Does the human X contain a third evolutionary block? Origin of genes on human Xp11 and Xq28

Comparative gene mapping of human X-borne genes in marsupials defined an ancient conserved region and a recently added region of the eutherian X, and the separate evolutionary origins of these regions was confirmed by their locations on chicken chromosomes 4p and 1q, respectively. However, two groups of genes, from the pericentric region of the short arm of the human X (at Xp11) and a large group of genes from human Xq28, were thought to be part of a third evolutionary block, being located in a single region in fish, but mapping to chicken chromosomes other than 4p and 1q. We tested this hypothesis by comparative mapping of genes in these regions. Our gene mapping results show that human Xp11 genes are located on the marsupial X chromosome and platypus chromosome 6, indicating that the Xp11 region was part of original therian X chromosome. We investigated the evolutionary origin of genes from human Xp11 and Xq28, finding that chicken paralogs of human Xp11 and Xq28 genes had been misidentified as orthologs, and their true orthologs are represented in the chicken EST database, but not in the current chicken genome assembly. This completely undermines the evidence supporting a separate evolutionary origin for this region of the human X chromosome, and we conclude, instead, that it was part of the ancient autosome, which became the conserved region of the therian X chromosome 166 million years ago.     

FISH analysis for apparently simple terminal deletions of the X chromosome: Identification of hidden structural abnormalities

We report on fluorescence in situ hybridization (FISH) analysis in 30 mosaic or nonmosaic females diagnosed as having apparently simple terminal X deletions by standard G‐banding analysis. FISH studies for DXZ1, the Xp and Xq telomere regions, and the whole X chromosome painting were carried out for the 30 females, indicating rearranged X chromosomes with signal patterns discordant with terminal deletions in 6 cases: one dic(X)(DXZ1++) chromosome, two der(X)(qtel++) chromosomes, one Xq? (qtel+) chromosome, and two der(X)(ptel++) chromosomes. Additional FISH studies were performed for the 6 cases using probes defining 12 loci on the X chromosome, showing large Xp deletion and small Xp duplication in the dic(X)(DXZ1++) chromosome, partial Xp deletions and partial Xq duplications in the two der(X)(qtel++) chromosomes, an interstitial Xq deletion in the Xq? (qtel+) chromosome, and partial Xq deletions and partial Xp duplications in the two der(X)(ptel++) chromosomes. Clinical assessment of the 6 cases revealed tall and normal stature in the two mosaic cases with the der(X)(ptel++) chromosomes that were shown to be associated with SHOX duplication. The results suggest that unusual X chromosome rearrangements are often misinterpreted as simple terminal X deletions, and that FISH analysis is useful for precise structural determination and better genotype‐phenotype correlation of the X chromosome aberrations. © 2001 Wiley‐Liss, Inc.     

Expression of common fragile sites on the X chromosome corresponds with active gene regions

The expression of common chromosomal fragile sites on human chromosomes has been proposed to be a cytogenetic expression of gene activity. Distinctive patterns of expression of two common fragile sites on the human X chromosome were observed in females. The fragile site at Xp22.31, located in a band region that contains genes which escape X inactivation, was expressed on both X chromosomes. By contrast, the fragile site at Xq22.1, in a region assumed to be subject to X inactivation, was expressed almost exclusively on one X, the active X chromosome. These findings provide evidence that common fragile site expression only occurs in regions with active genes.     

Loss of heterozygosity on chromosome XP22.2-p22.13 and Xq26.1-q27.1 in human breast carcinomas.

In an attempt to investigate the X chromosome harboring putative tumor suppressor genes (TSGs) in sporadic breast carcinoma, we performed loss of heterozygosity (LOH) studies on 23 breast carcinomas using 15 polymorphic markers covering the whole X chromosomes. Matched DNA extracted from tumor samples and corresponding normal tissues were analyzed by polymerase chain reactions (PCR) using microsatellite markers. In 10 cases (43.5%), LOH was detected for at least 1 of the 15 polymorphic markers of the X chromosome tested. Four cases carried a LOH at Xp, and three cases LOH on Xp and Xq. Three cases carried a LOH Xq. Percentage of LOH was relatively high in DXS987 (26.7%), DXS999(30.0%), HPRT(21.4%), DXS1062(23.1%) loci. Common regions of deletions were found on Xp22.2-p22.13 (30% of LOH) measuring about 4.5Mb and Xq26.1-q27.1 (23.1% of LOH) measuring 10 Mb. The deleted allele was an active copy of the X chromosome. The results indicate the TSGs on the X chromosome are involved in breast cancer.     

