X Inactivation Center Location

[Pricilla Igoe]

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X-chromosome inactivation was investigated cytologically using the modified Kanda method which differentially stains inactive X-chromosome material at metaphase in balanced 13 1/2-day female embryos heterozygous for four X-autosome rearrangements, reciprocal translocations T(X;4)37H, T(X;11)38H and T(X;16)16H (Searle's translocation) and the insertion translocation Is(7;X)1 Ct (Cattanach's translocation). In all cases non-random inactivation was found. In the reciprocal translocation heterozygotes only one translocation product ever showed Kanda staining. In addition in a proportion of cells from T(X;4)37H, T(X;11)38H and Is(7;X)1Ct the Kanda staining revealed differential staining of X-chromosome material and attached autosomal material within the translocation product. In a study of 8 1/2-day female embryos doubly heterozygous for Searle's translocation and Cattanach's translocation two unbalanced types of embryo were found. In one type of unbalanced female embryo of the karyotype 40(X(7)/X16;16/16) no inactivated X-chromosomal material is found. A second unbalanced type of female embryo, of the presumptive karyotype 40(X(7)/XN;16X/16) was found in which two inactivated chromosomes were present in the majority of metaphase spreads. A simple model for the initiation of X-chromosome inactivation based on the presence of a single inactivation centre distal to the breakpoint in Searle's translocation explains these findings.

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X-chromosome inactivation is responsible for sex chromosome dosage compensation in females (XX), and ensures that X-chromosomal genes are not expressed at twice the levels of expression in males (XY) [1]. It occurs during early female embryonic development [2], but the exact timing in humans is still elusive. Once the choice for the inactivation of either the maternal or paternal X-chromosome is made, it is stably inherited to all daughter cells through mitosis. The choice of which of the two X-chromosomes is inactivated is random and does not depend on paternal or maternal origin. Therefore, females are mosaic and consist of a population of cells with preferential expression of either paternal or maternal X-chromosome. Not all females have equal proportions of cells with the paternal or maternal X-chromosome inactivated. This so-called skewed X-inactivation can be explained in different ways [3]. Firstly, skewing might be caused by selective pressure: a variant on one of the X-chromosomes is associated with lethality or limited survival and will undergo negative selection [4]. This explains, to a certain degree, symptoms in female carriers of variants associated with X-linked recessive diseases. For example, in the X-linked recessive disorder Duchenne muscular Dystrophy (DMD), a number of female cases with translocations that forced the inactivation of the normal DMD allele, were already reported in the 1980s [5,6,7,8]. Secondly, the cause of skewing may be purely stochastic in nature: just by chance more cells inactivate the paternal or maternal X-chromosome [9]. Given that X-inactivation is occurring in an embryonic stage where there are limited number of cells giving rise to the different germ layers, this may lead to skewing in the compartment arising from these limited sets of precursor cells.

In this study, we investigated X-inactivation in the blood of a population of healthy daughters from the Genome of the Netherlands (GoNL) project [17] of which large-scale RNA-sequencing (RNA-seq) were generated. We took advantage of the availability of full genome sequences of the parents to unequivocally assign reads covering heterozygous single-nucleotide variants (SNVs) to the maternal and paternal alleles, and assessed the degree of skewing in the population and the genes that consistently escape X-inactivation in the population. Finally, we discuss the implications for the clinical diagnostic practice.

The null hypothesis in the test is that the median paternal ratio of the gene is not different from the overall median paternal ratio for that individual, consistent with absence of escapee behavior. The alternative hypothesis is that the median paternal ratio of the gene is closer to 0.5 than the overall median paternal ratio for that individual, consistent with escapee behavior.

The ethical approval for this study lies with the individual participating cohorts (CODAM, LL, LLS, and RS) and institutional review boards. A broad consent for participation in research, including research on genotypes, was obtained from all participants. Given the privacy-sensitive nature of the DNA and RNA data, the data have been deposited at the European Genome-Phenome Archive (EGA) under the accession number EGAS00001001077 and is under controlled access. Requests for the data can be filed in the EGA system and will be handled by the BIOS data access committee. The committee will provide access to researchers for studies with a solid scientific background.

Examples of the distributions of the allelic and paternal ratios across all SNVs with sufficient coverage in an individual are presented in Figure S3. The degree of skewing did not depend on the age of the individual (Figure S4).

