdown attests historical recombination between GAB3Int1 and the F8 gene. Through computational analysis of reference assembly sequence data, we note in the GD breakdown region and in the F8 gene a higher than average density of the 13-mer CCNCCNTNNCCNC consensus motif, commonly associated with recombination hotspots. Citing Literature Supporting Information Figure S1. Representative QF-PCR assays from unrelated females, for whom the tested markers are informative. Smaller peaks preceding the designated allele peaks represent stutter products, typically generated by PCR amplification of dinucleotide STRs (F8In21and F8Int9.2). Allele names are the length in base pairs of each fluorescence peak. The marker names are indicated above each electropherogram. Figure S2. Combined (blue bars) and absolute (red bars) frequency distribution of informative markers. The term 'combined informativeness' refers to the combined frequency distribution of informative markers in the tested sample population (i.e. the extent or power of informativeness) when typing several loci. Overall, the 0.28 cM-resolution marker-set enabled achieving a combined informativeness of 0.91 for at least one marker and 0.60 for a minimum of two informative markers, with at least one F8-intragenic locus. Table S1. STR loci, physical position, primer sequences, 5′-end modifications, and observed allele range, comprising the 0.28 cM-resolution marker-set assayed by QF-PCR for indirect tracking of HEMA. Table S2. Genotype frequencies in 100 unrelated healthy females from the Northern Region of the State of Rio de Janeiro, Brazil. Table S3. Haplotype classes and frequencies in 20 unrelated males screened positive for Inv22-1 and Inv22-2 mutations, for an Argentinean cohort. Filename Description HAE_2404_sm_FigS1.pdf58.3 KB Supporting info item HAE_2404_sm_FigS2.pdf601.4 KB Supporting info item HAE_2404_sm_TableS1.docx48 KB Supporting info item HAE_2404_sm_TableS2.doc136 KB Supporting info item HAE_2404_sm_TableS3.doc39.5 KB Supporting info item Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume17, Issue2March 2011Pages 257-266 RelatedInformation