Cytologic evidence for three human X-chromosomal segments escaping inactivation

Summary Early replication of prometaphasic human sex chromosomes was studied with the bromodeoxyuridine (BrdU)-replication technique. The studies reveal that two distal segments of Xp, including bands Xp 22.13 and Xp 22.3, replicate early in S-phase and therefore may not be subject to random inactivation. Furthermore, the replication of these distal segments of Xp occurs synchronously with those of the short arm of the Y chromosome including bands Yp 11.2 and Yp 11.32. These segments of Xp and Yp correspond well to the pairing segment of the X and Y chromosomes where a synaptonemal complex forms at early pachytene of human spermatogenesis. The homologous early replication of Yp and the distal portion of Xp may be interpreted as a remnant left untouched by the differentiation of heteromorphic sex chromosomes from originally homomorphic autosomes. A third early replicating segment is situated on the long arm of the X chromosome and corresponds to band Xq 13.1. This segment may be correlated with the X-inactivation center postulated by Therman et al. (1979).     

Molecular cytogenetic analysis of a duplication Xp in a male: further delineation of a possible sex influencing region on the X chromosome

We describe a male infant with severe mental retardation and autism with a duplication of the short arm of the X chromosome. Chromosome painting confirmed the origin of this X duplication. Molecular cytogenetic analysis with fluorescence in situ hybridization (FISH) identified one copy of the zinc finger protein on the X chromosome (ZFX) and two copies of the steroid sulfatase gene (STS), further delineating the breakpoints. Based on cytogenetic and molecular comparisons of cases from the literature of sex-reversal in dup(X),Y patients and our patient, we suggest that a possible secondary sexinfluencing gene involved in the regulation of sex determination or testis morphogenesis is present at the distal Xp21.1 to p21.2 region.     

Pairing of X and Y chromosomes,non-inactivation of X-linked genes,and the maleness factor

Summary In this paper observations are summarized and speculations discussed, and it is suggested that some loci on the distal short arm of the X chromosome (Xp) are not randomly inactivated in the female, because they are within the proximal part of the pairing segment between Xp and Yp. This peculiarity of gene expression may be a remnant of the evolutionary history of the sex chromosomes, the pairing segment of which may involve at least 27% of Xp and 95% of Yp. Crossing over seems to occur mostly in the terminal third of the X/Y pairing segment. However, crossing-over inhibition control may lapse, or may be on the X and Y (e.g. Xg, H-Y, STS, and perhaps others) might cross over with a variable frequency which is proportional to their distances from the telomeres of the short arms. It is postulated that the DNA of the pairing segment is composed in a way which may also permit unequal crossing over to occur between the X and the Y, thereby giving rise to exceptions to X-or Y-linked inheritance. The peculiarities of behaviour and the position of other loci on the sex chromosomes are also discussed briefly.     

Is selection responsible for the low level of variation in the last intron of the ZFY locus?

DNA variability was investigated in the last intron of the Y-chromosome-specific zinc finger gene, ZFY, and its X homolog on Xp21.3, ZFX. No polymorphisms were found in the 676-bp ZFY segment in a sample of 205 world-wide-distributed Y chromosomes, other than a solitary nucleotide variant in one individual (nucleotide diversity pi = 0.0014%). In contrast, 10 segregating sites (pi = 0.082%) were identified within 1,089 bp of the ZFX sequence in a sample of 336 X chromosomes. Four of these polymorphisms, which contributed most of the diversity, were located within an Alu insert disrupting the ZFY-ZFX homology (pi Alu = 0.24%). The diversity in the homologous portion of the ZFX intron, although higher than that in ZFY, was lower than that found in genomic segments believed to evolve neutrally; interspecies divergence in both segments was also reduced. Although this suggests that the evolution of both ZFY and ZFX homologs may not be entirely neutral, both Tajima and HKA tests did not reject neutrality. The lack of statistical significance may be attributed to a lack of power in these tests (the low divergence and variability values reduce the power of the HKA and Tajima tests, respectively); furthermore, Homo sapiens has recently undergone a rapid population growth, and selection is more difficult to detect in an expanding population. Therefore, the failure to reject neutrality does not necessarily indicate the absence of selection. In this context, the phylogenetic argument was given more weight in out interpretations. The high level of sequence identity in ZFY and ZFX segments, in spite of their separation 80-130 MYA, reflects a lower mutation rate as compared with other segments believed to undergo unconstrained evolution. Thus, the possibility of weak selection contributing to the low level of nucleotide diversity in the last ZFY intron cannot be excluded and should be kept in mind in the population genetics studies based on Y chromosome variability.     

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.     

Homologous early replication patterns of the distal short arms of prometaphasic X and Y chromosomes

Summary Replication studies on prometaphasic human sex chromosomes reveal a distinct early replicating segment on both distal Xp and Yp. These segments correspond to high resolution bands Xp22.3 and Yp11.3. The findings demonstrate synchronous replication of these parts of the sex chromosomes and correspond to the comparatively long stretches of Xp and Yp that participate in a synaptonemal complex. Furthermore these observations are compatible with Polani's view that suggests homologous segments with similar genetic information on both sex chromosomes (Polani 1982).     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

Genes on the short arm of the human X chromosome are not shared with the marsupial X.

