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Medical Genetics 1 2 - Lecture notes ALL
Medical Genetics (PATH10200)
University College Dublin
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! Medical Genetics Medical genetics: the study of genetic disease in humans! Clinical genetics: the medical speciality which deals with families affected by or at risk of genetic disease.! ! Darwins theory of evolution:! Minor random genetic variations can give selective advantages, according to the environment in which an organism is found.! ! Mendel’s laws of heredity: ! 1. Law of Uniformity: When 2 homozygotes for different alleles (different forms of a gene) are crossed, all of the F1 offspring are identical and heterozygous (two different alleles for a single trait.)! 2. Law of Segregation: Each individual possesses 2 genes for each characteristic, only one of which can be transmitted at any one time! 3. Law of Independent Assortment: Members of different gene pairs segregate to offspring independently of one another! ! Mendelian disease: diseases in which the phenotypes are largely determined by the action/ lack of action of mutations at individual loci. ! ! Eugenics: The improvement of a population’s genetic constitution by selective breeding. Today can be replaced by manipulation of individual genes e. alteration of the germline of plants and animals by microinjection, transgenics, and culture of embryonic stem cells.! ! Cell division: is the distribution of identical genetic material (DNA) to two daughter cells. ! • Cell division is vital for: reproduction, renewal and repair.! • Cells in the body are diploid – two copies of each chromosome ! • 23 pairs of chromosomes – 46 chromosomes in total! Mitosis preserves diploid number (opposite-meiosis)! ! Interphase prepares the cell for cell growth. Cell has increased metabolic activity, preparing for mitosis. Chromatin in duplicated into chromatids, which are held together at the centromere! G1: cell growth ! S phase: DNA synthesis, duplication of chromosomes! G2: cell growth! ! Mitosis: two sister chromatides containing identical copies of chromosome DNA are pulled apart and repackaged as two complete nuclei! Cytokinesis: following division of the nucleus and the cytoplasm divides! ! During Mitosis! Prophase - Chromatin in the nucleus begins to condense. Centrioles move to opposite ends of the cell and fibers extend and form spindles.! Prometaphase - Nuclear membrane dissolves. Kinetochore form on the centromeres. Microtubules attach and the chromosomes begin to move.! Metaphase - Spindle fibers align the chromosomes along the metaphase plate. (ensures that when separated each cell recieves one copy of each chromosome).! Anaphase - Paired chromosomes separate.! Telophase - Chromatides arrive at opposite poles of the cell, and the new membranes form around the daughter nuclei. Chromosomes disperse. ! Page 1 ! Medical Genetics Cytokinesis: Cleavage furrow aligned close to metaphase plate. Contractile actin microfilaments constrict cleavage furrow and two separated cells are produced.! ! Meiosis: is a specialised form of cell division, which produces gametes (ova or sperm). In humans, this form of division produces haploid (23 chromosomes) gametes from diploid (46 chromosomes) parent cells. Meiosis involves two divisions: meiosis Ι and meiosis ΙΙ. ! Meiosis I! Interphase I: Chromatids are formed by DNA replication! Prophase I: Homologous chromosomes pair to form bivalents and recombination occurs. In each bivalent chromosome material is exchanged between homologous chromatids, recombination. Then the recombinant chromosomes gradually separate but remain attached at points where recombination has occurred known as chiasmata.! Metaphase I: The paired chromosomes become attached to the cell spindle! Anaphase I: Homologous chromosomes separate to opposite poles! Telophase I: Two haploid cells are formed! Meiosis II! Metaphase II:! The chromosomes become attached to the cell spindle! Anaphase II:! The chromatids separate to opposite poles! Telophase II:! Each cell divides into two cells ! ! Meiosis introduces genetic variation in several ways! 1. The generation of haploid gametes with can recombine with a new gamete at fertilisation to be diploid and different from parent.! 2. Genetic diversity of each gamete is assured by the independent segregation of the chromosomes to daughter cells.! 