Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (1–4). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (5–7). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (9–14). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (9–15). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (10–13). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 14–18). This circumstance results in “autosomal” dosage compensation (14, 16–18). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 19–21). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 22–24). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 22–25). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).     

Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences

Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ～1.1-1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.     

Random X chromosome inactivation in a female with a variant of Wiskott-Aldrich syndrome

A 15-month-old female presented with eczema, thrombocytopenia, recurrent infections and failure to thrive. She had low serum IgM and IgG subclasses and an abnormal lymphocyte proliferative response to periodate in vitro. Molecular X chromosome inactivation analysis, using the polymorphic HUMARA DNA probe, showed that the infant has random X chromosome inactivation. We conclude that she has an atypical form of Wiskott-Aldrich syndrome which may be inherited in an autosomal recessive manner.     

Dynamics of telomere shortening in neutrophils and T lymphocytes during ageing and the relationship to skewed X chromosome inactivation patterns

Human haemopoiesis undergoes profound changes throughout life, resulting in compromised regenerative capacity of haemopoietic stem cells. It has been suggested that telomere shortening results in senescence of haemopoietic stem cell subsets and may influence the balance between stem cell renewal and proliferation. Telomere length and telomerase activity was measured in whole blood leucocytes, neutrophils and T cells from cord blood and individuals aged from 1 year to 96 years. Rapid telomere shortening [700 base pairs (bp)] was demonstrated in the first year of life, followed by a gradual decline of 31 bp/year. T cells were shown to have longer telomeres than neutrophils (mean difference 372 bp, P = < 0.001) but demonstrated similar rates of shortening (20 +/- 0.3 bp/year vs. 22 +/- 0.3 bp/year). Telomerase was detectable in T cells but not in neutrophils, suggesting that telomerase is not the rate-limiting step for regulation of telomere length in haemopoietic cells. Stem cell utilization as measured by X chromosome inactivation patterns was found to be independent of telomere length. This supports the concept that age-dependent skewed haemopoiesis is the result of random stem cell loss or X-allelic exclusion rather than telomeric senescence. These studies provide insight into the ageing process and a reference point for evaluating replicative stress in individuals of different age groups.     

Skewed X chromosome inactivation in fraternal female twins results in moderately severe and mild haemophilia B

Summary. Female carriers of haemophilia B are usually asymptomatic; however, the disease resulting from different pathophysiological mechanisms has rarely been documented in females. In this study, we investigated the mechanisms responsible for haemophilia B in fraternal female twins. We sequenced the factor IX gene (F9) of the propositus, her father, a severe haemophilia B patient and the other family members. X chromosome inactivation was assessed by the methylation‐sensitive HpaII‐PCR assay using X‐linked polymorphisms in human phosphoglycerate kinase 1 gene (PGK1) and glutamate receptor ionotropic AMPA 3 gene (GRIA3). The twins were found to be heterozygotes with a nonsense mutation (p.Arg384X) inherited from their father. The propositus, more severely affected twin, exhibited a significantly higher percentage of inactivation in the maternally derived X chromosome carrying a normal F9. The other twin also showed a skewed maternal X inactivation, resulting in a patient with mild haemophilia B. Thus, the degree of skewing of maternal X inactivation is closely correlated with the coagulation parameters and the clinical phenotypes of the twins. Furthermore, we identified a crossing‐over in the Xq25–26 region of the maternal X chromosome of the more severely affected twin. This crossing‐over was absent in the other twin, consistent with their fraternal state. Differently skewed X inactivation in the fraternal female twins might cause moderately severe and mild haemophilia B phenotypes, respectively.     

Stochastic epigenetic mutations (DNA methylation) increase exponentially in human aging and correlate with X chromosome inactivation skewing in females

In this study we applied a new analytical strategy to investigate the relations between stochastic epigenetic mutations (SEMs) and aging. We analysed methylation levels through the Infinium HumanMethylation27 and HumanMethylation450 BeadChips in a population of 178 subjects ranging from 3 to 106 years. For each CpG probe, epimutated subjects were identified as the extreme outliers with methylation level exceeding three times interquartile ranges the first quartile (Q1-(3 × IQR)) or the third quartile (Q3+(3 × IQR)). We demonstrated that the number of SEMs was low in childhood and increased exponentially during aging. Using the HUMARA method, skewing of X chromosome inactivation (XCI) was evaluated in heterozygotes women. Multivariate analysis indicated a significant correlation between log(SEMs) and degree of XCI skewing after adjustment for age (β = 0.41; confidence interval: 0.14, 0.68; p-value = 0.0053). The PATH analysis tested the complete model containing the variables: skewing of XCI, age, log(SEMs) and overall CpG methylation. After adjusting for the number of epimutations we failed to confirm the well reported correlation between skewing of XCI and aging. This evidence might suggest that the known correlation between XCI skewing and aging could not be a direct association but mediated by the number of SEMs.     