Eight genes located on the short arm of the human X chromosome (MAOA, SYN1, OAT, OTC, CYBB, DMD, ZFX, POLA) have been mapped in several marsupial species by cell hybrid analysis and/or in situ hybridization using probes derived from human cDNA. Seven appear to be autosomal in all marsupial species examined. The eighth, CYBB, detected a site on the X, as well as major autosomal sites. Although these genes are not conserved on the X chromosome in marsupials, at least some of them are arranged together in autosomal clusters. The autosomal location of human Xp genes in marsupials could mean that this region either was lost from a large ancestral X chromosome in the marsupial lineage or was acquired by a small ancestral X (and perhaps Y) in the eutherian lineage. Either explanation demands that the region was not subject to X chromosome inactivation in a common ancestor 120-150 MyrBP.     

Sex reversal due to Xp disomy by t(X;Y)(p21;q11).

A 20-month-old infant exhibiting psychomotor retardation, dysmorphisms and ambiguous external genitalia was found to have a 46-chromosome karyotype including a normal X chromosome and a marker Y with most of Yq being replaced by an extra Xp21-->pter segment. The paternal karyotype (G and C bands) was 46,XY. The marker Y composition was verified by means of FISH with a chromosome X painting, an alphoid repeat and a DMD probe. Thus, the final diagnosis was 46,X,der(Y)t(X;Y)(p21;q11)de novo.ish der(Y)(wcpX+,DYZ3+,DMD+). The patient's phenotype is consistent with the spectrum documented in 13 patients with similar Xp duplications in whom sex reversal with female or ambiguous genitalia has occurred in spite of an intact Yp or SRY gene. A review of t(X;Y) identifies five distinct exchanges described two or more times: t(X;Y)(p21;q11), t(X;Y)(p22;p11), t(X;Y)(p22;q11-12), t(X;Y) (q22;q12), and t(X;Y)(q28;q12). These translocations probably result from a recombination secondary to DNA homologies within misaligned sex chromosomes in the paternal germline with the derivatives segregating at anaphase I.     

Mapping the human ZFX locus to Xp21.3 by in situ hybridization

Summary In situ hybridization using a probe specific for the human ZFX and ZFY loci assigns the ZFX gene to Xp21.3 and the ZFY gene to Yp11.32.     

Characterization of the Xp21-23 region in the wood lemming, a region involved in XY sex reversal.

The wood lemming (Myopus schisticolor) harbors two types of X chromosome, a normal X and a variant X, designated X*. The X* chromosome contains a mutation that causes XY sex reversal. We have previously demonstrated that the Xp21-23 region is deleted from X* and is associated with XY sex reversal. To further analyze the deleted region, we have constructed and characterized seven X chromosome- and region-specific recombinant DNA libraries. Further, we have screened mouse fetal gonad cDNA libraries with the microdissected Xp21-23 DNA as a probe in an attempt to identify homologous and expressed sequences from the deletion. Fourteen positive clones were isolated, and sequence analyses showed that ten of these contained identical sequences homologous to mouse gamma-satellite sequences. One of the remaining four was perfectly homologous to the mouse gene Ccth (chaperonin containing t-complex polypeptide 1, eta subunit). Southern blot indicated that the Ccth cDNA was located on the X chromosome, not deleted from the X* but closely linked to the deletion region. Although the role of the Ccth containing region in sex determination of the wood lemming requires additional studies, the isolation of the mouse Ccth gene by the deletion Xp21-23 probe could be important since this gene is mainly expressed in testis.     

Genetic counselling in carriers of reciprocal chromosomal translocations involving short arm of chromosome X

A central concept in genetic counselling is the estimation of the probability of occurrence of unbalanced progeny at birth and other unfavourable outcomes of pregnancy (miscarriages, stillbirths and early death). The estimation of the occurrence probability for individual carriers of four different X-autosome translocations with breakpoints at Xp, namely t(X;5)(p22.2;q32), t(X;6)(p11.2;q21), t(X;7)(p22.2;p11.1), and t(X;22)(p22.1;p11.1), is presented. The breakpoint positions of chromosomal translocations were interpreted using GTG, RBG and FISH-wcp. Most of these translocations were detected in women with normal phenotype, karyotyped because of repeated miscarriages and/or malformed progeny. A girl with very rare pure trisomy Xp22.1-->pter and a functional Xp disomy was ascertained in one family and her clinical picture has been described in details. It has been suggested that not fully skewed X chromosome inactivation of X-autosome translocation with breakpoint positions at Xp22 (critical segment) could influence the phenotype and risk value. Therefore, the X inactivation status was additionally evaluated by analysis of replication banding patterns using RBG technique after incorporation of BrdU. In two carriers of translocations: t(X;5)(p22.2;q32) and t(X;7)(p22.2;p11.1), late replication state of der(X) was observed in 5/100 and 10/180 analysed cells, respectively. In these both cases the breakpoint positions were clustered at the critical segment Xp22.2. In two other cases, one with the breakpoint position within [t(X;22)(p22.1;p11.1)] and one outside the critical region [t(X;6)(p11.2;q21)], fully skewed inactivation was seen. Therefore, we suggest that neither the distribution of the breakpoint positions nor fully skewed inactivation influenced the phenotype of observed t(X;A) carriers. The occurrence probabilities of the unbalanced progeny were calculated according to Stene and Stengel-Rutkowski along with application of updated available empirical data. In the studied group the values of occurrence probability for unbalanced offspring at birth ranged from 2.1% to 17%. Information on the magnitude of the individual figures may be important for women carrying a reciprocal X;A translocation when deciding upon further family planning.     

