GENOMICS 31, 319–326 (1996) ARTICLE NO. 0054 Hereditary Hemochromatosis: Generation of a Transcription Map within a Refined and Extended Map of the HLA ClassI Region ANGELA T OTARO,J OHANNA M. R OM M ENS ,* , ² ANNA GRIFA,C LAUDIO L UNARDI,‡ M ASSIM O C ARELLA,J ACK J. HUIZENGA,* ANTONELLA R OETTO,§ C LARA C AM ASCHELLA,§ GIORGIO DE S ANDRE ,‡ AND P AOLO GASPARINI 1 Servizio di Genetica Medica, IRCCS-Ospedale ‘‘CSS,’’ I-71013 San Giovanni Rotondo, §Dipartimento di Scienze Biomediche ed Oncologia Umana, Ospedale S. Luigi, Orbassano, 10100 Turin, and ‡Istituto di Clinica Medica, Universita’ di Verona, 35100 Verona, Italy; * Department of Genetics, Research Institute, The Hospital for Sick Children and ² Department of Molecular and Medical Genetics, University of Toronto, Toronto, M5G 1X8 Canada Received August 21, 1995; accepted November 10, 1995 overload. The disease prevalence is estimated to be 2 – Hereditary hemochromatosis, a common severe in- 5/1000 in the Caucasian population, with a correspond- herited disease, maps to the short arm of chromosome ing carrier frequency of 0.045 – 0.071 (McKusick, 1994). 6 close to the HLA-A locus. Recently, linkage data on The underlying biochemical defect of the disease is un- Italian and French populations confirmed this loca- known, but the gene has been mapped to the short arm tion, while a similar analysis on Australian and British of chromosome 6, containing the histocompatibility an- populations located the gene closer to D6S105, a tigen (HLA) class I region (Simon et al., 1987). marker residing telomeric of HLA-A. To increase our Linkage disequilibrium studies in French and Italian knowledge on the region of highest linkage disequilib- populations initially suggested a narrow candidate re- rium in our population and possibly to identify the gion for the HFE gene, including a 400-kb stretch of disease gene, a 1.2-Mb detailed physical and transcrip- DNA flanking the HLA-A gene, spanning from I.82 to tion map was generated, spanning the HLA class I re- HLA-F (Boretto et al., 1992; Gasparini et al., 1993; gion. Thirty-eight unique cDNA fragments, retrieved Yaouanq et al., 1994). However, association studies, following the hybridization of immobilized YACs to performed in the Australian population (Jazwinska et primary pools of cDNAs prepared from RNA of fetal al., 1993), have extended this region to the D6S105 brain, adult brain, liver, placenta, and the CaCo 2 cell locus (Weber et al., 1991), which is estimated to be at line, were characterized. All cDNA fragments were po- sitioned in a refined and extended map of the human least 2 cM telomeric of HLA (Stone et al., 1994; Volz et major histocompatibility complex spanning from HLA- al., 1994). We recently reported new polymorphisms E to approximately 500 kb telomeric of HLA-F. The and sequence tagged sites from the HLA class I region localization of known genes was refined, and a new and identified a polymorphic marker, named Y52, gene from the RNA helicase superfamily was identi- which showed the highest allelic association in our pop- fied. Overall, 14 transcription units in addition to the ulation (Totaro et al., 1995). Allele 1 of Y52, together HLA genes have been detected and integrated in the with HLA-A serotype A3 and D6S265 allele 1, mark map. Thirteen cDNA fragments show no similarity the ancestral HFE haplotype in our population. Y52 with known sequences and could be candidates for the and D6S265 were positioned very close to the HLA-A disease. Their characterization and assessment for locus (Totaro et al., 1995). These data support this area involvement in hemochromatosis are still under inves- as being the most likely candidate region for the dis- tigation. Seven new polymorphisms, some tightly ease gene. Several novel coding sequences have already linked to the disease, were also identified and local- been obtained from this region (El Kahloun et al., 1993; ized.  1996 Academic Press, Inc. Wei et al., 1993; Goei et al., 1994), but despite the exten- sive effort, there has as yet been no indication that these coding sequences are hemochromatosis candi- INTRODUCTION dates. We have extended the cloned region by isolation of an overlapping YAC clone, 169H11, and applied the Hemochromatosis (HFE) is a common autosomal re- cDNA selection technique to the entire region of 1.2 cessive disease characterized by chronic dietary iron Mb containing the HLA-A locus, spanning from 90 kb proximal to HLA-E to about 550 kb distal to HLA-F. 1 To whom correspondence should be addressed at Servizio di Ge- Moreover, to refine and extend the physical and tran- netica Medica, IRCCS-Ospedale ‘‘CSS,’’ I-71013 San Giovanni Ro- tondo, Italy. Telephone: /39 882 410825. Fax: /39 882 411616. scription map beyond the distal part of the human HLA 319 0888-7543/96 $12.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.