Centromere retention and loss during the descent of maize from a tetraploid ancestor

Although centromere function is highly conserved in eukaryotes, centromere sequences are highly variable. Only a few centromeres have been sequenced in higher eukaryotes because of their repetitive nature, thus hindering study of their structure and evolution. Conserved single-copy sequences in pericentromeres (CSCPs) of sorghum and maize were found to be diagnostic characteristics of adjacent centromeres. By analyzing comparative map data and CSCP sequences of sorghum, maize, and rice, the major evolutionary events related to centromere dynamics were discovered for the maize lineage after its divergence from a common ancestor with sorghum. (i) Remnants of ancient CSCP regions were found for the 10 lost ancestral centromeres, indicating that two ancient homeologous chromosome pairs did not contribute any centromeres to the current maize genome, whereas two other pairs contributed both of their centromeres. (ii) Five cases of long-distance, intrachromosome movement of CSCPs were detected in the retained centromeres, with inversion the major process involved. (iii) The 12 major chromosomal rearrangements that led to maize chromosome number reduction from 20 to 10 were uncovered. (iv) In addition to whole chromosome insertion near (but not always into) other centromeres, translocation and fusion were found to be important mechanisms underlying grass chromosome number reduction. (v) Comparison of chromosome structures confirms the polyploid event that led to the tetraploid ancestor of modern maize.     

Analysis of FMR1 gene premutation and X chromosome cytogenetic abnormalities in 100 Tunisian patients presenting premature ovarian failure

Objective

To evaluate the prevalence of FMR1 premutations and X chromosome cytogenetic abnormalities in a large cohort of Tunisian women with premature ovarian failure (POF).

Patients and methods

The cohort consisted of 127 Tunisian women with POF referred by endocrinologists and gynecologists for genetic investigation in the context of idiopathic POF and altered hormonal profiles. Clinical information concerning the reproductive function in the family, previous hormonal measurements and/or possible fertility treatment were collected. Karyotype, FISH analyses, FMR1 and FMR2 testing were performed for all patients.

Results

Fifteen patients (11.81%) presented structural or numerical X chromosomal abnormalities. Moreover, we detected in 12 patients (10.71%) a high level of X mosaicism. Analysis of FMR1 gene in the 100 patients without X chromosomal abnormalities showed that five percent of the patients carried a FMR1 premutation allele. On the other hand, the FMR2 screening did not reveal any deletion.

Conclusion

Our study confirms the major role of X chromosome abnormalities in POF and highlights the importance of karyotype analyses and FMR1 screening. These investigations provide valuable information for diagnosis and genetic counseling for these women who still have a 5% chance of spontaneous conception.     

From the Cover: PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus

Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.     

Organization of the prolamin gene family provides insight into the evolution of the maize genome and gene duplications in grass species

Zea mays, commonly known as corn, is perhaps the most greatly produced crop in terms of tonnage and a major food, feed, and biofuel resource. Here we analyzed its prolamin gene family, encoding the major seed storage proteins, as a model for gene evolution by syntenic alignments with sorghum and rice, two genomes that have been sequenced recently. Because a high-density gene map has been constructed for maize inbred B73, all prolamin gene copies can be identified in their chromosomal context. Alignment of respective chromosomal regions of these species via conserved genes allow us to identify the pedigree of prolamin gene copies in space and time. Its youngest and largest gene family, the alpha prolamins, arose about 22–26 million years ago (Mya) after the split of the Panicoideae (including maize, sorghum, and millet) from the Pooideae (including wheat, barley, and oats) and Oryzoideae (rice). The first dispersal of alpha prolamin gene copies occurred before the split of the progenitors of maize and sorghum about 11.9 Mya. One of the two progenitors of maize gained a new alpha zein locus, absent in the other lineage, to form a nonduplicated locus in maize after allotetraplodization about 4.8 Mya. But dispersed copies gave rise to tandem duplications through uneven expansion and gene silencing of this gene family in maize and sorghum, possibly because of maize's greater recombination and mutation rates resulting from its diploidization process. Interestingly, new gene loci in maize represent junctions of ancestral chromosome fragments and sites of new centromeres in sorghum and rice.     

Resolution and evolution of the duck-billed platypus karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male sex chromosome constitution

The platypus (2n = 52) has a complex karyotype that has been controversial over the last three decades. The presence of unpaired chromosomes and an unknown sex-determining system especially has defied attempts at conventional analysis. This article reports on the preparation of chromosome-specific probes from flow-sorted chromosomes and their application in the identification and classification of all platypus chromosomes. This work reveals that the male karyotype has 21 pairs of chromosomes and 10 unpaired chromosomes (E1-E10), which are linked by short regions of homology to form a multivalent chain in meiosis. The female karyotype differs in that five of these unpaired elements (E1, E3, E5, E7, and E9) are each present in duplicate, whereas the remaining five unpaired elements (E2, E4, E6, E8, and E10) are absent. This finding indicates that sex is determined by the alternate segregation of the chain of 10 during spermatogenesis so that equal numbers of sperm bear either one of the two groups of five elements, i.e., five X and five Y chromosomes. Chromosome painting reveals that these X and Y chromosomes contain pairing (XY shared) and differential (X- or Y-specific) segments. Y differential regions must contain male-determining genes, and X differential regions should be dosage-compensated in the female. Two models for the evolution of the sex-determining system are presented. The resolution of the longstanding debate over the platypus karyotype is an important step toward the understanding of mechanisms of sex determination, dosage compensation, and karyotype evolution.