Introduction to the Human chromosome

Introduction to the Human chromosome
L11 Introduction to the Human chromosome

Define the following terms relating to chromosome morphology: sister chromatids, centromere, p arm, q arm, telomere and kinetochore. Define homologs. Describe genes and alleles in relationship to homologs. Define autosomes and sex chromosomes, gametes and somatic cells. Describe the chromosomal basis of gender determination in humans. Define and distinguish the characteristics by which chromosomes are classified. Be certain to use the following terms: metacentric, submetacentric, acrocentric and telocentric. Define the significance of mitosis and differentiate the phases and the significant events in each phase. Define the significance of meiosis and differentiate the phases and significant events in each phase including the sub-phases of metaphase I. Define crossing over including its effect on the alleles located on homologous chromosomes.

Eukaryotic Chromosome
Chromosome – distinguish between species and enable transmission of genetic information, facilitating reproduction and maintenance of a species (linear with ≈ 1010 bp in length) Genes interspersed along the chromosome with origins of replication about every 100,000 bases Made of a single molecule of DNA complexed with proteins (histones) Compacted into the cellular nucleus Occur in sets of two identical sister chromatids Centromere – contains recognition sites for kinetochore proteins Telomeres – specialized repetitive sequences that “cap” each end (maintained by telomerase)

Types of DNA Sequence Heating denatures DNA, causing the double helical structure to come apart Slow cooling allows for the stands to reassociate at a rate dependent on the unique and repetitive sequences contained within the DNA 60-70% of the human genome – composed of single (low copy number) DNA sequences 30-40% of the human genome – moderately to highly repetitive DNA sequences (that are not transcribed) (e.g., satellite and interspersed sequence DNA)

Nuclear Genes Nuclear DNA – ≈3×109 bp / 25,000-30,000 genes
Genes – largely unique DNA sequence that codes for a polypeptide with a cellular function or in combination with other polypeptides to form a functional unit (e.g., enzyme, hormones, receptors) Distributed variations between the chromosomal regions: Heterochromatic regions – contains genetically inert non-coding sequences Centromeric regions – contain non-coding sequences Sub-telomeric regions – highest gene density

Nuclear Genes Multigene family – code for proteins of similar functions, arising by gene duplication and subsequent divergence Classic gene family – high degree of DNA sequence similarity Gene superfamily –limited sequence homology but are functionally related, sharing similar structural domains Exons – coding sequences (remain after splicing) Introns – non-coding intervening sequences (spliced out before translation)

Pseudogenes Pseudogenes – DNA sequences that very closely resemble an expressed gene (but is not generally expressed) Glucocerebrosidase (GBA) gene – codes for a lysosomal enzyme that degrades glycosylceramide → glucose and ceramide GBA pseudogene (ΨGBA) – located 16 kb downstream of function GBA gene Arise through a duplication event inserted into the genome but is inactive due to a mutation in the coding or regulatory elements of the gene Insertion of a cDNA sequence (through reverse transcriptase activity) without promoter sequences for expression

Extragenic DNA Extragenic DNA (“junk DNA”) – non-coding sequence with evolutionary conservation, playing a role in gene regulation Tandem DNA repeats: Satellite DNA – repetitive DNA sequences clustered around the centromeres and kept separate from the main DNA sequences Mini-satellite – terminal telomeric sequences that maintain the integrity of the chromosomal ends, possessing hypervariable-short tandem repeats (for DNA fingerprinting) Microsatellites – nucleotide repeats located throughout the genome that are associated with disease (arise by slip strand mispairing or unequal crossover)

Extragenic DNA Interspersed repetitive DNA sequences:
Short interspersed nuclear elements (SINEs) – short repeated DNA sequences of <500 bp that rely on transposons for expression ALU repeats – 300 bp repeats with sequence similarity to a signal recognition particle from protein synthesis that is cut by Alu I restriction enzyme Long interspersed nuclear elements (LINEs) – long repeated DNA sequences of ≈6,000 bp LINE-1 (L1) – repeated 6,000 bp sequence that codes for a reverse transcriptase Both are implicated in inherited disease miRNA – repress gene expression through RNA/DNA hybrids, exhibiting many widespread functions (e.g., apoptosis, tumorigenesis, cell regulation, organogenesis)

Mitochondrial DNA (mtDNA)
Mitochondrial DNA – 16.6 kb / 37 genes Two types of rRNA and 22 types of tRNAs with 13 subunits of enzymes (e.g., CYT-b and CYT-oxidase) involved in energy production, using oxidative-phosphorylation Maternally inherited

Cytogenics Cytogenics – the study of chromosomes and cell division
During cell division, DNA is maximally contracted and arranged into sister chromatids Chromatids join at the central core (centromere region), diving the chromosome into p and q movement Kinetochore – proteins that assemble at the centromere, allowing for spindle fiber (microtubule) attachment and subsequent separation during replication Proteins: CenH3 (a specialized histone), microtubules (tubulins), motor proteins (dynein and kinesin)

Morphology of Chromosomes
Telomeres – repetitive TTAGGG DNA sequences Normal cells: steadily decrease in length, allowing for cell divisions Tumor cells: increased telomerase activity to increase cell survival Classified by the position of the centromere: Metacentric – located near the center of the p and q arms Submetacentric – located in an intermediate position Acrocentric – located at the terminal end (and may have satellites)

Karyotyping Chromosome-specific band patterns:
Euchromatin – light staining active genes Heterochromatin – dark staining non-coding DNA Diploid (2N): 22 autosomal pairs and 1 sex pair One member of each pair is derived from each pair – haploid (N) Homologs – members of a pair of chromosomes

Nomenclature At any given gene location:
Chromosome number: 1-22 Chromosome arm: p or q Chromosome band: 1-X Abnormality examples: +/- for gain or loss Normal: 46, XY and 46, XX Male with trisomy 21 (Down’s Syndrome): 47, XY+21 Female with Cri du Chat Syndrome: 46, XX, del 5p Translocation: 46, XY, t(2;4)(p23;q25) Karyotype Symbol Explanation Example cen centromere del deletion 46, XX, del(1q21) dup duplication 46, XY, dup (13q14) fra fragile site i isochrome 46, X,i(Xq) inv inversion 46, XX, inv(9p12q12) ish in situ hybridization r ring chromosome 46, XY, r(21) t translocation 46, XY, t(2;4)(q21,q21) ter terminal end pter or qter / mosaicism 46,XY/47,XXY

Stages of the Cell Cycle
Interphase (G1+S+G2): lasts ≈16-24 hours Gap 1 (G1) – duplication of cellular contents Synthesis phase (S) – duplication of the 46 chromosomes Gap 2 (G2) – checks for errors and makes repairs Non-dividing cells arrest in G0 Mitosis – replication of somatic cells, producing 2 identical haploid daughter cells Cytokinesis – division of the cytoplasm

Mitosis Prometaphase (prophase):
Formation of 2 centrioles with microtubules, moving to the opposite poles of the cells Condensation of the chromosomes into sister chromatids Disintegration of the nuclear membrane Attachment of the microtubules to the chromosomal centromeres Metaphase – chromosomes align at the metaphase plate using the spindle apparatus from the centrioles Anaphase – division of the centromeres and separation of the sister chromatids to opposite poles of the cells Telophase – development of new nuclear envelopes around independent chromosomes

Meiosis Meiosis – cellular division during gamete formation (in spermatocytes and oocytes), occurring in 2 stages Meiosis I – reductional division, deriving a haploid number of chromosomes In male X and Y segments, tips of the homologous short arms pair at the pseudoautosomal regions Meiosis II – a mitotic cell division with a haploid number of chromosomes Synaptonemal complex – protein structure that forms between homologous chromosomes (2 pairs of sister chromatids), mediating chromosome pairing, synapsis, and recombination Recombination – crossing-over between non-homologous chromatids, creating recombinant chiasmata

Meiosis – Stages: Leptotene – condensation of chromosomes
Zygotene – alignment of homologous chromosomes along a synaptonemal complex Pachytene – tight coiling of each pair of chromosomes (which can lead to bivalent crossing over between non-homologous chromosomes or chiasmata) Diplotene – separation of chromosomes still attached at the chiasmata May have 1-3 chiasmata depending on the size, generating diversity Diakinesis – continued separation of homologous chromosomes and condensations

Meiosis – Outcomes: Division of chromosomes so that each child receives a maternal and paternal set, preventing identical divisions (1/223 probability) Crossing over generates diversity through gene shuffling, alternating genomic regions from each parent

Chromosomal abnormalities I and II

Contrast/compare aneuploidy and polyploidy.
Using the proper nomenclature, identify the most common aneuploidy conditions Identify mitotic and meiotic nondisjunction and the effects of each. Delineate mosaicism and explain how it effects phenotypic expression of a chromosomal disorder Distinguish between the following chromosomal aberrations: reciprocal and non-reciprocal translocations, Robertsonian translocation, insertion, deletion, paracentric and pericentric inversions, ring chromosome and isochromosome. Summarize the potential complications, if any, that may occur at mitosis and/or meiosis for each aberration. Define chimerism and differentiate between the two most common causes/forms.