3. Recombination which generates new genetic combinations, introducing further diversity.! ! Gametogenesis! Oogenesis: the formation of the ova.! Mostly occurs prior to birth in the developing foetal ovaries. Primordial germ cells give rise to ova progenitors (oogonia), which undergo numerous mitotic divisions before maturing into primary oocytes. During prophase I they enter a stage of maturation arrest, known as dictyotene. With ovulation, meiosis is completed, with formation of a single secondary oocyte and the first polar body. Meiosis II is completed after fertilisation in the Fallopian tube, when the secondary oocyte divides into an ovum and a polar body.! ! Spermatogenesis: the formation of sperm! Spermatogonia undergo about 30 mitotic divisions by the onset of puberty. ! ! Thereafter rapid division leads to the formation of primary spermatocytes, which enter meiosis I and emerge 60 - 65 days later as mature haploid spermatids. These then mature into spermatozoa. ! ! Cytogenetics: study of structure, function and evolution of chromosomes.! Genomics: study of the structure and organisation of the genome.! ! Chromosomes: a long DNA molecule tightly packed around histone and non-histone core proteins. ! • The basic structural unit is a nucleosome.! • An average human chromosome has 6 x 105 nucleosomes and 108 base pairs of DNA.! • The human chromosome number is 46 (44 autosomes and 2 sex chromosomes) ! ! ! ! ! ! ! ! ! ! ! Medical Genetics Mosaicism: a mixture of two genetically different cell lines in a person derived from a single embryo.! ! Chimerism: is the presence of two genetically different cell lines in the one person, but the cell lines are from two different embryos. The commonest reason is after a bone marrow transplant, but very rarely a embryo can form from the fusion of two different (dizygotic) embryos, and give rise to a human chimera.! ! Structural Chromosomal Abnormalities! Inversions: Inversions occur when a chromosome breaks in two places and the resulting piece of DNA is reversed and re-inserted into the chromosome. ! Pericentric inversions: involve the centromere ! Paracentric inversions: do not involve the centromere.! Deletion: part of the chromosome is removed and the broken portions re-join.! Isochromosome: Arm completely deleted and other arm duplicated and replaces loss, causes loss of genes and incorrect of genes that isn’t needed.! Ring chromosomes: usually occur when a chromosome breaks in two places and the ends of the chromosome arms fuse together to form a circular structure. ! Robertsonian Translocation: The joining of two acrocentric chromosomes at the centromeres with loss of their short arms to form a single abnormal chromosome.! Reciprocal Translocation: a segment of one chromosome is exchanged with a segment of another chromosome of a different pair. Balanced, the correct amount of chromosome material is present. Unbalanced, there is too much or too little chromosome material present e. Cri-du-chat.! ! Cri-du-chat: deletion of short arm of chromosome 5. ! Symptoms: Small heads, significantly delayed development, poor growth, an unusual facial appearance, congenital heart disease. They can have an unusual mewing like cry as infants.! ! Numerical Chromosomal Abnormalities! Monosomy: loss of a chromosome e. Turner Syndrome! Trisomy: extra chromosome e. Down Syndrome! Tetrasomy: four copies of a chromosome! Polyploidy: multiple copies of each chromosome! Triploidy: three of each chromosome ! Marker: extra chromosome not identified by banding pattern. ! ! Non-disjunction at mitosis ΙΙ leads to the formation of a disomic and a nullisomic gamete which in turn could lead to abnormalities in offspring.! ! Down’s Syndrome: Trisomy 21. 47, XX, +21. 1/700.! Facial features of epicanthic folds (inside of eyes), slant to eyes, flat nasal bridge, prominent tongue, midfacial hypoplasia (upper jaw, cheekbones and eye sockets develop much slower than the rest of the face), single palmar creases, small low set ears, a flecks in the iris called Brushfield’s spots, babies are floppy, fingers curve in.! Developmental delay (to different extents), congenital heart disease in 40%, intestinal atresias (narrowing or absence of a portion of the intestine). Higher chance of developing: epilepsy, leukaemia, deafness, presenile dementia.! ! Aetiology (causation/origination) of Down Syndrome! Meiotic nondisjunction (95%): at mitosis the both copies go to one gamete. Probability increases as mother gets older.! ! Page 4 ! ! ! ! ! ! ! ! ! ! Medical Genetics Mosaicism (2%): only some cells, derived from the one embryo – mosaic Down syndrome some cells are 46,XX and some are 47,XX +21! Unbalanced Robertsonian Translocation (3%): fusion between the two acrocentric chromosomes 14 and 21. The child therefore has three chromosomes 21 -2 separate chromosomes 21, and a third as part of the fusion chromosome. Karyotype written as 46. People with balaced translocation are carriers. Gamete ‘fitness’ determines probability of child getting DS. If father carries mutation then probability is lower.! ! Patau’s Syndrome: Trisomy 13. 1/5000! Bilateral cleft lip and palate, no real development of nose or eyes, extra digit (Postaxial polydactylyl), holoprosencephaly (a single small forebrain), scalp defects, congenital heart disease.! Chromosome 13 much bigger than 21 so severe disabilities, usually neonatal deaths or stillbirths. Usually derives from meiotic non-disjunction but are rare forms of Robertsonian translocations. ! ! Edward’s Syndrome: Trisomy 18. More common in girls. 