Evolution of Human Genome - Sara V - PDF

Sara V MOL MED BLOCK 2 EVOLUTION OF HUMAN GENOME Life - Unified (governed by DNA). The perpetuation of DNA Genome – complete set of genetic information in an organism. Comprised of units (genes – DNA sequence coding for relevant m...

Sara V MOL MED BLOCK 2 EVOLUTION OF HUMAN GENOME Life - Unified (governed by DNA). The perpetuation of DNA Genome – complete set of genetic information in an organism. Comprised of units (genes – DNA sequence coding for relevant molecule) - Most DNA/largest genome = plants - Most genes = water flea (multicellular eukaryote) - Most genes = trichomonas vaginalis (single-celled eukaryote) Human genome - diploid, 46 chromosomes (22 pairs of autosomes & 1 pair of sex chrom...) - haploid genome comprises 25 molecules. 3 billion nucleotides - Nuclear genome = 22 autosomes + 2 sex - Supercoiled, fitting space of 6 micrometers - Roughly 20 000 genes, some being transcribed in many ways (one gene = many functional molecules), some transcripts join, and genes can combine Genome size variation (very little significance) - Accurate replication and mutation are “ideal” in DNA perpetuation - Size of genome changes when DNA mutates Gene number variation (not indication of complexity) - Same reasons size may vary. (accidentally duplicated or deleted genes, bacteria may insert genes into genome of other organisms, DNA sequence shuffled) Genome evolution - Life evolved from single-celled organisms to multi-celled - Humans @ end of evolution chain, our genome is modified versions of DNA in other organisms - Steps: 1. Small strands of nucleic acid floating in primordial soup 2. Single cell arose, DNA protected by cell membrane 3. DNA amounts expand through mutation 4. Single cells work together to form multicellular organisms 5. Introns accumulate, breaking genes into smaller exons (flexibly connect with each other) Shared DNA sequence - Most shared DNA sequences are turned off in human genome - Small sequence differences account for large functional differences 1 Sara V - 98-99% similar to other humans (remaining 2% accounts for differences) Studying human genetics 1. Macro level – chromosomes (number, structure, FISH, karyotyping) [chromosomes & their abnormalities] 2. “Micro” level – DNA sequence, mutations and polymorphisms, PCR, sequencing [DNA sequence and pathogenic mutations] 3. Epigenetic level – modifications on top of DNA sequence. Imprinting & “epi”mutations [epigenetic modifications and mistakes] HUMAN GENOME PROJECT Human Genome Project (HGP) - Completely sequence the entire human DNA complement & map every gene to chromosome placement - Coordinated by NIH, and publicly funded - Cost – 13 years and 3 billion USD - Cost now decreased due to machinery and technology. Now $1000 Aim - Develop improved sequencing technologies (Beyond Sanger) - Sequence model organisms - Store info in useful ways to improve software tools for analysis - Consider ethical, legal and social implications Whose genome? - Genome reference does not represent exact match for any person’s genome - First reference = composite genome from several different people - Sequencing was done concentrated in few centers, each sequencing individual chromosomes Genome data - Bermuda principle set out rules for public release of DNA data - Now available on genome browsers Findings - 25% of genome are genes. Only 1.5% are coding - Some chromosomes carry more genes than others (size relative) - Random distribution with gene deserts and gene islands (gene rich regions) Reference Human Genome - Digital database of haploid DNA sequence - A representative example of the genome of one idealized individual 2 Sara V - Standard for comparison Uses of human genome data - Basic biology – identify human gene homologues - Clinical genetics – analyze molecular basis of inherited childhood disorders - Technology leaps - sequencing technology, new era of bioinformatics TYPES OF GENETIC VARIATION How human genomes differ/normal variation includes: - Single nucleotide polymorphism (SNPs) - Repetitive DNA sequence (from short tandem repeats (STRs) to copy number variants (CNVs)) - Epigenetic differences Mutation vs Polymorphism - Mutation (any changes in DNA sequence) > labelled based on frequency in population. (Seen in less than 1%) - Polymorphism (DNA sequence variant commonly seen in population (More than 1%)) Single nucleotide polymorphisms - Occurs once in every 100-300 base pairs - 11 million SNPs in our genome, commonly observed - Most occur outside of gene sequence - Single nucleotide replication errors (passed down several generations) - Uncertainty about frequency = single nucleotide variants (SNVs) - SNPs are useful tools to identify and examine different genomes Repetitive sequence - 50% of genome is repetitive sequence - Replicating repetitive DNA is error prone. DNA polymerase can lose track of how many repeats have been successfully replicated and how many await replication - Interspersed vs tandem Size ranges of repetitive DNA - Size can range from 1 nucleotide to over 1 million nucleotides - Homopolymer or homopolymer tract 1 nucleotide repeat unit GGGGGGG = 1 nucleotide repeat unit, 7 repeat units TTTTTTTTTTTTTTTTTTTT = 1 nucleotide repeat unit, 20 repeats - Microsatellites 2-10 nucleotide repeat units ATATATAT = 2 nucleotide repeat units, 4 repeats - Minisatellites 11-50 nucleotide repeat units 3 Sara V - Repeat units occur in tandem & referred to as variable number tandem repeats (VNTRs) or short tandem repeats (STRs) - Large repeat units (>50 nucleotides) occur in either tandem or interspersed and called copy number variants (CNVs) making up 5-10% of the genome. Mostly found outside gene sequence. Pathogenic - The way in which genomes differ can become source of disease Allele - Alternative form of gene/DNA sequence found at the same locus (homologous chromosomes) - Homozygous = same allele on both copies of chromosome - Heterozygous = alleles are different - Males are hemizygous for genetic variants on X and Y chromosomes Locus (“position”) - Single nucleotide = which number on chromosome - Gene = which arm of chromosome - Mutation/polymorphism = which nucleotide on which chromosome Genotype - Combination of alleles at specific locus Phenotype - Observable characteristics of genotype POPULATION GENETICS Allele frequency - Rate of occurrence of gene variant at particular locus - High frequency - common alleles, observed in many individuals in a population - Low frequency - rare alleles observes in few individuals - Diploid population = 2 alleles (locus occupied by either) Calculating allele frequency - 2000 copies of gene - G allele/2000 = frequency of G Hardy-Weinberg Principle - Randomly mating populations maintain constant allele frequencies and genotype frequencies from one generation to the next - Only works under certain assumptions (population usually violates one) Factors influencing allele frequency - Mutation rates > number of new mutations in a gene over time. Introduces new allele 4 Sara V - Migration > (gene flow) Genes transferred from one area to another. Introduces a new allele. Reduces genetic differences, if extensive - populations merge. - Natural Selection > favours alleles offering fitness advantages (better adapted to suit physical, structural and physiological environment) - Genetic drift > reduces genetic variation, can occur by chance. Could result in beneficial allele loss or harmful allele may become fixed - Population bottleneck = event that drastically reduces the size of population. Allele frequency may be very different from those prior to the event, some alleles may be missing - Founder effect = occurs when a small group of individuals breaks off from a larger population, new population is isolated USING GENETIC VARIATION IN THE LAB Polymorphism - Change in sequence present in at least 1% of population - Found throughout the genome - If location is known, can serve as landmark for other genes or regions - Each polymorphic marker has different versions of alleles. Types of Polymorphism - Single nucleotide polymorphism (SNP) - Variable number tandem repeats - “Indel” polymorphism Short tandem repeats (microsatellites) - Tandem repeated nucleotide units of 2-6 base pairs - 3% of genome and most found on non-coding regions - Chromosome 19 = highest density of STRs - Most common STRs are A rich Classifying STRs 1. Length of major repeat unit (mono-,di-,tri-,penta-,hexanucleotide repeats) 2. Repeat structure (simple, compound and complex) Naming (eg. D3S1266) - D for DNA - 3 means chromosome 3 - S stands for STR - 1266 is unique identifier Typing STRs - PCR - Primers designed to flank repeated region - PCR generates products of different lengths depending on number of repeats - QF-PCR uses a fluorescent labelled primer for PCR - Primers for diff