Classical and Molecular Cytogenetics of the Pufferfish Tetraodon Nigroviridis

Because of its highly compact genome, the pufferfish has become an important animal model in genome research. Although the small chromosome size renders chromosome analysis difficult, we have established both classical and molecular cytogenetics in the freshwater pufferfish Tetraodon nigroviridis (TNI). The karyotype of T. nigroviridis consists of 2n = 42 biarmed chromosomes, in contrast to the known 2n = 44 chromosomes of the Japanese pufferfish Fugu rubripes (FRU). RBA banding can identify homologous chromosomes in both species. TNI 1 corresponds to two smaller FRU chromosomes, explaining the difference in chromosome number. TNI 2 is homologous to FRU 1. Fluorescence in-situ hybridization (FISH) allows one to map single-copy sequences, i.e. the Huntingtin gene, on chromosomes of the species of origin and also on chromosomes of the heterologous pufferfish species. Hybridization of total genomic DNA shows large blocks of (species-specific) repetitive sequences in the pericentromeric region of all TNI and FRU chromosomes. Hybridization with cloned human rDNA and classical silver staining reveal two large and actively transcribed rRNA gene clusters. Similar to the situation in mammals, the highly compact pufferfish genome is endowed with considerable amounts of localized repeat DNAs.     

Parental origin of normal X chromosomes in Turner syndrome patients with various karyotypes: implications for the mechanism leading to generation of a 45,X karyotype

The parental origin of the X chromosome of 45,X females has been the subject of many studies, and most of them have shown that the majority (60-80%) of the X chromosomes are maternal in origin. However, studies on the parental origin of normal X chromosomes are relatively limited for Turner syndrome (TS) females with sex chromosome aberrations. In this study, we used PCR-based typing of highly polymorphic markers and an assay of methylation status of the androgen receptor gene to determine the parental origin of normal X chromosomes in 50 unbiased TS females with a variety of karyotypes. Our results showed a higher paternal meiotic error rate leading to the generation of abnormal sex chromosomes, especially in the case of del(Xp) and abnormal Y chromosomes. Isochromosome Xq and ring/marker X chromosomes, on the other hand, were equally likely the result of both maternal and paternal meiotic errors. A thorough review of previous results, together with our data suggests, that the majority of TS karyotype are caused by paternal meiotic errors that generate abnormal sex chromosomes, and that most 45,X cells are generated by mitotic loss of these abnormal sex chromosomes, resulting in maternal X dominance in these cells.     

Opposite deletions/duplications of the X chromosome: two novel reciprocal rearrangements

Paralogous sequences on the same chromosome allow refolding of the chromosome into itself and homologous recombination. Recombinant chromosomes have microscopic or submicroscopic rearrangements according to the distance between repeats. Examples are the submicroscopic inversions of factor VIII, of the IDS gene and of the FLN1/emerin region, all resulting from misalignment of inverted repeats, and double recombination. Most of these inversions are of paternal origin possibly because the X chromosome at male meiosis is free to refold into itself for most of its length. We report on two de novo rearrangements of the X chromosome found in four hypogonadic females. Two of them had an X chromosome deleted for most of Xp and duplicated for a portion of Xq and two had the opposite rearrangement (class I and class II rearrangements, respectively). The breakpoints were defined at the level of contiguous YACs. The same Xp 11.23 breakpoint was found in the four cases. That of the long arm coincided in three cases (Xq21.3) and was more proximal in case 4 (Xq21.1). Thus class I rearrangements (cases 1 and 2) are reciprocal to that of case 3, whilst that of case 4 shares only the Xp breakpoint. The abnormal X was paternal in the three cases investigated. Repeated inverted sequences located at the breakpoints of rearrangements are likely to favour the refolding of the paternal X chromosome and the recombination of the repeats. The repeat at the Xp11 may synapse with either that at Xq21.3 or that at Xq21.1. These rearrangements seem to originate as the Xq28 submicroscopic inversions but they are identifiable at the microscopic level and result from a single recombination event.     

