3"Genetics" Fields: Heredity and its variation. Subfields: - "Human Genetics”:denotes the science of heredity andits variationin human.- ”Medical Genetics”:deals with human genetic variationsof medical relevance / significance .
4Molecular and biochemical Medical GeneticssubgroupsClinical Genetics-concerned withClinical manifestationOf genetic diseasesImmunogenetics -the study of thegenetics of theimmunesystemMolecular and biochemicalgenetics -the study of thestructure and functionof individual genes.Cytogenetics -the study of thestructure ofchromosomes.Population genetics -the study of geneticsof populations..Genetic epidemiology -the study ofepidemiology ofgenetic disease.
6Historical * Engravings (around 6,000 years) -Showed pedigree documenting the transmission of certaincharacteristics of some animals.*Aristotle and Hippocrates-Human characteristics determined by the semen(utilising the menstrual blood as a culture medium anduterus as an incubator).-Semen was thought to be produced by the whole body andhence it was explained that 'baldheaded fathers’ had'baldheaded' sons.* 17th century-‘Sperm' and 'ovum' were recognised byDutch scientists and it was explained that female could alsotransmit characteristics to her offspring.(Contd)
7Historical (Conti.) * 18th and 19th centuries * 19th century -There was a revival of interest in heredity andit was shown that several traits such as extradigits (polydactyly) were inherited in different ways.* 19th century-Joseph Adams published "A treatise on the Supposed Hereditary Properties" and indicateddifferent mechanisms of inheritance.-This book was intended as a basis for genetic counselling.
8* In 1865, Gregor Mendel Historical (Conti.) - An Austrian Monk, published his resultsof breeding experiments on Garden Peas.- His work can be considered as the discoveryof `genes' (traits) and how they are inherited.- He put forward patterns of inheritance of variouscharacteristics and single gene disorders.-These are known as ‘Mendels Laws of Inheritance.
9Historical (Conti.) * Mendel showed that some characteristics were: -"dominant" (e.g.tall height),- others were "recessive“ (e.g. short height).-each characteristic was controlled by a pair of "factors".* In 1909, a Danish botanist, Johannsen, namedthe hereditary factors as ‘genes’.- two identical genes was referred to as `homozygous',- two different genes for the same characteristic,were called `heterozygous'.
10Historical (Conti.)Multiple forms of the same gene that occupy the same lociand give rise to different forms of the same characteristicsare referred to as allelomorphs"or allelesAllelesHomozygous Heterozygous
11rediscovered by three workers: Historical (Conti.)* The 20th century ( development of genetics):- Mendels Laws were independentlyrediscovered by three workers:- Hugo De Vries ( in Holland)- Carl Correns (in Germany) and- Erich Von Tschermakin (in Austria).
12Historical (Conti.) * In 1902 : - Archibald Garrod and William Bateson (fathers of Medical Genetics), discovered `Alkaptonuria'and recognized it as an inherited disorder involving chemical processes.- Garrod called it an "Inborn Errors of Metabolism”- Todate several thousand of such disorders have been identified.
13Historical (Conti.) * In 1903 : - Sutton and Boveri proposed that ‘chromosomes’ carry the hereditary factors.Chromosomes( Chroma=color; soma=body)were recognised as thread like structures,(so called because of their affinity for certain stains).* In 1906 :- Bateson contributed the term "Genetics"for this new science.* In 1941:- Beadle and Tatum formulated the"one gene - one protein" theory.* In 1956 :- The correct number of chromosomeswas established as 46.
14Historical (Conti.) * By late 1950's : - Excellent techniques for the studyof chromosomes were developed.* In 1953:- James Watson and Francis Crick ( in Britain)described the structure of the geneticmaterial i.e. DNA, and were awarded Nobelprize in 1962.* Mid 1970's :-The field of Medical Geneticshas been transformed and significant newdiscoveries about the genes, their expressionand genetic diseases have been made.
15Historical (Conti.) * The 'Human Genome Project‘: - An International project, to map the entirehuman genome, was initiated in 1990 tobe completed by the year 2005( however,it was completed in 2003).* To-date:- extensive information has beengained about chromosomes, gene mapping,gene sequencing, functions and genetic disorders.
16The genetic knowledge is increasing exponentially and has extensive applications in clinical medicine
17* During the last three decades: - a decrease in frequency of infectious diseases.- improved nutrition, antibiotics and immunization.- almost one third of the patients in paediatricsuffer from genetic defects.
18It has become essential for all medical personnel's to have a clear knowledgeof human and medical genetics.
19Mendels Laws of Inheritance Three Laws of Inheritance:The Law of Unit Inheritance.The Law of Segregation.iii) The Law of Independent Assortment.
20The Law of Unit Inheritance The characteristics (traits i.e. genes) do not blend( mix), but are inherited as units, which might notbe expressed in the first generation off-springs,but may appear unaltered in later generations.First Generation Second GenerationTT t t Tt TtTt Tt Tt Tt TT Tt Tt ttAll tall in the first generation 75% Tall and 25% short in 2nd(As t is recessive & does not appear) generation.( T= Tall, dominant gene; t = Short, recessive gene)
21The Law of Segregation - The two members of a single trait (gene) i.e. alleles, are never found in the same gamete,but always segregate and pass todifferent gametesGameteZygote- The failure of two allelesto segregate due to chromosome Gametenon-disjunction give rise to geneticdefects(e.g. in Down’ssyndrome)
22The Law of Independent Assortment * Members of different gene pairs assort to the gametesindependently of one anotheri.e. random recombination of maternal and paternal chromosomes occur in gametes.Maternal Paternal Crossing-over Gametes
23The exceptions to Law of Independent Assortment (not recognised by Mendel) are closely "linked“genes on the same chromosome, which do notassort independently.Maternal Paternal Crossing-over Gametes
25What is the Genetic Material? Proteins ?RNA?DNA?