Types of Chromosomal Disorders
Chromosomal disorders – most do not result in live birth, leading to spontaneous abortions Single gene defects – Mendelian genetic disorders generating nuclear and mitochondrial gene defects (which typically result in live birth) Multifactorial disorders – most common disorders that lead to fewer congenital malformations, resulting in live births that typically manifest later during adulthood Somatic cell genetic disorders – arise after birth in somatic cells, commonly causing cancers or tumors that are not heritable

Nomenclature Locus – designates the position or location of a gene sequence on a chromosome Alleles – different versions of the gene at a specific locus Homozygosity – having the same allele of a given gene locus on both chromosomes Heterozygosity – having different alleles at the gene locus of a chromosome Polymorphism – significant variability in non-coding regions results from a mutation or random alteration in DNA, presenting 2+ sequence variants in a population with a frequency >1%

Karyotyping – Process:
Isolation of WBCs from a peripheral blood sample, culturing onto a medium containing PHA to stimulate cell division and growth Addition of colchicine, preventing mitotic spindle formation to arrest cell division during metaphase (for maximal visibility) Cells are places in a hypotonic solution to stimulate cell lysis on a prepared slide Digestion with trypsin and subsequent staining with Giemsa, creating unique banding patterns of light and dark bands Euchromatin – light staining of active genes Heterochromatin – dark staining of inactive genes Analysis of “metaphase spread” for karyotyping

Comparative Genomic Hybridization (CGH)
Comparative genomic hybridization (CGH) – reveals the loss or gain of chromosomal regions in test samples relative to normal controls Test and reference sample DNA is labeled and hybridized with green and red fluorochromes, respectively, on either metaphase chromosomes or an array of BAC clones Green chromosomal areas have gained areas while red chromosomal areas have lost areas

Abnormalities Abnormalities arise as a consequence of errors in chromosome replication and division (identified through karyotyping and molecular methods) Numerical: Aneuploidy – deviations from normal 46, XY (e.g., monosomy, trisomy, tetrasomy) Polyploidy – gain of another complete set of chromosomes (e.g., triploidy, tetraploidy) Structural – result from the breaking and rejoining of segments into a different configuration Translocations: reciprocal, Robertsonian Deletions Insertions Inversions: paracentric, pericentric Rings Isochromosomes Mixoploidy: mosaicism, chimerism

Non-disjunction Arise from an error in chromosomal separation during meiosis I or meiosis II Meiosis I error – gamete contains both homologs of 1 chromosomal pair Meiosis II error – gamete contains 2 copies of 1 homolog Mosaicism – an embryo with 2 cell types in very early zygotes during mitosis Cause: unclear Hypothesis: Problem with spindle formation in aging females Recombination failure in some of the female’s fetal primary oocytes

Non-disjunction Monosomy trisomy
Absence of a single chromosome due to an error during anaphase Survival: Turner Syndrome 45, X Presence of an extra chromosome Survival (60%): Down Syndrome 47, XY, +21 Patau Syndrome 47, XY, +13 Edwards Syndrome 47, XY, +18

Aneuploidy – Trisomy: Patau Syndrome – presence of an extra CHR-13 (trisomy 13), causing a deficit in growth and development Edwards Syndrome – presence of an extra CHR-18 (trisomy 18), causing distinctive malformations of the craniofacial area, hands, and feet Craniofacial area: low-set and malformed ears, micrognathia, small mouth with an unusually narrow palate, upturned nose, narrow eyelid folds due to palpebral fissures, widely spaced eyes due to ocular hypertelorism Hands and feet: overlapped, flexed fingers, webbing of the 2nd and 3rd toes, clubfeet

Aneuploidy – Trisomy: Downs Syndrome – presence of an extra CHR-21 (trisomy 21), causing intellectual and developmental disabilities Development: flattened nose and face, upward slanting eyes, single palmer crease, increased toe creases, widely separated 1st and 2nd toes Risk: increases with maternal age (36 years) Non-disjunction in maternal meiosis I Robertsonian translocations – a break between 2 acocentric chromosomes near the centromeres with subsequent fusion of the longer arms (through centric fusion)

Aneuploidy – Partials:
Cat Eye Syndrome (Schmid-Fraccaro Syndrome) – results from an inverted duplication of the long arm inv dup 22q11, causing coloboma of the iris, anal atresia with fistula, downslanting palpebral fissures, heart and renal malformations Normal or near-normal mental development Wolf-Hirschhorn Syndrome (WHS) – results from a partial deletion of the short arm 4p-, causing severe development delays and characteristic facial appearance 85-90% of cases occur due to de novo deletion

Aneuploidy – Partials:
Cri du Chat Syndrome – results from a deletion of the short arm del5p, causing a “cat-like” cry from an underdeveloped larynx Others: severe cognitive and motor delays, behavioral problems, unusual facial features, small head with wide eyes 90% of cases occur due to de novo deletion DiGeorge Syndrome – results from a deletion of the long arm 22q11.2, causing developmental defects Developmental defects: palatal defects, conotruncal heart defects, abnormal ear exams, hypocalcemia, microcephaly, mental retardation

Polyploidy Created non-viable fetuses that are either spontaneously aborted or die shortly after birth Triploidy – 69 Tetraploidy – 92 Causes: Retention of a polar body in oocyte division Formation of a diploid sperm Fertilization of an oocyte by 2 sperm (dispermy)

Translocations Translocations – transfer of genetic material from 1 chromosome to another due to the breaking and exchanging of fragments Can be an equal balanced or reciprocal exchange, preserving all chromosomal material (e.g., CHR-11 ↔ CHR-22) Identified by FISH Robertsonian translocation – reciprocal translocation in which the breakpoints are located at or near the centromere of 2 acocentric chromosomes, creating a large metacentric chromosome and a much smaller fragment (e.g., CHR-13, -14, -15, -21, -22) Can produce balanced or unbalanced bivalent, trivalent, or quadrivalent translocations, leading to normal translocations, partial trisomy, or partial monosomy

Translocations – Quadrivalent:
The pattern of chromosome segregation (adjacent or alternate) determines whether the gametes have a balanced or unbalanced complement of genetic material 2:2 segregation – segregation of the quadrivalent during the later stages of meiosis I If alternate chromosomes segregate to each gamete, the gamete will carry a normal or balanced haploid complement With fertilization, the embryo will either have normal chromosomes or carry a balanced arrangement 3:1 segregation – segregation of 3 chromosomes to 1 gamete with only 1 chromosome to the other gamete, leading to an unbalanced trisomy or monosomy

Translocations Downs syndrome – can result from a balanced Robertsonian translocation to cause a partial 21 trisomy of 13q21q or 14q21q Parents with one child with a partial trisomy 21 from a balanced location may be at risk for a 2nd child with Down Syndrome Translocation tracing – identifies translocation carriers in a family All gametes will be disomic for 21 or nullsomic for 21 66% of cases occur due to de novo deletion

Deletions Deletions - result in a loss of genetic material, generating a monsomic region ( >2% deletion of a haploid genome is lethal) Large chromosomal deletions: Cru du Chat Syndrome (4p del) and Wolf-Hirschorn Syndrome (5p del) Seen under a light microscope Submicroscopic deletions: Angelman Syndrome (15p del) and Prader-Willi Syndrome (15p del) Seen by high resolution prometaphase cytogenic and FISH

Insertions Insertions – when one segment of a chromosome is inserted into another chromosome Balanced – if moved from another chromosome and inserted Unbalanced – if lost from another chromosome and inserted 50% chance of inheriting the insertion or deletion randomly

Inversions Inversions – two-break rearrangement within a single chromosome where the segment is reversed in position Pericentric inversion – includes the centromere Crossover during meiosis I: inversion loop forms, resulting in 1 duplication of the non-inverted segment and deletion of the other end (while the other has the opposite arrangement with the inversion chromosome) Paracentric inversion – does not involve the centromere If crossover loop occurs, the products are an acentric fragment or dicentric (which fails to undergo mitosis and leads to zygote failure)

Others Isochromosomes – loss of 1 chromosomal arm with the duplication of the other (2p’s or 2q’s) Ring chromosome – created when a strand break occurs at both ends of the same chromosome, generating “sticky ends” that form a ring (while the material from the ends is deleted)

