New FACTOR IX linked marker alleles in African Haemophilia B patients* C. MITCHELL, C. L. MITCHELL and A. KRAUSE Division of Human Genetics, School of Pathology, the National Health Laboratory Service (NHLS) and the University of the Witwatersrand, Johannesburg, South Africa Summary. Three markers, one restriction length polymorphism (RFLP) (MseI) and two microsatellite markers (Intron 1 and 3ÕUTR), linked to the FACTOR IX gene, were assessed for the purpose of genetic testing for Haemophilia B families in South Africa. This was carried out using seven Haemophilia B families and fifty random control samples. We observed five new alleles for the Intron 1 marker within the black control and patient sample groups, and informativity in 89% (8/9) of all carrier * females for at least one of the three markers observed. These markers are useful for carrier detection and prenatal diagnosis of Haemophilia B in the great majority of South African black and white families. Keywords: carrier detection, factor IX gene, haemo- philia B, linked markers, prenatal diagnosis, South African populations Introduction Haemophilia B is the rarer of the X-linked recessive haemophilias. It is characterized, like Haemophilia A, by prolonged or renewed bleeding after injury, spontaneous muscle and joint haematomas and intracranial bleeding [1]. It affects approximately one in 20 000 males world wide [1]. Haemophilia B is caused by mutations within the FACTOR IX gene, located at Xq27.1–27.2. The gene is 35 kb in length and transcribes eight exons (a–h) [1]. To date over 500 mutations have been characterized within the FACTOR IX gene and are listed in an annually updated database, most of which are point mutations but also include deletions, insertions and repeat sequences [2]. Due to this heterogeneity, direct mutation detection as a molecular diagnostic tool can be relatively expensive. Thus, routine molecular genetic testing for Haemophilia B through linkage analysis is more practical, especially where resources are more limited. One is able to track the Ôhigh-riskÕ chromosome in a particular pedigree, using a linked marker, provided that linked markers have been identified and are informative in a particular pedi- gree. The most informative markers are those with high heterozygosity rates and a large amount of genetic variation; prime examples of these are microsatellite markers as they tend to have many alleles. Genetic testing for Haemophilia B has not been available in South Africa. Thus a study, aimed at identifying useful linked markers for Haemophilia B testing within the South African black and white populations, was initiated. We selected three of the previously described markers, namely the 1 MseI restriction fragment length polymorphism (RFLP); a C to T transition in the 5¢ flanking region of the FACTOR IX gene and two microsatellite markers, 3¢untranslated region (UTR) and Intron 1 (also referred to as the DdeI polymorphism), based on published high heterozygosity rates in other popula- tions [3,4,5]. The 3¢UTR marker is described as a ÔcrypticÕ long tandem dinucleotide repeat consisting of alternating purines and pyrimidines [RY(i)], labelled cRY(i). It contains two short dinucleo- tide repeats (GT) a (ATGCGT) 4 AG(AC) b GCAT(AC) 3 (AT) 2 , where a and b vary [4]. As a result of these differing repeat sizes, four alleles I–IV with the repeats sequences a 6 b 4 (162 bp), a 5 b 4 (160 bp), a 5 b 3 (158 bp) and a 4 b 4 (158 bp) respectively, have been described [4]. The Intron 1 marker involves two repeat sequences, labelled A and B, which differ only *Mothers of affected sons or daughters are at high risk of being carriers but not confirmed carriers. Correspondence: Cathrine Mitchell, Division of Human Genetics, NHLS, 1st Floor, James Murray Building, Cnr of DeKorte and Hospital Street, Braamfontein, PO Box 1038 Johannesburg 2000, South Africa. Tel.: 2711 489 9225; fax: 2711 489 9226; e-mail: cathrinemitchell@gmail.com Accepted after revision 11 April 2007 Haemophilia (2007), 13, 642–644 DOI: 10.1111/j.1365-2516.2007.01486.x Ó 2007 The Authors 642 Journal compilation Ó 2007 Blackwell Publishing Ltd