STR locations labelled with diff colour dye Interpreting an electropherogram 5 Sara V - Consists of a series of peaks representing the amplification products - Height of peaks measured in relative fluorescent units (RFU) and is proportional to amount of PCR product detected - Peaks of homozygous loci are expected to be taller than those of heterozygous loci - Numbers along the x-axis represent the length of DNA fragment in no. of base pairs - 2 peaks of same colour appear directly next to each other = heterozygous locus - When peaks stand alone = homozygous genotype at that locus (height double that of heterozygous peaks) Amelogenin (AMEL) - Amplifies non-polymorphic sequence on the X and Y chromosomes - Presence or absence of Y chromosome - Relative amount of X to Y sequence - Females have XX thus a single peak is formed, males possess XY exhibiting 2 peaks TAF9L and SRY - TAF9L has sequences on chromosomes X and 3 - The chromosome 3 specific peak can be used as a reference peak to determine number of chromosomes present - The Y specific marker, SRY, peaks in normal males but not amplified in females Percentage testing - Identify DNA fragments in child that are absent in mother, therefore inherited from biological father - Done by home affairs/ due to legal disputes / forensics/ swapped babies at hospital - Outcomes = alleged parent excluded OR alleged parent not excluded Forensics - Blood, hair, saliva, semen, bone - DNA profiling can solve crimes CHROMOSOMES I: KARYOTYPING AND FISH Structure of a chromosome - Homologous > one maternal and one paternal that pair up during cell division - Sister chromatids > identical copies formed by DNA replication of chromosome (common centromere) - Centromere > essential to cell division & ensure accurate chromosome segregation - Telomeres > Region of repetitive nucleotide sequences at each end of chromosome, protecting it from deterioration/fusion with neighboring chromosomes - Short arm = p & long arm = q Types of chromosomes (position of centromere) 6 Sara V - Telocentric > centromere found at end of chromosome (no p arm) (not in humans) - Acrocentric > centromere severely offset from centre thus much shorter p arm - Submetacentric > centromere off centre, shorter p arm relative to q arm - Metacentric > centromeres in the middle, p and q comparable in length Karyotyping - Collection of chromosomes (their number and appearance) - Resolution = number of bands visible. Depends on how condensed chromosomes are, 350-550 (limited resolution) - Time consuming, small abnormalities not visible, costly 1. CULTURE > Sample collection (blood, bone marrow, amniotic fluid), Culture in growth medium (sterile flow hood, 5 drops blood + growth medium, stimulates mitosis), Incubate for 3 days (37 C for 72 hrs) 2. HARVEST > Stop mitosis in metaphase (mitotic arrest, spindle inhibitor), Add hypotonic solution (KCL, causes cell to burst, metaphase spread for visualization), Wash supernatant with fixative (wash to kill cells and remove debris, methanol and acetic acid, 3 times) 3. ANALYZE > Prepare slides (ensure adequate spreading, frozen slide, drop onto slide, humidity), Stain slides (different stain techniques, Giemsa, C banding, G banding), Analyse (analysis done microscopically, 10-20 cells analyzed, photographed and reported according to karyogram, report according to ISCN) Nomenclature ISCN (Int Standard for Cytogenic Nomenclature) - Revise list of abbreviations (slide 23, module 2, lecture 1) FISH (Fluorescent In-Situ Hybridization) - Detects small deletions/duplications which are not visible using conventional techs - Detect & localise presence or absence of specific DNA sequences on chromosomes - Utilizing complementary base pairs of sequences you are looking for - Uses: microdeletions & duplications, confirm arrangements & ID origin of material - Higher resolution, number of probes limited, cost depends on probes, short TAT 1. DENATURE > to generate single stranded DNA, formamide & ethanol, heat 2. PROBE > designed for short section of single stranded DNA, fluorescently labelled 3. HYBRIDISATION > binding (re-annealing) of probe and sample, heat & humidity 4. ANALYZE > visualise using fluorescent microscope, each probe visualised as a distinct signal, analyze multiple cells (deletion < 2 signals & duplic. > 2 signals) CHROMOSOMES II: NUMERICAL CHROMOSOME ABNORMALITIES Types of numerical abnormalities - Polyploidy - More than 2 haploid chromosome sets - Eg, triploidy (3 haploid sets = 69 chromosomes) - Aneuploidy - Gain or loss of one (or more) chromosomes - Eg. Trisomy (46+1 = 47 chromosomes) 7 Sara V - Eg. Monosomy (46-1= 45 chromosomes) - Techniques for detecting: karyotype or QF-PCR (fluorescent dye for STR) Nomenclature Number of chromosomes, sex chromosomes, +/- the extra/missing chromosomes - Triploidy male = 69, XXY - Trisomy = 47, XY, +21 Non-disjunction during meiosis Meiosis I > 2 homologous chromosomes fail to separate, trisomy or nullisomic embryos Meiosis II > 2 sister chromatids don’t separate, trisomy or nullisomic embryos Mechanism resulting in polyploidy (fertilization abnormalities) Digyny > diploid ovum (non-expulsion of second polar body) fertilized by haploid spermatozoa Diandry > fertilization of one oocyte by 2 spermatozoa, 4x more frequent than digyny Mosaicism & chimerism - MOSAICISM - 2 or more different cell lines present, different cell lines have different karyotypes (chromosome arrangements) - CHIMERISM - 2 or more cell lines present originating from 2 or more zygotes (early fusion) Common aneuploidies Trisomy 21 (down syndrome) - Non-disjuction of chromosome 21 or robertsonian translocation or mosaic trisomy 21 - Clinical features = dysmorphic, intellectual delay, hearing loss, visual impairment Trisomy 13 (prev. Patau syndrome) - Severe intellectual delay, congenital abnormalities of organ systems, cleft, poor prognosis (rare survival rate beyond 1 year) Trisomy 18 (prev. Edwards syndrome) - Severe intellectual disability, clenched fists with overlapping digits, rockerbottom feet, poor prognosis (rarely live beyond 1 year) Turner syndrome (45, X) - Short stature, delayed puberty, infertility, kidney & cardiac abnormalities, lymphoedema Klinefelter syndrome (47, XXY) - Tall stature, reduced muscle tone, small testes, delayed puberty, decreased facial and body hair, speech and language defects CHROMOSOMES III: STRUCTURAL CHROMOSOME ABNORMALITIES Structural chromosome abnormalities 8 Sara V - Chromosome imbalance due to structural changes (deletion, duplication, translocation, inversion, insertion, ring chromosomes, isochromosomes) - One chromosome can have multiple abnormalities - Total number of chromosomes is normal but changes in structure (missing, extra, switched) Deletions - Part of chromosome is missing or deleted (terminal/interstitial deletion) - Some genetic material is missing (syndromic features, depends on genes involved) - Sporadic or dominant (dominant = 50% recurrence risk) - Test: FISH, Array, MLPA or karyotype - 15q11 deletion = Angelman & Prader Willi syndrome Duplication - Part of chromosome is duplicated (terminal/interstitial) or (direct/inverted) - Results in extra genetic material (severity depends on genes involved, less severe than deletions) - Sporadic or inherited (inherited = 50% recurrence risk) - Test: FISH, Array, MLPA or karyotype - 22q11 duplication = 22q duplication syndrome Translocation: Reciprocal - Exchange of material between chromosomes - Balanced - no loss or gain of genetic material, phenotypically normal - Unbalances - gain or loss of genetic material, can be inherited from balanced parent (recurrence is complicated) - Array if unbalanced, Karyotype if balanced Translocation: robertsonian - Exchange between 2 acrocentric chromosomes to form one large metacentric chromosome - Eg. 45, XX, rob(13;13)(q10;q10) - Test: karyotype Inversion - Portion of chromosome broke off, inverted and reattached - Pericentric or paracentric - If isolated the individual is often phenotypically normal (normal variant) - Test: karyotype Insertion - Portion of chromosome broke off and is inserted elsewhere - Interchromosomal or intrachromosomal - Direct insertion or inverted insertion 9 Sara V - Test: karyotype Ring chromosome - Portion of chromosome broke off and formed a ring - Can happen with or without loss of genetic material - Very rare and often severe - Usually sporadic - Test: karyotype Isochromosome - Formed by mirror image copy of a chromosome segment - Results in a gain and loss in genetic material - test : FISH, Array, MLPA or karyotype - Isochromosome 12 = pallister killian syndrome MOSAICISM AND CHIMERISM Mosaicism - Condition where tissues of genetically different types occur in the same organism but all the cells have arisen from a single zygote - Presence of 2 (or more) populations of cells with