1/3000! Growth retardation, “elfin” face, aged look, unusual ears, rocker bottom feet, clenched hands, congenital heart disease, exomphalos (intestinal contents outside abdomen), renal abnormalities. ! Usually meiotic non-disjunction, 18 not acrocentric so don’t usually get translocation form. ! ! Turner Syndrome: 45, X. 1/10000 females! Most 45, X conceptions miscarry.! Short stature, ovarian dysgenesis (failure to develop), primary amenorrhoea (absent periods), infertility, webbed neck, peripheral lymphoedema (swollen feet/hands), no secondary sex characteristics, Coarctation (narrowing) of descending aorta.! ! Klinefelter Syndrome: 47, XXY. 1/ 1000 males! 1 in 10 azoospermic males (produce no sperm), advanced parental age. Long limbs, short trunk, breast development (gynaecomastia), hypogonadism (body doesn't produce enough testosterone), small genitalia, mild developmental and educational problems.! ! Autosomal Aneuploidy! The term aneuploidy refers to cytogenetic abnormalities in which all or part of one or more chromosomes is added or deleted. Autosomal aneuploidy refers to all such abnormalities that do not involve the sex chromosomes! • Increased foetal loss! • Poor growth (prenatal & postnatal)! • Abnormal dysmorphic (difference in body structure that suggests genetic syndrome/birth defect etc.) appearance! • Structural malformations (e. congenital heart disease)! • Developmental delay! • Monosomy (single copy) more severe than trisomy (three copies)! ! Indications for Chromosomal analysis! 1. Multiple congenital anomalies ! 2. Ambiguous genitalia! 3. Primary amenorrhea! 4. Primary male infertility! 5. Recurrent (3 or more) miscarriages! ! Page 5 ! ! ! ! ! ! ! ! ! ! Medical Genetics Sequence variant: a base pair change that does not change the amino acid sequence (a type of polymorphism)! Synonymous substitution: is silent. Changed base pair doesn’t alter protein the triplet codes for.! Missense: base change causes code different amino acid, (can be harmful mutation or neutral polymorphism)! Nonsense: stop codon, premature termination! Conservative: new amino acid is in the same group, no effect.! Non-conservative: altered activity, function, or stability.! Deletion/Insertion: altered reading frame (when isn’t a multiple of three), premature termination of protein, altered amino acid sequence, loss of function, activity, stability ! Splice-site mutation: a change that results in altered RNA sequence.! Others: mutations in regulatory regions of the gene, large deletions or insertions, chromosomal translocations or inversions, submicroscopic ! ! Analysis Techniques in Medical Genetics! • Restriction Endonuclease/Enzyme: DNA is cut at sequences of nucleotides called restriction sites and then they are separated based on size/charge by gel electrophoresis. In mutations cuts occur at different places so is used as diagnostic tool. ! • DNA Cloning: Is the selective amplification of a specific DNA fragment or sequence. DNA fragment is cleaved by endonuclease, transferred to the corresponding base pair alignment of suitable vector (bacterial plasmid). Recombinant vector is inserted into a specially modified bacterial host cell (where it has the ability to be replicated) and cultured on an agar plate in various conditions and the survival of the host indicates the nature/ functionality of the target DNA. Vector used is size dependant.! • Polymerase Chain Reaction: A technique which amplifies a single or a few pieces of copies of DNA across several orders of magnitude. Is copied in multiple cycles of denaturation and re synthesis of the DNA strand using DNA polymerase. Amplification results in sufficient quantities of target DNA for direct visualisation by ultraviolet fluorescence staining (no probe needed).! • Southern Blotting: Restriction enzyme cleaves DNA, separated by gel electrophoresis and denatured with alkali making it single stranded. Transferred to blotting paper and is labelled with a DNA probe that can be detected by use of radiation.! ! Mutation Detection Techniques! • Direct Sequencing: DNA denatured into single strands, primer binds to one of the strands, DNA polymerase synthesises complimentary DNA strand, stopped by the incorporation of a dye-labeled terminator nucleotide, which identifies the base at the position where strand extension stopped, when many strand termination reactions are performed together, each of the bases in a DNA strand can be identified.! • Single Strand Conformational Polymorphism (SSCP): DNA single strands fold to make 3D conformations, which under gel conditions result in different electrophoretic mobility.! • Conformational Sensitive Gel Electrophoresis (CSGE): DNA is denatured, reannealed with probe and ran through gel. ! • Denaturing Gradient Gel Electrophoresis (DGGE): probe hybridises with denatured target DNA strand, if mutation is present will separate out during electrophoresis. ! • Protein Truncation Assay (PTT): cDNA is made from RNA and translated. The protein product is analysed on a gel, good to detect frameshift mutations. ! • Denaturing High-Performance Liquid Chromatography (DHPLC)! • Microarray (Chip) Assay: contains synthetic DNA probes to which complimentary DNA strands bind which are then made visible on the chip when read. Can have; expression arrays, SNP chip arrays, re-sequencing arrays, BAC arrays, tissue arrays.! ! ! Page 7 ! ! ! ! ! ! ! ! ! ! Medical Genetics **Need to