MS523.L13.Sex-Linked Inheritance and Disorders.Q3.23 2.pdf

Concepts: A) While the general developmental program and genetics are qualitatively similar amongst mammals, there are subtle differences within mammals that require knowledge of human/primate specific development and genetics. B) Because of post-zygotic developmental differences, mouse models are o...

Concepts: A) While the general developmental program and genetics are qualitatively similar amongst mammals, there are subtle differences within mammals that require knowledge of human/primate specific development and genetics. B) Because of post-zygotic developmental differences, mouse models are of modest relevance; however, studies of human embryos via IVF or induced progenitor germ cells are informative. C) Current reproductive practices in industrialized countries are associated with reduced fertility and increased potential for sex-linked disorders mainly resulting from an age-associated increase in aneuploidies. D) Current practice of defining ambiguous biological sex is as a disorder of sexual development (DSD) either involving mono/polygenic variants or chromosome numerical aberrations. E) Mono/polygenic causes of disorders of sexual development are the most prevalent and involve variants in transcription factors, sex steroid synthesis, sex steroid receptors, or other gene that cause syndromic diseases. F) Numerical chromosome aberrations are about 15% of disorders of sexual development and result from in sex chromosomes with too little or too much of either X or Y causing infertility and too little of X resulting in short stature and too much of X or Y resulting in tall stature. G) Inheritance of disease variants from the sex chromosomes differ from autosomes since the male is hemizygous and most result from variants in the X-chromosome because the Y is gene poor. H) Variable expressivity (analog phenomes) are common in many sex-linked diseases owing either to mosaicism at the numerical chromosome level (post-zygotic/somatic) or in X-chromosome inactivation. I) Inheritance of imprinted autosomal disease variants does not follow standard Mendelian inheritance patterns. J) These imprinted/epigenetically modified genes are read in a parent of origin manner and can cause disease even though the patient is heterozygous for a recessive disease or if the genes are inherited by uniparental isodisomy. 11/7/23 Dr. Darl Ray. Swartz 1 II) Biological Sex Development Programs A) Biological sex is not binary but is analog 1) Shades of grey between male and female phenome B) Biological male and female sex is common in animals and there are several different genetic paths to male and female phenomes 1) X and Y chromosomes for mammals (a) Male is hemizygotic (XY instead of XX or AA) 2) Z and W chromosomes for birds (a) Female is hemizygotic (WZ instead of ZZ or AA) (b) Cell gender is cell autonomous, not necessarily controlled by steroids (i) Can have a bird that is male on one side and female on the other 3) X and none (O) or insects (a) XX for females 4) Temperature (during development) in many reptiles 11/7/23 Dr. Darl Ray. Swartz 2 II) Biological Sex Development Programs C) Human (mammalian) program of genetic > hormonal > biological 1) Cell non-autonomous development of sexually di-morphic organs via estrogens and androgens 2) Allows for hormonal manipulation of biological sex and potentially gender D) Bipotential gonad forms very early then differentiates upon hormonal stimulation (cell non-autonomous) at about 6 weeks 1) Primordial germ cells (PGCs) migrate from yolk sac to gonadal ridge in gut cavity adjacent to primitive kidney (mesonephros) 2) Proliferate and form bi-potential gonad containing PGCs (a) Can develop into spermatogonia or oogonia (b) Undergo different epigenetic program than somatic cells and differ between spermatogonia and oogonia 3) Developmental program biased to male or female gonad development by Y-chromosome gene and potentially dosage of X-chromosome 11/7/23 Dr. Darl Ray. Swartz Embryology of the Female Reproductive Tract Andrew Healey DOI: 10.1007/174_2010_128, 3 II) Biological Sex Development Programs E) SRY (sex-determining region Y) gene on the Y chromosome codes for master transcription factor for downstream transcription factors giving male biased proteome 1) Consider the master switch of sex development 2) SRY > SOX9 > FGF9, PDG2, Anti Mullerian hormone > male reproductive system (a) SOX9 = Sex-determining region Y box 9 protein, FGF9 = fibroblast growth factor 9, PDG2 = prostaglandin G2 (i) FGF9 and PDG2 function as local factors (paracrine) that also upregulate SOX9 expression to drive conversion of cells (in a wave like fashion) (b) SRY expressions is transient but SOX9 expression maintained by positive feedback to maintain expression 