Tissues analyzed: Carrel - primary human fibroblast cell lines, rodent/human somatic cell hybrids [14] Park - primary human fibroblast cell lines, rodent/human somatic cell hybrids [14] Zhang - immortalized human B-cells [22] Cotton - human fibroblast cell lines [20] Tukiainen - diverse human tissues [23]

We report that skewed X-inactivation is common in the general female population. The degree of skewed X-inactivation reported earlier varies considerably [23, 24]. This is partly due to the differences in the assays used to assess the X-inactivation status. The most commonly used assay examines the DNA methylation status of the polymorphic AR locus (cf. HUMARA assay). Another group of assays analyzes allele-specific RNA expression at distinct heterozygous loci by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The latter assays provide a more direct output measurement of X-inactivation. A direct comparison between allele-specific expression and HUMARA assay [25] demonstrated a number of inconsistencies and suggested that methylation status does not always reflect expression and that the HUMARA assay may be influenced by preferential amplification of AR alleles with shorter repeats. Moreover, expression of a single X-linked locus may not reflect the expression status of the entire X-chromosome as there are genes with variable levels of escape from X-inactivation in the healthy population. The combination of genome and RNA-seq-based analysis presented here can be regarded as an aggregate of all allele-specific expression measurements over the entire X-chromosome, and a robust and direct way of assessing X-inactivation status and skewing per individual. This makes it a useful clinical diagnostic tool for assessing X-inactivation status. In case the cost of RNA-seq are prohibitive, allele-specific quantitative RT-PCR assays could serve as an alternative, in particular when heterozygous loci have been identified by Sanger sequencing, gene panel, whole-exome sequencing, or whole-genome sequencing. However, based on the presented results, we strongly recommend not relying on single SNVs for the assessment of the X-inactivation status, but to at least include SNVs in several different genes.

Our current RNA-seq-based results stand out from previous papers, as we have observations along the entire X-chromosome and can uniquely assign each of these observations to the maternal or paternal chromosome, given the availability of the full parental haplotypes. Nevertheless, most of our results are consistent with earlier reports. In the largest study so far, Amos-Landgraf et al. [24] determined the distribution of X-inactivation patterns in blood samples from 1005 phenotypically unaffected newborn infants and adult women, using the AR methylation assay. In the resulting data set, 25% of the individuals demonstrated skewing ratios >0.7 or 0.8 or

In a recently published RNA-seq-based paper from the GTEx consortium assessing X-inactivation in the general population across tissues, only 1 out of 449 individuals demonstrated extreme skewing (>95% across 16 tissues) [23], where we find already 2 in our population of 79 individuals. This may be partly explained by the fact that we are able to provide an accurate assignment of each allele to the paternal or maternal X, where parental genotypes are not available in the GTEx cohort. In the GTEx paper, it is nicely demonstrated that, despite variable escapee behavior across tissues, the X-inactivation patterns are usually consistent across tissues. Together with results from other studies [26, 31], this suggests that X-inactivation status in the blood is at least partly predictive for X-inactivation status in other tissues.

Assessment of the X-inactivation status has important implications for clinical diagnostics. Monoallelic or preferential expression of one of the alleles (skewing) is often seen as an indication of the presence of a nonsense mutation that induces nonsense-mediated decay. However, monoallelic expression needs to be seen in the context of the inactivation status of the entire X-chromosome. We show here that the mere fact that expression of only one allele is observed provides insufficient proof for its pathogenicity. This is further corroborated by the lack of correlation between the X-inactivation status of mothers and daughters, in line with the stochastic nature of the embryonic X-inactivation process. Proof for pathogenicity is only obtained when other (non-escapee) genes demonstrate biallelic expression. If this is not the case, the individual may just be a case of extreme skewing of X-chromosomal expression, which is also observed in the normal population. Knowledge of the X-inactivation status is also important for the classification of the increasing number of variants of unknown significance (VUS) identified by genome-wide sequencing technologies. Often, inheritance helps to classify VUS, but X-linked segregation patterns may be clouded by skewed X-inactivation. Skewing of X-inactivation may also explain the phenomenon of symptomatic female carriers of X-linked recessive disorders and differences in penetrance of dominant disorders. There have been a number of conflicting reports on the association of the X-inactivation status with clinical symptoms in these disorders [9, 32,33,34,35,36]. The assessment of X-inactivation status may explain why these relationships are difficult to consolidate: the frequently used AR methylation status may not be entirely predictive for the inactivation status of the disease locus. Moreover, the sole assessment of the AR methylation status does not tell whether the disease or the normal allele is preferentially inactivated in a given individual.

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