know:! • General Info about DNA , replication etc.! • Mutation, autosomal dominant (x3 forms), autosomal recessive (x2) Germline vs Somatic mutation! • Polymorphism, point mutation, sequence variant, synonymous substitution, missense, nonsense, conservative, non-conservative, deletion, insertion, splice-site mutation.! • Restriction endonuclease, DNA cloning, PCR, Southern blotting. ! • Direct sequencing, SSCP, CDGE, DGGE, PTT, DHPLC, Microarray assay.! ! Single gene disorders: which occur due to alterations in one or both copies of a single gene! ! Autosomal Dominant disease! • Autosomal gene is located in one for the 22 non-sex chromosomes! • It is said to show dominant inheritance if its effects are manifest in the heterozygous state i. an alteration in a single copy of an autosomal dominant gene has a recognisable clinical outcome. ! • In male-to-male transmission: cannot be an X chromosome related and few Y linked (because of Y effects fitness of sperm). Thus has to be autosomal disease. If it survives 2 generations then most likely that it is dominant.! • Each offspring of an affected parent has a 50% chance of being affected ! ! Incidence: the rate at which new cases occur.! Prevalence: the proportion of a population affected at any one time, is usually lower than the incidence.! Congenital: that the condition is present at birth, not all congenital orders are genetic, not all genetic disorders are congenital.! Penetrance: the percentage of gene carriers who manifest a disorder.! If often age dependent.! Expression: the way in which the genetic disorder is manifest. There is variable expression in many autosomal dominant disorders. E. in one breast cancer gene the woman may develop ovarian cancer, two types of breast cancer or only one type of breast cancer. Can also be sex dependant.! ! Familial Hypercholesterolaemia 1/500, common ADD! High cholesterol levels and so develop early coronary artery disease, skin depositions of lipids (xanthomas) are visible.! Cholesterol: 20-30% ingested in the diet. The rest is synthesised and secreted from the liver. Removed from circulation by a low density lipoprotein (LDL) complex (containing cholesterol) binding to an LDL receptor in the hepatocyte (in liver) and degraded.! Mutations in: the synthesis of the LDL receptor in the endoplasmic reticulum, its transport to the Golgi apparatus, abnormality in LDL binding to the receptor, abnormality of the receptor clustering in a coated pit (to carry out endocytosis). Treatment: Diet changes and HMG Coenzyme A reducaste inhibitors (statins), targets cholesterol production in the liver.! ! Type 1 Neurofibromatosis 1/2,000! Characterised by multiple brown skin patches (develop early) then later multiple benign skin tumours (neurofibromas). Carry one normal gene and one mutated gene. The condition is fully penetrant, but has very variable expression. Rare complications include scoliosis (curvature of the spine), and brain and eye tumours. The disorder is caused by mutations in the NF1 gene on chromosome 17.! ! ! Page 8 ! ! ! ! ! ! ! ! ! ! Medical Genetics E. Osteogenesis Imperfecta: inherited brittle bone disease. Short stature, recurrent fractures, blue sclera, deafness, joint laxity. Caused by mutations in the Type I collagen genes that produce collagen proteins and are expressed in bone. Col1A1 (chromosome 17q21) and COl1A2 (chr7q22), form a heterotrimer of 1 Col1A1 and 2 Col1A2 in a “rope– like” triple helical coil of proteins. Missense mutations in Col1A1 destabilise triple protein helix! ! Gain of Function Mutations! A. Increased expression of a protein: Charcot-Marie-Tooth disease type 1a (CMT1A) is a type of inherited neurological disorder that affects the peripheral nerves. Affected individuals experience weakness and wasting of the muscles of the lowers legs and later they experience hand weakness and sensory loss. CMT disease. Is caused by a duplication of the PMP22 gene on chromosome 17. Overexpression leads to abnormal myelin with poor conduction of electrical impulses through peripheral nerves! B. Increased activity of a protein: Achondroplasia. Mutations in the FGFR3 (fibroblast growth factor receptor 3) gene (provides instructions for making a protein that is involved in the development and maintenance of bone and brain tissue). FGFR3 protein is overly active and always switched on, which interferes with skeletal development and leads to the disturbances in bone growth seen with this disorder.! C. New function of gene product: Huntington’s disease. Additional CAG in coding region of gene i. additional Gylcine which aggregates in brain tissue, causing cell damage.! ! A disorder showing autosomal recessive inheritance is manifest only in the homozygous state, affected individual has to altered copy of the gene, but the same altered copy. Parents are at least heterozygous carriers of the same mutant/abnormal allele.! • If both parents have alteration, child 25% chance in being affected with both, 50% chance that child will be carrier, 25% normal.! • The children of a normal person and an affected parent all of the children will be carriers.! • If one parent is