11/7/23 Dr. Darl Ray. Swartz Development 137, 3921-3930 (2010) 4 II) Biological Sex Development Programs E) SRY (sex-determining region Y) gene on the Y chromosome 3) SRY program stimulates cells (some) to differentiate into Sertoli cells (a) Nurse cell for spermatogonia and developing spermatocytes (b) Secretes anti-Mullerian hormone to inhibit development of uterus (c) Stimulates development of interstitial cells of Leydig (i) Secrete testosterone (d) Stimulates development of Wolffian duct into epididymis/vas Deferens 4) SRY transcriptional program inhibits some of the components of the female developmental program via inhibition of Wnt4 signaling 5) SRY program (mainly via testosterone) stimulates PGCs to proliferate and form prespermatogonia then arrest in mitosis until puberty 6) Mutations in any of the genes in the pathway or signaling molecules can result in XY sex reversal (a) Chromosomally XY but phenotypically female, typically not fertile (i) Cbx2 (transcription co-factor) (ii) Gata4/Fog2 (transcription factor/cofactor) (iii) Map3k4 (kinase) 11/7/23 Dr. Darl Ray. Swartz 5 II) Biological Sex Development Programs F) Female developmental program driven by un-inhibited transcriptional program 1) WNT4, RSPO1, FOXL2, transcription factors and follistatin stimulate ovary program (a) Exact pathway not understood and may require all components plus some other genes (b) Deletion of SRY results in gonad dysgenesis (failure to develop) so female sex is not the default program 2) Oogonia undergo mitosis to proliferate then enter meiosis I (a) Arrest in meiosis I at the end of prophase 1 at the diplotene stage (b) Resume meiosis at puberty in an ovulation- and fertilization-dependent manner (c) Selected oocyte and associated follicular cells grow, mature, and are released each cycle (i) Unknown mechanism for selection (ii) About 85 days for primordial follicle/primary oocyte to develop into mature (Graafian) (d) Loose primary oocytes throughout pre-menopausal life mostly through apoptosis (e) Can have early-onset menopause because of accelerated loss of oocytes (pre7 11/7/23 Dr. Darl Ray. Swartz mature ovarian failure) II) Biological Sex Development Programs G) Testis and ovary-specific cells differentiate and produce hormones 1) Balance of androgens and estrogens determines the development (completion) of the reproductive system and the secondary sex characteristics (a) Anatomical developmental errors and changes can be addressed by surgery (b) Many secondary sex characteristics can be modulated by hormone levels (i) Hormone therapy 2) Testosterone (androgen) stimulates development of male reproductive system (a) Spermatogenesis (b) Testis formation (c) Maturation of vas Deferens (d) Glands (seminal vesicle and prostate) (e) Testis descent into scrotum from lower abdominal cavity 3) Estrogens stimulate development of the female reproductive system (a) Oocyte maturation and release (b) Gland/uterine tube maturation (c) Mammary gland development (along with other hormones) (d) Ovary formation and descent 11/7/23 Dr. Darl Ray. Swartz 8 II) Biological Sex Development Programs H) Anatomical development of the excurrent path involves androgens and estrogens to form the glands, ducts, and external genitalia for each sex 1) External anatomical features present at about 16 weeks and used to define sex at birth (a) Can have ambiguous genitalia suggesting a disorder of sexual development 2) Further and final development occurs at puberty (a) Can be partially reversed by hormone treatment over a long period of time 11/7/23 Dr. Darl Ray. Swartz 9 II) Biological Sex Development Programs H) Steroid receptors are transcriptional regulators that recruit chromatin remodeling complexes to form euchromatin (transcriptionally permissive) 1) Estrogen and androgen (testosterone/DHT) receptors (ES and AR) function as homo-dimers 11/7/23 Dr. Darl Ray. Swartz 10 II) Biological Sex Development Programs H) Steroid receptors are transcriptional regulators 1) Estrogen and androgen (testosterone/DHT) receptors (ES and AR) (a) Receptor has 4 domains (from N > C) 11/7/23 (i) Activity function 1 (modulating region/AF1) - Bind transcription co-activators or co-repressors independent of ligand - Random coil structure that varies between ER and AR (ii) DNA binding domain - Binds unique sequences with three nucleotide spacer 1. Estrogen response element (ERE) and androgen response element (ARE) a. ERE: AGGTCAnnnTGACCT b. ARE: AGAACAnnnTGTTCT (iii) Hinge region - NLS signals and links to ligand binding domain (iv) Ligand binding domain - Binds ligand in a pocket and closes pocket via conformational changes - Activity function 2 (AF2) dependent upon bound ligand Dr. Darl Ray. Swartz 11 II) Biological Sex Development Programs H) Steroid receptors are transcriptional