Reflections on the history of genetic medicine at Johns Hopkins University.

Two members of the faculty-who witnessed the birth of Genetic Medicine and remained to see it evolve-present their reflections about the history of genetic medicine at the Johns Hopkins Medical Institutions. They tell how the genetic units in Pediatrics and Medicine that were initiated by Barton Childs and Victor McKusick, respectively, became the McKusick Nathans Department of Genetic Medicine in 2020.

Choosing the Active X: The Human Version of X Inactivation.

Humans and rodents differ in how they carry out X inactivation (XI), the mammalian method to compensate for the different number of X chromosomes in males and females. Evolutionary changes in staging embryogenesis and in mutations within the XI center alter the process among mammals. The mouse model of XI is predicated on X counting and subsequently choosing the X to 'inactivate'. However, new evidence suggests that humans initiate XI by protecting one X in both sexes from inactivation by XIST, the noncoding RNA that silences the inactive X. This opinion article explores the question of how the active X is protected from silencing by its own Xist locus, and the possibility of different solutions for mouse and human.

X-linked diseases: susceptible females.

The role of X-inactivation is often ignored as a prime cause of sex differences in disease. Yet, the way males and females express their X-linked genes has a major role in the dissimilar phenotypes that underlie many rare and common disorders, such as intellectual deficiency, epilepsy, congenital abnormalities, and diseases of the heart, blood, skin, muscle, and bones. Summarized here are many examples of the different presentations in males and females. Other data include reasons why women are often protected from the deleterious variants carried on their X chromosome, and the factors that render women susceptible in some instances.

An overview of X inactivation based on species differences.

X inactivation, a developmental process that takes place in early stages of mammalian embryogenesis, balances the sex difference in dosage of X-linked genes. Although all mammals use this form of dosage compensation, the details differ from one species to another because of variations in the staging of embryogenesis and evolutionary tinkering with the DNA blueprint for development. Such differences provide a broader view of the process than that afforded by a single species. My overview of X inactivation is based on these species variations.

Silencing XIST on the future active X: Searching human and bovine preimplantation embryos for the repressor.

X inactivation is the means of equalizing the dosage of X chromosomal genes in male and female eutherian mammals, so that only one X is active in each cell. The XIST locus (in cis) on each additional X chromosome initiates the transcriptional silence of that chromosome, making it an inactive X. How the active X in both males and females is protected from inactivation by its own XIST locus is not well understood in any mammal. Previous studies of autosomal duplications suggest that gene(s) on the short arm of human chromosome 19 repress XIST on the active X. Here, we examine the time of transcription of some candidate genes in preimplantation embryos using single-cell RNA sequencing data from human embryos and qRT-PCR from bovine embryos. The candidate genes assayed are those transcribed from 19p13.3-13.2, which are widely expressed and can remodel chromatin. Our results confirm that XIST is expressed at low levels from the future active X in embryos of both sexes; they also show that the XIST locus is repressed in both sexes when pluripotency factors are being upregulated, during the 4-8 cell and morula stages in human and bovine embryos - well before the early blastocyst (E5) when XIST on the inactive X in females starts to be upregulated. Our data suggest a role for DNMT1, UHRF1, SAFB and SAFB2 in XIST repression; they also exclude XACT and other 19p candidate genes and provide the transcriptional timing for some genes not previously assayed in human or bovine preimplantation embryos.

The single active X in human cells: evolutionary tinkering personified.

All mammals compensate for sex differences in numbers of X chromosomes by transcribing only a single X chromosome in cells of both sexes; however, they differ from one another in the details of the compensatory mechanisms. These species variations result from chance mutations, species differences in the staging of developmental events, and interactions between events that occur concurrently. Such variations, which have only recently been appreciated, do not interfere with the strategy of establishing a single active X, but they influence how it is carried out. In an overview of X dosage compensation in human cells, I point out the evolutionary variations. I also argue that it is the single active X that is chosen, rather than inactive ones. Further, I suggest that the initial events in the process-those that precede silencing of future inactive X chromosomes-include randomly choosing the future active X, most likely by repressing its XIST locus.

Stochastic gene expression and chromosome interactions in protecting the human active X from silencing by XIST.

Mammals use X chromosome inactivation to compensate for the sex difference in numbers of X chromosomes. A relatively unexplored question is how the active X is protected from inactivation by its own XIST gene, the long non-coding RNA, which initiates silence of the inactive X.  Previous studies of autosomal duplications show that human chromosome 19 plays a critical role in protecting the active X. I proposed that it genetically interacts with the X chromosome to repress XIST function on the future active X.  Here, I show that the type of  chromosome 19 duplication influences the outcome of the interaction: the presence of three chromosome 19s is tolerated whereas duplications affecting only one chromosome 19 are not. The different outcomes have mechanistic implications for how chromosome 19 interacts with the future active X, pointing to a role for stochastic gene expression and possibly physical interaction.