affected and the other is a carrier, there is a 50% they will be affected and a 50% chance of being a carrier. Called ‘pseudodominant inheritance’ as it mimics the traits of the autosomal dominant pattern. ! ! Sickle Cell Anaemia! • Glu6Val mutation in both copies of β-globin gene on chromosome 11. Is a mutation of the shape of the β-hemoglobin chains in red blood cells.! • Red blood cells become more fragile, and break up (haemolyse).! • Chronic anaemia can give rise to sickling crises due to blockage of small blood vessels with fragmented red cells.! • Common in West Africa (up to 1 in 4 carrier frequency), Caribbean, and African Americans! ! Thalassemia common in Middle and Far East.! α-Thalassemia: Underproduction of α-globin from α-globin gene cluster on chromosome 16 (common in South East Asia).! β-Thalassemia: Underproduction of b-globin from b-globin gene on chromosome 11 (common in Mediterranean).! ! Cystic Fibrosis 1/2000-3000 in Western Europe, 1/1600 Ireland.! Serious chronic life threatening disorder where the body produces abnormally thick and sticky mucus which builds up in the breathing passages of the lungs and in the pancreas! Lungs: large volumes of infected sputum (mucus from lower airways), chronic infection with damage to bronchi (bronchiectasis) and fibrosis of lung (production of excess fibrous connective tissue). Need antibiotics, physiotherapy, bronchodilators (to open airways) for some, in extreme conditions heart lung transplant.! ! Page 10 ! ! ! ! ! ! ! ! ! ! Medical Genetics Digestive system: blocked pancreatic ducts, failure to absorb food (malabsorption), In infants – blocked bowel due to thickened meconium (meconium ileus/earliest stools produced by a infant)! Late complications: cirrhosis (scarring and poor function) of liver, diabetes mellitus! Infertility in males: bilateral absent vas deferens (sperm duct failed to form properly)! Diagnosis: Parents notice delayed growth, excess levels of sodium and chloride in sweat and genetic testing.! Mutation: Most are homozygous mutations of the Cystic Fibrosis Transmembrane Regulator (CFTR) gene on 7q31 which provides instructions for making channels that regulate the transport of Chloride (Cl-) outward through cell membrane, with influx of sodium (Na+) into the cell.! Most common mutation is c_1523delCTT in exon 10. Protein effect is the deletion of amino acid Phe position 508 (p or DF508).! ! Compound Heterozygote: a person who is affected by an autosomal recessive disorder, with two different mutations, one in each copy of the disease gene e. in cystic fibrosis.! Double heterozygote: someone who isn’t affected but carriers two different mutations for two different autosomal recessive diseases e. in profound childhood deafness.! Locus Heterogeneity: more than one gene which causes the same clinical disorder e. oculocutaneous albinism and severe/profound congenital/prelingual deafness. ! ! Oculocutaneous Albinism 1/50 are carriers.! Is characterised by a lack of pigment in skin, and in the iris and pigmentary layer of the eye (which leads to problems with sight). There are two different genes, the tyrosine hydroxylase gene (chromosome 10) and the P gene (chromosome 15), mutations in either of which can cause oculocutaneous albinism. Genetic background can influence the extent of the disorder.! ! Profound Childhood Deafness 1/1000 infants (about 1/2 is genetic)! Most commonly autosomal recessive. At least 30 genes involved (DFNA genes) the most common being connexin 26 (10-20% of genetic deafness)! • If a deaf couple with AR deafness due to the same gene have children – all children will be also affected! • If a deaf couple with AR deafness due to two different genes have children, all children will be hearing, and double heterozygotes! ! Consanguinity: marriage between relatives. Increases the risk of having a child with an autosomal recessive disorder because of the autosomal recessive gene mutation carried by a common ancestor.! 1/3 of the world’s marriages are consanguine.! ! Heterozygote Advantage: Carrying genetic disorder may confer! small historical advantage over non-carriers which may exaplin carying disease frequency.! Sickle Cell Disease in West Africa: Carriers of sickle cell disease! more resistant to severe forms of malaria (cerebral Malaria) so more likely to survive malarial infection. Advantage only in areas with endemic malaria.! Cystic Fibrosis in Western Europe: Were carriers more resistant to intestinal infection (cholera, dysentery) than non carriers, during times of endemic gastrointestinal illness infections.! ! Caused by a mutation in a single gene on the X chromosome! • A male with the mutation is affected, he does not have a second X chromosome to compensate and is hemizygous. (i. only one copy of the gene is present)! ! Page 11 ! ! ! ! ! ! ! ! ! ! Medical Genetics Causes of female affected by an X-linked recessive disorder (rare)! 1. Carrier mother has a child with an affected father! E. X linked red-green blindness – 8% of males affected. Woman whose father has RG colour blindness marries man with RG colour blindness. Going to get mutation from father (second X chromosome)! 