ESS gene expression of X-linked imprinted genes subject to sexual selection

We define ESS (Evolutionary Stable Strategy) conditions for the evolution of genomic imprinting at an X-linked locus. The system analysed is designed for mammalian imprinting in which X-linked genes typically undergo random X-inactivation and lack Y-linked homologues. We consider two models that map cellular gene expression to fitness in females subject to random X-inactivation. In the first model, female fitness is simply a function of the average gene expression across all cells. In the second model, each cell contributes independently to fitness, and female fitness is assessed as the average of these contributions across all cells. In both models, imprinting readily evolves when sexual selection favours different levels of gene expression in the two sexes. Imprinting is beneficial as it improves adaptation in both sexes. There are limits to the improvement in adaptation when sexual selection is strong and favours greater gene expression in males (the heterogametic sex). We also consider the consequences of an active Y-linked homologue on the evolution of imprinting. Our analysis suggests that restrictive conditions apply for the evolution of polymorphic ESSs at an X-linked imprinted loci.     

Dosage Regulation of the Active X Chromosome in Human Triploid Cells

In mammals, dosage compensation is achieved by doubling expression of X-linked genes in both sexes, together with X inactivation in females. Up-regulation of the active X chromosome may be controlled by DNA sequence–based and/or epigenetic mechanisms that double the X output potentially in response to autosomal factor(s). To determine whether X expression is adjusted depending on ploidy, we used expression arrays to compare X-linked and autosomal gene expression in human triploid cells. While the average X:autosome expression ratio was about 1 in normal diploid cells, this ratio was lower (0.81–0.84) in triploid cells with one active X and higher (1.32–1.4) in triploid cells with two active X''s. Thus, overall X-linked gene expression in triploid cells does not strictly respond to an autosomal factor, nor is it adjusted to achieve a perfect balance. The unbalanced X:autosome expression ratios that we observed could contribute to the abnormal phenotypes associated with triploidy. Absolute autosomal expression levels per gene copy were similar in triploid versus diploid cells, indicating no apparent global effect on autosomal expression. In triploid cells with two active X''s our data support a basic doubling of X-linked gene expression. However, in triploid cells with a single active X, X-linked gene expression is adjusted upward presumably by an epigenetic mechanism that senses the ratio between the number of active X chromosomes and autosomal sets. Such a mechanism may act on a subset of genes whose expression dosage in relation to autosomal expression may be critical. Indeed, we found that there was a range of individual X-linked gene expression in relation to ploidy and that a small subset (∼7%) of genes had expression levels apparently proportional to the number of autosomal sets.     

Evidence for X-Linkage of Steroid Sulfatase in the Mouse: Steroid Sulfatase Levels in Oocytes of XX and XO Mice

The steroid sulfatase (STS) levels in mature oocytes of XX and XO mice were assayed along with lactate dehydrogenase (LDH), an autosomal marker, and glucose-6-phosphate dehydrogenase (G6PD), a known X-linked gene. LDH levels in XX and XO oocytes were equal, whereas STS and G6PD levels were approximately twice as high in XX oocytes as in XO oocytes. These results indicate that the STS gene is X-linked in the mouse just as it is in humans. Assays of STS in kidney tissue of XX and XO mice indicated dosage compensation for the gene, which is different from that observed in humans.     

Disruption of the serine/threonine kinase 9 gene causes severe X-linked infantile spasms and mental retardation

X-linked West syndrome, also called "X-linked infantile spasms" (ISSX), is characterized by early-onset generalized seizures, hypsarrhythmia, and mental retardation. Recently, we have shown that the majority of the X-linked families with infantile spasms carry mutations in the aristaless-related homeobox gene (ARX), which maps to the Xp21.3-p22.1 interval, and that the clinical picture in these patients can vary from mild mental retardation to severe ISSX with additional neurological abnormalities. Here, we report a study of two severely affected female patients with apparently de novo balanced X;autosome translocations, both disrupting the serine-threonine kinase 9 (STK9) gene, which maps distal to ARX in the Xp22.3 region. We show that STK9 is subject to X-inactivation in normal female somatic cells and is functionally absent in the two patients, because of preferential inactivation of the normal X. Disruption of the same gene in two unrelated patients who have identical phenotypes (consisting of early-onset severe infantile spasms, profound global developmental arrest, hypsarrhythmia, and severe mental retardation) strongly suggests that lack of functional STK9 protein causes severe ISSX and that STK9 is a second X-chromosomal locus for this disorder.     

Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.     