Mixoploidy Mosaicism – individual with 2+ somatic chromosome numbers derived from the same zygote Chimerism – presence in an individual of 2+ distinct cell lines of different genetic origin Dispermic chimeras – result of double fertilization of 2 ova which fuse to 1 embryo Hermaphrodite – fusion of different sex embryos (XX/XY) Blood chimeras – exchange of blood between twins in utero

Uniparental Disomy Uniparental disomy – presence of 2 homologous chromosomes inherited from only 1 parent Isodisomy – parent passes on 2 copies of the same chromosome (through non-disfunction in meiosis II) Heterodisomy – parent passes on 1 copy of each homolog (through non-disfunction in meiosis I)

Abnormalities – Sex Chromosome:
Gender specific: Female abnormalities – variation in the number of X chromosomes Turner Syndrome (45, X) and Triple X Syndrome (47, XXX or 48, XXXX) Male abnormalities – variations in the X and Y number Klinefelter Syndrome (47, XXY or 48, XXXY or XY/XXY mosaic) or XYY Syndrome (47, XYY) of “super-males”

Instability Syndromes
Rare single gene (autosomal recessive) syndromes where there is a characteristic cytogenic abnormality, generating a broad molecular defect (and increased risk of cancer) Bloom Syndrome – defect in DNA ligase, increasing somatic recombination and sister chromatid exchange ICF Syndrome – deficiency of 1 of the DNA methyltransferases that maintains pattern of genome methylation, leading to centrometric instability and immunodeficiency E.g., Fragile X Syndrome

Patterns of inheritance: mendelian

Define the following basic genetics terms: dominant allele, recessive allele, phenotype, genotype, mutation, premutation and wild type. Distinguish why it is more difficult to determine inheritance patterns in humans than in experimental animals such as fruit flies. Describe how such patterns are ascertained in humans. Calculate genetic risk by using a Punnett square. Define pleiotrophy. Compare/contrast variable expressivity and penetrance and their clinical presentations. Explain why most lethal autosomal dominant traits are inherited from a mosaic parent or result from de novo mutations. What kinds of lethal dominant traits can be directly passed through several generations? Define and give an example of codominance. Define consanguinity and explain why it increases the probability of having a child with a genetic disorder. Define locus heterogeneity and distinguish its effect on genetic inheritance. Explain why males are more commonly affected by a recessive trait carried on the X chromosome. Define the tenants of the Lyon hypothesis and recognize the results of these tenants in females. Define skewed inactivation. Distinguish why it is difficult to determine an X-linked dominant trait from a pedigree. Recognize the pedigree for a Y-linked trait. Name the two traits inherited via this mode. Distinguish the clinical presentation of traits inherited via partial sex linkage. Distinguish between the pedigrees for the following modes of inheritance: autosomal dominant, autosomal recessive, X-linked recessive, X-linked dominant and Y-linked recessive. Define multiple alleles and give an example. Calculate genetic risk using a Punnett square. Distinguish between the following non-Mendelian patterns of inheritance: anticipation, mosaicism (somatic and gonadic), uniparental disomy Distinguish between polygenic and multifactorial inheritance. Compare/contrast the presentations of monogenic and polygenic traits present within a population. Given a specific trait, choose its mode of inheritance. Determine if a trait is polygenic or multifactorial.

Nomenclature Mendelian inheritance – single gene traits caused by mutations in nuclear genes Locus – a segment of DNA occupying a defined position (frequently called “gene”) Alleles - alternative variants of a locus/gene Wild-type – common allele Mutant or variant allele – differ from wild-type due to a mutation Haplotype – a given set of alleles at a locus or cluster of loci on a chromosome Polymorphism – at least 2 reasonably common loci variants Genotype – set of alleles that make up an individual’s genome Phenotype – observable expression of the genotype as a morphological characteristic or biochemical trait Single Gene Disorder – determined by the alleles at a given locus Homozygous – all alleles the same at a given locus Heterozygous – alleles at a given locus are different Compound heterozygote – mutant alleles of the same loci present on each chromosome Pleiotrophy – genes that have 1+ discernible effect

Nomenclature Hemizygous – abnormal allele on male X chromosome Proband – the member of a family who appears to have the disease (index case) Kindred – extended family diagramed in a pedigree Pedigree – graphical representation of a family tree with constant symbols 1st degree relative: parents, siblings, and offspring of the proband 2nd degree relatives: grandparents, aunts, and uncles, etc. Consanguineous – a couple who have 1+ common ancestors Penetrance – probability that a gene will have any phenotypic expression at all (% of people with the genotype who are affected) Reduced penetrance – <100% expression of the disease genotype Expressivity – the severity of expression of the phenotype among individuals with the disease causing genotype Variable expressivity – severity of disease differs among people with the genotype

Mendelian Inheritance
Unifactorial inheritance – disorders or traits attributed to a single gene disorder Autosomal inheritance – a gene that is located on an autosome Sex-linked inheritance – a gene on an X or Y chromosome X pairs with the pseudoautosomal region on Y (vice-versa) Patterns of inheritance: Dominant expression – only 1 copy of a mutant gene pair is needed (HH/Hh) Recessive expression – 2 copies of the same mutant allele is present (hh)

Autosomal Inheritance
Male and females are equally affected Dominant inheritance: Expressed in both homozygous dominant (HH) and heterozygous (Hh) individuals Co-dominance expression of 2 alleles at the same locus (e.g., ABO blood groups) Possible incomplete dominance of heterozygotes, leading to less severe symptoms Recessive inheritance: Expressed only in homozygous recessive individuals (hh) Not expressed in carriers but can contribute to loss of function as a result of the mutation in future generations (Hh) Pseudoautosomal inheritance – exchange of X and Y genetic material, mimicking autosomal inheritance Dyschondrosteosis – dominant mutation in SHOX gene skeletal dysplasia with forearm deformity and short stature

Onset of Disease Congenital disorder – recognized at birth
Fetal disorder – shown by multiple miscarriages in a family (proving reduced fertility) Late-onset disorder – can reproduce (but the presence of the allele may be masked by the death of a parent) Genetic heterogeneity – diseases that show similar phenotypic variations Allelic heterogeneity – related phenotypes as a result of different mutations at the same locus CFTR has 1,400 mutations that all produce a clinical spectrum of the disease (Classic Cystic Fibrosis), causing progressive lung disease, pancreatic insufficiency, and congenital absence of the male vas deferens Loci heterogeneity – overlapping phenotypes as a result of mutations at different loci Retinitis Pigmentosa – causes visual impairment due to degeneration of the photoreceptors (through autosomal dominance, autosomal résistance, and X-linkage)

Onset of Disease Phenotypic (or clinical) heterogeneity – distinct phenotypes in different families with mutations in the same allele, impacting genetic counseling Hirschprung Disease – dominant loss of function mutation in RET (receptor Tyr kinase), causing a failure to develop colonic ganglia defect in colon motility and constipation Other RET mutations: multiple endocrine neoplasia type 2A/2B – inherited cancer of the thyroid and adrenal glands due to unregulated hyperfunction LMNA gene – encodes lamin A/C (a nuclear envelope protein) Associated with: Emery-Driefuss Muscular Dystrophy, Charcot-Marie-Tooth Peripheral Neuropathy or Lipodystrophy, Hutchinson-Gifford Progeria

Pedigree Analysis of gene transmission (diagramed in a Punnett square): Carrier × Carrier: Rr × Rr – generates RR, 2 Rr, or rr offspring (1:2:1) Carrier × Affected: Rr × rr – generates 2 Rr and 2 rr offspring (1:1) Affected × Affected: rr × rr – generates rr only Influenced by the sex of individual with sex-influenced traits HFE gene mutation: Hemochromatosis – affects females by lowering the dietary intake of iron, lowering alcohol usage, and increased iron loss during menstruation Carrier frequency – important in calculating disease risk → genetic counseling R r RR Rr rr R r

Consanguinity Consanguinity – chance of a mutant allele at the same locus increases if the parents are related (due to possessing a single common ancestor) Measured by the coefficient of inbreeding (F) to identify the identity of descent Xeroderma Pigmentosum – defective DNA repair (commonly in 1st cousin marriages) Inbreeding – mating between individuals of an geographically restricted area (or for cultural reasons), increasing the risk of obtaining a mutant allele Tay-Sachs Disease – autosomal recessive neurological degenerative disorder resulting in death within 6 months in Ashkenazic Jews (frenquency: 1/30)

Autosomal Dominant Disorders
Huntingtons Disease – completely autosomal dominant disease Incomplete dominant diseases: Achondroplasia – skeletal disorder, causing short stature dwarfism Familial Hypercholesteremia – premature coronary disease Sex-limited dominant diseases – autosomally transmitted within one gender LCGR mutation: Familial Testotoxicosis – male-limited precocious puberty at 4 years of age