different genotypes in one individual who has developed from a single fertilized egg - CAUSE - mitotic mutation arises early in development, resulting in an individual with a mix of cells (some with and some without the mutation) - DIAGNOSIS - cytogenetic analysis of blood can detect (percentage of cells will show abnormal karyotype while the rest will show a normal karyotype). Sometimes more difficult, may need to look at different tissues (skin as well as blood) Mechanism of trisomic mosaicism - Most common form of mosaicism found through prenatal diagnosis and in neonates involved trisomies - Individual now carries 2 cell lines, one normal and one trisomic Mechanism 1: nondisjunction in early normal embryo - Nondisjunction in early cell division results in fraction of the cell with a trisomy Mechanism 2: Trisomic Rescue - Trisomic embryo undergoes nondisjunction and some cells revert to normal chromosomal arrangement (trisomic rescue) Abnormalities - (Mosaic Down Syndrome - 46,XX/47,XX,+21) Mosaicism leads to milder phenotype than non-mosaic patients with the same disorder - Although complete trisomy 14 or 9 is not compatible with postnatal life, trisomy mosaic for these chromosomes been diagnosed in newborns with multiple congenital anomalies Sex chromosome mosaicism 10 Sara V - Turner Syndrome (45,XO) - usually infertile - Turner Syndrome (45,XO/46,XX) - 30% of Turner females, often multiple miscarriages X chromosome activation - One X inactivated in all cells of female mammals - Which X is inactivated is initially a completely random process - All female mammals are genetic mosaics Germline Mosaicism (gonadal mosaicism) - Mutation confined to portion of germ cells - Can be transmitted to offspring - If offspring is germline mosaicism, parents will not be affected. (blood samples will test negative for mutation) - Most common = autosomal dominant (achondroplasia) and X linked disorders (Duchenne Muscular Dystrophy) - Most people unaware until they have children that are affected - If autosomal dominant mutation, child will be affected, but not mosaic, and has a 50% chance of spreading to offspring Somatic mosaicism - Normal and abnormal cell lines within cells of the body (may or may not be present in germline) - Mutation only transmitted to offspring if present in germline Chimerism - An organism carrying cell populations derived from 2 or more diff zygotes of same/diff species - Transplant and transfusion patients are also chimeras - Non-identical twins may be chimeras (shared blood supply in placenta allowing blood stem cells to pass from one and settle in bone marrow of the other) ANEUPLOIDY TESTING USING STRs Prenatal testing - Testing for diseases/conditions before birth - Types: chorionic villi sampling or amniocentesis (fetal cells in amniotic fluid) Process 1. Collect sample 2. Extract DNA 3. Amplify DNA: QF-PCR 4. Perform capillary electrophoresis 5. Electropherogram 11 Sara V CONSEQUENCES OF MUTATIONS Mutations - Change in genomic sequence - Can be single bp, duplication of entire segment or chromosome - Not always pathogenic - Possible consequences: silent (no effect), beneficial (good effect), deleterious (bad effect) - Mutations occur at rate of 1 in every 50 million nucleotides added to chain - With 6 x 109 nucleotides in human cell, each cell contains 120 new mutations (excluding exogenous causes of mutation) Most in non-coding regions Acquired vs germline mutations - Acquired > occurring in non germline tissues and cannot be inherited - Germline > present in egg or sperm, can be inherited, cancer family syndrome Static vs dynamic mutations - Static > do not change when passed to offspring (point, deletion/insertion, splice) - Dynamic > have potential to change when passed to offspring (triplet repeats) Point mutation (static) Single nucleotide substitution (one base changed to another) occurring in less than 1% of population If substitution is seen in more than 1% = polymorphism Include : ○ silent mutation ~ do not change amino acid coded for No effect on aa sequence. Can influence kinetics of protein folding & DNA splicing ○ nonsense mutation ~ mutation results in stop codon instead of amino acid No protein (nonsense mediated decay) or premature termination of protein with altered function (truncated) ○ missense mutation ~ amino acid change (eg. sickle cell disease) Nature of the aa substitution. Chemistry (polar/non-polar), location (conserved/ non-conserved region of the gene), size (can aa chain still fold correctly) Insertions/deletions (INDELS) Multiples of three -> in frame mutation (usually milder) If not multiples of three -> frameshift mutations (usually more severe) ○ Impact of deletions: the entire gene/part of the gene. Frameshift or in-frame ○ Impact of insertions: insertion of 3bp or multiples of 3bp = extra aa in protein. Insertion not 3 bp or x3 then, frameshift during translation = altered protein sequence 3’ of the mutation ○ Frameshift indels generally lead to: possible introduction of stop codon, altered/abolished protein function 12 Sara V Splice site mutations ~15% of disease causing mutations in humans affect RNA splicing SD = splice donor. SA = splice acceptor SD is stretch of conserved sequence around beginning of intron (first nucleotides of most introns are “GT”) SA is stretch of conserved sequence around end of intron (last two nucleotides of most introns are “AG”) Known as the “GT-AG” rule ○ Inactivation of donor/acceptor splice site prevents splicing ○ Exon skipping/intron retention (difficult to predict) Other Impacts - Disruption of gene structure by translocation (Philadelphia chr) or inversion (Haemophilia) - Prevent by promoter working by mutation or methylation MUTATION NOMENCLATURE Why? To write about, discuss and compare mutations in diff patients around the world Nomenclature - c. ~ coding DNA seq - g. ~ genomic DNA seq - p. ~ protein seq - m. ~ mitochondrial DNA - r. ~ RNA Coding DNA (cDNA) - Nucleotides before first coding nucleotide have a minus (ie, -3,-2,-1) - No nucleotide numbered 0 - First coding nucleotide of gene investigated is numbered 1, all others are counted in sequence - Ionic, non-coding sequences counted in relation to nearest coding nucleotide - Sequence after translation stop site is numbered with * (*1,*2,*3) - Eg. c.372A>G : begin with letter, A is 372nd nucleotide in sequence, changes to G Genomic DNA - “g” nomenclature refers back to a reference genome sequence and assigns a more specific number - Eg. g.44921A>G : begin with letter, A is 44921st nucleotide in reference genome sequence, changes to G Protein - Some DNA mutations affect corresponding protein, write about mutation at protein level. - Both 3 and 1 letter abbreviations for aa can be used (3 is preferred) - Begins with affected aa, uses numbers to indicate aa position in the protein chain 13 Sara V - When transcribing, rewrite sequence but replace T with U - Eg. p.Ala23Pro : aa 23 (codon 23) Ala has changed to Pro Nomenclature 1 - Silent mutations > describe at coding/genomic level - Missense mutations > coding and protein levels - Nonsense mutation > Stop codon is denoted by the abbreviation “Ter” for termination Nomenclature 2 - Nucleotide substitution in introns > specify the coding nucleotide number first, then the distance from the splice site (c.9+4G>T) - Deletions and insertions > del for deletions and ins for insertions. Nucleotide position or aa symbol comes first (range indicated by _ or -) - Duplications > use dup to indicate. Note: Always number from perspective of normal/original sequence PEDIGREE DRAWING Family pedigree - Genetic representation of a family tree that indicates the relationship between family members and illustrates the inheritance of a trait/disease through several generations - Why? Promote risk assessment, establish inheritance patterns, identify at-risk family members, directs medical management, educate patient and explore understanding - Advantages: easy to read, fast, permanent and clear record, combines medical data and biological relationships, standard symbols Family history info to be collected - Age, date of birth and death (+ cause of death) - Relevant health info + diagnosis (age at diagnosis) - Ethnic background - Infertility or no children by choice - Pregnancy complications Pedigree symbols - Square = male, circle = female. Diamond = unknown - Horizontal line relationship - marriage or reproductive relationship - 2 horizontal lines relationship - consanguinity (related by blood) - // - relationship no longer exists - vertical descending line - offspring - Upside down Y (non-identical twins) / upside down Y + - (identical twins) - Ombre shade = carrier - Coloured dot in center of circle = carrier female of X linked condition MENDELIAN INHERITANCE: AUTOSOMAL DOMINANT 14 Sara V Mendelian inheritance - Biological