2. Non-random X chromosome inactivation! Just by chance, >90% of relevant cells (e. liver cells in Haemophilia) have non-random Xinactivation. If the active X chromosome has a mutated gene on it, the woman will manifest the X linked disorder! 3. Turner’s syndrome! If her only X chromosome has a mutation it will be active! 4. X – autosome translocation! If X2 inactivated, then autosome gene expression is not impaired. Therefore in an Xautosome translocation, the translocated X will always be active, and the normal X inactive. However, the woman can be affected with X-linked recessive disorder due to breakage of gene D on the X1 chromosome, or because she carries a mutation in a gene elsewhere on the active X1 chromosome! ! X-Linked Dominant Inheritance! X linked dominant disorder which affects both males and females but females may be less affected! e. X-Linked Hypophosphataemic Rickets! Short stature, bowed bones. Deficiency of enzyme which converts vitamin D into active 1,25 Vitamin D. Most Rickets is due to dietary or skin deficiency of Vitamin D however X-linked patients don’t respond to standard Vitamin D supplement treatment. Males generally more severely affected. Treat effectively with active 1,25 form of Vitamin D! ! X linked dominant which affects females only, mutation is lethal in hemizygous male pregnancies.! e. Incontinentia Pigmenti! Penetrant in females with variable expression due to random x! Inactivation. Neonatal patchy blisters on skin at birth, Heal to pink scarred areas. Some have eye abnormalities, hypoplastic (incomplete formation) teeth, seizures and developmental delay.! Mutation in NEMO gene on Xq28! ! ! FISH – Fluorescent in Situ Hybridisation! FISH: allows chromosomes to be studied in more detail, so that a segment of DNA (a probe) which corresponds to a specific chromosome region can be hybridised to a specific chromosome area, to determine if a chromosome deletion of duplication is present. Flourescent antibodiesrecognise the DNA probe with then stain the portion of chromosomeswhich can then be examined through a special microscope.! ! Fibre Fish: interphase chromosomes are attached to a slide in such a! way that they are stretched out in a straight line, rather than being tightly! coiled, as in conventional FISH, or adopting a random conformation.! Spread out chromosome: disadvantage tends to stretch the internal part! of chromosome less at telomeres! ! ! Page 13 ! ! ! ! ! ! ! ! ! ! Medical Genetics Interphase FISH: is a ‘direct’ approach where single locus probes (SLPs)! are use to probe cell nuclei to assess the gross numerical and structural ! characteristics of a tumor cell population. Can be used to see how many copies of each ! chromosome are in a nucleus (identify trisomy etc.)! ! Chromosome Painting: allows the visualization of individual chromosomes in metaphase or interphase cells. Computer-generated "false colour", small variations in fluorescence wavelength among probes are enhanced as distinct primary colours. The combination of probes that hybridize to a particular chromosome produces a unique pattern for each chromosome. This makes it particularly easy to detect segmental deletions and translocations among chromosomes (limited) and identifying the origin of small supernumerary markers or rings.! ! M FISH: permit the simultaneous visualization of all human chromosomes in different colors, considerably facilitating karyotype analysis. If there is a rearrangement of chromosome material then a single chromosome would have different colours.! ! Colour Bar Codes: used to produce a highly defined colored banding pattern of chromosomes in a multi-color format. Allows for sub-regional definition of the chromosomes.! ! Multicolour high resolution banding mBand: allows the differentiation of chromosome region specific areas at the band level. unique colours for bands so rearrangement can be ! easily seen.! ! Bivariate flow karyotype: analysis is performed using data from chromosomes stained with two fluorescent dyes and measured in a flow cytometer/cell sorter. Histogram can be used to identify chromosome and measure the purity of that chromosome.! ! Laser Micro dissection and Pressure catapulting: allows the fast isolation of single chromosomes for the generation of chromosomespecific paint probes. Burn out material around chromosome, box off chromosome, use air to lift chromosome, analyse.! ! Metaphase Comparative Genomic Hybridization (CGH) ! The test DNA is labeled with a fluorescent dye of a specific color, while DNA from a normal control sample is labeled with a dye of a different color and are then mixed together and applied to a microarray. The DNAs have been denatured, they are single strands; thus, when applied to the slide, they attempt to hybridize with the arrayed singlestrand probes. Next, digital imaging systems are used to capture and quantify the relative fluorescence intensities of the labeled DNA probes that have hybridized to each target. The fluorescence ratio of the test and reference hybridization signals is determined at different positions along the genome, and it provides information on the relative copy number of sequences in the test genome as compared to the normal genome. Doesn’t detect balanced rearrangements. If no loss or gain then cannot detect the rearrangement.! ! Genomic Microarrays! 