X inactivation, female mosaicism, and sex differences in renal diseases.

A good deal of sex differences in kidney disease is attributable to sex differences in the function of genes on the X chromosome. Males are uniquely vulnerable to mutations in their single copy of X-linked genes, whereas females are often mosaic, having a mixture of cells expressing different sets of X-linked genes. This cellular mosaicism created by X inactivation in females is most often advantageous, protecting carriers of X-linked mutations from the severe clinical manifestations seen in males. Even subtle differences in expression of many of the 1100 X-linked genes may contribute to sex differences in the clinical expression of renal diseases.

The Non-random Location of Autosomal Genes That Participate in X Inactivation.

Mammals compensate for sex differences in the number of X chromosomes by inactivating all but one X chromosome. Although they differ in the details of X inactivation, all mammals use long non-coding RNAs in the silencing process. By transcribing XIST RNA, the human inactive X chromosome has a prime role in X-dosage compensation. Yet, the autosomes also play an important role in the process. Multiple genes on human chromosome 1 interact with XIST RNA to silence the future inactive Xs. Also, it is likely that multiple genes on human chromosome 19 prevent the silencing of the single active X - a highly dosage sensitive process. Previous studies of the organization of chromosomes in the nucleus and their genomic interactions indicate that most contacts are intra-chromosomal. Co-ordinate transcription and dosage regulation can be achieved by clustering of genes and mingling of interacting chromosomes in 3D space. Unlike the genes on chromosome 1, those within the critical eight MB region of chromosome 19, have remained together in all mammals assayed, except rodents, indicating that their proximity in non-rodent mammals is evolutionarily conserved. I propose that the autosomal genes that play key roles in the process of X inactivation are non-randomly distributed in the genome and that this arrangement facilitates their coordinate regulation.

X inactivation in triploidy and trisomy: the search for autosomal transfactors that choose the active X.

Only one X chromosome functions in diploid human cells irrespective of the sex of the individual and the number of X chromosomes. Yet, as we show, more than one X is active in the majority of human triploid cells. Therefore, we suggest that (i) the active X is chosen by repression of its XIST locus, (ii) the repressor is encoded by an autosome and is dosage sensitive, and (iii) the extra dose of this key repressor enables the expression of more than one X in triploid cells. Because autosomal trisomies might help locate the putative dosage sensitive trans-acting factor, we looked for two active X chromosomes in such cells. Previously, we reported that females trisomic for 18 different human autosomes had only one active X and a normal inactive X chromosome. Now we report the effect of triplication of the four autosomes not studied previously; data about these rare trisomies - full or partial - were used to identify autosomal regions relevant to the choice of active X. We find that triplication of the entire chromosomes 5 and 11 and parts of chromosomes 1 and 19 is associated with normal patterns of X inactivation, excluding these as candidate regions. However, females with inherited triplications of 1p21.3-q25.3, 1p31 and 19p13.2-q13.33 were not ascertained. Thus, if a single key dose-sensitive gene induces XIST repression, it could reside in one of these locations. Alternatively, more than one dosage-sensitive autosomal locus is required to form the repressor complex.

Why females are mosaics, X-chromosome inactivation, and sex differences in disease.

At every age, males have a higher risk of mortality than do females. This sex difference is most often attributed to the usual suspects: differences in hormones and life experiences. However, the fact that XY males have only one X chromosome undoubtedly contributes to this vulnerability, as any mutation that affects a gene on their X chromosome will affect their only copy of that gene. On the other hand, cellular mosaicism created by X inactivation provides a biologic advantage to females. There are 1100 genes on the X chromosome, and most of them are not expressed from the Y chromosome. Therefore, sex differences in the expression of these genes are likely to underlie many sex differences in the expression of diseases affected by these genes. In fact, this genetic biology should be considered for any disease or phenotype that occurs in one sex more than the other, because the disease mechanism may be influenced directly by an X-linked gene or indirectly through the consequences of X inactivation.

Embryonic loss of human females with partial trisomy 19 identifies region critical for the single active X.

To compensate for the sex difference in the number of X chromosomes, human females, like human males have only one active X. The other X chromosomes in cells of both sexes are silenced in utero by XIST, the Inactive X Specific Transcript gene, that is present on all X chromosomes. To investigate the means by which the human active X is protected from silencing by XIST, we updated the search for a key dosage sensitive XIST repressor using new cytogenetic data with more precise resolution. Here, based on a previously unknown sex bias in copy number variations, we identify a unique region in our genome, and propose candidate genes that lie within, as they could inactivate XIST. Unlike males, the females who duplicate this region of chromosome 19 (partial 19 trisomy) do not survive embryogenesis; this preimplantation loss of females may be one reason that more human males are born than females.

