ABP-280 is a ubiquitous actin binding protein present in the cytoskeleton of many different cell types. ABP-280 was mapped to distal Xq28, 50-60 kb downstream of the Green Colour Pigment (GCP) genes. To establish if ABP-280 may be a candidate for one of the muscle disease localized by linkage analysis to distal Xq28 we looked for alternative forms of ABP-280 mRNA. Several different ABP-280 mRNAs were indeed identified: two are X-linked and are produced by alternative splicing of a small exon of 24 nucleotides. At least one additional gene encoding a RNA more than 70% identical to ABP-280 in the 1700 bp sequenced has also been found. It was mapped to chromosome 7. While both forms of the X-linked ABP-280 are ubiquitous, the gene on chromosome 7 is highly expressed only in skeletal muscle and heart. The two genes were therefore excellent candidates for the X-linked and for the autosomal dominant form of the Emery-Dreifuss Muscular Dystrophy (EDMD) both of which have been described. So far, however we were unable to demonstrate mutations in the coding region or affecting the alternative splicing of the X-linked form of ABP-280, in several patients studied, and we think that it is quite unlikely that this is the gene responsible for EDMD.

After interaction of CD40L with CD40 and costimulation by appropriate cytokines (interleukins 4 and 10), 13 cells proliferate and perform unoglobulin isotype switch (6). Mutations in CD40L transcripts in 12 cases ofHIGMi have been characterized (8-11). Among these, 11 have been detected in the extracellular domain, with the 12th being located in the transmembrane domain. Nine patients demnstrated point mutations; three patients reported by a single group had deletions of 8, 10, and 63 bp, respectively (10). Interestingly, these three deletions clustered within a limited region of the coding sequence. Although a splicing defect has been hypothesized, it could not be demonstrated since the genomic structure ofthe CD40L gene was not known. In additon, two other incompletely characterized mutations have been briefly reported (12, 13), one of which had a deletion of 58 nucleotides adjacent to those absent in the 63-bp deletion.These small deletions in mRNA might result from true genomic deletions or mutations that affect splcing of the transcript. Here we report the CD40L genomic structure and a more precise characterization of these abnormaites. We also show that knowledge of the CD40L exonintron structure can be applied to prenatal diagnosis of HIGSi using PCR amplification of genomic DNA derived from fetal samples. MATERIALS AND METH-OSolation ofGenoic Clones for CD40L. Two parallel strategies were used to isolate .CD40L genomic sequences. The first utilized PCR amplification ofgenomic DNA using primer pairs corresponding to the coding regions. The second approach involved isolation of clones from an X chromosomeenriched cosmid library gridded onto nylon filters (14) using a full-length CD40L cDNA (7). Both approaches proved successful and provided identical data regarding sequence and structural organization.The following primers were used to amplify the whole coding region of CD40L in six steps.

Thirty-two probes for CpG islands of the distal long arm of the human X chromosome have been identified. From a genomic library of DNA of the hamster-human cell hybrid X3000.1 digested with the rare cutter restriction enzyme EagI, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5' end of genes of the X chromosome has been used to distinguish between EagI sites in CpG islands versus isolated EagI sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5' end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated EagI site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylation of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.

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