• 2nd Pearson-White lecture
• Website http://faculty.virginia.edu/mammgenetics
• 3/31/2004
• X chromosomes, abnormalities
• X inactivation
• imprinting

• in diploid cells, only one X chromosome remains active, no matter how many there are present.
• a single X chromosome remains active for every two sets of autosomes, there must be a counting mechanism analogous to Drosophila sex determination

• phenotypic female with gonadal dysgenesis and sexual immaturity
• have primary amenorrhea (failure to menstruate), infertility, short stature, webbed neck, increased carrying angle at the elbow, cardiovascular and renal abnormalities
• 45,X in more than half the patients

• 46,X,abnormal X of the following types:
• Deletion X,Xp-
• Isochromosome X,i(Xq) monosomy short arm, trisomy long arm
• Ring X,r(X)
• Mosaicism
• X/XX
• X/XY virilization, gonadoblastoma
• X,abnormal X
• X/XXX
• X/XX/XXX etc.
• 46,XY/45,X arise as male zygotes, then undergo a mitotic nondisjunctional event resulting in conversion to female with Turner syndrome

• There are two active X chromosomes during ovarian development, and certain genes appear to need to be active for normal ovarian function.
• The inactive X chromosome is reactivated in oogonia when meiosis begins during fetal life.

• fetal ovary 7 million oocytes
• ovary at birth 3 million
• menarche 400,000
• menopause 10,000

• menopause has occurred before menarche
• Families in which menopause occurs early have a deletion of Xq26 portion of the X chromosome
• This suggests that genes in this region of the X are required for maintenance of ovarian function and prevention of premature menopause.
• Suggests that these genes are not inactivated on the inactive X.

• 1 per 1000 live male births
• male with small testes, hyalinized testicular tubules, and azoospermia (failure to produce normal amounts of sperm), resulting in infertility and variable signs of hypogonadism, social pathologies, somewhat reduced IQ
• postpubertal testicular failure
• may have additional X chromosomes, if so, more likely to be mentally retarded (when also have Y)
• first demonstration in humans that sex determined by presence or absence of Y chromosome, rather than number of X chromosomes

• found as 47,XYY, or 48,XXYY
• 47,XYY
• occurs 1/1000 in male live births
• occurs 4-20 per 1000 inmates
• 48,XXYY 50 times higher in prison inmates than in newborn population
• aneuploidy of the Y chromosome must arise from meiotic nondisjunction in the father

• incidence 1 in 20,000
• have X-Y interchange
• Sry transgenic mice, XX become male

• biallelic genes are those with two alleles, contributed by father and mother
• all autosomal genes
• X-linked genes in females
• in males (one X and one Y chromosome), the sex-linked genes are monoallelic
• there are a few genes on the Y chromosome with functional homologues on the X chromosome, e.g. GM-CSF receptor
• monoallelic expression of biallelic genes occurs by genomic imprinting
• small number of autosomal genes

• and by X chromosome inactivation in females
• occurs in all cells in which genes are expressed
• many genes are expressed only on the active X chromosome
• Xist is expressed only from the inactive X chromosome
• some X chromosome genes escape X inactivation
• this can also happen in allelic exclusion in immunoglobulin gene expression in B lymphocytes, and in T cell receptor gene expression in T lymphocytes

• Both entail homologous genes or chromosomes behaving differently within the same cell.
• In imprinting, the control is mainly at the single gene level.