X-linked Inheritance X-linked inactivation of normal females: random inactivation of an X in somatic cells, equalizing the amount of product expressed in each sex One X active in one set of somatic cells and the other X active in a second set of somatic cells (e.g., Barr bodies), acting as mosaics for 2 cell populations to manifest possible heterozygotes Manifestation of heterozygotes or skewed X-linked inactivation: Hunter’s Syndrome – X-linked lysosomal storage disorder in which cells can make iduronate sulfatase (active normal X), secreting the enzyme into the cellular space, while mutant X cells can export the active enzyme

X-linked Dominance Regularly expressed in heterozygotes, lacking male-to-male transmission and affecting all daughters of affected males Due to X-linked inactivation, almost all females express incomplete X- linked dominance Hypophosphatemic Rickets – impaired ability of the kidney tubules to reabsorb PO4 Rett Syndrome – mutation in a DNA-binding protein (MECP2), causing the rapid onset of neurological problems (which leads to spastic children with purposeless flapping of arms and legs and autism)

Mosaicism “Pure” somatic mosaic – mutant cells are not present in the gametes, manifesting as a segmental or patchy abnormality (e.g., Segmental Neurofibromatous 1 (NF1)) Depends on when the mutation occurred and what cell lineage it occurred in “Pure” germline mosaic – mutant cells are restricted to the gametes (e.g., Hemophilia A/B and Osteogenesis Imperfecta) Must rule out autosomal dominant, X-linked disease, and carrier status Mixed mosaic – could be present in both somatic and gametic cell lineages (depending on when the embryonic mutation occurred)

Characteristics: Autosomal Recessive
Characteristics: Autosomal Dominant Phenotype seen in siblings of the proband Phenotype appears in every generation with each affected individual having an affected parent (exceptions low penetrance and new mutation) Males and females equally affected Any child of an affected parent has a 50% of disease Both parents of an affected child are asymptomatic as carriers of mutation Phenotypically normal family members do not transmit the disease phenotype to their children (exceptions low penetrance or variable expressivity) Parents may be from consanguinous mating (especially if the condition is rare in the general population) Males and females equally likely to transmit phenotype to children of either sex Recurrence risk is ¼ for sibling of the proband Isolated cases may be due to a new mutation (especially if fitness is low)

Characteristics: X-linked Recessive Characteristics: X-linked Dominant
Incidence of traits is much higher in males than females Affected males with normal mates: No affected sons with all daughters affected Heterozygous females are unaffected (but may express the condition depending on X-linked inactivation pattern) Offspring of carrier females (males and females equally) have a 50% chance of inheriting disease Affected males transmit to all daughters and each carrier daughter has a 50% chance of transmitting to her sons Affected females are 2X as common as affected males and tend to have milder disease and variable expression of phenotype No male to son transmission Carrier females transmit the trait while affected males in a pedigree are related via female relatives A high % of isolated cases are due to new mutations

Unstable Repeat Expansions
Generation of expanded repeats beyond a normal range can lead to a disease phenotype: Mechanism: “slipped mispairing” – a DNA replication error Repeat CAG (poly Q): CAGCAGCAG Repeat CCG (poly R): CCGCCGCCG All conditions have neurological symptomology Parental bias – anticipated susceptibility Founder effect – loss of genetic variation that occurs when a new population is established by a very small number of individuals Transmission: autosomal, recessive, or X-linked Differences: Length and base sequences of the repeated unit Number of repeats in a normal, presymptomatic or affect individual Location of the repeated unit within gene Pathogenesis of disease Degree to which instability occurs in mitosis and meiosis Parental bias where expansion occurs

Huntingtons Disease – autosomal dominant neuropathic degeneration of the cortex and striatum, leading to death (repeat CAG (poly Q) >39 expansions) Appears at earlier ages when transmitted by an affected male Premutation: poly Q expansions Other poly Q diseases: Spinobulbar Muscular Atrophy (X-linked) and Spinocerebellar Ataxias (autosomal dominant) Fragile X Syndrome (Xq.27.1) – improper condensation of chromatin during mitosis (repeat CCG (poly R) >1000 expansions) Premutation: poly R expansions

Myotonic Dystrophy – autosomal dominant (through the mother) disorder, causing myopathy, cataracts, and hypogonadism (repeat CTG (poly L) >2,000 expansions) Premutation: expansions Friedreich Ataxia (FRDA) – autosomal recessive expansion in the mitochondrial intron of frataxin, causing uncoordinated limb movements, cardiomyopathy, speech difficulties, and scoliosis (repeat AAG (poly K) >100 expansions)

Genetic Modification Modifier – a gene that alters the phenotype in a non-allelic gene Cystic Fibrosis: explanation for pulmonary insufficiency Mannose binding lection 2 (MBL2) – prevents phagocytosis induced through complement activation on the surface of carbohydrates Transforming growth factor-β1 (TGFβ1) – promotes lung fibrosis and scarring after infection β-Thalassemia: coinheritance of α-Thalassemia is linked to milder disease expression, reducing the amount of excess α- chain produced to offset the β-chain imbalance in hemoglobin

Gene Pleiotrophy Pleiotrophy – genes with 2+ discernible effects or a gene that expresses effects on multiple aspects of physiology and anatomy Marfan Syndrome – autosomal dominant of Fibrillin 1 gene in connective tissue, causing abnormalities in the skeleton, eye, and cardiovascular system Others: Cystic Fibrosis – affects the sweat glands, lungs, and pancreas Osteogenesis Imperfecta – affects the bones, sclera, and teeth Albinism – affects pigmentation and optic fiber development

Patterns of inheritance: mitochondrial

Identify the organization and structure of mitochondrial genome DNA and give examples of what type of proteins it encodes. Distinguish homoplasmy and heteroplasmy. Understand maternal input (transmission) on disease Identify the interaction between nuclear genome and mitochondrial genome. Identify role of mitochondrial in disease (ex: mutations in mtDNA genome LHON, MERRF) and mutations in nuclear DNA (Leigh Syndrome, secondary OXPHOS disorders)that result in mitochondrial dysfunction. Identify examples of nuclear gene defects that cause mitochondrial disease.

Mitochondria Cross-talk:
Nuclear genes – provide mitochondrial proteins (1,500) which can have mutations, conveying mutant proteins to the mitochondria (e.g., heme biosynthesis, protein import and assembly, mtDNA transcription/replication) >60% of diseases result from nuclear gene mutations of proteins Mitochondrial genes – encode 13 proteins functioning in respiratory complexes 15% of diseases result from mitochondrial gene mutations

Mitochondria Compartmentalization:
Cytosol: glycolysis Innermembrane: Fatty-acyl CoA transport & oxidative phosphorylation Matrix: TCA cycle & β-oxidation Oocyte production: small number of mother’s mitochondria are randomly selected to enter the early egg cells Bottleneck effect – random selection can contribute a greater ratio of mutant mitochondria per egg (which replicate), increasing the possibility of heteroplasmy in the child If the level of mutant mitochondria exceeds a certain threshold  mitochondrial dysfunction

mtDNA transcription and replication
Map of Disease MELAS – Mitochondrial Myopathy and Encephalomyopathy  lactic acidosis and stroke-like symptoms KSS – Kearns-Sayre Syndrome LHON – Leber’s Hereditary Optic Neuropathy NARP – Neuropathy, Ataxia, and Retinis Pigmentosa MERRF – Myoclonic Epilepsy with ragged red fibers LSP – Leigh Syndrome mtDNA transcription and replication

Mitochondrial Disease
Mitochondrial Myopathy – subclass of disease with neuromuscular deficiency Symptoms: poor growth, muscle weakness and loss of coordination, visual problems, hearing problems, learning disabilities, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems (e.g., seizures and neurodevelopmental disorders), autonomic dysfunction, dementia, diabetes mellitus

LHON – Leber’s Hereditary Optic Neuropathy – causes painless, bilateral and subacute visual failure Diagnosis: visual findings of legal blindness (which significantly impacts quality of life) Mutation: m.3460G>A, m G>A, or m.14484T>C Affect different Complex I genes Treatment: provision of visual aids and occupational therapy with support At-risk: alcoholics and smokers LSP – Leigh Syndrome – causes heterogeneous respiratory chain complex defects Mutation: NDUFA12 (nuclear) of Complex I or ATP6 (mitochondrial) of Complex V Others: MTTV, MTTK, MTTW, or MTTL1 of mitochondrial tRNA proteins Inheritance: X-linked recessive and autosomal recessive