Functional disomy of Xp including duplication of DAX1 gene with sex reversal due to t(X;Y)(p21.2;p11.3)

Translocations involving the short arms of the X and Y in human chromosomes are uncommon. One of the best-known consequences of such exchanges is sex reversal in 46,XX males and some 46,XY females, due to exchange in the paternal germline of terminal portions of Xp and Yp, including the SRY gene. Translocations of Xp segments to the Y chromosome result in functional disomy of the X chromosome with an abnormal phenotype and sex reversal if the DSS locus, mapped in Xp21, is present. We describe a 7-month-old girl with severe psychomotor retardation, minor anomalies, malformations, and female external genitalia. Cytogenetic analysis showed a 46,X,mar karyotype. The marker was identified as a der(Y)t(Xp;Yp) by fluorescence in situ hybridisation analysis. Further studies with specific locus probes of X and Y chromosomes made it possible to clarify the break points and demonstrated the presence of two copies of the DAX1 gene, one on the normal X chromosome and one on the der(Y). The karyotype of the child was: 46,X,der(Y)t(X;Y)(p21.2;p11.3). The syndrome resulted from functional disomy Xp21.2-pter, with sex reversal related to the presence of two active copies of the DAX1 gene located in Xp21. Few cases of Xp disomy with sex reversal have been reported, primarily related to Xp duplications with 46,XY karyotype, and less often to Xp;Yq translocations. To our knowledge, our patient with sex reversal and a t(Xp;Yp) is the second reported case.     

Differentially regulated and evolved genes in the fully sequenced Xq/Yq pseudoautosomal region

Human sex chromosomes, which are morphologically and genetically different, share few regions of homology. Among them, only pseudoautosomal regions (PARs) pair and recombine during meiosis. To better address the complex biology of these regions, we sequenced the telomeric 400 kb of the long arm of the human X chromosome, including 330 kb of the human Xq/YqPAR and the telomere. Sequencing reveals subregions with distinctive regulatory and evolutionary features. The proximal 295 kb contains two genes inactivated on both the inactive X and Y chromosomes [ SYBL1 and a novel homologue ( HSPRY3 ) of Drosophila sprouty ]. The GC-rich distal 35 kb, added in stages and much later in evolution, contains the X/Y expressed gene IL9R and a novel gene, CXYorf1, only 5 kb from the Xq telomere. These properties make Xq/YqPAR a model for studies of region-specific gene inactivation, telomere evolution, and involvement in sex-limited conditions.     

Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes

In therian mammals (placentals and marsupials), sex is determined by an XX female: XY male system, in which a gene (SRY) on the Y affects male determination. There is no equivalent in other amniotes, although some taxa (notably birds and snakes) have differentiated sex chromosomes. Birds have a ZW female: ZZ male system with no homology with mammal sex chromosomes, in which dosage of a Z-borne gene (possibly DMRT1) affects male determination. As the most basal mammal group, the egg-laying monotremes are ideal for determining how the therian XY system evolved. The platypus has an extraordinary sex chromosome complex, in which five X and five Y chromosomes pair in a translocation chain of alternating X and Y chromosomes. We used physical mapping to identify genes on the pairing regions between adjacent X and Y chromosomes. Most significantly, comparative mapping shows that, contrary to earlier reports, there is no homology between the platypus and therian X chromosomes. Orthologs of genes in the conserved region of the human X (including SOX3, the gene from which SRY evolved) all map to platypus chromosome 6, which therefore represents the ancestral autosome from which the therian X and Y pair derived. Rather, the platypus X chromosomes have substantial homology with the bird Z chromosome (including DMRT1) and to segments syntenic with this region in the human genome. Thus, platypus sex chromosomes have strong homology with bird, but not to therian sex chromosomes, implying that the therian X and Y chromosomes (and the SRY gene) evolved from an autosomal pair after the divergence of monotremes only 166 million years ago. Therefore, the therian X and Y are more than 145 million years younger than previously thought.     