inheritance following Mendel’s principle of hereditary - Inheritance patterns for single gene disorders Autosomal dominant - Trait that manifests in heterozygous state (one mutant allele and another normal one) - 2 copies of every autosomal gene (one copy from each parent) - Only one copy needed to express the condition - Occurs in every generation & number of affected males = affected females Probability of inheritance - Punnett square - mathematical probability of inheriting a specific trait/mutation - Predict genotypes of offspring based on parental genotype Achondroplasia - Short limbed dwarfism - Cartilage not converted to bone - breathing problems, recurrent ear infections - Double are lethal MENDELIAN INHERITANCE: AUTOSOMAL RECESSIVE Autosomal recessive - Trait which manifests in homozygous state, in a person possessing both copies of abnormal/mutant allele - Parents of affected children are usually phenotypically normal - Affected individuals are not seen in every generation - Eg. cystic fibrosis, sickle cell anemia Carriers - Heterozygous individuals - Protein is halved but adequate - One gene mutation does not usually cause health conditions Allelic heterogeneity - For some recessive conditions, one specific mutation gives rise to the phenotype - For others, it is possible that many diff mutations can arise and will result in the same genetic condition - Results in clinical variability depending on how mutation disrupts the gene structure or function - Disease severity is determined by position of mutation - Genetic testing complications - mutation analysis difficult. Population genetic info is essential. Allows screening of most common mutations Compound heterozygosity - Deleterious mutation unlikely to happen repeatedly at the exact same place in a gene - If both are loss of function alleles - clinical effect is same as a homozygous - Most individuals with recessive disease are compound heterozygous 15 Sara V Consanguinity - Increased chance that 2 individuals are carriers of same allele - Rare alleles more likely to “meet up” in offspring of cousins than in offspring of parents who are unrelated - Risk of having child with birth defect = 2-3% - 1st cousin marriages = 5-6% SEX-LINKED INHERITANCE Sex-linked inheritance - Females (XX), males (XY) - X linked genetic condition is caused by a mutation in a gene on the x chromosome - Affects males and females differently - Mostly X linked and usually recessive (dominant X linked are rare) - Males predominantly affected. Females are usually carriers. X - linked recessive in females - Females have 2 X chromosomes and if one of the genes on an X chromosome has a mutation then the normal gene on the other X compensates - Healthy carriers of the x linked condition - Females show mild signs of the condition some times - Carrier females can pass on to sons and daughters X - linked recessive in males - If one of the genes on male X, there is no copy to compensate - He will be affected by the condition - Affected father can only pass mutation on to daughters Haemophilia - Bleeding disorder that affects males - Deficiency in clotting factors - Recurrent bleeding in joints - Female carriers may have some bleeding manifestations Duchenne Muscular Dystrophy (DMD) - Muscle weakness begins in legs and pelvis - Progressive difficulty walking, most wheelchair bound by 12 - Females rarely affected but may have symptoms of muscle weakness X-linked dominant (rare) - For certain conditions, males affected with an X-linked dominant disorder may not survive and will spontaneously abort (eg. Rett syndrome) Rett Syndrome - Girls almost exclusively (mutation to MECP2 gene) - Early development is normal but then slows down - Intellectual disability & inability to perform motor functions & walk 16 Sara V POPULATION SPECIFIC CONDITIONS Importance of studying diversity of diseases in diff populations - Research opportunity - Major relevance to diagnostic testing: - Frequencies of disease may differ between groups & mutational basis may be diff - Inappropriate testing may be performed, missing important diagnoses Founder effect - New population established by small number of individuals from larger pop - New pop distinctively diff genotypically and phenotypically - Small = more inbreeding and less genetic variation - Rare alleles move to one of 2 extremes - Allele is lost/survives and becomes more dispersed - Increases frequency of recessive alleles and increases homozygous recessive individuals - Afrikaner population in SA Heterozygotic advantage - Heterozygous genotype has higher fitness than homozygous dominant or recessive genotypes - Sickle cell anemia - affecting haemoglobin - Causes anaemia, fatigue, shortness of breath, jaundice, organ damage - Carriers are resistant to malaria thus distinct advantage Consanguinity - Marriage or reproductive relationship between 2 closely related individuals - Higher risk of genetic disorders - Closer the relationship, higher probability of inheriting identical copies of faulty recessive genes African pop: albinism - Fair skin and light hair - Incr risk of sun damage and skin cancer - Genes associated with melanin production Caucasians: cystic fibrosis - Most common in northern european heritage - One of most common autosomal recessive disorders - Poor movement of water across epithelial cell surface, causing secretions to be dry and sticky Ashkenazi Jewish pop - Autosomal recessive disorders - Consanguineous marriages encourages pop bottlenecks - Decreases genetic diversity - Tay-sachs disease 17 Sara V - rare autosomal recessive condition, Destroys nerve cells in brain and spinal cord - Only survive into early childhood Sclerosteosis in Afrikaners - autosomal recessive disorder characterised by bone overgrowth Thalassaemia in Indians - Fewer RBC in body and less haemoglobin in RBC - Severe forms need blood transfusions or donor stem-cell transplant Genetic Testing - Carrier screening - prevent recessive conditions - Prenatal Genetic Diagnosis - carrier parents wish to confirm child is unaffected - Prenatal Genetic Testing - pregnant lady determining status of fetus - Predictive testing - adult onset conditions UNUSUAL GENETIC PHENOMENA Penetrance - Consider possibility of reduced penetrance when considering inconsistencies in family history (skipped generations) - Prop of pop who carry disease-causing allele and express the disease phenotype - Complete = mutation, when present, expressed in all members of population who have that mutation Reduced/incomplete penetrance - Carry mutation but not develop the disorder - Mutation, when present, only expresses in part of the population - Eg. Dominant retinoblastoma. Cancer of retina affecting children. 90% penetrant. 10% of carriers will not develop tumor but may pass on the gene - Eg. Familial cancers. Mutations in BRCA 1 and 2 genes develop breast cancer. 85% penetration of BRCA1 by age 70 Possible mechanisms underlying the phenomenon of reduced penetrance - Mutation type - severe vs mild mutations - Environmental - age at first pregnancy, breast feeding, BMI - Age - late onset - Gender - breast cancer if female - Modifier genes - genes at other loci influence trait Variable Expressivity - Varying degrees of severity - Range of symptoms that can occur in different people with same condition - Same genotype leading to range of phenotypes - Eg. Neurofibromatosis. Spots on skin to benign tumors growing along nerves - Eg. Marfan Syndrome. Connective tissue disorder. Tall and thin with long slender fingers. FBN1 gene mutation. Some life threatening complications. 18 Sara V New Mutations - Novel genetic changes - de novo mutations - 44-82 single nucleotide mutations (compared to parent) - 1-2 in coding sequence - Recurrence risk is exceptionally low (child with disorder from new mutation) - 50% of having affected child (individual with autosomal dominant disorder) - Either during gamete formation or post-zygotically in early embryo - Eg. Achondroplasia. 