Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.

Closely linked restriction fragment length polymorphisms (RFLPs) are potentially useful as diagnostic markers of genetic defects, and, in principle, RFLPs can be employed to construct a complete linkage map of the human genome. On the X chromosome, linkage studies are particularly rewarding because in man more than 120 X-linked genes are known. Thus, it is probable that each X-specific RFLP will be of use as a genetic marker of one or several X-linked disorders. To facilitate the search for closely linked RFLPs, we have regionally assigned 16 cloned DNA sequences to various portions of the human X chromosome, employing a large panel of somatic cell hybrids. These probes have been used to correlate genetic and physical distances on Xp, and it can be extrapolated from these data that the number and distribution of available Xq sequences will also suffice to span the long arm of the X chromosome.     

X chromosome inactivaton and SV40 transformation of mammalian cells.

Five embryonic mouse cultures and one human fibroblast culture were transformed with SV40. The cultures were studied cytologically to see if the normal pattern of sex chromosome replication was maintained in SV40 transformed cells. Characteristic late replication patterns were observed for both the X and Y chromosomes, and there was no evidence for loss of the inactive X chromosome, even in cells with 4 or more X chromosomes. The human line was heterozygous at two X-linked loci and a clonal analysis showed that the expression of X-linked genes was not affected by SV40 transformation.     

X Chromosome Duplications Affect a Region of the Chromosome They Do Not Duplicate in Caenorhabditis Elegans

X chromosome duplications have been used previously to vary the dose of specific regions of the X chromosome to study dosage compensation and sex determination in Caenorhabditis elegans. We show here that duplications suppress and X-linked hypomorphic mutation and elevate the level of activity of an X-linked enzyme, although these two genes are located in a region of the X chromosome that is not duplicated. The effects do not depend on the region of the X chromosome duplicated and is stronger in strains with two doses of a duplication than in strains with one dose. This is evidence for a general elevation of X-linked gene expression in strains carrying X-chromosome duplications, consistent with the hypothesis that the duplications titrate a repressor acting on many X-linked genes.     

The human phosphoglycerate kinase multigene family. HLA-associated sequences and an X-linked locus containing a processed pseudogene and its functional counterpart

Several phosphoglycerate kinase genes were previously detected in the human genome by blot hybridization with a phosphoglycerate kinase cDNA probe. Using subcloned fragments of the cDNA we estimate the presence of four independent phosphoglycerate kinase genes. These genes have been mapped to both the human X chromosome (band q13) and chromosome 6 (p12-21.1) using a panel of human-rodent somatic cell hybrids and by chromosomal in situ hybridization. The genomic distribution of phosphoglycerate kinase sequences is conserved in man and mouse, not only for the X chromosome, but also for linkage to the respective major histocompatibility complexes. Molecular cloning of X-linked phosphoglycerate kinase sequences led to the identification of a novel intronless phosphoglycerate kinase pseudogene which is localized proximal to the active gene on the X chromosome.     

X‐chromosome‐located microRNAs in immunity: Might they explain male/female differences?

In this paper, we hypothesize that X chromosome-associated mechanisms, which affect X-linked genes and are behind the immunological advantage of females, may also affect X-linked microRNAs. The human X chromosome contains 10% of all microRNAs detected so far in the human genome. Although the role of most of them has not yet been described, several X chromosome-located microRNAs have important functions in immunity and cancer. We therefore provide a detailed map of all described microRNAs located on human and mouse X chromosomes, and highlight the ones involved in immune functions and oncogenesis. The unique mode of inheritance of the X chromosome is ultimately the cause of the immune disadvantage of males and the enhanced survival of females following immunological challenges. How these aspects influence X-linked microRNAs will be a challenge for researchers in the coming years, not only from an evolutionary point of view, but also from the perspective of disease etiology.     

Determination of a molecular map position for Hyp using a new interspecific backcross produced by in vitro fertilization.