Gene Expression in Adult Metafemales of Drosophila Melanogaster

The expression of selected X-linked and autosomal genes was examined in metafemales (3X:2A) compared to diploid sisters. Three enzyme activities (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, beta-hydroxyacid dehydrogenase) encoded by X-linked genes are not significantly different in the two classes of flies. In contrast, three autosomally encoded enzyme activities (alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, isocitrate dehydrogenase) are reduced in metafemales. Protein and DNA comparisons between metafemales and diploid sisters show a lowered level of total protein whereas the total DNA measurements are similar. Thus, the total cell number in metafemales is basically unchanged but gene expression is reduced. Phenotypic analysis of three autosomal loci, glass (gl), purple (pr) and pink-peach (pp), show that all three have lowered expression in metafemales while the X-linked loci, white-apricot (wa) and Bar (B), are dosage compensated. Quantitative dot blot analysis of messenger RNA levels of the second chromosomal locus, alcohol dehydrogenase (Adh), and the X chromosomal locus, rudimentary (r), show that Adh has reduced expression and r is partially compensated per total RNA in metafemales. It is proposed that the increased dosage of the X chromosome inversely affects both the X and autosomal gene expression but the simultaneous increased dosage of the structural genes on the X results in dosage compensation. The reduced levels of expression of autosomal genes could contribute to the great inviability of metafemales.     

Genetic localization and phenotypic expression of X-linked cataract (Xcat) in Mus musculus

Linkage data relative to the markers tabby and glucose-6-phosphate dehydrogenase are presented to locate X-linked cataract (Xcat) in the distal portion of the mouse X-chromosome between jimpy and hypophosphatemia. The human X-linked cataract-dental syndrome, Nance-Horan Syndrome, also maps closely to human hypophosphatemia and would suggest homology between mouse Xcat and human Nance-Horan Syndrome genes. In hemizygous males and homozygous females penetrance is complete with only slight variation in the degree of expression. Phenotypic expression in Xcat heterozygous females ranges from totally clear to totally opaque lenses. The phenotypic expression between the two lenses of a heterozygous individual could also vary between totally clear and totally opaque lenses. However, a correlation in the degree of expression between the eyes of an individual was observed. A variegated pattern of lens opacity was evident in female heterozygotes. Based on these observations, the site of gene action for the Xcat locus is suggested to be endogenous to the lens cells and the precursor cell population of the lens is concluded to be small. The identification of an X-linked cataract locus is an important contribution to the estimate of the number of mutable loci resulting in cataract, an estimate required so that dominant cataract mutagenesis results may be expressed on a per locus basis. The Xcat mutation may be a useful marker for a distal region of the mouse X-chromosome which is relatively sparsely marked and the X-linked cataract mutation may be employed in gene expression and lens development studies.     

HGPRT mutation induction by N-ethyl-N-nitrosourea as measured by 6-thioguanine resistance is higher in male than in female Syrian hamster fetuses

BACKGROUND: The consequences of mutations in embryonic and fetal cells are serious and contribute to high prenatal sensitivity to mutagenic agents. An understanding of the factors that influence the yield of such mutations is important for management of adverse effects of perinatal exposures. Resistance to 6-thioguanine (6-TG) can be utilized to study mutational events at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. HGPRT is X-linked and recessive. According to the Lyon hypothesis, male cells have only one X-chromosome and female cells randomly inactivate the second X-chromosome. This leads to the prediction that X-linked genes should be equally sensitive to the mutagenic effects of toxicants in male and female fetuses. METHODS: We tested this supposition by in utero exposure of Syrian hamster fetuses to N-ethyl-N-nitrosourea (ENU) at day 12 of gestation. ENU is a strong carcinogen and mutagen. HGPRT mutations were detected by selection with 6-TG. RESULTS: Surprisingly. the male cells had 4 to 5 times more 6-TG mutants than female cells, in two separate experiments (p<0.001). Ouabain resistance, reflecting a co-dominant autosomal locus, was used as a control, and we found that there was no significant difference between male and female cells (p=0.549). CONCLUSIONS: Possible reasons for the sex difference in mutations include escape of the second X-chromosome from inactivation in some of the female cells, or higher mutability in male cells. In any event, there is a gender difference in vulnerability to mutation of an X-linked gene that has previously not been appreciated, and that may be relevant to toxicological studies of such genes. HGPRT is frequently used to monitor mutagenic events in human fetuses.     

Serological evidence for H-Y antigen in XO-female mice

Summary H-Y antigen was examined in XX-, XY-, and XO-mice using spleen, kidney, and liver cells of the animals for the absorption of the anti-H-Y antiserum produced in the rat. The cells of the XY- and XO-mice were found to be H-Y antigenpositive while the cells of the XX-mice were negative. As in Turner syndrome patients with 45,X, in the XO-female mice the H-Y antigen titre was reduced as compared to normal XY-male mice; intermediate values between those of normal male and female mice were obtained. These results clearly indicate that as in man, in the mouse the structural gene for H-Y antigen is not Y-linked but is located on an autosome. Furthermore, the concept of the regulation of the H-Y antigen gene expression in the human (Wolf et al. 1980a, b) by an X-linked repressor gene, escaping X-inactivation in the XX-female and an Y-linked inducer gene also seems to hold true in the mouse.     