80% of new cases are due to de novo dominant mutations - Eg. Apert syndrome. Almost always a new mutation (mitten hands) TRIPLET REPEAT DISORDERS Mendelian laws of inheritance - One allele from each parent - Phenotype controlled by action of one (and only one) egene - Stable inheritance of mutations Outliers (triplet repeat disorders) - Disease in offspring could be more severe than parent - Further age at onset in offspring * non-mendelian Repeats - DNA polymerase can make mistakes and daughter cells can contain more repeats than parents. Repeat size changes between generations (dynamic) Mendel's rules & TRD - Referred to as non-mendelian inheritance - Does not disober mendelian principles but cannot be explained by them alone TRDs molecular basis Threshold concept > repeat number above a threshold results in disease. Diff for diff disorders. Mutation is dynamic > increase repeat number between generations. Instability becomes manifest > individuals within single fam have diff repeat numbers, In same individual, diff tissues have diff repeat Position > intragenic and extragenic If present within exons > encode a series of identical aa, translated to protein (gain-of-function) If present in UTR > disrupt transcription, translation or protein function (loss-of-function) Myotonic dystrophy - Type one muscular dystrophy - Autosomal dominant inheritance with marked anticipation - Muscle weakness, onset 20s to 30s - Individuals with CTG expansions in prematation range have no reported symptoms - Age at onset is inversely correlated with repeat number 19 Sara V Huntington disease - Autosomal dominant inheritance - 35 to 44 onset - Median survival - Movement disorder and psychiatric disturbances - Onset age inversely prop with repeat number Huntington disease like 2 (HDL2) MITOCHONDRIAL INHERITANCE Mitochondria - Provides ATP via krebs and oxphos - Regulate apoptosis - Calcium homeostasis Genome - Circular - 16500 bp DNA - 37 genes - All coding except d-loop - No histones and no introns and no beginnings of replication - Highly mutable, mtDNA replication is error prone (no proofreading) - Undergoes more rounds of replication, throughout cell cycle - Sperm do not contribute (mtDNA found in tail, lost during fertilization) Matrilineal inheritance - All children from affected female can be affected - No children from affected male will be affected Homoplasmy - One type of mtDNA in cell - All wild type or all mutant Heteroplasmy - Coexistence of wild type and mutant mtDNA in diff cells/within single cell/within single mitochondrion Bioenergetic threshold - % mutant mtDNA incr, ATP prod decr - When % mutant mtDNA reaches critical threshold, cell manifests abnormalities of respiratory chain - Prop mutant mtDNA determines penetrance and severity - % may differ in diff tissues Mitochondrial disorders - Neonatal childhood or adult 20 Sara V - Clinically heterogeneous - Isolated organs but frequently multisystem disorder MELAS > mtDNA, matrilineal Friedreich Ataxia > Frataxin, nuclear DNA, autosomal recessive Leigh Syndrome > mtDNA/nuclear, most= autosomal recessive, 20% = matrilineal 3 parents baby - Nucleus from mother - Mitochondria from donor egg - And male sperm NEW TECHNOLOGIES FOR GENETIC TESTING 1: MICROARRAYS Microarray - 2D array on solid substrate that tests large amounts of biological material using high throughput screening miniaturized, multiplexed and parallel processing and detection methods - Gene/DNA chip = microarray slide - Probe = specific DNA sequence attached to each spot on slide - Target = where probe hybridizes to patients DNA sample - Fluorescent signal = manner which hybridization is detected Comparative genomic hybridization (CGH) array - 2 colour approach (patient and reference with 2 diff colours) - Identify copy number variants by comparing dosage of patient DNA (green) relative to reference (red) - When there is no change = equal binding, net yellow emission, equal amounts of coloured fluorescence - When duplicated = more green than red, overall green emission - Deletions = reduced green, net red emission - Dosage measured by converting signal ratio to a log2 ratio acting as proxy for copy number - Increased log2 ratio represents a gain in copy number in patient sample and a decrease indicates a loss Single nucleotide polymorphism (SNP) array - One colour approach - Detects SNP and copy number variants - Small fragments of DNA - Each position on array corresponds to locus - Copy number changes are determined by relative intensity of bound DNA at each allele - Dosage determined by calculating B allele frequency. Normalized ratio of quantity of B allele to total quantity of both Microarray limitations - inability to detect balanced rearrangements (normal copy number) - Low-level mosaicism may be missed 21 Sara V - CNVs not represented, will not be detected - Small CNVs may be missed - Polyploidy may be diff to detect (CGH) - Cannot exclude disease caused by single-gene mutations Indications for microarray testing - Developmental delay/intellectual disability - Congenital abnormalities - Autism spectrum disorder Pathogenic CNV (clinically significant) - Detectable change in copy number of chromosome segment which has been previously described or can be shown to be causal of clinical phenotype in patient Uncertain clinical significance - Insufficient evidence available to determine clinical significance - May be reclassified at later stage Benign CNV (clinically insignificant) - Detectable change in copy number of chromosome segment which has been described in healthy individuals with no known abnormal phenotype - Contributes to human diversity Pre-test counselling - Describe test, benefits, limitations, possible outcomes, consent Post-test counselling - Implications of test result, incidental findings, recurrence risks, management, patient resources, support NEW TECHNOLOGIES IN GENETIC TESTING II: NGS Targeted gene panel testing - Analyse multiple related genes simultaneously - May be 2-100+ genes - High yield if carefully selected - Less ambiguous and unwanted results - Useful if phenotype is relatively distinct, multiple genes known to cause similar phenotype (locus heterogeneity) Whole exome sequencing (WES) - Sequence entire coding sequence of human genome (exons) - Conder if: poorly defined phenotype, suspect new syndrome - Interpretation is challenging - generates new data and difficult to prove causation Whole Genome sequencing (WGS) - Sequences all DNA of individual - Massive data set 22 Sara V - May detect intronic mutations, breakpoints, structural rearrangements - Increases chance of uncertain findings NGS Challenges - Analysis is complex > requires expertise, interpretation influenced by experience and knowledge about the gene, discrepancies between labs - Variants of uncertain significance > knowledge limitations, classification is complex, additional studies may prove diff outcomes - Incidental findings > unrelated to indication for testing, may be of medical value to patient, counselling issues - Missed mutations > larger rearrangements, technical issues, epigenetic changes - Heterogeneity > Mutations in diff genes cause same disease and some in same gene cause diff diseases Role of genetic counsellor - Explain technologies and communicate uncertainties with patients MULTIFACTORIAL INHERITANCE - Human biology runs on pathways and systems, run by proteins being coded for by genes - Genetic variation modifies how these systems operate between individuals - Polygenic traits: metabolism, height, skin pigmentation, IQ - Continuous distribution Discontinuous traits - All diseases are discontinuous - Mendelian disease > mutation means disease. No underlying susceptibility - Polygenic > need several DNA variants in many genes before you get disease. Underlying susceptibility, having high risk variants increasing risk for getting disease. (normal distribution). Underlying susceptibility is continuous. Disease state is discontinuous. Multifactorial inheritance - Environmental factors can influence disease outcome - Gene expression patterns can change - Polygenic inheritance + environmental factors = multifactorial inheritance Types of inheritance - summary Mendelian > mutation on one gene Polygenic > variants in many genes (rare) Multifactorial > variants in many genes + environmental factors Liability shift - Chance of disease is modified by family history of disorder. - Not case for mendelian (only consider mom & dad) - Your liability shifts with each affected family member as well as the severity of their disease 23 Sara V - Liability curve shifts to the right & shifts more with each family member Recurrence vs relative risks - punnet square = exact recurrence for Mendelian - Cannot calculate exact for multifactorial - Relative risk based on no. of affected family members and environmental factors - Disease clusters in families but no distinct pattern (may resemble Mendelian) Relative risk A - Low environmental and genotypic risk B - High environmental and low genotypic risk C - low environmental and high genotypic risk D - high environmental and high genotypic risk - Risk is never 0 nor 100 irrespective of level of risk Heritability studies - estimate relative contribution of genetic and environmental influences to multifactorial trait 1. Population/migration > disease shows diff prevalence per ethnicity.