We have established a Mus spretus/Mus musculus domesticus interspecific backcross segregating for two X-linked mutant genes, Ta and Hyp, using in vitro fertilization. The haplotype of the recombinant X chromosome of each of 241 backcross progeny has been established using the X-linked anchor loci Otc, Hprt, Dmd, Pgk-1, and Amg and the additional probes DXSmh43 and Cbx-rs1. The Hyp locus (putative homologue of the human disease gene hypophosphatemic rickets, HYP) has been incorporated into the molecular genetic map of the X chromosome. We show that the most likely gene order in the distal portion of the mouse X chromosome is Pgk-1-DXSmh43-Hyp-Cbx-rs1-Amg, from proximal to distal. The distance in centimorgans (mean +/- SE) between DXSmh43 and Hyp was 2.52 +/- 1.4 and that between Hyp and Cbx-rs1 was 1.98 +/- 1.39. Thus closely linked flanking markers for the Hyp locus that will facilitate the molecular characterization of the gene itself have been defined.     

X-linked premature ovarian failure: a complex disease

Involvement of the X chromosome in premature ovarian failure was demonstrated by the relatively frequent chromosomal rearrangements in patients, but the requirement of two X chromosomes for ovarian function was quite unexplained until recently. Review of the data on chromosomal rearrangements suggests that several genes along the X chromosomes contribute to ovarian function. In most instances, no single X chromosome gene has a causative role in premature ovarian failure, and the phenotype is likely to derive from the additive effect of X-linked and non-X-linked factors. Recent data on a small group of balanced X-autosome translocations showed that X-linked premature ovarian failure might also be caused by a different mechanism, namely position effect of the X chromosome on non-X-linked genes, and suggest a peculiar organization of the X chromosome during oogenesis.     

Polymorphic X-chromosome inactivation of the human TIMP1 gene.

X inactivation silences most but not all of the genes on one of the two X chromosomes in mammalian females. The human X chromosome preserves its activation status when isolated in rodent/human somatic-cell hybrids, and hybrids retaining either the active or inactive X chromosome have been used to assess the inactivation status of many X-linked genes. Surprisingly, the X-linked gene for human tissue inhibitor of metalloproteinases (TIMP1) is expressed in some but not all inactive X-containing somatic-cell hybrids, suggesting that this gene is either prone to reactivation or variable in its inactivation. Since many genes that escape X inactivation are clustered, we examined the expression of four genes (ARAF1, ELK1, ZNF41, and ZNF157) within approximately 100 kb of TIMP1. All four genes were expressed only from the active X chromosome, demonstrating that the factors allowing TIMP1 expression from the inactive X chromosome are specific to the TIMP1 gene. To determine if this variable inactivation of TIMP1 is a function of the hybrid-cell environment or also is observed in human cells, we developed an allele-specific assay to assess TIMP1 expression in human females. Expression of two alleles was detected in some female cells with previously demonstrated extreme skewing of X inactivation, indicating TIMP1 expression from the inactive chromosome. However, in other cells, no expression of TIMP1 was observed from the inactive X chromosome, suggesting that TIMP1 inactivation is polymorphic in human females.     

Disruption of a conserved region of Xist exon 1 impairs Xist RNA localisation and X-linked gene silencing during random and imprinted X chromosome inactivation

In XX female mammals a single X chromosome is inactivated early in embryonic development, a process that is required to equalise X-linked gene dosage relative to XY males. X inactivation is regulated by a cis-acting master switch, the Xist locus, the product of which is a large non-coding RNA that coats the chromosome from which it is transcribed, triggering recruitment of chromatin modifying factors that establish and maintain gene silencing chromosome wide. Chromosome coating and Xist RNA-mediated silencing remain poorly understood, both at the level of RNA sequence determinants and interacting factors. Here, we describe analysis of a novel targeted mutation, Xist(INV), designed to test the function of a conserved region located in exon 1 of Xist RNA during X inactivation in mouse. We show that Xist(INV) is a strong hypomorphic allele that is appropriately regulated but compromised in its ability to silence X-linked loci in cis. Inheritance of Xist(INV) on the paternal X chromosome results in embryonic lethality due to failure of imprinted X inactivation in extra-embryonic lineages. Female embryos inheriting Xist(INV) on the maternal X chromosome undergo extreme secondary non-random X inactivation, eliminating the majority of cells that express the Xist(INV) allele. Analysis of cells that express Xist(INV) RNA demonstrates reduced association of the mutant RNA to the X chromosome, suggesting that conserved sequences in the inverted region are important for Xist RNA localisation.     