• in somatic cells, X inactivation occurs early in embryonic life
• the inactivation is random, i.e. either the paternal or maternal X chromosome is inactivated in each cell
• X inactivation is complete; virtually all of the X chromosome is inactivated
• now know of examples where this is not true
• X chromosome inactivation is permanent and clonally propagated
• occurs in all mammals

Calico cat

• calico cats are always female
• almost true, some XXY males have been calico
• one X chromosome carries the gene for black coat color
• the other X chromosome carries the gene for yellow coat color
• in 64-cell embryos, one of each pair of X chromosomes and its genes are randomly silenced
• daughter cells inherit active or inactive X chromosomes, creating a cat with patches of coat color

• inactive X chromosome condenses to form a Barr body, attached to nuclear membrane
• also called a sex chromatin body
• The number of Barr bodies equals the number of X chromosomes minus one
• e.g. 49,XXXXX have 4 Barr bodies
• inactive X chromosome is late replicating

• a structurally abnormal X chromosome (i.e. with a deletion) is preferentially inactivated, leaving the normal X active
• but with balanced X-autosome translocations, usually the normal X chromosome is inactivated
• inactivation of the X-autosome translocated chromosome probably lethal
• X inactivation is permanent in most somatic cells, but must be reversible in the development of germ cells

• occurs transiently during gametogenesis
• then reactivated
• occurs early in development
• late blastula stage in mice
• each cell inactivates one X chromosome, randomly chosen
• pattern is clonally inherited thereafter
• female mammals are thus mosaics

• the Y chromosome carries only a few functional genes
• some are Y-specific, e.g. SRY, the testis determining gene
• some are also found on the X chromosome
• X and Y partially pair during meiosis in male cells, and can exchange sequence information
• there are two pseudoautosomal regions, PAR1 and PAR2

• 43 out of 233 human X-linked genes escape inactivation, mostly on Xp arm
• As noted in Turner syndrome, some genes on Xq must remain active to maintain ovarian function until menopause
• some genes that escape inactivation are in the pseudoautosomal (PAR) region, but some are not
• there are some mouse-human differences
• the human homolog escapes inactivation, e.g. ZFX, RPS4X, UBE1
• the mouse homologs are all inactivated, Zfx, Rps4, and Ube1X

• metatherian mammals, noneutherian
• kangaroos, wombats, bandicoots, opossums,
• no placenta, + pouch
• absolute paternal X chromosome inactivation
• DNA methylation is not associated with inactivation of HPRT and G6PD genes in the inactive X chromosome in marsupials

• Two genes (SYBL1 and H-SPRY-3) are subject to X inactivation, and have Y-linked homologs that are also silenced
• Mechanism of Y chromosome gene silencing is not understood

• XO humans have Turner syndrome
• only 1% survive to birth
• due to monosomy for a gene or genes common to the X and Y chromosome
• normal XX and XY individuals will be biallelic for these genes
• XO individuals will be monoallelic at these loci
• XO mice are viable, no prenatal lethality

• mapping XY females who carry different deletions of the Y that remove SRY
• depending on the deleted region they may or may not have Turner’s syndrome
• mapped to a 90 kb region between SRY and ZFY
• region carries a very highly conserved 40S ribosomal S4 protein, RPS4Y
• both RPS4Y and its X-linked homolog, RPS4X are both known to be functionally active, suggesting that this gene has a role in Turner’s syndrome

• called XIC, mapped to Xq13
• The evidence is that if the X chromosome is broken by a translocation, only one of the two resulting segments undergoes inactivation. The location of the inactivation center has been mapped

• signal from X inactivation center, spread along chromosome, promoted by booster elements (Fig 3)
• signal spreading is less effective in autosomal material than in the X chromosome itself
• LINEs may represent booster sequences
• X chromosome is strikingly LINE-rich
• Efficiency of spread into autosomal regions correlates with their LINE content
• inactive X replicates late, the late replication may help maintain the inactive signal, in which late replication prevents transcription and transcription is required for early replication

• signal may be methylation of the DNA, but methylation may be a consequence rather than a cause of the inactivation
• differential methylation not found in marsupials, in which X inactivation always occurs on the paternal X

• Xist= X Inactivation Specific Transcript
• pronounced exist
• Xist is expressed only from the inactive X chromosome, not the active X chromosome
• Xist expressed after the 2 cell stage.
• Xist maps to the XIC
• has been cloned in mouse and human