regulators 2) Bind many different chromatin remodelers when ligand bound (a) P300/CBP > histone acetyl transferases (b) Histone methylases and de-methylases (c) SRCs (steroid receptor cofactors) > linker/docking site for a gaggle of transcription factors and signal transduction proteins 3) Pleotropic effects of gonadal hormones (and other steroidal hormones) explained by local chromatin euchromatization (a) Opens the chapter of a book and other cell-type specific transcription factors determine the sequences/sentences that are read (b) Generally are growth/proliferation promoters and antiapoptotic 4) Can bind DNA element in the absence of ligand and recruit heterochromatin forming complexes (a) Repressor function 11/7/23 Dr. Darl Ray. Swartz 12 II) Biological Sex Development Programs I) Steroid receptors are targets of many signal transduction pathways through phosphorylation, acetylation, and ubiquitination to modulate interaction with cofactors J) Activated steroid receptors can signal through activation of small GTPase and kinase pathways to give a rapid cellular response Int. J. Mol. Sci. 2017, 18(8), 1713 11/7/23 Dr. Darl Ray. Swartz 13 II) Biological Sex Development Programs K) Variants in ER and AR genes associated with disease 1) ER has ER1 (Chr 6) and ER2 (Chr 14) while AR has one (Chr X) 2) Can have constitutively active variants that form via somatic mutation upon hormone deprivation treatment for breast and prostate cancer (a) Artificial selection for variants within the population of cancer cells (b) Common in breast cancer (ER) and prostate cancers (AR) 3) ER1 inactivating variants have been found but very low frequency (3 recorded familial cases) (a) Miss-regulated hypothalamic-pituitary-gonadal axis giving very high estrogen levels (b) Failure of development of some secondary sex characteristics in females (c) Low bone density for age independent of biological sex 4) ERa (ER1 protein product) up-regulated in many breast cancers and level used as a diagnostic for treatment (a) Upregulation may be via upstream signal transduction pathway that enhances expression or mutation in transcription regulatory sites (b) Upregulation may result from increased copy number 11/7/23 Dr. Darl Ray. Swartz Int J Endocrinol. 2015; 2015: 298107. 14 II) Biological Sex Development Programs K) Variants in ER and AR genes associated with disease 5) AR N-terminal has variable number of CAG (glutamine) repeats that can expand and cause a disease called Kennedy Syndrome or spinal and bulbar muscular dystrophy (a) Classical X-linked recessive rare disease with adult onset (b) Rarely found in females but if homozygous for variant have lesser phenotype because of lower testosterone (c) Phenotype presents at > 40 yo as loss of motor function/tremors and muscle loss (d) Disease has variable penetrance related to CAG# and patient age (i) < 34 repeats normal (ii) 35 to 46, age- and CAG#-dependent penetrance (iii) >47 age-dependent penetrance (e) Molecular mechanism is by accumulation of AR protein plaques in motor neurons in the brain stem and spinal cord (i) N-terminal domain mediated aggregation that is lyso/proteasome resistant (ii) Mis-targeting of co-activators Dr. Darl Ray. Swartz 11/7/23 https://www.mda.org/disease/spi nal-bulbar-muscular-atrophy 15 III) Sex Chromosome Inheritance and Variants A) X and Y chromosomes evolved from autosomes 1) X retained function and Y lost (and continues) many genes (a) Thought that SRY gene lost from autosome (primordial X) (b) Y lost genes to minimize gene dosage effects (c) Retained pseudo-autosomal region 29 OCTOBER 1999 VOL 286 SCIENCE 11/7/23 Dr. Darl Ray. Swartz 16 III) Sex Chromosome Inheritance and Variants B) X and Y synapse during meiosis at pseudoautosomal regions (PARs) at the tips of the chromosomes 1) PAR 1 at Xp22 and Yp11 (a) 2.7 Mbp and relatively gene rich (ca 24 genes in humans) (b) Contains SHOX (short stature homeobox) gene associated with short stature in XO females 2) PAR 2 at Xq28 and Yq12 (a) 155 Kbp on X and 57 Kbp on Y and gene poor (ca 6 genes) 3) Do recombine/crossover at PAR 1 4) Do not get inactivated on the Xi 11/7/23 Dr. Darl Ray. Swartz 17 III) Sex Chromosome Inheritance and Variants C) Sex-linked single gene variants generally follow different inheritance pattern than autosomal variants 1) Male zygote receives Xm (maternal) and Y from father 2) Female zygote receives Xp (paternal) and Xm 3) Y-specific genes inherited only in males 4) XX with disease variants mostly observed in male offspring since hemizygous for X zXp Y zXm zXpzXm zXmY zXm zXpzXm zXmY 11/7/23 Z = Gene(s) of interest on X chromosome Dr. Darl Ray. Swartz 18 III) Sex Chromosome Inheritance and Variants C) Sex-linked single gene variants generally follow different inheritance pattern than autosomal