Exclusion mapping of the gene for X-linked neural tube defects in an Icelandic family

Various polymorphic markers with a random distribution along the X chromosome were used in a linkage analysis performed on a family with apparently Xlinked recessive inheritance of neural tube defects (NTD). The lod score values were used to generate an exclusion map of the X chromosome; this showed that the responsible gene was probably not located in the middle part of Xp or in the distal region of Xq. A further refining of these results was achieved by haplotype analysis, which indicated that the gene for X-linked NTD was located either within Xp21.1-pter, distal from the DMD locus, or in the region Xq12–q24 between DXS106 and DXS424. Multipoint linkage analysis revealed that the likelihood for gene location is highest for the region on Xp. The region Xq26–q28, which has syntenic homology with the segment of the murine X chromosome carrying the locus for bent tail (Bn), a mouse model for X-linked NTD, is excluded as the location for the gene underlying X-linked NTD in the present family. Thus, the human homologue of the Bn gene and the present defective gene are not identical, suggesting that more than one gene on the X chromosome plays a role in the development of the neural tube.     

Sex-linked mammalian sperm proteins evolve faster than autosomal ones

X-linked genes can evolve slower or faster depending on whether most recessive, or at least partially recessive alleles are deleterious or beneficial due to their hemizygous expression in males. Molecular studies of X chromosome divergence have provided conflicting evidence for both a higher and lower rate of nucleotide substitution at both synonymous and nonsynonymous sites, depending on the nucleotide sites sampled. Using human and mouse orthologous genes, we tested the hypothesis that genes encoding male-specific sperm proteins are evolving faster on the X chromosome compared with autosomes. X-linked sperm proteins have an average nonsynonymous mutation rate almost twice as high as sperm genes found on autosomes, unlike other tissue-specific genes, where no significant difference in the nonsynonymous mutation rate between the X chromosome and autosomes was found. However, no difference was found in the average synonymous mutation rate of X-linked versus autosomal sperm proteins, which along with corresponding higher values of Ka/Ks in X-linked sperm proteins suggest that differences in selective forces and not mutation rates are the underlying cause of higher X-linked mammalian sperm protein divergence.     

Comparative mapping of Z-orthologous genes in vertebrates: implications for the evolution of avian sex chromosomes

Sex chromosomes of birds and mammals are highly differentiated and share several cytological features. However, comparative gene mapping reveals extensive conserved synteny between the chicken Z sex chromosome and human chromosome 9 but not the human X sex chromosome, implying an independent origin of avian and mammalian sex chromosomes. To better understand the evolution of the avian Z chromosome we analysed the synteny of chicken Z-linked genes in zebrafish, which is the best-mapped teleost genome so far. Existing zebrafish maps do not support the existence of an ancestral Z linkage group in the zebrafish genome, whereas mammalian X-linked genes show at least some degree of synteny conservation. This is consistent with in situ hybridisation mapping data in the freshwater pufferfish, Tetraodon nigroviridis where mammalian X-linked genes show a much higher degree of conserved synteny than human chromosome 9 or the avian Z chromosome. Collectively, these data argue in favour of a more recent evolution of the avian Z chromosome, compared with the mammalian X.     

Localization of the human X-linked gene for chronic granulomatous disease to the mouse X chromosome: implications for X-chromosome evolution

The gene encoded at the human X-linked chronic granulomatous disease locus (cytochrome b245 beta subunit) has been mapped to the mouse X chromosome using an interspecific Mus domesticus x M. spretus cross. The localization of this gene provides detailed information on one of the proposed ancestral breakpoints that account for the divergent evolution of the mouse and human X chromosomes.     

Assignment of genes to the human X chromosome by the two-dimensional electrophoretic analysis of total cell proteins from rodent-human somatic cell hybrids.