Mitochondrial depletion syndromes: Alpers Syndrome (4A) and MNGIE (4B): DNA polymerase gamma-2 (PLOG2) – c-terminal polymerase domain with an n- terminal exonuclease domain for proof-reading function and increased fidelity of mtDNA replication Mutations in exonuclease function: high frequency of randomly distributed mutations in the mtDNA genome (due to decreased polymerase activity) Mutation: A467T and repeat CAG (poly Q) expansion Progressive External Opthalmoplegia – autosomal dominant and recessive mutation in PLOG

KSS – Kearns-Sayre Syndrome – causes opthalmoplegia, pigment degeneration of the retina, and cardiomyopathy (with associated facial and muscle weakness, deafness, small stature, and increased CSF protein) Mutation: mtDNA deletions in the muscle mitochondria (e.g., MTTL1) MERRF – Myoclonic Epilepsy with ragged red fibers – causes an elevation in pyruvate and lactate due to deficiencies in the Complex I (NADH-CoQ reductase) and IV (cytochrome c oxidase) subunits Mutation: 8344A>G of MTTK (80-90%), MTTL1, MTTS1/2, MTTF, or MTND5 NARP – Neuropathy, Ataxia, and Retinis Pigmentosa – causes sensory neuropathy, muscle weakness, ataxia, and vision loss Mutation: ATP6 (a subunit of ATP synthase) (70-90%)

MELAS – Mitochondrial Encephalomyopathy – causes muscle weakness, recurrent headaches, loss of appetite, vomiting, and seizures resulting in brain damage (with associated lactic acidosis) Mutation: Complex I (NADH-CoQ reductase) and tRNA MT-TL1 (80%), MT-TH, or MT-TV MNGIE – Mitochondrial Neurogastrointestinal Encephalopathy Syndrome (POLIP Syndrome) – autosomal recessive mutation in TYMP (thymidine phosphorylase) (100%), causing poor GI mobility, pseudo-obstruction with peristalsis, and subsequent malabsorption Mutation: Complex IV (cytochrome c oxidase)

Acquired Mitochondrial Disease
Polymerase gamma (POLG) – replicates mitochondrial DNA (similar to reverse transcriptase in HIV) Antiretroviral drugs will also inhibit POLG in AIDS patients, stopping the replication of host mitochondrial DNA and leading to a decreased number of organelles and function → metabolic acidosis Treatment: IVF of donated female eggs with affected mother’s maternal genome and father’s sperm creates an embryo with no mitochondrial disease

Patterns of inheritance: multifactorial

Distinguish between polygenic and multifactorial inheritance.
Describe how polygenic traits present within a population. Give examples. Describe the tests to determine if a trait is polygenic or multifactorial.

Complex Disease Inheritance
Non-genetic factors (e.g., the environment) play a crucial role in disease causation: Multifactorial or complex inheritance pattern Family members have more shared gene patterns (2-4%) and environmental exposures than an individual chosen at random from the population Gene-to-gene interactions: polygenicity of multigenic effects – additive or synergistic amplification by genes at multiple loci Gene-to-environment interactions: raise or lower susceptibility, triggering disease acceleration Examples: common congenital malformations or acquired childhood and adult diseases

Complex Disease Inheritance
Qualitative traits – presence or absence of a genetic disease Quantitative traits – measurable physiological or biochemical quantities (e.g., height, blood pressure, serum cholesterol concentration, and BMI) Relative risk ratio (λr) – measured of familial aggregation by comparing the frequency of the disease in the relatives of an affected proband with its prevalence in the general population λ 𝑟 = 𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑖𝑛 𝑟𝑒𝑙𝑎𝑡𝑖𝑣𝑒𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑟𝑜𝑏𝑎𝑛𝑑 𝑝𝑟𝑒𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑖𝑛 𝑡ℎ𝑒 𝑔𝑒𝑛𝑒𝑟𝑎𝑙 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛 Value of λr = 1 (low): indicates that a relative is no more likely to develop the disease than any other individual in the population

Genetic Studies Gene mapping studies identify genes and associated coincidence of mutation: Concordance – 2 related individuals with the same disease (through inheritance or shared environmental factors) Can occur even when the 2 affected relatives have different predisposing genotypes (with a “phenocopy” or “genocopy” of the disease) Discordant – only 1 member has the disease in a comparison of 2 family members (in which the unaffected doesn’t possess the predisposing genotype or has not experienced the “trigger” for the disease) Case control studies compare patients with disease with selective criteria that direct the choice of individuals without the disease (controls): Ascertainment bias – family with the disease can report better than a control family Recall bias – family with affected members that are more motivated to report

Alleles in Common with the Proband
Genetic Studies Selective criteria: occupation, environmental exposure, geographic location, ethnicity Unrelated family members act as controls: Spouses matching in age, ethnicity, location, and exposure Alleles in Common with the Proband RELATIONSHIP PROPORTION Monozygotic twins 1 1st degree relative: dizygotic twin, sibling, or parent 2nd degree relative: grandparent 3rd degree relative ⅛

Twin Studies Digozytic (DZ) twins – 2 eggs fertilized upon conception, sharing 50% commonality between alleles Greater concordance for disease in MZ twins compared to same sex DZ twins presents a strong argument for genetic influence in disease development Monozygotic (MZ) twins – split from the same fertilized egg and, thus, sharing identical genes (0.3%) Allows for comparison of identical individuals raised in different environments Disease concordance of <100% presents a strong argument for the influence of non-genetic factors (e.g., exposure to infection, dietary differences, somatic mutations, aging, X-linked inactivation in females) Limitations: MZ share the same genotype but not the exact same gene expression due to somatic rearrangements or X-linked inactivation Environmental exposure will not be the exact same into adulthood Measurement of concordance give an average estimate: relative predisposing factors may differ and skew interpretation Ascertainment bias: Volunteer-based ascertainment – one twin recruits the other after diagnosis of the disease Population-based ascertainment – recognized first as twins before the health assessment

Multifactorial Inheritance
Multifactorial inheritance – combination of several genes or variants Variety of environmental conditions: prenatal exposure in utero, childhood environment, and adult environment Polygenic (quantitative traits) – exhibit a Gaussian distribution Position of the graph peak and shape are governed by the mean (μx) and the variance (σx2) Mean (μx) – arithmetic average of the values Variance (σx2) – measure of the spread of values on either side of μx Trait is considered normal or abnormal depending on how far it is above or below μx Basic statistical theory – when a quantitative trait is normally distributed in a population, only 5% of the population will have measurements more than 2 standard deviations above or below μx Approximately 68%, 95%, and 99.7% of observations fall within the mean ± 1, 2, or 3 standard deviations, respectively

Polygenic Inheritance
Binomial expansion: (p + q)(2n): where p = q = ½ and n = # of loci As the number of loci increases, the distribution increasingly resembles a normal Gaussian curve If the trait is determined by 2 equally frequent alleles at a single locus: phenotypic distribution of three groups 1:2:1 If the trait is determined by 2 alleles at 2 additive loci: distribution phenotypic distribution of five groups 1:4:6:4:1

Polygenic Inheritance
Correlation: statistical measure of the degree of the relationship between 2 parameters Example: 1st degree relatives share 50% of their genes and should correlate as 0.5 if polygenic Physiological parameters with continuous normal distribution: BP, head circumference, height, intelligence, skin color Regression of the mean – observed tendency for offspring to deviate from the mean (due to certain characteristics being influenced by the environment or by non-additive genes)

Liability/Threshold Model
Liability/threshold model - all of the factors that influence the development of a multifactorial disorder (genetic or environmental) can be lumped together as a single phenomenon known as liability, accounting for discontinuous multifactorial traits Population incidence – proposes that a threshold exists above which the abnormal phenotype is expressed in a general population Familial incidence – proposes that a threshold exists above which the abnormal phenotype is expressed among relatives Curve is shifted to the right for relatives and the extent of the shift depends on relatedness

Liability/Threshold Model
Deleterious liability – combination of several “bad” genes and adverse environmental factors The mean liability of a group can be determined from the incidence of the disease in the group using the statistics of the normal distribution, estimating the correlation between relatives Generates a threshold that, once reached, determines those that are affected (based on familial risk) Examples: cleft lip or palate, club foot, congenital heart defects, hydrocephaly, neural tube defects, pyloric stenosis