variants 5) XX heterozygous for recessive disease variant (carrier) by non-diseased Y – classic muscular dystrophy example of X-linked recessive inheritance (a) 50% chance in male offspring (b) Minimal chance in female offspring (c) Note that “recessive” gene may have dosing effect giving mild phenotype in mother and heterozygote (carrier) female offspring (i) Called a manifesting carrier RXmrXm by RXpY RXp Y RXm RXpRXm RXmY rXm RXprXm rXmY 11/7/23 Dr. Darl Ray. Swartz 19 III) Sex Chromosome Inheritance and Variants C) Sex-linked single gene variants generally follow different inheritance pattern than autosomal variants 6) XX homozygous for recessive disease variant by Y w/o disease (a) 100% chance of male offspring with disease (b) Minimal chance of female offspring with disease 7) XX heterozygous for dominant disease variant by Y w/o disease for X-linked dominant inheritance (a) 50% of chance of male offspring with disease (i) Generally greater effect on phenome likely mediated by lack of extra X (b) 50% of chance of female offspring with disease (c) Example of fragile X disease being less severe 11/7/23 Dr. Darl Ray. Swartz in females rXmrXm by RXpY RXp rXm RXprXm rXm RXprXm RXmrXm Y rXmY rXmY by rXpY rXp RXm rXpRXm rXm rXprXm Y RXmY rXmY 20 III) Sex Chromosome Inheritance and Variants C) Sex-linked single gene variants generally follow different inheritance pattern than autosomal variants 8) Y with recessive X disease by XX w/o disease gene (a) Minimal chance of male offspring with disease (b) Minimal chance of female offspring with disease 9) Y with recessive X disease by XX heterozygous for recessive disease (a) 50% of chance of male offspring with disease (b) 50% of chance of female offspring with disease 10) Y with dominant X disease by XX w/o disease gene (a) Minimal chance of male offspring with disease (b) 100% chance of female offspring with disease 11/7/23 Dr. Darl Ray. Swartz RXmRXm by rXpY rXp RXm rXpRXm RXm rXpRXm RXmrXm by rXpY rXp RXm rXpRXm rXm rXprXm rXmrXm Y RXmY RXmY Y RXmY rXmY by RXpY RXp rXm RXprXm rXm RXprXm Y rXmY rXmY 21 III) Sex Chromosome Inheritance and Variants D) X-linked hypophosphatemia is an X-linked dominant disease 1) Phenotypic features (a) Low serum phosphate (b) Normal calicitriol (functional form of Vit. D) even though serum phosphate is low (c) Long bone abnormalities of bowed of legs at an early age (ca 2 yo), short stature, and other bone malformations (similar to rickets) (d) Joint pain in adulthood resulting from calcification of tendons, ligaments and joint capsule at points of insertion into bone (enthesopathy) (e) Dental abnormalities associated with reduced dentin formation (f) No difference in disease severity between biological males and females (g) Diagnosis by morphology (skeletal dysmorphias), serum analysis (Phosphate, calcitriol), and urine (reduced phosphate resorption – relatively high urine phosphate) (h) Genetic testing via single gene analysis, multigene panel (for bone diseases), or genome sequencing 11/7/23 Dr. Darl Ray. Swartz 22 III) Sex Chromosome Inheritance and Variants D) X-linked hypophosphatemia is an X-linked dominant disease 2) Genetics and mechanism of pathogenicity (a) Frequency of ca 1/20,000 (b) 100% penetrance with variable expressivity (c) Inheritance can be from affected parents or via de-novo somatic mutation or germ-line mutations in parent (d) Caused by inactivating mutations in the PHEX (phosphate-regulating endopeptidase homolog) gene (i) Numerous (350 to date) variants that result in loss of protein function (enzymatic activity to membrane targeting) (e) PHEX gene product normal function (i) Membrane protein that degrades proteins, especially osteopontin Ø Excessive osteopontin inhibits bone formation (ii) Inhibits phosphatonin (FGF23) production via regulation of expression (?) Ø Excessive FGF23 results in increased urine phosphate by downregulating a kidney Na/PO4 transporter Ø Inhibits vit D synthesis and thus intestinal absorption even though blood phosphate is low 11/7/23 Dr. Darl Ray. Swartz 23 III) Sex Chromosome Inheritance and Variants D) X-linked hypophosphatemia is an X-linked dominant disease Beck-Nielsen et al. Orphanet Journal of Rare Diseases (2019) 14:58 3) Treatment focuses on correcting bone deformation and decreasing pain (a) High oral phosphate and calcitriol during long bone growth period (b) Orthopedic treatments or surgery to correct bone dysmorphias (c) Burosmab (crysvita) transfusion > antibody that blocks FGF23 action (i) Costs of $160,000 – 200,000/year 11/7/23 Dr. Darl Ray. Swartz 24 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development 11/7/2 3 Dr. Darl Ray. Swartz 25 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development A) Disorders of sex development (DSD) is the preferred clinical term and