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The chromosomal abnormality represented by an isodicentric X chromosome [idic(X)(q13)] is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females. A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene. Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region. In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs. Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations. FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML. Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region.

Source/Description: Primers for the DXS983 locus (AFMO78zal) (1) were used to screen the ICRF Human Yeast Artificial Chromosome (YAC) library. These primers identified the same 6 YAC clones that had been isolated with the primers for the microsatellite repeat locus DXS453 (2) (Mfd66). The reverse (R) and forward (F) primers for DXS4S3 were previously shown to amplify fragments from 160—170 bp with 5 alleles (one of these alleles only has a frequency of 0.01), the PIC is 0.63 (2). Similarly, the R and F primers for the locus DXS983 identifies 4 alleles in the size range 173-183 bp with a PIC of 0.62 (1). When the primers for DXS453 and DXS983 were mixed in all 4 pairwise combinations and used in the polymerase chain reaction (PCR), an amplified product in the correct size range was observed with primer pairs, DXS983R/DXS453F and DXS983F/DXS453R, in addition to the controls, DXS983F/DXS983R and DXS453F/DXS453R. To ensure that these amplified products were in fact recognising the same locus, a Southern blot was prepared and hybridized with the primer DXS453R. The primer was labelled with •yPATP using a standard phosphorylation labelling procedure with polynucleotide Irinase (3). The primer hybridized to all the amplified products irrespective of whether they were derived from the locus DXS4S3 or DXS983. We conclude that the dinucleotide repeat at the locus DXS453 is identical to that designated at locus DXS983.

X chromosome inactivation is a mechanism of dosage compensation that regulates the expression of mammalian X-linked genes between XY males and XX females. This phenomenon is cis-acting, clonally heritable, and requires the presence of an X inactivation center (XIC). In our attempts to characterize this phenomenon, we have focused on the physical organization of the human XIC localized to Xq13. From previous studies, we had determined that the candidate XIC interval contained two loci (DXS128 and XIST) and was bound by the breakpoints of two structurally abnormal inactivated X chromosomes, a t(X;14) and an idic(Xp). Here we present a refined mapping of the XIC-containing region using the breakpoint of a late replicating rearranged X (rea(X)), and the initial characterization of a set of 40 yeast artificial chromosomes (YACs) derived from the XIC-containing region. These YACs form a 2.6 Mb contig which completely covers the XIC, and physically links the RPS4X, PHKA1, XIST, and DXS128E genes, as well as a laminin receptor pseudogene (LAMRP4). Furthermore, we have determined the relative orientations of these four genes, and have derived a restriction map of the region using the rare cutter enzymes BssHII, EagI, MluI, NruI, SalI, SfiI, SstII (or SacII), and NotI. We have identified at least 9 CpG-rich islands within this region, and have discovered a large (approximately 125 kb) inverted duplication proximal to the XIC based on symmetrical restriction patterns and homologous probes. We estimate the maximum size of the XIC-containing interval to be between 680 kb and 1200 kb, based on the localization of the breakpoints of the rearranged X chromosomes mentioned above. This lays the groundwork for the further characterization of the XIC region and the isolation of other expressed sequences therefrom.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a potent regulator of glycolysis. Previous studies assigned the gene for human liver PFK-2/FBPase-2 (HGMW-approved symbol PFKFB1) to the X chromosome; however, precise localization remained ambiguous, with the gene variously placed between Xcen-q13, Xq27-q28, and Xp11.22-p11.21. We have localized the gene within a YAC contig clustered around ALAS2 (human erythroid delta-aminolevulinate synthase) in Xp11.21 and have identified eight YACs positive for the gene. Four of these overlapping YACs were mapped using rare-cutter restriction enzymes to provide in-depth characterization of an 820-kb region encompassing the PFK-2/ FBPase-2 and ALAS2 genes. PFK-2/FBPase-2 was found to lie close (within approx. 250 kb) and telomeric to ALAS2. Three putative CpG islands were also detected in the region.

We demonstrate that all the repeat elements representing the conserved loci DXF34 and DXS390 lie between the X;9 and the X;17 translocation breakpoints associated with incontinentia pigmenti type 1 (IP1). Sequence-tagged sites (STSs) at DXF34S1, DXS14, and DXS390 have been used to isolate YAC clones containing these loci, and a contig of approximately 2 Mb has been constructed. Patterns of hybridization observed in the YAC clones indicate that DXS390 comprises two distinct regions (A and B). The STS at DXS390 detects the A region and includes a polymorphic CA repeat (PIC = 0.25). This expansion of the cloned region around DXF34 and DXS390 will enable the isolation of additional conserved sequences that will help in understanding both the lesions underlying the pathogenesis of IP1 and the size and extent of the man-mouse homologous block defined by DXF34.

In order to study the distribution of genes that escape X chromosome inactivation, a high density yeast artificial chromosome (YAC) contig and STS map spanning approximately 6 Mb has been constructed in Xp11.21-p11.22. The contig contains 113 YACs mapped with 53 markers, including 10 genes. Four genes have been assayed for their expression status on both the active and inactive human X chromosomes, and these data have been combined with previous results on two other genes in the contig. Three of these genes escape X inactivation and have been localized to a single YAC clone of approximately 1075 kb. The other three genes are subject to inactivation, with two of them lying among the genes that escape inactivation. These results suggest that there are both regional control signals as well as gene-specific elements that determine the X inactivation status of genes on the proximal short arm of the human X chromosome.