• RNA is expressed, but does not code for protein
• longest ORF (open reading frame) is 400 bp
• mouse 15 kb RNA, human 17 kb RNA
• RNA remains trapped in nucleus
• expressed only from the inactive X chromosome
• expressed in all mature female cells, and briefly in testes when an inactive X chromosome is present
• stabilization of Xist RNA mediates initiation of X inactivation

• XIST RNA may form a "cage" around the chromosome, acting as a barrier to RNA polymerases and blocking transcription
• The act of transcribing XIST RNA may induce a conformational change in the chromosome that inactivates it
• XIST RNA, alone or interacting with a nuclear factor, binds to the chromosome, inducing conformational change that inactivates it
• The act of transcribing XIST RNA "opens" a site on the X chromosome that allows it to attach to some inactivation machinery on the nuclear membrane
• to explain why Barr bodies are on the nuclear membrane
• XIST RNA, alone or interacting with a nuclear factor, binds to a site on the X chromosome that allows it to attach to some inactivation machinery on the nuclear membrane

Tsix and Xist have a yin and yang relationship

• Tsix is an antisense transcript to Xist, is transcribed through the Xist locus
• Tsix is expressed during the pre-inactivation period

• at CpG islands on the inactive X
• DNA cytosine methylation inhibitors such as 5-azacytidine can reactivate X-linked genes
• demethylation precedes transcription during the reactivation of HPRT by 5-azacytidine
• in marsupials X-linked CpG islands are not heavily methylated, nor are these genes kept stably inactivated
• DNA methyltransferase -/- transgenic mouse embryos are not able to maintain genomic imprinting, nor can they properly control Xist expression, which is required for the establishment of X inactivation

• in some cases imprinting is involved in the choice of X chromosome for inactivation, with the paternally derived X chromosome being preferentially inactivated.
• DNA methylation is known or believed to be involved in the mechanism in both cases.
• However, imprinting affects individual genes in different ways, although it appears global in the sense that it will affect the haploid complement of chromosomes derived from one parent. X chromosome inactivation involves coordinated regulation of a whole chromosome.

• for most genes there is no difference in expression whether the allele has been inherited from the mother or from the father
• but some genes are influenced by their parental origin. They have a 'imprint' of their gametic origin.
• Its occurrence in mammals has only recently been recognized, and was deduced from a number of different lines of research, including classical genetic studies, studies on X-inactivation, and the development of diploid parthenogenetic (gynogenetic, and androgenetic) embryos.

• maternal allele but not paternal allele is expressed in some cases
• paternal but not maternal allele is expressed in other cases
• disease can occur if the normally expressed allele is mutant
• disease can occur if there is uniparental disomy
• an individual may receive two copies of a specific chromosome from one parent instead of one maternal and one paternal homolog
• if two copies of the chromosome with the nonexpressed allele are inherited, that locus will be nonfunctional by this epigenetic mechanism

• how are maternal and paternal alleles distinguished?
• must carry an imprint to signal the difference
• must be stably inherited for many rounds of DNA replication
• imprint must be erased when it passes through the germline
• a man may transmit a chromosome inherited from his mother
• a woman may transmit a chromosome inherited from her father

• described twice
• 1. 2003 years ago
• 2. once in rabbits, but never seen again
• experiments on parthenogenesis reveal imprinting

• early explanations for why these failed to develop:
• haploid female pronucleus gets diploidized, but many homozygous mutant genes lethal, that had been hidden even in inbred mice
• require male genetic information to go further

• can then make eggs with two male pronuclei, or two female, so they will be diploid
• they do not develop
• male ­ female nucleus or genomes
• therefore, something is unique about the genome from each sex

• androgenotes:
• male male have embryonic part small, retarded, or absent
• extra-embryonic part is OK
• only go to about 8 d, seldom even see somites, although extra-embryonic development looks better
• gynogenotes:
• female female have embryonic part OK
• extra-embryonic part is small, retarded, or absent
• only go to 10 d
• thus the female genome is required for embryonic development
• the male part is required for extraembryonic development

• If reconstitute with same manipulations and a nucleus from each parent, they develop normally.
• If make chimeras between parthenogenotes and normal embryos, can get development to term, although the chimeric offspring tend to be small.
• Parthenogenetic cells are selected against in latter part of gestation, and under-represented in skeletal muscle and liver.