involves chromosomal numerical aberrations or single/multiple gene disorders B) Variant phenome is detected late gestation/at birth, at puberty, or from fertility issues C) Current estimates a bit fuzzy but may be 1/100 (1%) of population D) Current classification has three main groupings: 1) Sex chromosome numerical aberrations, about 15% of cases 2) 46, XX DSD > chromosomally female but has androgen excess via variants in enzyme, or testis/ovitestis, about 35% of cases 3) 46, XY DSD > chromosomally male but has disorder of gonadal development or disorder in androgen (synthesis or receptor) action, about 50% of cases 11/7/23 Dr. Darl Ray. Swartz 26 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development NAture revIeWS | GENETICS volume 22 | September 2021 | 589 E) Mono/polygenic variants causing DSD are numerous and difficult to diagnose 1) Many genes involved in sex developmental pathways 2) Other genes causing syndromes that also affect sex development F) Diagnosis and treatment involves a multidisciplinary team during the lifetime of the patient 1) OBGYN/Primary care physician 2) Geneticist 3) Surgeon 4) Psychologists 5) Support groups G) Considering the complexity of biological sex determination and the complexity of brain development, development of gender identity is highly complex 1) Multifactorial > many genes + environment 11/7/23 Dr. Darl Ray. Swartz 27 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development H) Chromosomal numerical aberrations resulting from aneuploidies 1) All can occur via zygotic mitotic errors 2) Most occur from meiotic errors in meiosis I (MI) or meiosis II (MII) giving disomic or nullisomic gametes from one of the parents 3) Meiotic errors more frequent in chromosomes with only two chiasmata (a) X, Y, 21, 22, 16 4) Meiotic errors more frequent in oocytes than spermatozoa (a) Oocytes at 20% (i) Increase with increasing age likely associated with years in prophase 1 (ii) Likely overestimated from IVF/clinical data - Not sampling many w/o clinical complications (iii) Gamete be disomic or nullisomic for the chromosome (b) Spermatocytes at 1-2% (i) Likely underestimate as # based on zygote genotype/phenotype - Survival of the fittest zoa that fertilize the egg - Survival of zygote via mitotic rescue of aneuploidies 11/7/23 Dr. Darl Ray. Swartz 28 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development H) Chromosomal number aberrations resulting from aneuploidies 5) Frequency of all types at 1/500 livebirths (a) 1/375 for male (b) 1/600 females Orphanet Journal of Rare Diseases 2010, 5:8 6) Sex chromosome aneuploidies have variable expressivity (a) Mosaics (b) Variable X-chromosome inactivation (c) Epigenetics (d) Androgen receptor CAGn variability (e) Variants in other chromosomes (i) May need more information than just the karyotype 11/7/23 Dr. Darl Ray. Swartz 29 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development I) XXY > Klinefelter syndrome 1) Mostly from paternal non-disjunction MI error giving XY gamete with some being from MI or MII maternal errors giving XX gamete Normal gametes are monosomic Those w/o chromosome are nullisomic 2) 1/500 – 1,000 frequency in male births (likely much higher because of undiagnosed cases) 3) Other Xn>2Y numerical variants have similar features as Klinefelter syndrome 4) Mild phenome resulting in many un-diagnosed but features are: (a) Slowed physical and intellectual development (b) Lowered male phenome (secondary sex characteristics) and infertility (c) Clinodactyly (curved little finger) 11/7/23 Dr. Darl Ray. Swartz (d) Taller 30 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development J) XXX > Generally taller stature 1) Mostly from maternal MI errors rest from MII errors or post-zygotic mitotic errors 2) 1/1000 frequency in female births (a) Likely higher as phenome not unique 3) Other Xn with n>3 have similar phenomes but lower frequency 4) Variable expressivity in the phenomes but major features are: (a) Taller (maybe dosage of SHOX gene) (b) Epicanthal folds (c) Clinodactyly (d) Hypotonia at infancy (e) And others at lower levels including genitourinary malformations, seizures, tremors, hip dysplasia 11/7/23 Dr. Darl Ray. Swartz Orphanet Journal of Rare Diseases 2010, 5:8 31 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development K) XYY 1) Only from non-disjunction MII error giving YY gamete 2) 1/1000 frequency in male births (likely higher because of undiagnosed cases) 3) Variable expressivity likely from mosaics in the phenomes but major features are: (a) Tall stature (b) Learning and developmental disabilities (i) High prevalence in patients with autistic spectrum and ADHD (c) Large head and teeth (d) Clinodactyly 11/7/23 Dr. Darl Ray. Swartz 32 IV) Sex Chromosome Numerical Variants and