(migrant pop that integrates with local pop) Keep prevalence in new environments (genetic component larger). Changes prevalence between populations (environmental component larger) 2. Family studies > does disease occur at a higher frequency in family history than it does for the average population? If yes, the genetic component is larger. (untrue? - inherit environment) 3. Twin studies > concordant for disease or discordant for disease. Monozygotic or dizygotic. Sample both and compare concordance between two types. If genetics are large, concordance in monozygous would be high. If concordance is equal, environment larger. - ascertainment bias. Adopted separately and reunited at 30. Same name, dogs name, favorite subject, marital status, same job type, smokers 4. Adoption studies > compare frequency of disease between individuals remaining with biological families and those adopted out of them (gold standard) 5. Biochemical studies > metabolite and enzyme activity levels. Only useful if biochemical basis is known 6. Animal study > present disease similar to humans. Model organism disease traces back to a single gene unlike humans 24 Sara V APPROACHES TO STUDYING MULTIFACTORIAL DISEASES Purpose - average doctor rarely encounters unifactorial diseases - Most fatalities caused by common/multifactorial diseases Fibrodysplasia Ossificans Progressiva (stone man syndrome) Association study - Candidate gene association study (CGAS) - Hypothesis driven - Basic knowledge about disease genetics is known/suspected - Specific candidate genes are selected and tested for association with disease status Association study - Genome-wide association study (GWAS) - Hypothesis free - Entire human genome scanned - No prior knowledge needed Collect 2 samples (one of healthy people [control] and another diagnosed with disease [case group]) Smile = healthy. Frown = diagnosed people. Red = high genetic risk Tag Single nucleotide polymorphisms (SNPs) - Search through entire genomes (>6 billion nucleotides) - Tag snips for proxy for larger regions - Single nucleotide that suggests info about surrounding nucleotides - Allele 1 of tag SNP represents a section of DNA - Allele 2 of tag SNP represents the same section but slightly modified sequence Population stratification - Separate population by ethnicity - Case and control have diff allele frequencies attributable to diversity in background population, which is unrelated to disease status, a study is said to have population stratification Wellcome Trust association study - Genome-wide - 500 568 tag SNPs - Excluded if genetics revealed non-caucasion ancestry Bipolar disorder - Manhattan plot shows results - Each dot is a single tag SNP (x axis shows which chromosome) - Green dots are SNPs that occur statistically more often in affected individuals than control - Higher dot on axis, the stronger the association Coronary artery disease - Region on chromosome 9 - Can’t be true because it is polygenic Crohn’s disease 25 Sara V - Association found on 1,2,5,10,16,18 - Polygenic Hypertension - One marker on X chromosome Rheumatoid arthritis - Strong signals on chromosome 1 and 6 Diabetes - 6 associated with both rheumatoid arthritis and T1D - More associated signals expected Limitations - Assume all control individuals are completely healthy but may have developed the disease with later onset - Red faces are high risk, so may develop disease (healthy at time of disease) - Can we assume all British Caucasians are the same? Subcategories may affect results - If tag SNP is strongly associated, you have to further analyze to find disease risk SNP (indirect association). Tag SNP found on same gene segment as disease risk SNP - Association signal may occur in areas with no genes, genes of unknown function (non-coding region) DIRECT TO CONSUMER GENETIC TESTING Definition - Tests that are freely available to public through media - Without approval of doctor or insurance company - Eg 23andme PHARMACOGENOMICS I: INTRODUCTION - Variation in responses to medication - Due to environmental factors and genetic factors Drug response - Multifactorial trait Variation in drug response 1. Efficacy of drug - Drug resistance/non-response - Allows development of new drugs 2. Safety - Adverse drug reactions (mild to fatal) - Many drugs fail clinical trials 3. Dose - Narrow therapeutic range. - Too high -> toxicity and dose related ADR - Too low -> not effective 26 Sara V - Diff patients require diff doses of same drugs Aims to - Ensure maximum efficacy with minimal adverse effects Pharmacogenomics - Study of interaction between genetic makeup and response to a drug - Pharmacogenetics - study of effect of one variant on one gene Approach - Genotype of each group - Identify variants specific to group - Used to predict response in future Haplotype - Groups of variants associated with particular trait - Groups of variants inherited together from one parent on one chromosome - Inherited together because physically close to each other on same chromosome ADME Absorption, distribution, metabolism, excretion pharmaADME database - 32 core + 340 extended genes Drug response heterogeneity between populations Inconsistent patient responses to drug therapies Poor metabolises -> build up of drug in liver, lack of efficacy (give less drug) - Two non-functional alleles Extensive metabolizer -> as expected (give as prescribed) - Two functioning alleles (100% protein production) Ultra-rapid metabolizers -> cleared too quickly from liver (give more drug) - Gene duplication Drug metabolizing enzymes (>30 families) CYP 450 cytochrome P450 superfamily - Metabolise 60% of prescribed drugs - Expressed primarily in liver - 57 genes known to date - CYP 1, CYP 2, CYP 3 - Subfamilies a-e - Variants described - CYP2C9*2 (allele 2) Metabolic pathways affected by diff dna variants - SNPs, mutations, VNTR, deletions and duplications CYP2D6 - gene with most variants (over 80) Aim of pharmacogenomics - Inform personalised medicine and precision medicine 27 Sara V Precision medicine - Medical care designed to optimise efficiency or therapeutic benefit for particular individual/group of patients, by using genetic or molecular profiling - considerations : availability of tech and cost Precision public health - Decisions based on populations as opposed to individuals - Most cost effective approach for developing countries - Treatment tailored to groups Hurdles in implementing pharmacogenomic testing: - Limited drug alternatives - Disincentive to make other drugs available - Educating healthcare providers PHARMACOGENETICS IN CLINICAL PRACTICE Warfarin (anticoagulant) - Prevents/treat bleeding disorders - Narrow therapeutic index > safety of drug - CYP2C9 metabolises warfarin - Test for variants specific to ethnicity - VKORCI vitamin epoxide reductase Codeine - Opiate for pain - Selectively relieves pain - ADR: drowsiness, nausea Statins - Reductase inhibitors - ADR: statin induced myopathy Concomitant drugs - Existing drugs affecting reactions to other drugs PHARMACOGENETICS IN AFRICA Challenges in africa - High disease burden, with low resources and many political challenges - Extensive genetic diversity with very little data - SA doing bulk of research - Safety, efficacy and dosage PRENATAL INVESTIGATIONS - Investigations of fetus during pregnancy 28 Sara V - Detect abnormalities in development - Offer option of termination in case of poor outcome Who should consider? Pregnant women (of all ages) if: - Close relative/previous child with serious genetic condition - One of the partners of couple have serious condition that may be passed onto baby - One or both parents are carriers of known genetic disorder Pregnant women if: - Mother is 35 years or older - Some teratogenic exposure during the pregnancy (alcohol/ chemotherapy) - Positive screening test indicating increased risk of an abnormality Trimesters - 1st = from conception to 13th week - 2nd = 14th week to 27th week - 3rd = 28th week to 40th (birth) Prenatal investigations 1. Prenatal screening - Separating high risk pregnancies from low risk - Results are not definitive - not used to make diagnosis 2. Prenatal diagnostic - Investigate fetus during pregnancy to detect definite abnormalities - Results are definitive - Usually invasive Types of prenatal screening 1. Family history taking (pedigree) and pregnancy history - Medical & pregnancy history (teratogenic exposure, stillbirths) - Age of conception (35+ advanced age = high risk) - Family history (3 generations) 2. Fetal ultrasound - Sound waves to produce image - Generates picture of baby in womb - identify abnormalities - Utilization: determine fetus size, growth and development, detect structural features, recognise and detect fetal abnormalities 3. Biochemical testing (blood - 1st and 2nd trimester) - 11-13.6 weeks - Blood test measuring proteins in mothers bloo - 16-20 weeks - Maternal serum screening 4. Non-invasive prenatal testing (NIPT) Prenatal diagnosis (PND) 29 Sara V - Manage risk - Enable couples to have healthy children - Plan prenatal and postnatal treatment - Psychological preparation Types 1. Chorionic villi sampling - Invasive, early in pregnancy - Placental tissue (shares DNA with fetal tissue) - Identify chromosomal abnormalities - Miscarriage risk of 1-2% - Week 11-13 - Injection to numb, ultrasound guidance, insert needle to obtain biopsy - Pro: earlier test and TOP - Cons: invasive, miscarriage risk, bleeding and cramping, infection, risk of mother to child transmission, maternal contamination 2. Amniocentesis - Ultrasound guidance, needle inserted - Retrieve amniotic fluid (baby fibroblast cells) - Lower miscarriage risk - Week 16-22 - Minimal infection risk - Pros: lower miscarriage risk and less risk of sample failure than CVS - Cons: invasive, infection risk, later stage psychosocial issues, TOP more complex 3. Cordocentesis - Fetal blood taken from umbilical cord - Not common - highly skilled doctor needed - Done later in pregnancy - 18th week onwards - Miscarriage risk= 2-3% - Pros: convenient sample for analysis - Cons: invasive, infection risk, psychosocial issues, TOP more complex Fetal sampling - CVS: chorionic villi (placental sample) > does not require cells to be grown - Amniocentesis: amniotic fluid (fibroblast cells) F> cells grown to be tested - Cordocentesis: fetal blood > direct analysis for genetics Genetic testing - Specific single gene disorder - QF-PCR (aneuploidy screen) - Chromosome analysis (karyotype) (trisomy and structural rearrangements) - Microarray analysis Termination of pregnancy act (1996) 30 Sara V - TOP up until week 12 - Between 12 and 20, allowed with opinion of one medical practitioner - Physical or mental abnormality with fetus - Emotional distress of parents (psychologist advocate for TOP) - Pregnancy due to rape - After week 20, allowed with opinion of 2 medical practitioners - Endangerment to mother - If continuing pregnancy with lead to malformations of fetus NON-INVASIVE PRENATAL TESTING Need for non-invasive tests - Invasive testing carries a risk for procedure related miscarriage - NIPT refers to techniques that evaluate fetal calls in blood sample drawn from the mother Cell free fetal dna (cffDNA) - Genetic material released by placenta and circulates within mothers blood - Not encapsulated - Reflects genetic make-up of developing baby - Combines with cell free maternal DNA forming cell free DNA (maternal blood) - 3-10% of cfDNA comes from fetus (up to 10-20% near end of pregnancy) [amount depends on body weight, maternal diseases, aneuploidy, twin pregnancies] - Enrichment of cfDNA in blood caused by apoptosis of fetal blood cells and trophoblast cell breakdown - Can be detected from week 4/ 5 until delivery - Eliminated after labour, does not persist into next pregnancy Fetal fraction - Proportion of cfDNA in maternal blood that comes from placenta is known as the fetal fraction - Must be above 4% (around 10th week of pregnancy) - Increases with gestational age - Low fetal fraction can lead to inability to perform test or false negative How NIPT performed - Free fetal cell DNA found in mothers DNA - Blood sample taken from mother - Should be performed when fetal fraction is at least 4% - Usually around 10 weeks gestation Who needs to consider NIPT - First trimester risk assessment > 11-14th week - Ultrasound and biochemical testing - Usually offered to women with increased chance of fetus with aneuploidy NIPT applications - Fetal gender determination 31 Sara V - Fetal RhD genotyping - Detection of aneuploidies and microdeletions - Trisomy, 21,18,13 - Triploidy - Monosomy x - Microdeletions and gender Protocol principle - Next generation sequencing - Distinguish fetal and maternal DNA: - Counting (CNV), epigenetic difference, genetic difference Copy number differences between chromosomes - 2 rows of each - When trisomy is present, amount of certain chromosome is higher, 5 rows - More maternal cfDNA, but if aneuploidy exists in fetus, proportions are different compared to other fetal chromosomes Factor affecting NIPT outcome - Insufficient amount of cfDNA - Fetal fraction is below acceptable level - Insufficient numbers of fragments sequenced and.or aligned Pros - Most accurate screening test - 90% - Can be done early in pregnancy - Non-invasive (intermediate step between normal screening and invasive diagnostic testing) - No risk of miscarriage Cons - Not diagnostic but rather indicates need for invasive Availability - Global availability - Rare in SA, mainly found in private sector - 7.5 k for test > expensive Ethical implications - Easy and safe and early in pregnancy - Feared that informed consent may be more difficult - Feared that testing and selective abortion will become normalized - Feared that there will be a trend towards accepting testing for minor abnormalities and non-medical traits as well RECURRENT MISCARRIAGES Miscarriages 32 Sara V - Loss of intrauterine pregnancy before foetus reaches viability - Most common complication of early - 15% of pregnancies lead to miscarriage (1st trimester is most common) - 2nd trimester - 2-3% of pregnancy losses Common causes of miscarriages 1st trimester - Genetic factors - Endocrine abnormalities - Immunological disorders - Infection 2nd trimester - Maternal medical illnesses - Anatomic abnormalities Signs and symptoms of miscarriages - Vaginal discharge - Loss of pregnancy symptoms - Abdominal cramping - Passing clots Recurrent miscarriages - RM is defined as loss of 3 or more early ( changed shape of uterus - acquired > polyps, fibroids - Cervical incompetence >> 2nd trimester loss, cervix shortens and causes preterm labour 2. Endocrine - Affects implantation and maintenance of embryo 3. Immunological factors - Antiphospholipid syndrome (APS) > autoimmune disorder. Antibodies attacking phospholipids 4. Genetic factors - Single gene conditions > autosomal recessive and dominant conditions, X linked conditions - Numerical chromosomal abnormalities > foetal aneuploidies (trisomies, polyploidy and monosomies). Not inherited, but de novo. Non-recurring - Structural chromosomal abnormalities > unbalanced chromosomal translocations, from parents carrying balanced chromosome rearrangements. (2-5%) 5. Idiopathic - cause unknown (most common) 33 Sara V POC - Products of conception (fetal tissue remaining in uterus) - Karyotype or chromosomal microarray (CAM) - CAM can detects smaller abnormalities (not offered) - Parental karyotyping Genetic counseling - Recurrence risk is difficult to predict - If due to balanced translocation > couple keep trying, offer prenatal testing (CVS or amnio) or preimplantation testing ASSISTED REPRODUCTIVE TECHNOLOGIES Infertility - Failure to achieve clinical pregnancy after 12 months of regular unprotected sexual intercourse - 15-20% of population - Higher in developing countries - Surrogacy and adoption are more affordable - ⅓ - issue with man and vice versa Male infertility - Abnormal sperm production or function > caused by undescended testicles, or infection or genetics - Problems with sperm delivery > genetic diseases or structural problems - Overexposure to certain environmental factors - Damage related to cancer and its treatment > radiation and chemo Female infertility - Ovulation disorders > hormonal or thyroid abnormalities or eating disorders - Cervical abnormalities > shape of uterus, polyps - Fallopian tube damage or blockage > inflammation - Endometriosis - Primary ovarian insufficiency (early menopause) 34 Sara V - Pelvic adhesions - Cancer and treatment Risk factors - Age, tobacco, alcohol, overweight, underweight, exercise issues Assisted Reproductive Technology - Treat infertility - IVF - most common and effective type of ART - Sometimes use donor eggs, donor sperm, surrogate, frozen embryos - 1.5% of infants - Higher order births attributed to ART Types of ART 1. In Vitro fertilization (IVF) Preimplantation genetic testing (PGT) > identify genetic defects in embryos created by IVF before pregnancy. If affected with a condition them embryo is not implanted PGT-M (monogenic) - monogenic condition where parents are known carriers or infected. Specific single gene is tested PGT-SR (structural rearrangement) - if parents is carrier for balanced translocation PGT-A (aneuploidy) - advanced maternal age, recurrent pregnancy loss, male infertility Advantages - avoids TOF, affected child, recurrent miscarriages - Retrieves eggs and fertilizes with sperm, egg grows in a petri dish becoming an embryo and implanted into the uterus. - Woman takes fertility drugs (ovarian stimulation hormone therapy) to ovulate on predictable timeline and produce multiple eggs - IVF produces multiple embryos increasing likelihood of having multiple babies 2. Intrauterine insemination (IUI) - Sperm placed directly into uterus - Sperm washed and concentrated - More affordable, lower success rate, less invasive - One procedure - Some women opt for fertility drugs to increase chance of success 