Mapping DNA sequences in a human X-chromosome deletion which extends across the region of the Duchenne muscular dystrophy mutation.

A somatic cell hybrid has been constructed and characterized using fibroblasts from a phenotypically normal woman who possesses an X chromosome with an interstitial deletion of the short arm. High-resolution banding indicates that the deleted segment is either Xp22.13-p11.4 or Xp22.11-p11.23. Southern blot hybridization to previously mapped DNA sequences confirms that the missing segment of the X chromosome is a deletion and not an interstitial translocation and supports the cytogenetic interpretation that the deletion extends proximal of Xp11.3 and therefore probably comprises Xp22.11-p11.23. Three further DNA sequences have been localized to the region of the deleted segment. The following order has been assigned to the seven probes used: Xpter-RC8-pXUT22-(OA1,C7,M2C)-L1.28-RD6 -Xcen.     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

Cloning and comparative analysis of the bovine, porcine, and equine sex chromosome genes ZFX and ZFY.

A growing body of evidence suggests the involvement of sex chromosome genes in mammalian development. We report the cloning and characterization of the complete coding regions of the bovine Y chromosome ZFY and X chromosome ZFX genes, and partial coding regions of porcine and equine ZFX and ZFY genes. Bovine ZFY and ZFX are highly similar to each other and to ZFX and ZFY from other species. While bovine and human ZFY proteins are both 801 amino acids long, bovine ZFX is 5 amino acids shorter than human ZFX. Like in humans, both bovine ZFY and ZFX contain 13 zinc finger motifs and belong to the Krueppel family of C2H2-type zinc finger proteins. The internal exon-intron organization of the bovine, porcine and equine ZFX and ZFY genes has been determined and compared. Within this region, the exon lengths and the positions of the splice sites are conserved, further suggesting a high evolutionary conservation of the ZFX and ZFY genes. Additionally, new alternatively spliced forms of human ZFX have been identified.     

Regional assignments of the zinc finger Y-linked gene (ZFY) and related sequences on human and mouse chromosomes

Recent chromosome walking experiments have identified a candidate gene (ZFY) for the testis-determining factor on the human Y chromosome (Page et al., 1987). We report here the regional assignments of the ZFY gene and related sequences in the human and the mouse. By in situ hybridization, we assigned ZFX and ZFY to human chromosome bands Xp21 and Yp11.3, respectively. Although the mouse harbors two Zfy genes, only one site at band A1 of its Y chromosome was significantly labeled. The mouse Zfx gene and the Zfa gene on chromosome 10 were assigned to bands XD and 10B5, respectively. These assignments of the ZFX gene in human and mouse add another marker to the conserved syntenic group for evaluating the evolutionary relationship of the human and mouse X chromosomes.     

X-Y crossing over in the chimpanzee

Summary Single-copy DNA sequences defining several pseudoautosomal loci on the human sex chromosomes are shown to be highly conserved in the genome of the chimpanzee. Segregation analysis of polymorphic pseudoautosomal probes in a chimpanzee pedigree revealed that the transmission of the paternal alleles was not strictly sex-linked. In situ hybridization localized the pseudoautosomal probe 29C1 specifically to Xp22-Xpter and to Yq12.2-Yqter on the chimpanzee sex chromosomes. Thus, our results demonstrate the existence of homologous segments on the chimpanzee X and Y chromosomes, which regularly undergo recombinatory exchange in male meiosis. The chimpanzee is now the third mammalian species, besides man and mouse, in which there is genetic evidence for a pseudoautosomal segment on the sex chromosomes.     

Nullisomy for the distal portion of Xp in a male child with a X/Y translocation

Summary An unbalanced X/Y translocation was found in a male child with malformed external genitalia and in his mother, who are respectively nullisomic and monosomic for the distal portion of Xp and have the translocated distal segment of Yq in excess. The loss of the distal portion of Xp is supposed to be the cause of the phenotypic abnormalities present in these subjects. The phenotype of our subjects is compared with those of the other cases of X/Y translocation described in the literature.