Heritability Heritability (h2) – defined as a fraction of total phenotypic variance of a quantitative trait that is caused by genes (not the environment) and measures the extent that different alleles at a variable number of loci are responsible for variable expression across a population (by quantifying the role genetic differences play in determining variability across a quantitative trait in a population) No genetic contribution: h2 = 0 Genetic contribution: h2 = 1 Difficulties: Relatives share more than genes: an h2 value alone may not be a good estimate Even if h2 is high (closer to 1), it tells you nothing about the mechanism underlying the trait (i.e., the number of alleles) or how the alleles at the different loci interact Cannot be considered in isolated populations Degree of familial clustering: λ 𝑠 = 𝑟𝑖𝑠𝑘 𝑡𝑜 𝑡ℎ𝑒 𝑠𝑖𝑏𝑙𝑖𝑛𝑔𝑠 𝑜𝑓 𝑎𝑓𝑓𝑒𝑐𝑡𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑟𝑖𝑠𝑘 𝑡𝑜 𝑡ℎ𝑒 𝑔𝑒𝑛𝑒𝑟𝑎𝑙 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛 Multifactorial diseases are common and pose a significant risk to morbidity and mortality

Identification Method 1: Linkage Analysis – map single gene disorders by studying co-segregation of other known genetic markers Complications: Difficult to look for additive linkages of several genes that minimally contribute to a disease phenotype in polygenic traits Age of onset can impact the genetic status of family members Requires excess information (e.g., mode of inheritance, gene frequencies, and penetrance) that is difficult to acquire Method 2: Affected-Sib Pair Analysis – look for “identity by descent” in affected sibling pairs Whether the affected siblings inherit a specific allele more or less often than by mere coincidence (as a random incidence in the population)

Identification Method 3: Linkage Disequilibrium Mapping – mapping a specific region of a chromosome expected to contain the disease gene (by reducing it to a finite area, constructing SNP haplotypes within the region, defining historial crossover points, and sequencing possible variants associated with the disease) Affected by mating preferences Method 4: Association Studies – compare frequencies of a specific variant in affected parents with its frequency in a carefully matched control group (in a case control study) Odds ratio – measures the strength of association (evidence for association if the frequencies differ significantly) Disadvantages (that grant false positives): small sample sizes, weak statistical support for SNP or loci, low a priori probability of selected SNPs or varients associated with the disease, and population stratification

Identification Method 5: Genome Wide Association Studies – compare multiple variants across the genome in a case control study HAPMAP Project – typing of SNPs to different ethnicities in order to show that SNPs are strongly correlated with SNPs on a nearby genetic region Advantages: allows for a comprehensive unbiased scan of the genome that can identify novel susceptibility factors (by capturing all meiotic events in a population and disease genes with only small associated risks) Identifies multiple interacting disease genes and their respective pathways, providing a comprehensive understanding of the disease etiology Associated results: allow for a better understanding of disease susceptibility, identification of low and high risk genes in a polygenic disease, identification of new drug targets directly related to disease causation (in personal and proactive therapy), and susceptibility and expected onset of disease ascertained earlier in life (for better detection and safer treatment to prevent significant damage or for administration of therapeutics to prevent the disease entirely)

Multifactorial Disease
Digenic Retinitis Pigmentosa – causes retinal degradation due to heterozygous mutations in 2 unlinked genes, resulting in photoreceptor membrane defects Idiopathic Cerebral Brain Thrombosis – causes clots to form in the venous system of the brain, leading to catastrophic occlusion and death (5-30%) Factors: Factor V – Arg 506 Gly substitution increases stability to sustain a longer anticoagulant effect Prothrombin – mutation (g.2021G>A) in 3’URT Oral contraceptives – contain synthesis estrogen, effecting Factor X and prothrombin Placental Artery Disease – causes severe preeclampsia, premature separation of the placenta from the uterine wall, and intrauterine growth retardation and stillbirth Factors: oral contraceptives, mutation (g.2021G>A) in prothrombin gene, methylene tetrahydrofolate reductase (MTHFR) variant

Deep Vein Thrombosis – due to Factor V and prothrombin mutations Factors: trauma, orthopedic surgery, malignant disease, prolonged periods of immobility, oral contraceptives, increased age Can develop to a pulmonary emboli  death Hirschprung Disease (HSCR) – complete absence of the intrinsic ganglion cells of the mesenteric and submucosal complexes of the colon, preventing peristalsis and causing constipation and intestinal obstruction Factors: RET gene at 10q11.2 of tyrosine kinase receptor, EDNRB gene at 13q22 of G-coupled protein endothelial receptor B, EDN3 gene at 20q13- ligand (acting in parallel pathways to interact and develop ganglion cells)

Alzheimer’s Disease – neurogenerative disease that causes dementia in individuals >65 years of age (1-2%), characteristically exhibiting an accumulation of amyloid plaques in the brain Factors: associated with ApoE allele e4/e4 (19%), copper interaction with the blood-brain barrier At-risk: age, gender, family history, African-American or Caribbean Hispanic ethnicity

Principles of epigenetics and epigenetics mechanisms

Epigenetics Epigenetics – encompasses all the heritable changes and alterations of gene expression (without changing the DNA sequence) Stably transmitted from one cell generation to next, forming “cellular memory” Effects are reversible (through reprogramming) Effects involve modifications (e.g., genomic imprinting, X-linker inactivation, position effects) Changes in regional chromatin structure: Gene silencing – inaccessibility to the condensed heterochromatin prevents access to transcription factors, preventing gene expression Position effect – permanent silencing of a gene due to inversion or translocation of a DNA sequence, leaving it permanently condensed near the centromere

Epigenetics Epigenetic marker enzymes: Epigenetic markers:
“Writers” – add covalent chemical marks to DNA or histones “Erasers” – remove covalent chemical marks in DNA or histones “Readers” – effector proteins that bind to interpret epigenetic markers Recruit additional proteins to create different chromatic states: Compaction Chromatic remodeling – changes in nucleosome spacing and structure, allowing access to transcription factors Histone substitution (of non-standard histones) Epigenetic markers: Patterns of methylation in cis-acting regulatory elements of DNA strands determine transcription (playing a role in genomic imprinting and X-link inactivation) Highly methylated regions exhibit no gene expression Acetylation of histones in the nucleosome triggers gene activation Others: ubiquination, sumoylation, phosphorylation

Epigenome Epigenome – total collection of epigenetic changes across a genome (cell- specific, varying with the current cell-state) Characteristic of individual cell types Plasticity (influenced by environment, development, aging) Relatively stable trans-generational transmission mCpG islands in DNA can change with the cell cycle Histone changes are more dynamic, changing within hours Reprogrammable within differentiated cells: Somatic nuclear transfer (“Dolly” experiment): transplanting of a differentiated nucleus into an oocyte cytoplasm allowed the development of a normal adult Induced pluripotent stem cells (iPSC): only requires 4 transcription factors to initiate reprogramming from a differentiated fibroblast to a stem cell Global effort: International Human Epigenetic Consortium (IHEC)

Epigenome Sources of genetic changes that can produce disease:
Mutation in DNA Uniparental disomy Epimutation – abnormal chromatin formation at 1+ loci in the genome Primary epimutation – immediate cause of disease (e.g., chemical or environmental) through DNA methylation or histone acetylation Secondary epimutation – controls epigenetic processes (e.g., deacetylase)

Disease - Chromatin Modifiers
Arise from a defect in “writers”: DNA methylases (DMNTs) and chromatin modifiers (HDAC or HAT) Produces abnormal chromatin domains/loci across the genome, illiciting phenotypic variability Outcomes: incompatible with life, causing neurodevelopmental diseases with intellectual disabilities Rhett Syndrome – causes a rapid loss of language and motor skills which eventually stabilizes (primarily in girls), leading to characteristic handwringing movements with growth retardation, seizures, autistic behaviours, and microencephaly Causal agent: loss of methyl-binding protein 2 (MeCP2)

Disease – Dysregulation of Heterochromatin
Heterochromatin: contains repetitive DNA or silencer elements, expanding across the entire chromosome, to convert “open” chromatin to condensed transcriptionally silent chromatin (facilitated by communication between nucleosomes) Silencing prevention: barrier elements that protect genes from the surrounding environment and nucleosome free areas Facioscapulohumeral Dystrophy (FSHD): Variations leadings to toxic expression of DUX4 transcription factors: FSHD1 – reduction in heterochromatin at telomeric tandom microsatellite repeats at 4q35 FSHD2 – creation of polyadenylation sites at the end of tandem repeats (due to >DUX4 mutation) Types of dysregulation: Heterochromatination of normally expressed genes, leading to silencing of active genes Position effect – silencing of a gene due to inversion or translocation of a DNA sequence, leaving it permanently condensed near the centromere (in the heterochromatin region) Reduction of gene silencing and activation of previously silent genes BRAC1 tumor suppressor protein – maintains constitutive heterchromatin Mutations can cause a loss of heterochromatin organization  mitotic recombination and genome instability

Disease – Environmental Changes
Fluidity of epigenomic chromatin status is determined by cytosine methylation patterns, histone modification patterns, and nucleosome spacing Environmental signals can significantly alter gene expression: Illumina Infinium Human Methylation 450 Bead Chip Aging: marked by progressive inefficiency in cell, tissue, and organ function by changing somatic cells of organs and stem cells that regenerate tissue