Imprint switching for non-random X-chromosome inactivation during mouse oocyte growth

In mammals, X-chromosome inactivation occurs in all female cells, leaving only a single active X chromosome. This serves to equalise the dosage of X-linked genes in male and female cells. In the mouse, the paternally derived X chromosome (X(P)) is imprinted and preferentially inactivated in the extraembryonic tissues whereas in the embryonic tissues inactivation is random. To investigate how X(P) is chosen as an inactivated X chromosome in the extraembryonic cells, we have produced experimental embryos by serial nuclear transplantation from non-growing (ng) oocytes and fully grown (fg) oocytes, in which the X chromosomes are marked with (1) an X-linked lacZ reporter gene to assay X-chromosome activity, or (2) the Rb(X.9)6H translocation as a cytogenetic marker for studying replication timing. In the extraembryonic tissues of these ng/fg embryos, the maternal X chromosome (X(M)) derived from the ng oocyte was preferentially inactivated whereas that from the fg oocyte remained active. However, in the embryonic tissues, X inactivation was random. This suggests that (1) a maternal imprint is set on the X(M) during oocyte growth, (2) the maternal imprint serves to render the X(M) resistant to inactivation in the extraembryonic tissues and (3) the X(M) derived from an ng oocyte resembles a normal X(P).     

The mode of genetic transmission of a gouty family with increased phosphoribosylpyrophosphate synthetase activity

Summary The mode of genetic transmission of gout and increased activity of phosphoribosylpyrophosphate synthetase (PRPPS) was studied in one family. Among 15 members of Family F, two male members had gout and had PRPPS activity of erythrocyte lysates three times higher than normal subjects. Five female members had activity 2.5 times higher than normal. The difference between the activities of male and female affected members was statistically significant (P<0.05). To examine the genetic trait of this abnormal PRPPS, the incorporation of ³H-adenine into erythrocytes or lymphocytes was studied using autoradiography. The number of grains which show the uptake of labeled adenine into cells revealed a normal distribution pattern in two normal persons and in two male patients, and a mixed pattern of the two cell populations in two female affected members. These results suggested mosaicism in female members and X-linked dominant transmission of this trait. Thermal inactivation of PRPPS of an affected female was intermediate between that from a normal subject and that from the affected males. This result showed the heterogeneity of the PRPPS from the hemolysate of an affected famale. The genotype of PRPPS on the X-chromosome was assumed and the lod score between PRPPS and Xg was also estimated. From these findings and electrophoretical study, it was suggested that the abnormal enzyme was a mutant enzyme transmitted in an X-linked dominant trait, and that the mutation occurred on the structural gene of the PRPPS.     

A novel cell model to study the function of the adrenoleukodystrophy-related protein

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder due to mutations in the ABCD1 (ALD) gene. ALDRP, the closest homolog of ALDP, has been shown to have partial functional redundancy with ALDP and, when overexpressed, can compensate for the loss-of-function of ALDP. In order to characterize the function of ALDRP and to understand the phenomenon of gene redundancy, we have developed a novel system that allows the controlled expression of the ALDRP-EGFP fusion protein (normal or non-functional mutated ALDRP) using the Tet-On system in H4IIEC3 rat hepatoma cells. The generated stable cell lines express negligible levels of endogenous ALDRP and doxycycline dosage-dependent levels of normal or mutated ALDRP. Importantly, the ALDRP-EGFP protein is targeted correctly to peroxisome and is functional. The obtained cell lines will be an indispensable tool in our further studies aimed at the resolution of the function of ALDRP to characterize its potential substrates in a natural context.     

Expression of an X-linked muscular dystrophy in a female due to translocation involving Xp21 and non-random inactivation of the normal X chromosome

Summary A young female was diagnosed as having X-linked muscular dystrophy of the Duchenne type. Chromosome studies, including trypsin-Giemsa banding, Quinacrine fluorescence, and nucleolus organizer region (NOR) silver staining revealed an X-autosome reciprocal translocation t(X;21) (p21;p12). Utilizing both [³H] thymidine autoradiography and the BrdU-Hoechst 33258-Giemsa technique, lymphocytes and fibroblasts were found to show a preferential inactivation of the normal X suggesting the presence of a single mutant gene on the translocated X. This patient is one of seven reported cases of an X-linked muscular dystrophy associated with an X-autosome translocation. In all seven cases the exchange point in the X chromosome is in band p21 at or near the site of the Duchenne gene.