• primary gametic imprint acquired by one gamete
• in zygote and pre-implantation embryo, imprint is maintained on one chromosome
• in somatic embryonic cells, gametic imprint is translated to functional difference
• in germ cells, imprints are erased
• imprinted and maternally repressed
• Igf2, SnRPN, SP2, Ins1 and Ins2, Xist
• imprinted and paternally repressed
• H19, Igf2/MPR

• may be a conflict between maternally and paternally derived genes that affect growth. It may be selectively advantageous for the father's genes to promote growth of the fetus, and thus give it a better chance of survival, and advantageous for the mother's genes for the fetus to remain relatively small, to promote the mother's chances of having further offspring.
• Differential susceptibility of two homologues (maternal and paternal) to inactivation would reduce the risk that both will simultaneously be inactivated or activated (a presumably lethal error).

• Prader-Willi (PWS) and Angelman syndrome (AS) are both due to deletion of 15q11-13, but manifest differently depending on whether the allele was inherited from the mother or the father
• Failure to inherit the paternal region gives PWS
• Failure to inherit the maternal region gives AS
• usually arise sporadically, deletion in paternally transmitted chromosome 15, or maternal non-disjunction of chromosome 15 leads to trisomy, then loss of the paternal homologue
• SNRPN gene maps to this locus, and shows imprinting, and is thus a candidate gene for PWS
• inherited as autosomal dominant

• characteristic of chronic myelogenous leukemia
• resulting in translocation between chromosome 9 and 22
• chromosome 9 part is preferentially of paternal origin, and the 22 preferentially maternal.

• expansion of the trinucleotide repeats occurs preferentially on maternally transmitted X chromosomes that bear the FRAXA premutation, and probably takes place after fertilization.
• Recent experiments have demonstrated that chromosomal domains containing imprinted genes, including the FRAXA locus, are replicated asynchronously, with the maternal allele being replicated later than the paternal allele.
• Not sure whether a delay in replication leads to imprinting, or whether it is the imprinting of alleles that disrupts the timing of replication.

• maternal allele, H19 activation, elements unavailable to interact with Igf2, leaving it silent
• paternal allele, methylation prevents H19 activation, leaving enhancers free to turn on Igf2 or Ins-2
• deletion of H19 leads to activation of Igf2 and Ins-2, 30% higher weight in mice
• DMD (differentially methylated domain) appears to act as a boundary or insulator

• Mammalian development
• both parental genomes needed to complete development
• Gene expression
• Igf2/MPR and H19 are exclusively maternally expressed
• Igf2 and SnRPN are exclusively paternally expressed
• X chromosome inactivation
• paternal X chromosome is preferentially inactivated in some tissues of female mammals
• Human genetic disease
• maternal deletion of chromosome 15 results in Angelman’s disease

• Uniparental disomy phenotypes
• inheritance of two paternal copies of mouse chromosome 11 results in mice that are larger than littermates
• Chromosome loss in tumors
• Wilms’ tumour and osteosarcoma show loss of maternal chromosome
• Timing of chromosome replication
• maternally-inherited chromosome is late-replicating in some regions, while the paternally inherited copy replicates early

• Chromosome translocations
• chromosome 9 component of the Philadelphia translocation chromosome is of paternal origin
• Methylation of non-expressed transgenes
• transgenes that are methylated on maternal, but not paternal, inheritance
• Frequency of meiotic recombination
• meiotic recombination is more frequent in females than in males
• Expansion and retreat of triplet repeats
• preferential expansion in males of the repeat associated with Huntington’s disease