Chromosomal Localization of the Adrenoleukodystrophy-Related Gene in Man and Mice

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Abstract

We report here on the chromosomal mapping of the adrenoleukodystrophy-related (ALDR) gene on both the human and the mouse genomes. This gene encodes a peroxisomal ATP binding cassette transporter, closely related to the transporter identified as responsible for the adrenoleukodystrophy phenotype. ALDR maps on the syntenic region on murine and human autosomes. In addition, we could determine its position in relation to known microsatellite framework markers; this will allow to test its role in Zellweger syndrome and/or related peroxisomal disorders.

Adrenoleukodystrophy is a severe X-linked neurodegenerative disorder whose clinical phenotype results from impaired peroxisomal beta-oxidation of very-long-chain fatty acids [1, 2]. This is due to the dysfunction of the ALD gene located on Xq28 and encoding a peroxisomal membrane protein (ALDP) [3]. ALDP belongs to the family of ATP-binding cassette (ABC) transporters, which are involved in the transport of a wide variety of substrates across cell membranes [4]. Functional ABC transporters form a pore-like structure spanning the membrane several times and are composed of two highly similar halves. These can be part of a single, internally symmetrical polypeptide; alternatively, the final functional moiety can arise from the posttranslational association of two similar half transporters. ALDP is a hemitransporter and the identification of its functional dimerization partner is so far lacking [5]. Two other peroxisomal ABC hemi-transporters closely related to ALD have been identified in mammals. The first, PMP70, is a 70-kD protein with an overall identity of 38% with ALDP [6, 7]. More recently, we reported on the identification of an even closer relative of ALD, named ALDR (for adrenoleukodystrophy-related) [8]. The ALDR gene was identified by a PCR-based approach targeting novel mammalian ABC transporters expressed in mouse macrophages [9]. Among the amplified products, two sequences closely related to ALD were found. One corresponds to the mouse PMP70 (cfr. GB ac#L28836) since it encodes a protein with more than 90% identity with the available sequences from human and rat PMP70 [7, 10]. The second showed 66% identity with ALD, clearly too distant to be considered its cognate murine sequence. It corresponds in fact to a novel ABC transporter, present in the mouse and human genome [8]. Its sequence similarity to ALD and the specific pattern of expression suggest that the three peroxisomal ABC transporters might behave as facultative or nonequivalent partners in some tissues.

The chromosomal localization of the two amplification fragments belonging to PMP70 and ALDR transcripts was carried out on mouse genome by in situ hybridization analysis. In both cases the probe was a fragment of 340 bp spanning the region of the ATP-binding cassette. Experiments were carried out using metaphase speads from a WMP male mouse in which all the autosomes except 19 were in the form of metacentric Robertsonian translocations. Concanavalin-A-stimulated lymphocytes were cultured at 37° C for 72 h with 5-bromodeoxyuridine added for the final 6 h of culture, to ensure R-banding of good quality. The hybridization protocol was as already described [9]. The PMP70 probe hybridized specifically to mouse chromosome 3 band G-H1 (not shown), in line with previous results based on the analysis of interspecific backcrosses which positioned the murine pxmp-1 locus in the distal region of chromosome 3, between Egf (epidermal growth factor) and amy-2 (pancreatic amylase) [11].

In the 100 metaphase cells examined after hybridization with the ALDR probe, the distribution of chromosome-associated grains was not random and 91.4% of them mapped to the 15E-15F bands of the murine genome (fig. 1, left panel). In order to map the ALDR gene on the human genome, interspecific hybridization was carried out, with the longest cDNA clone containing the whole open reading frame for murine ALDRP [8]. Phytohemagglutinin-stimulated human lymphocytes were used to prepare chromosome spreads. On the human genome, the ALDR gene mapped to the q11-q12 bands of chromosome 12 (fig. 1, right panel). This region corresponds indeed to the region of synteny to mouse chromosome 15 (map position 47 to 65 cM) [12].

Fig. 1
figure1

Localization of the ALDR gene on the mouse and human genomes by in situ hybridization. Left panel: Diagram of WMP mouse Rb (13; 15) chromosome indicating the distribution of labeled sites on chromosome 15. Right panel: Idiogram of the human G-banded chromosome 12 illustrating the distribution of labelled sites on bands q11-q12.

To further refine the map position of the human ALDR gene, pairwise linkage analysis was carried out on a panel of whole genome radiation hybrids as described by Gaypay et al. [13]. PCR analysis was performed by standard hot start protocols using two primers from exon 1 of the human ALDR gene and the framework markers. The sequence of the two ALDR primers used for mapping purposes were as follows: forward: 5′CACGCCTAT-GAAACCTATTTTA 3′; reverse: 5′CCTTTTCTATGT-GCTTCCTCT 3′. Results were analyzed semiautomatically. Eight framework markers were linked and the closest markers were D12S1589 (lod score of 23.73 at 33 cR), D12S1604 (11.97 at 67 cR) and D12S87 (9.37 at 86 cR). Given the distances between these markers [13] this indicates the order pter-D12S87-ALDR-D12S1589-D12S1604-qter. The D12S87-D12S1589 is known to span the centromere [14], This result is thus consistent with in situ hybridization. The identification of a micro-satellite interval of 5 cM (D12S87-D12S1589) containing the ALDR gene will allow the testing of this gene as a candidate in Zellweger syndrome families or for other peroxisomal disorders.

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Acknowledgments

This work was supported by institutional grants from INSERM and CNRS and by a specific grant from the GREG.

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Correspondence to Giovanna Chimini.

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Key Words

  • ATP binding cassette transporters
  • Adrenoleukodystrophy
  • In situ mapping
  • PMP70