The role of X inactivation and cellular mosaicism in women's health and sex-specific diseases.

Sex-specific manifestations of disease are most often attributed to differences in the reproductive apparatus or in life experiences. However, a good deal of sex differences in health issues have their origins in the genes on the sex chromosomes themselves and in X inactivation-the developmental program that equalizes their expression in males and females. Most females are mosaics, having a mixture of cells expressing either their mother's or father's X-linked genes. Often, cell mosaicism is advantageous, ameliorating the deleterious effects of X-linked mutations and contributing to physiological diversity. As a consequence, most X-linked mutations produce male-only diseases. Yet, in some cases the dynamic interactions between cells in mosaic females lead to female-specific disease manifestations.

Familial nonrandom inactivation linked to the X inactivation centre in heterozygotes manifesting haemophilia A.

A basic tenet of the Lyon hypothesis is that X inactivation occurs randomly with respect to parental origin of the X chromosome. Yet, nonrandom patterns of X inactivation are common - often ascertained in women who manifest recessive X-linked disorders despite being heterozygous for the mutation. Usually, the cause of skewing is cell selection disfavouring one of the cell lineages created by random X inactivation. We have identified a three generation kindred, with three females who have haemophilia A because of extreme skewing of X inactivation. Although they have both normal and mutant factor VIII (FVIII) alleles, only the mutant one is transcribed; and, they share an XIST allele that is never transcribed. The skewing in this case seems to result from an abnormality in the initial choice process, which prevents the chromosome bearing the mutant FVIII allele from being an inactive X.

Titles and abstracts of scientific reports ignore variation among species.

An analysis of more than 1000 research articles in biology reveals that the name of the species being studied is not mentioned in the title or abstract of many articles. Consequently, such data are not easily accessible in the PubMed database. These omissions can mislead readers about the true nature of developmental processes and delay the acceptance of valid species differences. To improve the accuracy of the scientific record, I suggest that journals should require that authors include the name of the species being studied in the title or abstract of submitted papers.

Differential X reactivation in human placental cells: implications for reversal of X inactivation.

X inactivation--the mammalian method of X chromosome dosage compensation--is extremely stable in human somatic cells; only fetal germ cells have a developmental program to reverse the process. The human placenta, at term, differs from other somatic tissues, since it has the ability to reverse the X-inactivation program. To determine whether reversal can be induced at other stages of placental development, we examined earlier placental specimens using a cell-hybridization assay. We found that global X reactivation is also inducible in villi cells from first-trimester spontaneous abortions but not from first-trimester elective terminations. These differences in inducibility are not associated with detectable variation in histone H4 acetylation, DNA methylation, or XIST expression--hallmarks of the inactivation process--so other factors must have a role. One notable feature is that the permissive cells, unlike nonpermissive ones, have ceased to proliferate in vivo and are either beginning or in the process of programmed cell death. Cessation of mitotic proliferation also characterizes oocytes at the stage at which they undergo X reactivation. We suggest that, along with undermethylation, the apoptotic changes accompanying cessation of cell proliferation contribute to the reversal of inactivation, not only in placental cells, but also in oocytes entering meiosis.

Species differences in TSIX/Tsix reveal the roles of these genes in X-chromosome inactivation.

Transcriptional silencing of the human inactive X chromosome is induced by the XIST gene within the human X-inactivation center. The XIST allele must be turned off on one X chromosome to maintain its activity in cells of both sexes. In the mouse placenta, where X inactivation is imprinted (the paternal X chromosome is always inactive), the maternal Xist allele is repressed by a cis-acting antisense transcript, encoded by the Tsix gene. However, it remains to be seen whether this antisense transcript protects the future active X chromosome during random inactivation in the embryo proper. We recently identified the human TSIX gene and showed that it lacks key regulatory elements needed for the imprinting function of murine Tsix. Now, using RNA FISH for cellular localization of transcripts in human fetal cells, we show that human TSIX antisense transcripts are unable to repress XIST. In fact, TSIX is transcribed only from the inactive X chromosome and is coexpressed with XIST. Also, TSIX is not maternally imprinted in placental tissues, and its transcription persists in placental and fetal tissues, throughout embryogenesis. Therefore, the repression of Xist by mouse Tsix has no counterpart in humans, and TSIX is not the gene that protects the active X chromosome from random inactivation. Because human TSIX cannot imprint X inactivation in the placenta, it serves as a mutant for mouse Tsix, providing insights into features responsible for antisense activity in imprinted X inactivation.