GENOMICS 43, 149–155 (1997) ARTICLE NO. GE974793 Determination of the Genomic Structure of the XNP/ATRX Gene Encoding a Potential Zinc Finger Helicase Laurent Villard,* Anne-Marie Lossi,* Carlos Cardoso,* V. Proud,² Pierre Chiaroni,* Laurence Colleaux,* Charles Schwartz,‡ and Michel Fonte ´s * ,1 * Ge ´ne ´tique Me ´dicale et De ´veloppement, Faculte ´ de Me ´decine de la Timone, INSERM U 406, 27 Boulevard J. Moulin, 13385 Marseille Cedex 5, France; ² Laboratory of Medical Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294; and ‡J. C. Self Research Institute, Greenwood Genetic Center, Greenwood, South Carolina 29646 Received November 18, 1996; accepted May 8, 1997 1994). It is related to the SNF2/SWI family of transcrip- The XNP/ATR-X gene is involved in several X-linked tional regulators. mental retardation phenotypes: the ATR-X syndrome, XNP has been shown to be mutated in 13 patients the Juberg – Marsidi syndrome, and some severe men- with the a-thalassemia mental retardation (ATR-X) tal retardation phenotypes without a-thalassemia. Us- syndrome (Gibbons et al., 1995). Recently we described ing a vectorette strategy, we have identified and se- three mental retardation phenotypes without a-thalas- quenced the intron/exon boundaries of this gene. The semia resulting from mutations in the XNP gene. One gene is composed of 35 exons. It encodes a potential was due to a mutation in the pyrimidine stretch of a protein of 2492 amino acids. A search of the databases splicing site that led to the production of both normal identified three zinc finger motifs within the 5 end and abnormal XNP transcripts (Villard et al., 1996a). of the gene. Expression analysis in different tissues The second case was a missense mutation affecting the indicated that an alternative splicing event that in- helicase domain II (Villard et al., 1996c). The third mu- volves exon 6 is occurring. One of these alternatively tation consisted of single changes in the helicase do- spliced transcripts is predominantly expressed in em- main V, segregating in a large family with the Juberg – bryonic tissues. These data led us to search for muta- Marsidi syndrome (Villard et al., 1996b). Additionally, tions in the 5 region in ATRX patients without other removal of the last 100 amino acids of the putative mutations in the 3  region. In one patient a mutation XNP protein has been associated with genital abnor- was found in which part of exon 7 was removed from the XNP transcript, as a result of a mutation creating malities in three patients with ATR-X and sex reversal a novel splice site that is substituted for the natural (Gibbons et al., 1995; Ion et al., 1996). splice site. This new splicing event removed one zinc A compilation of the different mutations described finger motif. This is the first example of a mutation to date led us to make the following observations: a in XNP within the 5 coding region. It suggests that mutation in the region coding for the helicase domains mutations will be predominantly found in the helicase (about 3 kb) causes either the classical ATR-X pheno- region as well as in the zinc finger regions and leads type, if it does not affect an helicase domain, or a severe us to propose a large screening of additional patients. mental retardation phenotype, without a-thalassemia  1997 Academic Press (e.g., Juberg – Marsidi syndrome), if it alters a residue within a helicase domain. Mutations removing the last 100 amino acids likely result in ATR-X associated with INTRODUCTION sex reversal. To strengthen these genotype/phenotype correlations During the construction of a transcriptional map of further, it is important to perform a more thorough the DXS56-PGK1 region (Gecz et al., 1993), we isolated search for mutations in patients whose X-linked mental a gene just telomeric to DXS56 (Villard et al., 1995). retardation phenotype is potentially due to a mutation We named this sequence XNP (Gecz et al., 1994). The of XNP. Since only DNA is available for many patients, putative protein encoded by this gene contains helicase we have identified and characterized the exon/intron domains and is partially homologous to the brahma boundaries of XNP. The size of the detected introns transcriptional regulator of Drosophila (Stayton et al., was determined using long-range PCR, with primers from the flanking exons. As a result, we were able to demonstrate that XNP is composed of at least 35 exons, Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. U75653, encompassing a genomic region of at best 200 kb. U97080 – U97103, and AF000153 – AF000160. We also examined XNP expression, using RT-PCR, 1 To whom correspondence should be addressed. Telephone: 33-04 91 78 44 77. Fax: 33-04 91 80 43 19. E-mail: fontes@ibdm.univ-mrs.fr. and found alternative splicing involving exon 6. Fi- 149 0888-7543/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.