Disorders of Sexual Development L) XO > Turner syndrome 1) Mostly from paternal non-disjunction XY (MI or MII error) giving nullisomic gamete 2) 1/2,500 frequency in female births 3) Most conceptus lost, thought to be leading cause of miscarriage (a) 1/90 survive to term and develop 5) Variable expressivity in the phenomes but major features are: (a) Short stature (b) Webbed neck and low neck hairline (c) Lymphedema of hands and feet (d) Heart defects (e) Premature ovarian failure/infertility 6) Phenome features thought to result lack of PAR1 region genes and/or requirement for both X chromosomes during embryonic development (a) Short stature via SHOX gene for live birth (b) Placenta/trophoblast development likely use both X chromosomes (i) Do not inactivate X chromosome in trophoblast cells 11/7/23 Dr. Darl Ray. Swartz 33 V) X-Chromosome and Inactivation A) Led to the discovery of linked genes and eventually linkage disequilibrium that underpins GWAS via haplotypes B) Is essential for survival as no OY in the population C) Inheritance pattern follows the Fibonacci sequence with fewer possible ancestors than autosomes 1) One of my daughters’ X is from my mother 11/7/23 Dr. Darl Ray. Swartz 34 V) X-Chromosome and Inactivation D) Moderate sized chromosome of 153 Mbp (ca 3X Y) with about 800 coding genes 1) List at https://en.wikipedia.org/wiki/Category:Genes_on_human_chromosome_X 2) Many involved in X-linked variant diseases both recessive and dominant 3) Some recessive “carriers” have mild forms of the disease with phenotype dependent upon mosaicism in X-linked inactivation (a) Observed in muscular dystrophy and called manifesting carriers if have classic symptoms 4) Few genes involved in sex determination but does carry the AR gene important for both male and female development E) Many numerical, structural, CNV, and SNP variants associated with disease 11/7/23 Dr. Darl Ray. Swartz 35 V) X-Chromosome and Inactivation F) X-chromosome inactivation occurs such that one X – chromosome is used (Xa – active) and the other (for the most part) is heterochromatic (Xi – inactive) 1) Maintain one Xa relative to autosome set (Xa:2A) even in X chromosome numerical aberrations (XXY, Xn>2) 2) Parts of Xi are active especially in PAR1 (a) SHOX gene haploinsufficiency likely responsible for short stature of XO 3) May be skewing as to the origin of which X is inactivated (Xp or Xm) 4) Non-PAR1 genes on the Xi that are activated, called escapees (a) Highly variable expression of escapees from cell to cell and between females 11/7/23 Dr. Darl Ray. Swartz 36 V) X-Chromosome and Inactivation G) Xi is formed via a lncRNA that induces heterochromatinization in cis 1) LncRNA of XIST gene is processed like an mRNA but functions to induce heterochromatin as RNA 2) Somehow targets heterochromatin forming complexes to the chromosome it is transcribed from (in cis) 11/7/23 Dr. Darl Ray. Swartz 37 V) X-Chromosome and Inactivation G) Xi is formed via a lncRNA that induces heterochromatinization in cis 3) Human X inactivation begins randomly in the human blastocyst at about the time of implantation as assessed by single cell RNAseq (expression) data 4) In the post-implantation embryo, Xi is formed in somatic cells giving the bar body of the female nucleus (a) Once formed, maintained in cell progeny by other heterochromatic maintenance mechanisms (XIST is not expressed) http://www.mun.ca/biology/scarr/Barr_Bodies.html 11/7/23 Dr. Darl Ray. Swartz 38 V) X-Chromosome and Inactivation G) Xi is formed via a lncRNA that induces heterochromatinization in cis 5) Xi can get reactivated (a) Germ-line cells of the female and in oocytes during development (i) Idea is that the double dose is required for oocyte development (b) Human pluripotent stem cells irreversibly lose Xi during propagation (Xi erosion) (i) Involves expression of XACT and occurs randomly after the first few passage (ii) Required to give “pluripotent” potential of cells (c) Cancer cells – known for years that the bar body is lost in cancer cells (i) Histology/nuclear morphology is informative! 6) Reactivation of specific chromosome locations (outside of PAR1) may occur in some cell types and or disease 7) Loss of inactivation via loss of methylation may occur during old age (methylome erosion) associated Dr. with aging 11/7/23 Darl Ray. Swartz FEBS Letters 588 (2014) 2514–2522 39 VI) Y-Chromosome A) Small chromosome at 57 Mbp with about 568 predicted genes 1) Most are pseudogenes and lncRNA 2) 27 genes code for bonafide proteins in the male specific region (MSY, non-PARs) (a) 9 ubiquitously expressed (b) 14 testis or organ specific expression (c) 4??? 