Characterization of a supernumerary small marker X chromosome in two females with similar phenotypes

We describe two female patients mosaic for a cell line with an extra marker X chromosome in addition to a normal 46,XX cell line. To our knowledge, these cases are the first reports of females who had a cell line with a supernumerary marker X chromosome in addition to a normal cell line. They also had strikingly similar manifestations, including small hands and feet, minor facial anomalies, obesity, and mental retardation. The DNA content of the mar(X) chromosomes was investigated by fluorescent in situ hybridization using pericentromeric probes. The XIST gene, which is necessary for initiation of X-inactivation, was deleted from both marker chromosomes, suggesting that these chromosomes were not subject to inactivation. The short arm breakpoints of the mar(X)s were between the DNA markers DXS423E on Xp11.21 and UBE1 on Xp11.23. In Patient 1, mar(X) contained the androgen receptor gene and the DNA marker DXS1, both mapping to Xq11.2, whereas in Patient 2 the chromosome breakpoint was proximal to these markers. We suggest that the similar phenotypes of these patients may be due to the overexpression of genes in the common pericentromeric region of the X chromosome. Am. J. Med. Genet. 76:45–50, 1998. © 1998 Wiley-Liss, Inc.     

Characterization and use of somatic cell hybrids with interspecific translocations involving the human X chromosome

Two hybrid cell lines, whose only human material was a portion of the X translocated on to a mouse chromosome, have been characterized by cytogenetics, in situ hybridization and Southern blotting. In one hybrid (HORL911R8B) the region Xpter to Xq2(2–4) was identified. In the other (PIP) the single human fragment was found to contain sequences from two separate X chromosomal regions (corresponding approximately to Xp11.4–Xp22.1 and Xq26–Xqter). These two hybrids in combination with a third (WAG 8) retaining Xqter to Xp21 as a human X-autosome translocation chromosome, form a mapping panel for rapid subregional assignments to the human X chromosome. This mapping panel has been used to provide information about the order of DNA sequences derived from the X chromosome and to provide an assignment for an anonymous DNA segment, M201γ, to Xp11.4–Xp21.1.     

MECP2 duplications in six patients with complex sex chromosome rearrangements

Duplications of the Xq28 chromosome region resulting in functional disomy are associated with a distinct clinical phenotype characterized by infantile hypotonia, severe developmental delay, progressive neurological impairment, absent speech, and proneness to infections. Increased expression of the dosage-sensitive MECP2 gene is considered responsible for the severe neurological impairments observed in affected individuals. Although cytogenetically visible duplications of Xq28 are well documented in the published literature, recent advances using array comparative genomic hybridization (CGH) led to the detection of an increasing number of microduplications spanning MECP2. In rare cases, duplication results from intrachromosomal rearrangement between the X and Y chromosomes. We report six cases with sex chromosome rearrangements involving duplication of MECP2. Cases 1-4 are unbalanced rearrangements between X and Y, resulting in MECP2 duplication. The additional Xq material was translocated to Yp in three cases (cases 1-3), and to the heterochromatic region of Yq12 in one case (case 4). Cases 5 and 6 were identified by array CGH to have a loss in copy number at Xp and a gain in copy number at Xq28 involving the MECP2 gene. In both cases, fluorescent in situ hybridization (FISH) analysis revealed a recombinant X chromosome containing the duplicated material from Xq28 on Xp, resulting from a maternal pericentric inversion. These cases add to a growing number of MECP2 duplications that have been detected by array CGH, while demonstrating the value of confirmatory chromosome and FISH studies for the localization of the duplicated material and the identification of complex rearrangements.     