1) Clone! cDNA – use test and reference DNA, compare. Good for parallel copy number and expression analysis. Not good for gene desert regions and regulatory sequences not represented (introns, promoters etc.)! 1MB BAC Array - Bacterial Artificial Chromosome! 32K, BAC Array - ! Repeat free Arrays - ! ! Page 14 ! ! ! ! ! ! ! ! ! ! Medical Genetics Epigenetic Marks: chemical additions to the genetic sequence. Regulate open/closed state of genomic regions. The addition of methyl groups to the DNA backbone is used on some genes to distinguish the gene copy inherited from the father and that inherited from the mother (imprinting). The marks both distinguish the gene copies and tell the cell which copy to use to make proteins.! 1. RNA:! • Small interfering (si) RNAs: Involved in establishing ‘closed’ configuration at certain sites. Act on DNA repeats at centromeres & elsewhere in genome.! • Non-Coding (nc) RNAs: involved in establishing ‘open’ configuration in specific genomic regions others function in establishing the ‘closed’ configuration specific regions or even over a whole chromosome.! 2. The Nucleosome! Consists of a central core of 8 histone proteins (two molecules of each of histones H2A, H2B, H3 + H4) around which about 147 base pairs of DNA are coiled. Histone – H1 – helps bind further into 30nm fibre. Although histones do not interact with polymerase enzymes directly, their modification can affect the way DNA wraps around them and thereby influence which genes are expressed. Histone modifications are necessary for recruiting cofactors and for polymerase binding, and for maintaining chromatin stability. Important histone modifications include acetylation (at lysine residues) and methylation (at lysine and arginine residues). Each type of histone protein has its own repertoire of variants that differ in their amino acid sequence mostly in the N-terminal region. They mark a specific region of the DNA by replacing canonical histone from the nucleosomes present at that site. This highlighting of special regions in the genome has a significant role in recruitment of different factors to that site resulting in differential treatment.! 3. DNA Methylation! Is involved in; transcriptional gene silencing, chromatin compaction (contraction of DNA strand alters the shape of the strand and in doing so makes some genes inaccessible), genome stability, suppression of homologous recombination between repeats, genome defence (response to a stress), X chromosome inactivation (females).! ! Many imprinted genes are only expressed in the brain i. for cognitive functions.! Paternally expressed genes favor cellular proliferation/increase the growth of placenta and rate of transfer of nutrients from mother to embryo through placenta. Maternally expressed genes favor opposite.! ! The X chromosome - 1021 genes mapped - ‘gene rich’! Important growth & development genes, Major muscle protein (dystrophin), Blood clotting protein genes, Genes involved in intelligence development! When faulty, specific genetic conditions may result! • Haemophilia! • Duchenne & Becker muscular dystrophy! • Fragile X syndrome ! ! X inactivation – ensures that males and females have the same number of active Xchromosome genes. In each Female (somatic) cell one X chromosome shortens & condenses, most of its genes are not ‘readable’.! Genes on the Pseudo-Autosomal regions (homologous to a region of the X chromosome and which is responsible for sex chromosome pairing). Other genes in females are expressed on both X-chromosome and this increased expression of gene products may be a differentiating factor between the sexes.! With skewed inactivation more cells have the faulty X-chromosome active = show symptoms of the disorder.! ! Page 16 ! ! ! ! ! ! ! ! ! ! Medical Genetics Cells with structural changes to the X-chromosome are unlikely to survive as necessary genes aren’t being expressed.! ! Biparental inheritance: one copy of a chromosome inherited from the father, the other from the mother! Uniparental inheritance: Individual inherits both copies of a chromosome from one parent only.! Where the chromosomes inherited in Uniparental disomy (UPD) are imprinted there are implications for that individual.! ! E. UPD for chr 15! • If both Mother (maternal UPD) —> Prader-Willi syndrome! • If both Father (paternal UPD) —> Angelman syndrome! E. UPD for chr 11! • If both Father (paternal UPD) —> Beckwith-Wiedemann syndrome (an overgrowth! condition)! • If both Mother (maternal UPD) —> Russell-Silver syndrome (featuring growth delay)! ! Imprinted genes tend to cluster in large regions. Chromosome 11p15 & 15q11-13 carry extensive clusters controlled by one cis-acting imprinting center (IC), which coordinates imprinted expression of all genes in cluster.! 