3. Intrafallopian transfer (IFT) - Sperm placed with sperm into a tube, injected into fallopian tubes (laparoscopy) - Fertilized egg inside fallopian tube - Fertilization inside the body - Fertility drugs prior 4. Intracytoplasmic sperm injection (ICSI) - Sperm are injected into egg cell - Using fine glass needle - Similar to IVF (early stages) - Each egg injected with sperm, several embryos available 35 Sara V Risk associated with ART - Perinatal risks include: multifetal gestations, prematurity, low birth weight, small for gestational age, perinatal mortality, cesarean delivery, placenta previa, placenta abruption, preeclampsia and birth defects - Risks are much higher for multifetal gestations, singletons achieved with ART are at higher risk than singletons from natural pregnancies - Unclear association between risk and underlying causes of infertility - Multifetal pregnancies are the greatest risk - counseled about risk Multifetal pregnancies - More than one fetus in the uterus - Recommended that only one embryo is implanted - Embryo can split into identical twins Fetal reduction - Termination of one or more fetuses to lower fetal number - Increases chance of survival - Usually done for triplets to higher order - 4% risk of miscarriage for other fetuses Birth defects - Studies have documented increase in birth defects - May be due to oocyte manipulation - May also be associated with underlying infertility cause EPIGENETICS I: PRINCIPLES AND MECHANISM Cellular differentiation - 210 different types of cells in the human body, what process controls genetic differentiation if all cells contain same genetic complement - Arises through changes in gene expression profile during stepwise differentiation of pluripotent stem cells into fully committed and terminally differentiated cells - Process driven by epigenetic mechanisms, determining which genes become deactivated - All cells contain identical genome, but contain many different epigenomes determining cell fate, function and phenotype Epigenetics - Structural adaptation of chromosomal regions (chromatin remodeling) to alter activity states - Phenomenon of epigenetics describes additional, heritable layer of info on top of DNA nucleotide sequence, that influences the expression level of subsets of genes - “Epi” - over - Factors independent of DNA sequence itself - Epigenetics studies changes in regulation of gene activity an expression (not dependent on DNA) - Reversible 36 Sara V Chromatin - DNA - Histone proteins - Other non-histone proteins - Nucleosome units Histone proteins and nucleosomes - Scaffold around which DNA coils is a unit of 8 proteins, called histone proteins - Nucleosomes are basic unit of DNA packaging in eukaryotes - Histone tails can undergo modification Gene expression controlled by epigenetic mechanisms of: Histone modification - Histones are globular proteins and their amino terminal tails protrude from the nucleosome core structure - They can carry diff post translational covalent modifications: acetylation, methylation, phosphorylation, ubiquitylation - Acetylation >> +ve charged lysine residues loose charge, weakens DNA:histone interactions, DNA loses compaction (euchromatic), transcription activation - Methylation >> directly interfered with binding of transcription factors of DNA, DNA more compact (heterochromatic), transcription silencing DNA methylation - Addition of methyl group to DNA at the site of a cytosine (C) base - C base found in context of a 5’ - CG - 3’ sequence - No methylation (hypomethylated) = transcribed > spaces, for transcription factors to reach DNA - Methylation (hypermethylated) = not transcribed > distributed across the genome, and correlates with transcriptional silencing. DNA compacted, genes remain unexpressed Non-coding or interfering RNAs - In higher eukaryotes ncRNA regulate gene expression - Partially complementary to one or more messenger RNA molecules - Down regulate gene expression by rendering message as non-functional - Gene transcribed but not translated Imprinting - Epigenetic processes are crucial for embryonic development - Imprints certain genes - Some genes only expressed on chromosome inherits from the mother (maternal allele expressed, paternal allele silenced) - mono-allelic expression - Opposite true for other genes (paternal allele expressed) - Necessary for male and female genome needs to be present for normal funtion (asexual reproduction impossible) 37 Sara V - 80 imprinted genes in human genome (rare circumstance) - Maternal & paternal genomes are functionally different and both essential for development - If bi allele expression happens > incorrect dosage for normal functioning of protein leading to disease Imprinted locus Normally: one allele is functional, the other non-functional. Set during gametogenesis - Imprint erased and reprogrammed during gametogenesis (reversed, erased and reprogrammed) - Imprint established through DNA methylation (monoallelic expression, parent-of-origin expression) Epimutation (disorders) - Can lead to both genes being active (too much protein) - Both alleles inactivated (too little protein) - Manifested in disease and developmental disorders EPIGENETIC DISORDERS Epigenetics in disease - Disturbance of normal pattern can cause developmental disorders, cancer and complex diseases Imprinting disorders Prader Willi syndrome > (first to be described) maternal gene expressed - Severe hypotonia (floppiness in infancy and childhood) - Feeding difficulties in infancy - Excessive eating and development of morbid obesity in later infancy (don’t feel full) - Motor milestones and language development is delayed - Hypogonadism - No expression of SNRPN - 70% caused by deletions and 25% by UPD Angelman syndrome > paternal gene contribution, no maternal - Severe developmental delay and intellectual disability - Inappropriate laughter - Microcephaly - Seizures - Severe speech impairment - No expression of UBE3A - 70% caused by deletion and UPD rare Chromosome 15 Gene UBE3A PWS cluster SNRPN 38 Sara V Mechanisms that cause - Deletions on chromosome 15 (in paternal = PWS, if in maternal = angelman) - Uniparental disomy > both chromosome 15 from one parent Beckwith wiedemann syndrome > no maternal contribution of chromosome 11 - Overgrowth syndrome - Hemihyperplasia - Omphalocele - Macroglossia (large tongue) - Increase risk of cancer - Abnormal methylation and UPD 20% Russel silver syndrome > no maternal contribution of chromosome 11 - Feeding difficulties - Failure to thrive - Small - Triangle face, prominent forehead, small jaw - Risk of delayed development and learning disabilities (not always apparent) - Abnormal methylation and UPD Paternal genes lent to promote fetal growth Maternal genes restrain fetal growth X CHROMOSOME INACTIVATION X and y chromosomes - X a lot bigger - 1000 genes (155 million bp) on x, 100 genes on y Problem - Females have more genes than males - Female cells must be functionally equivalent to male > x inactivation Silencing - Of one of 2 X is randomly inactivated (random within cells, but progeny maintain pattern) - Epigenetic mechanism - Occurs at early stage in development (15-16 days) Steps 1. Cell counts number of X chromosomes 2. Random choice of one X to remain active 3. Silencing of future inactive X > recruitment of specialized factors 4. Inactivated chromosome condenses into Barr body, stably maintained in a silent state Inactive x - Condensed chromatin - Hypermethylated 39 Sara V - Hypoacetylated How many barr bodies - All but one are inactivated - If more than 1 X chromosome in cell, number of Barr bodies is one less than total x chromosomes - Mosaic (some have paternal X, others have maternal X) Mechanisms of x inactivation - Complex, achieved by differential methylation - X-inactivation center (XIC) - If XIC missing - no inactivation - XIST gene responsible for silencing of chromosome - triggers inactivation, expressed only from inactive x - Spreads inactivation methylation signal up and down chromosome causing methylation signals - Transcriptional silencing of genes How does one x remain active - Blocking factor joins inactivation factor - No other blocking factor to provide protection to other chromosomes Why do people with aneuploidies have abnormal phenotypes? - Not all of the x chromosome is inactivated - 15-20% of genes remain active - Genes in pseudoautosomal region (tip of short arm) > found on tip of x and y chromosomes - SHOX genes involved in stature (short stature homeobox gene) Skewed X-inactivation - More than 50% of cells have same X active - Problematic when carriers of x-linked conditions - More than 50% of mutated chromosome = express phenotype 2 reasons Physiological > chance Chromosome abnormalities involving X chromosome > structurally abnormal or have a translocation (x chromosome with autosome 40