The technique of two-dimensional (2-D) gel electrophoresis was sued to identify five human X-linked gene products in crude cell extracts of mouse-human and Chinese hamster-human somatic cell hybrids. The human origin of these five polypeptides was demonstrated by their comigration with human fibroblast proteins and their failure to comigrate with polypeptides in extracts from the mouse or hamster parental cells. All five polypeptides were present in extracts of rodent-human hybrids that contained a human X chromosome, but were not found in extracts of cells that lacked a human X chromosome. Chromosome analysis of the hybrid clones revealed that the human X chromosome is both necessary and sufficient for the expression of the five polypeptides, designated pX-24, pX-27, pX-37, pX-40, and pX-56. pX-56 can be identified as the human X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) (E.C.1.1.1.49), while polypeptides pX-24, pX-27, pX-37 and pX-40 have molecular properties unlike those of known human X-linked gene products. pX-24 appears to be a membrane-bound protein that maps to the distal portion of the long arm of the human X chromosome, while pX-27, pX-37, and pX-40 are soluble proteins that map to the proximal long arm or to the short arm of the human X chromosome. 2-D gel electrophoretic analysis of extracts from somatic cell hybrids provides a general method for identifying polypeptides in crude cell extracts coded for by any specific chromosome and can be used to study primary gene products not previously amenable to genetic analysis.     

Microsatellite variation and evolutionary history of PCDHX/Y gene pair within the Xq21.3/Yp11.2 hominid-specific homology block

To better understand the evolutionary dynamics of repetitive sequences in human sex chromosomes, we have analyzed seven new X/Y homologous microsatellites located within PCDHX/Y, one of the two recently described gene pairs in the Xq21.3/Yp11.2 hominid-specific homology block, in samples from Portugal and Mozambique. Sharp differences were observed on X/Y allele distributions, concerning both the presence of private alleles and a different modal repeat length for X-linked and Y-linked markers, and this difference was statistically significant. Higher diversity was found in X-linked microsatellites than in their Y chromosome counterparts; when comparing populations, Mozambicans showed more allele diversity for the X chromosome, but the contrary was true for the Y chromosome microsatellites. Evolutionary patterns, relying on intragenic PCDHX/Y SNPs, also revealed distinct scenarios for X and Y chromosomes. Greater microsatellite diversity was displayed by African X chromosomes within the most common haplotypes shared by both populations, whereas higher microsatellite diversity was found in Portugal for the ancestral Y chromosome haplotype. The most frequent PCDHY haplotype in Portuguese was the derived one, and it was not found in Mozambicans. TMRCA estimated by the rho parameter resulted in 13,700 years (7,500-20,000 years), which is consistent with a recent, post-Out-of-Africa origin for this haplotype. In conclusion, the newly described microsatellite loci generally displayed greater X-linked to Y-linked diversity and this pattern was also detected with slower evolving markers, with a remarkable differentiation between populations observed for Y chromosome haplotypes and, thus, greater divergence among Y chromosomes in human populations.     

Heterozygous expression of X-linked chondrodysplasia punctata

Summary Two females showing partial expression of X-linked chondrodysplasia punctata were identified in a family. Bone dysplasia was caused by an aberrant X chromosome that had an inverse duplication of the segment Xp21.2–Xp22.2 and a deletion of Xp22.3-Xpter. To characterise the aberrant X chromosome, dosage blots were performed on genomic DNA from a carrier using a number of X-linked probes. Anonymous sequences from Xp21.2–Xp22.2 to which probes D2, 99.61, C7, pERT87-15, and 754 bind were duplicated on the aberrant X chromosome. The proposita was heterozygous for all these markers. Dosage blots also showed that the loci for steroid sulfatase and the cell surface antigen 12E7 (MIC2) were deleted as expected from the cytogenetic results. Mouse human cell hybrids were constructed that retained the normal X in the active state. Analysis of these hybrid clones for the markers from Xp21.2–Xp22.2 revealed that all the alleles of the informative markers, present in a single dosage in the genomic DNA, were carried on the normal X chromosome of the proposita. The duplicated X chromosome therefore had two identical alleles, indicating that the aberration resulted from an intrachromosomal rearrangement.