15q11-q13! ~2 Mb segment, IC located @ 5’-end of SNRPN gene! IC deletions ! • Prader-Willi syndrome (PWS): Paternally expressed genes aberrantly silenced ! • Angelman syndrome (AS) - Maternally-silent genes aberrantly reactivated! ! Epigenetics and Disease! • Some Imprinting disorders; Prader-Willi syndrome (PWS) / Angelman syndrome (AS), Beckwith-Wiedemann Syndrome (BWS), Albright hereditary osteodystrophy (AHO), Transient neonatal diabetes mellitus (TNDM), Russell-Silver syndrome! • Cancer! • Parent of Origin effects; Coronary Heart Disease, Type II Diabetes, Obesity, Embryo transfer technologies! • Perhaps; Transexualism (male), memory formation, Schizophrenia, bipolar disorder, Alzheimer disease, autism-spectrum disorders.! ! Angelman-Syndrome (AS)! Result from the loss of function of the UBE3A gene on chromosome 15. People normally inherit one copy of the UBE3A gene from each parent. In certain areas of the brain, however, only the copy inherited from a person's mother (the maternal copy) is active. If the maternal copy of the UBE3A gene is lost because of a chromosomal change or a gene mutation, a person will have no active copies of the gene in some parts of the brain.! Primarily affects the nervous system. Have delayed development, intellectual disability, severe speech impairment, and problems with movement and balance (ataxia) and a happy affect. Some also have recurrent seizures (epilepsy) and a small head size (microcephaly).! ! Prader-Willi Syndrome (PWS)! Paternal copy of certain genes on chromosome 15 that is imprinted or silenced, while the maternal copy is expressed in PWS. Babies have developmental delay, trouble eating/ gaining weight, hypogonadism, are floppy, short stature, almond shaped eyes, and narrow bifrontal skull. Later they develop a craving for food, which can lead to overeating and obesity. ! ! Page 17 ! ! ! ! ! ! ! ! ! ! Medical Genetics • Archival samples - paraffin, formalin! ! Molecular genetics tests are tailored to the underlying mutation type! Sensitivity: the probability that the test will be positive if the patient actually has the disease.! Specificity: the probability that the patient actually has (or will develop) the disease if the test is positive! ! To analyse the disease! Target Detection: when you know what you are looking for, specific array to pick up a particular mutation.! Scanning Detection: analysis of the whole gene for a disorder to determine whether there is a mutation present that could be causing the disease. When not sure where the mutation is and can be used when target detection finds no mutation.! -> Most analyses start with a PCR step.! ! Polymerase Chain Reaction (PCR):! A technique which amplifies a single or a few pieces of copies of DNA across several orders of magnitude. Is copied in multiple cycles of denaturation and re synthesis of the DNA strand using DNA polymerase. Amplification results in sufficient quantities of target DNA for direct visualisation by ultraviolet fluorescence staining (no probe needed). ! Applications:! • Used in target detection for Duchene Muscular Dystrophy deletions Amplify exons along the dystrophin gene. By comparison with normal sample missing exons can be detected.! • Used in fragment sizing for deletion detection e. with cystic fibrosis.! • Used in fragment sizing for repeat number e. Triple Repeat Expansion Disorders; ! ! Fragile X Syndrome: repeat of CCG (loss of function) when the repeats get greater than 170 the gene is methylated and there is no protein expressed. When repeats get to large PCR doesn’t work, primers can copy the large region. Southern Blotting is then employed. Friedreich Ataxia: GAA repeat (loss of function), affects nervous system. Fragile X Ataxia tremor syndrome: is a late onset neurodegenerative disorder associated with problems with movement, memory, and the autonomic nervous system. Involves repeat in fragile X mental retardation 1 gene.! ! Fluorescent-energy transfer (FRET) Technologies: Reactions can be monitored quantitatively in real time which helps in the diagnosis of numerical aberrations, deletions, duplications, unbalanced translocations in carrier states of autosomal and X-linked diseases, and amplifications and loss-of-heterozygosity in cancer. Unlike other quantitative PCR methods, real-time PCR monitoring obviates post-PCR sample handling. This prevents potential contamination from product carry-over and allows fast, high-throughput assays. The real-time PCR method has a very large dynamic range of initial target molecule quantity.! ! DNA Sequencing ! Sanger dideoxy method! PCR is the first step: Amplification of exon fragments! PCR-like reaction, but dNTPs are mixed with fluorescent ddNTPs, which block further extension. Entire sequence can then be read.! Multiplex Ligation Probe Amplification: widely used dosage analysis method. Used for analysis of disorders where a deletion or duplication is the underlying mechanism! !• • ! Genomics - study of the structure and organisation of the genome! Genetics - study of properties of single genes or groups of genes ! Page 19
Medical Genetics 1 2 - Lecture notes ALL
Module: Medical Genetics (PATH10200)
University: University College Dublin
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