Disease – Genomic Instability
Universal characteristic of cancer cells: genomic instability (due to defects in chromosome segregation and DNA repair deficiencies) Weakens the capacity to maintain genomic integrity, leading to chromosomal instability with abnormal karytopes: Deletions – missing chromosomes Duplications or partial duplications – extra chromosomes Inversions and translocations – structural rearrangements Epigenetic patterns help maintain genome stability by suppressing the excess activity of transposons and maintaining the functions of centromeres and telomeres Loss allows cells to revert to less differentiated states: prototype cancer allows for more flexibility to adapt to the changing environment after reverting to a tumor-inducing phenotype (through hypomethylation of highly repetitive DNA strands) p53 mutated cells: show chromothripsis, shattering chromosomes into multitudes of fragments before subsequent end-joining repair Allows proto-cancer cells to attain plasticity to escape normal growth controls

Disease – Development Genomic imprinting – phenomenon where certain genes can be expressed in a parent-of-origin manner At an imprinted diploid locus, there is unequal expression of the maternal or paternal allele The parent specific imprinted locus “marks” are reprogrammed and susceptible to errors Uniparental disomy (UPD) – inheritance of 2 copies of chromosomes from a single parent Trans-regulation – a protein product acts at several chromosome promoters to increase the activity (transcription) of multiple genes Cis-regulation – a protein products acts on the same chromosome promoter to increase the activity (transcription) of the respective gene

Disease – Genomic Imprinting
Mechanisms Chr. Loci Genes Prader-Willi deletion, UPD, genomic imprinting 15q11/15q13 snoRNAs Angelman deletion, UPD, imprint defect, duplication point mutation 15q11/ 15q13 UBE3A Beckwith-Wiedemann imprint defect, UPD, 11p15.5 duplication, translocation, point mutation 11p15.5 IGF2, CDKN1C Silver-Russell UPD, duplication translocation, inversion epimutation 7p11.2/ 11p15.5 several candidates in region/ biallelic expression of H19 and decrease of IGF2 Pseudohypopara-thyroidism UPD, imprint defect, point mutation 20q13.2 GNAS1

Disease – Trans-regulation
Gene Effect Rubinstein-Taybi CREBBP/EP300 protein defect in co-activator of cAMP activity and change in HAT activities Rhett MECP2 X-linked for females: activity binding to methyl CpGs α-Thalassemia/ X-linked Mental Retardation ATRX Xq13: ATRX essential for survival of cortical neurons ICF Syndrome DNMT3B loss of function mutations in methyl transferase

Disease – Cis-regulation
Gene Effects αδβ- and δβ-Thalassemia deletion of locus control region (LCR) leads to decreased globin expression Fragile X expansion of CCG repeat abnormal methylation and silencing of FMR1 causes a neurodegeneration disorder repeats) FSH Dystrophy contraction of D4Z4 repeats exposed less repressive chromatin Multiple Cancers MLH1 (mismatch DNA repair) germline epimutation: abnormal methylation of the promoter

Clinical genetics: single gene disorders

Identify the clinical features of Huntington’s disease and distinguish its unique genetic features.
Differentiate the genetic basis for inheritance of Myotonic Dystrophy. Explain clinical features. Correlate the genotype-phenotype relationship in Myotonic Dystrophy Identify clinical features associated with Hereditary Motor and Sensory Neuropathy and identify the genes involved and the functions of their protein products. Identify clinical features associated with Neurofibromatosis. Distinguish between the genetics of neurofibromatosis I (NF1) and Neurofibromatosis 2. Identify clinical features associates with cystic fibrosis. Differentiate the biochemical and molecular basis for manifestation of cystic fibrosis. Identify genes involved and correlate the genotype-phenotype relationship. Identify clinical features associated with Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMB). Distinguish inheritance pattern of Duchenne Muscular Dystrophy and the role of dytrophin gene mutations. Identify clinical features associated with Hemophilia A and B. Distinguish the inheritance of these diseases and current treatments

Hemoglobinopathies Hemoglobin – 4 subunit globular protein that contains Fe2+ and acts in O2-transport Heme – Fe2+-containing porphyrin ring that binds O2, forming oxyhemoglobin Deoxyhemoglobin (with Fe3+) does not bind O2 Adult Hb (HbA) – consists of 2 α and 2 β chains designated as Hb α2β2 Fetal Hb (HbF) – consists of 2 α and 2 γ chains designated as Hb α2γ2 Possesses a higher affinity for O2, extracting from maternal circulation Porphyrias – genetic or acquired disorders affecting the enzymes of porphyrin or heme synthesis, causing neurological dysfunction, photosensitivity, and cardiac arrhythmias

Molecular basis of cancer
L19-L20 Molecular basis of cancer

Describe the basis of three molecular mechanisms that can predispose to cancer: Oncogenes (c-onc), Tumor Suppressor genes (TSG), and DNA repair genes. Describe the role of genetic mechanisms that underlie cancer phenotypes: Germline mutations, Epigenetic signatures, and miRNAs. Understand how cellular processes are impacted and promoted by neoplastic changes: Apoptosis, new angiogenesis, growth factors, genetic instability, loss of cell adhesion, and loss of cell cycle control. What are heritable cancer syndromes and how are they identified? Examples?

Cancer Cancer – rapid and uncontrolled cellular proliferation (neoplasia) that produces a malignant mass or tumor (neoplasm) Malignant – cell growth is no longer controlled by normal boundaries and is capable of progressing and invading neighboring (and more distant) tissues Benign – noninvasive or metastatic cancers Statistics: 2nd leading cause of death in the U.S., contributing to 20% of annual deaths and 10% of medical care costs (in developed countries) Affects all ethnicities and genders regardless off age Acting as a genetic disease: Gene dysfunction: initiates and drives cancerous changes in all tissues No 2 cancers will be the same, generating personalized medicine: Tyr Kinases are uniquely targeted by candidate drugs Heritable cancer syndrome genes: linked to sporadic cancer development Analysis and documentation of common mutations in cancer tissue, creating a signature or profile Genomic studies: identify changes associated with cancer

Nomenclature Hereditary cancer syndrome – first inherited cancer (present in all cells) causing gene mutations which is subsequently followed by somatic mutation at a later point in time Sporadic cancer – a mutation occurring in a single cell that progresses to a tumor via the accumulation of other mutations and clonal evolution Oncogene (Onc) – a mutation of a proto-oncogene, stimulating cell division and proliferation (neoplastic transformation) Proto-oncogene – a normal cellular protein coding gene that is involved in growth or cell survival Tumor suppressor genes (TSGs) – encode proteins that protect the transition of cells through checkpoints (as gatekeepers) or protect cell division and integrity of the genome (as caretakers) Gatekeepers: proteins regulators of mitosis and mediators of apoptosis Caretakers: proteins that detect and repair DNA mutations Loss of function: allows for the accumulation of deleterious mutations in proto-oncogenes and gatekeepers, promoting the development of cancer

Cancer Types: Sarcoma – originate in the mesenchymal tissues (e.g., bones, nervous system, muscle, connective tissue) Carcinoma – originate in the epithelial tissues (e.g., GI tract, lung bronchi) Hemopoietic/Lymphoid – originate in the bone marrow, lymphatic system, and peripheral blood Tumor classification: site, tissue type, histological appearance, degree (or stage) of malignancy Cancer phenotypes: altered cell proliferation, invasiveness, apoptosis, generation of new vasculature, increase in cellular signaling, genetic profile of “driver” mutations, and increasing incidence with age Multi-step process resulting from the dysregulation of genes  oncogenesis: involved in cell growth and regulation, resistivity to cell apoptosis, and maintenance of genetic stability

Oncogenesis Carcinogenesis – the process that produces genetic mutations induced by chemicals or physical agents Initiation – irreversible genetic change in a single cell Activation of oncogenes or anti-apoptotic genes – dominant (requires a single mutation) Mutations in tumor suppressor genes – recessive (requires mutation in both alleles) Promotion – increased proliferative ability in the initiated cell through clonal growth Progression – accumulation of more genetic damage, allowing the cells to develop a malignant phenotype Mechanism: proto-oncogene activation  progressive loss of tumor suppressor genes  activation of anti-apoptotic genes  loss of pro-apoptotic gene expression

Oncogenesis Tumorigenesis – driven by the accumulation of irreversible mutations (or drivers) in key genes that encode functions for cell cycle regulation, cell growth, and programmed cell death Require cooperativity: contributions from more than one defective oncogene, allowing for uncontrolled cell division and growth (without regulation) Clonal evolution: each successive mutation in a cell confers a growth advantage, outgrowing their “normal” neighboring cells Tumor progression: accumulation of additional drivers after more mutations (or epigenetic silencing of caretakers) that maintain DNA/genome integrity or cytogenetic normality (along with altered vascularization control, allowing for local clonal invasion or distant metastasis) K