3) 12 genes are paralogous to X-chromosome genes (X-Y pairs) required for survival (a) Most on the Y not essential as some XO in population (b) Involved in chromatin structure, splicing, and translation 4) Some paralogs/homologs not required or have gene dosage effects (a) Amelogenin for making enamel is present on both but only X-used (i) Variant on X can result in X-linked amelogenesis imperfecta (ii) Some expression of Y homolog gives variation in expressivity (iii) Different sized genes on X and Y that can be used to determine 11/7/23 Dr. Darl Ray. Swartz sex of cells via PCR 40 VI) Y-Chromosome B) Several Y-specific genes involved in testis development and spermatogenesis resulting in infertility 1) SRY (a) Can be translocated onto an autosome giving an XX male DSD (b) Can be mutated with loss of function to give XY female DSD (i) 15% of XY female cases (ii) Undeveloped gonad with female reproductive track and general phenome 2) Deleted in Azoospermia 1 (DAZ) > pre-meiotic translational regulation 3) RNA-binding motif protein, Y-chromosome (RBMY) > transcriptional regulation 4) Testis specific protein, Y-linked (TSPY2) > testis tumor suppressor 5) Ubiquitin specific peptidase 9, Y-linked (USP9Y) > Sertoli cell only syndrome 11/7/23 Dr. Darl Ray. Swartz 41 VI) Y-Chromosome C) Used for paternal genealogy and forensics via the male specific region 1) Used when autosomes are not informative 2) Y-specific short tandem repeats (STR/CNV) of known loci (DSY###) 3) Because of slow mutation, can use to trace lineage if data is available in criminal cases 4) Can use to rule out lineage of perpetrators in cases associated with social unrest where ethnic group may be accused D) Variants in Y-specific genes give Y-linked inheritance 1) Vertical transmission from father to son a) Does not skip generations and males only b) Known examples are genes involved in spermatogenesis 11/7/23 Dr. Darl Ray. Swartz Afr. J. Biotechnol. Vol. 14(27), pp. 2175-2178, 8 July, 2015 42 VII) Mosaicism A) Assumption that the genome of all cells in human are the same is incorrect 1) Programmed somatic mutagenesis in T and B-cell receptor via transposase-based mechanism 2) Increasing evidence for somatic variants in different tissues especially in the brain (a) May be a feature of cerebral development?? 3) May explain differences in heritability/expressivity of many diseases 4) End questions is what is the genome sequence at the single cell level and in the end what is the allelic exome/transcriptome at the single cell or cell type level 11/7/23 Dr. Darl Ray. Swartz Nature Reviews Immunology volume18, pages35–45 (2018) 44 VII) Mosaicism B) Mosaicism is a mixture of cellular genomes in a patient resulting from various causes 1) Random mutations caused by environment, errors in repair and replication, or spontaneous mutations 2) If lethal, mutant cells are lost 3) If not, survive and can contribute to phenome (a) Cancer is a unique form of mosaicism (i) Typically increase mosaicity with increasing duration of cancer developing drug resistant clones 4) Rare and difficult to trace disease origin through pedigrees 5) Can result in reversion (loss of) of inherited disease C) A chimera is an individual formed essentially from two zygotes or different individuals 1) Highly divergent genomes in the different cell types 11/7/23 Dr. Darl Ray. Swartz 45 VII) Mosaicism D) Blastocyst/epiblast/embryoblast likely has significant numbers of variants that develop during proliferation with loss of lethal mutants 1) Cell cycle controls relaxed during zygote > blastula then turned back on 2) Just starting to look at apoptosis (movie) (a) Oct4 gene is known pluripotent marker 3) Survival/selection of the fittest cells before committing to cell differentiation 4) Can delay proliferation of cells resulting from repair 5) Studies on IVF pre-implantation embryos show that 22% diploid and 73% were mosaics (a) Most mosaics were diploid + aneuploid (haploid or polyploid for various chromosomes) (b) Thought that aneuploids lost during early embryonic divisions to result in successful pregnancy 11/7/23 Dr. Darl Ray. Swartz 46 VII) Mosaicism E) Earlier it occurs and the cells survive, more likely to have in a higher proportion of mosaic somatic and germ line cells 1) If in germ line cells (primordial germ cells), found in spermatogonia and oogonia and resultant meiotic products of these cells (a) Termed germ-line mosaicism 2) Germ-line mosaicism is difficult to detect as cause but indicative if the same de novo mutation occurs in siblings 11/7/23 Dr. Darl Ray. Swartz https://www.sciencedaily.com/releases/2017/06/170616083126.htm 47 VII) Mosaicism F) Somatic mosaicism (non-germ line) effects fewer cells/organs the later they form (sans cancer) 1) Variation in expressivity related to proportion of variant cells in tissue, organ, or organ system G) Obvious examples in skin pigmentation disorders 1) Pigment lines follow lines of Blaschko related