Mother and daughter with 45,X/46,X,r(X)(p22.3q28) and mental retardation: analysis of the X-inactivation patterns

We report on a mother and daughter both with a 45,X/46,X,r(X)(p22. 3q28) karyotype and mental retardation. Fluorescence in situ hybridization (FISH) and microsatellite analyses for 14 loci/region at Xp22.3 and seven loci/region at Xq28 indicated that the ring X chromosome was missing a roughly 12-Mb region from Xp22.3 with the breakpoint between DXS85 and DXS9972, and another region of less than 100 kb from Xq28 with the breakpoint distal to the region defined by the FISH probe c8.2/1. X-inactivation analysis, using the methylation status of the AR gene (exon 1) as an indicator, showed that the normal and ring X chromosomes in the X,r(X)(p22.3q28) cell lineage were randomly inactivated. The Xp22.3 deleted region partially overlaps with the regional intervals of MRX19, MRX21, MRX24, MRX37, MRX43, and MRX49 associated with heterozygote manifestation. Therefore, it is likely that one or more of these MRX genes, subject to X-inactivation, are lost from the ring X chromosome, and that reduced expression of the MRX gene(s) caused by random X-inactivation has resulted in mental retardation in the mother and daughter.     

XY Sex Reversal in the Wood Lemming is Associated with Deletion of Xp21–23 as Revealed by Chromosome Microdissection and Fluorescence in situ Hybridization

In the wood lemming (Myopus schisticolor), XY sex reversal occurs naturally because of the presence of an X chromosome variant designated X*. The two types of X chromosome, X and X*, can be distinguished by G-banding, and analyses have demonstrated complex rearrangements of the short arm of X*. Here, chromosomal microdissection, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and fluorescence in situ hybridization (FISH) techniques have been used to generate and map DNA probes for different parts of the X and X* chromosomes. The results showed that the region of Xp21–23 is deleted from the X* and some of the deleted DNA sequences are homologous to the mouse gamma-satellite. The deletion must be associated with the sex reversal in this species. FISH experiments with dissected probes of X and distal half of Xq provided evidence for presence of homologous sequences between large regions of the X and Y chromosomes, including euchromatic and heterochromatic parts of the sex chromosomes. The findings of this study will be of significance for further cloning of important candidate gene(s) responsible for the XY sex reversal.     

Parental origin and mechanism of formation of X chromosome structural abnormalities: four cases determined with RFLPs

Parental origin and mechanism of formation of X chromosome structural abnormalities were studied in one each case of dup(X)(pter----p11.4::p22.1----qter), del(X)(qter----p11:), i(X)(qter----cen----qter), and inv dup(X) (pter----q22::q22----pter) using various X-linked RFLPs as genetic markers. Segregation and densitometric analyses on polymorphic DNAs revealed that the dup(Xp) and the del(Xp) are both of paternal origin and the i(Xq) and i dic(X) are of maternal origin. The dup(Xp) had arisen by an unequal sister chromatid exchange and the del(Xp) had occurred through an intrachromosomal breakage-reunion mechanism, both in the paternal X chromosome. The i(Xq) had arisen either through centromere fission of a maternal X chromosome, followed by duplication of its long-arm, or through a translocation between two maternal X chromosomes after meiotic crossing-over. The inv dup(X) arose through sister chromatid breakage and reunion in a maternal X chromosome. These results, together with those of previous studies, suggest that the de novo abnormalities due to events involving centromere disruption arise predominantly during oogenesis, while those due to simple breakage-reunion events occur preferentially during spermatogenesis.     

Organization and expression of human telomere repeat binding factor genes

The ends of mammalian chromosomes terminate in structures called telomeres. Recently a human telomere repeat binding factor (TRF1) that binds the vertebrate TTAGGG telomeric repeat in situ was isolated by Chong et al. (1). TRF1 regulates telomere length (2), which is often altered in cancer cells. To understand their genetic organization, TRF1 genes were localized to human chromosomes 13 cen, 21cen, and Xq13 by analysis of human monochromosomal hybrids, and by fluorescent in situ hybridization. We also confirmed the recent localization of a human TRF1 gene to chromosome 8, and provide evidence that this locus is alternatively spliced. In contrast to the TRF1 genes on chromosomes 8 and X, the chromosomes 13 and 21 TRF1 genes contained a 60 bp deletion in the coding region. The results suggest that two distinct forms of TRF1 are expressed and that the TRF1 gene family includes at least three pseudogenes whose dispersal in the human genome may have occurred via cDNA intermediates.