Fearon-Vogelstein Adenoma-Carcinoma Model
Class model for multi-stage progression of colorectal cancer: Normal colon cell Hyperproliferation Early adenoma Intermediate adenoma Late adenoma Carcinoma Metastasis Mutation of APC DNA hypomethylation Mutation of K-ras Loss of DCC Loss of p53 Accumulation other genetic mutations

Phenotypes of Molecular Cancer
Immortality: indefinite proliferation due to protection over the replication of telomeres, preventing chromosomal shortening, and loss of growth control after the loss of tumor suppressor activity Decreased dependence on growth factors for proliferation: self- generated production of tumor cell growth factors through autocrine signaling Loss of anchorage dependent growth/altered cell adhesion: allows for metastasis (through proteolytic cleavage of the basement membrane) Mechanism: detachment of tumor cells from the primary tumor, penetrating through the basement membrane (by degrading the ECM)  migration to foreign tissue sites through the blood and lymph Matrix metalloproteins (MPPs) – overexpressed in tumor cells

Loss of cell cycle control: multifactorial regulation of mitosis is controlled through oncogenes and tumor suppressor genes, irreversibly committing the cell to divide after an increase in mutations and resulting genome instability Cyclin-dependent kinase inhibitors (CKIs): Cip/Kip family: p21/Cip1/waf1/Sdi1, p21/Kip1, and p57/Kip2 – maintain a broad specificity, binding and inactivating most of the cyclin/CDK complexes needed for cell cycle progression (and upon detection of damaged DNA, p53 (stimulated by p21) halts the cell cycle) INK4 family: p16/INK4a, p15/INK4b, p18/INK4c, and p19/INK4d – regulate phosphorylation of Rb (a critical cell cycle checkpoint) Reduced sensitivity to apoptosis: overcomes promoters of apoptosis through overexpression of Bcl-2 and mutation or suppression of Fas-R/CD95, inhibiting the activity of the caspase family and associated p38 induced Bim/Bax interactions Phases: initiation phase after the activation of pathways from external signaling through death receptor ligands (e.g., CD59—Fas-L)  decision and effector phase involving the disruption of the mitochondrial membrane and release of cytochrome c into the cytoplasm (for protease and nuclease activation)  degradation and execution phase

Increased genetic instability: confers a selective growth advantage through translocation and associated rearrangements, acting as oncogenic drivers Chromosomal aneuploidy – gain or loss of 1+ chromosomes Chromosomal polyploidy – gain of extra sets of chromosomes Examples: Burkitt’s Lymphoma (t8,14) – translocation that results in transcriptional fusion protein, leading to the expression of c-myc proto-oncogene expression Philadelphia Chromosome (CML) (t9,22) – translocation that splices c-Abl+BCR kinase + GAP, forming consecutively active chimeric kinase) that drives cell proliferation) Gene amplification – multiple rounds of DNA replication at a single site (followed by FISH) Angiogenesis: hypoxia inducible factor 1 (HIF1) interacts with vascular endothelial growth factor (VEGF) (secreted by tumor cells) to stimulate neo-vascularization, stimulating hyperpermeability in proliferating cancer cells Anti-tumor experiments target angiogenesis to reduce tumor growth

Oncogenes Oncogenes – deregulated mutated derivatives of proto-oncogenes, contributing to cell proliferation and decreased sensitivity to cellular apoptosis, that confer a selective growth advantage Classification based on cell location and activity: Growth factors and growth factor receptors: stimulate tumor cell growth through self-generated autocrine signaling (and affect normal cells via paracrine mechanisms) Membrane-associated G-proteins: Ras oncogenes function in cell proliferation and survival, activating other oncogenes through constitutive intracellular signaling to promote unregulated cell proliferation (through kinase cascades) Serine-threonine protein kinases: after Ras recruitment, Raf activates MAPKs to activate transcription factors (containing Elk-1) and protein kinase C (to activate c-jun), acting as an intracellular signaling molecule

Oncogenes Cytoplasmic tyrosine kinases: Nuclear proteins:
SRC family – exhibits kinase activity (SH1) and promotes protein-to-protein interactions (SH2/SH3), generating constitutive signaling Bcr-Abl (chimeric fusion of t9,22 in Philadelphia chromosome) – exhibits kinase activity (c- Abl) and activates GTPase (Bcr), promoting unregulated cell growth (through Ras signaling)  reduces GF dependence, alters cell adhesion properties, and enhances cell viability Nuclear proteins: Transcription factors: Burkitt’s Lymphoma (t8,14) moves the Ig heavy chain (IgH) promoter driving c-myc (regulates by binding to E-box sequences) expression, inducing uncontrolled cell proliferation (as an oncogenic switch for oncogenes and tumor suppressor APC) Tumor suppressor APC – mediates the degredation of β-catenin which, in turn, acts as a transcription factor for Tcf, activating c-myc expression Telomerases – synthesize the hexamer (TTAGGG) that caps chromosomal ends (telomeres), allowing tumor cells to proliferate indefinitely (due to a mutation in the telomerase gene) Cytoplasmic proteins engaged in cell survival: increased expression of Bcl-2 oncogene disrupts apoptosis

Tumor Suppressor Genes (TSGs)
“Loss of function” mutations: point or small insertions/deletions (must lose heterozygosity to induce tumor development) Gatekeepers – directly involved in cell growth and death Caretakers – promote genetic stability through DNA repair, increasing the mutation rate of DNA and accelerating tumor formation Loss of Rb protein (recessive)  deregulation of cell cycle and uncontrolled cell division, continually activating target genes Normal mechanism: dephosphorylation of Rb allows for binding to E2F transcription factor, preventing transcription of E2F transcription target

Tumor Suppressor Genes (TSGs)
p53 tumor suppressor gene – transcription factor that promotes DNA repair and apoptosis while inhibiting growth (for genetic stability) Loss of function  tumorigenesis: mutation of p53 gene (in sporadic tumors and Li-Fraumeni Syndrome), vital transforming antigens (by SV40 T antigen), and cytoplasmic sequestration (preventing entrance into the nucleus to activate target genes) Adenomous Polyposis Coli (APC) gene: initial mutation in early colon carcinogenesis, leading to the activation of c-myc (can be inherited) Phosphatase and Tensin Homologue (PTEN): first identified in glioblastoma multiforma (aggressive brain tumor), inhibiting cell cycle progression and inducing apoptosis (as a negative regulator of Akt activation, opposing VEGF)

Translocations Chromosomal translocation can rearrange the genome and activate “new” promoter/enhancer elements, dysregulating normal gene expression Chronic Myelogenous Leukemia (CML) – Philadelphia chromosome t(9,22), moving Abl to the “break point cluster” Bcr, resulting in chimeric fusion (Bcr-Abl) that enhances Abl kinase activity Drug target: imatinib – inhibits kinase activity Burkitt’s Lymphoma – B cell-derived tumor of the jaw (in African children) due to t(8,14), positioning Ig heavy chain (IgH) enhancer to drive defunct c-myc expression  uncontrolled cell growth and proliferation Follicular B cell Lymphoma t(14,18) – IgH promotion of translocated Bcl-2, increasing the B cell population and inhibiting apoptosis

Heritable Cancers Li-Fraumeni: p53 gene mutation (70%)
Breast and/or Ovarian Cancer: BRCA1 (DNA repair and genomic stability as a cell cycle checkpoint), BRCA2, RAD51C (DNA repair), and RAD51D mutations (10-15%) Lynch Syndrome: MLH1 and MLH2 mutations (DNA repair), causing colorectal cancer Bloom’s Syndrome: BLM (a recQ helicase) mutation, causing short stature and increased light sensitivity Autosomal recessive: Xeroderm Pigmentosum (XP): XPC mutation (DNA repair), causing increased light sensitivity and subsequent skin cancer Franconi’s Anemia (FA): FRACB (DNA repair) mutation (in Ashkenazi Jews and South Africans), causing acute myelogenous leukemia and subsequent bone marrow failure

Others miRNA – post-transcriptionally degrade or inhibit the expression of target mRNA, acting as gene regulators and tumor suppressors Functions: cell proliferation and apoptosis Carcinogenesis: Planar chemicals – integrate into DNA, forming adducts that introduce mutations when replicated E.g., vinyl chloride, dimethylnitrosamine (DMN), benzopyrene, UV light, benzene, 3-MCA, nitrogen mustards

Introduction to the Human chromosome

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Introduction to the Human chromosome

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