to epidermal development fields (a) Akin to variated pigments (streaks) in flowers and leaves where formation of mutant can be traced H) Somatic mosaicism observed in numerical, structural, CNV, SNV, and XaXi variants 11/7/23 Dr. Darl Ray. Swartz 48 VIII) Uniparental Disomy and Imprinting A) Diseases resulting from uniparental disomy and imprinting have a very low frequency in human population ranging from 1/18,000 – 980,000 depending on the particular variant B) For some autosomal genes, the expression is dependent upon parental origin 1) Only the maternal or paternal gene is expressed making these genes “sex-linked” even through they are on autosomes 2) Mono-allelic expression instead of bi-allelic expression C) Uniparental disomy results from errors in gamete formation (meiotic errors) or mitotic events 11/7/23 Dr. Darl Ray. Swartz 50 VIII) Uniparental Disomy and Imprinting D) Gametogenic errors result when sisterS of one parental gamete or both autosomes from one parent form the zygote instead of one homolog from each gamete 1) If homologs are of same origin (ApAp or AmAm) called uniparental isodisomy (a) Mostly from MII errors > sisters do not separate 2) If homologs are of different origin (ApAm – i.e. homologous but from one parent) called uniparental heterodisomy (a) Mostly from MI errors > homologs do not separate E) Both idiosomies and heterodisomies can result in mosaics depending on mechanism of formation 11/7/23 Dr. Darl Ray. Swartz 51 VIII) Uniparental Disomy and Imprinting F) Mechanisms of formation upon fertilization or zygotic development: 1) Gamete complementation > nullisomic gamete fuse with disomic gamete (A) 2) Trisomy rescue > monosomic gamete fuses with disomic gamete then loss of one chromosome to return to diploid zygote (B) (a) Mosaics with trisomic cells 3) Nullisomic rescue > nullisomic gamete fuses with monosomic gamete with mitotic duplication of the monosome to return to disomy (C) 4) Post-fertilization repair > loss of aberrant chromosome followed by replication of normal chromosome (D) (a) Mosaics with normal cells 5) Can be forms where homologous recombination occurs (somatic recombination) mixing up segments or gene conversion (segmental uniparental disomy) (E) (a) Mosaics with normal cells 6) Failure of maternal X chromosome replication and replaced by paternal Xchromosome (F) 11/7/23 Dr. Darl Ray. Swartz 52 VIII) Uniparental Disomy and Imprinting G) Imprinting is the mono-allelic expression of a gene dependent upon parental origin 1) Involves heterochromatinization of paternal or maternal allele or loci/region that occurs during gamete formation (a) Haploid/mono-allelic expression for the gene or loci of interest 2) Imprinted gene is methylated during formation of gamete and maintained during development (a) Imprinted gene is inactivated and not expressed in zygote > adult (b) In females, involves egg-specific proteins associated with the subcortical maternal complex (SCMC) proteins as variants in these gene result in imprinting disorders (i) Secondary epimutations 3) Is tissue/organ, developmental, and organism specific (a) We are different than mice 11/7/23 Dr. Darl Ray. Swartz 54 VIII) Uniparental Disomy and Imprinting G) Imprinting is the mono-allelic expression of a gene dependent upon parental origin 4) Best detected by RNAseq data using haplotype SNPs to determine parental origin and a variety of tissues from population of adults – Genotype-Tissue Expression (GTEx) project (a) Observed (conservatively) 42 imprinted genes with variations in tissue and organ allelic expression (i) IGF2 female allele in brain and paternal elsewhere (ii) General paternal bias for allele expression in muscle (iii) Differences between many tissues and cells (transformed and primary culture) as well as individual differences - Least imprinting in cultured primary cells and transformed cells - Most imprinting in endocrine organs (iv) Several previously identified and several new genes identified - Some genes imprinted in all tissues - Only certain chromosomes contain imprinted genes 1. 1, 4, 6, 7, 10, 11, 14, 15, 19, and 20 55 11/7/23 Dr. Darl Ray. Swartz - Chr 11 and 15 imprinted genes most associated with disease VIII) Uniparental Disomy and Imprinting H) Several unique disease genes imprinted and disease phenome results from uniparental disomy 1) Expression of recessive disease form of gene even though heterozygous 2) Lack of expression of functional gene even though not a variant 3) Over expression of a gene (two maternal copies of a paternally imprinted gene or visa versa) I) Explains why cloning from somatic cells has a very low success rate and if successful the organisms have genetic diseases 1) Do not go through a re-writing of the epigenome during gametogenesis 11/7/23 Dr. Darl Ray. Swartz 57