26Griffith’s Experiment In live animal( Smooth &Virulent)- due to polysaccharide capsule(Non-Virulent)(Non-Virulent)Due to absenceof polysaccharidecapsuleTransformationOf rough to smooth form(Non-Virulent)(Virulent)(Non-Virulent)What is the Transforming Factor?
27Griffith’s Experiment Conducted in 1928 On a bacteria that produces pneumonia:- R(Rough) strains were non-virulent(did not produce disease)- S(Smooth) strains were virulent (produced disease)- Heated R and S strains were both non-virulent_The experiment:R injected in rats No diseaseHeated R injected in rats No diseaseS injected in rats Disease (Rat Died)Heated S injected in rats No diseaseHeated S + live R injected in rats Disease (Rat Died)Some substance in heated S transformed the R to SWhat was the Transforming Principle?
28In culture Growth of S colonies Experiment of Avery, Macleod and McCarty (1944)In culture(Culture)Smooth coloniesNo coloniesGrowth of S coloniesWhat is the TransformingFactor?
29The Transforming Principle Experiment of Avery, Macleod and McCarty (1944)1. Took extract from virulent(S) cells + R cells S ColoniesAs the bacteria was destroyed, but DNA was not.2. Treated the extract with:(a) Proteases Mixed with R cells S Colonies(b) Ribonuclease----Mixed with R cells S ColoniesDNase Mixed with R cells No Colonies of SConcluded that the transforming principle in the extract was DNA
31These and many other experiments proved that DNA is the carrier of geneticinformationin all living organisms except RNA viruseswhich have RNA as the carrier of genetic
32Genetic Material in the Living Cells * All living organisms are made up of cells.* Cells contain a nucleus surrounded by a nuclear membrane in eukaryotic cells, and a nuclear region in the prokaryotic cells.* Chromatin is made up of DNA and proteins (mainly histones(basic) and non-histone (acidic) proteins.
33Genetic material…contd The study of chromosomes, their structure and their inheritance is known as Cytogenetics.Each species has a characteristic number of chromosomes and this is known as karyotype.Prior to 1950's it was believed that humans had 48 chromosomes but in 1956 it was confirmed that each human cell has 46 chromosomes (Tjio and Levan, 1956).The genes are situated on the chromosomes in a linear order. Each gene has a precise position or locus.
35Chromosomes* One member of each chromosome pair is derived from each parent.* Somatic cells have diploid complement of chromosomes i.e. 46.* Germ cells (Gametes: sperm and ova) have haploid complement i.e 23.* The chromosomes of dividing cells are most readily analyzed at the `metaphase' or prometaphase stage of mitosis .
36The Normal Human Chromosomes * Normal human cells contain 23 pairs of homologous chromosomes:- 22 pairs of autosomes (numbered as 1-22 in decreasing order of size)- 1 pair of sex chromosomes.* Autosomes are the same in males and females* Sex chromosomes are:- XX in females- XY in males.* Both X are homologous. Y is much smaller than X and has only a few genes.pq
37Chromosome Structure* At the metaphase stage each chromosome consists of two chromatids joined at the centromere or primary constrictionThe centromere divides chromosomes into short (p i.e. petit) and long (q e.g. g=grand) arms. The tip of each chromosome is called telomere.The exact function of the centromere is not clear, but it is known to be responsible for the movement of the chromosomes at cell division.TelomerepCentromereq
38Chromosomes … contdIn a non-dividing cell the nucleus is filled with a thread-like material known as "chromatin".Before cell division, the chromatin multiplies (replicates), loses the relatively homogenous appearances and condenses to form rod like structures ."Genes" ,are units of genetic information present on the DNA.MitosisG2G1GoSThe Cell Cycle
39Each species has a characteristic gene map i. e Each species has a characteristic gene map i.e. the chromosomal location of the genes, and it is the same in all normal individuals of each species
40Classification Of Chromosomes Chromosomes are classified (analysed) accordig to:1. Shape and2. Staining1. Morphologically (shape)According to the position of the centromere as:(i) metacentric,(ii) sub-metacentric,(iii) acrocentric,(iv) telocentric (with centromere at one end.This occurs in other species, but not in man).
42* Acrocentric chromosomes (13, 14, 15, 21 and 22) have a small mass of chromatin knownas satellite attached to their short arm bynarrow stalks (secondary constrict).* The stakes contain genes for 18S and 28S rRNA.SatelliteStalk
43Staining Methods for cytogenetic analysis of chromosomes There are several staining methods for cytogenetic analysis of chromosomes.Each stain produces specific banding patterns known as "Chromosome Banding"G banding,- Q banding,R banding,C banding.The pattern is specific for each chromosome, and is the characteristics utilized to identify each chromosome.
44Staining Methods for Cytogenetic Analysis G Banding:Treat with trypsin and then with Geimsa Stain.R Banding:Heat and then treat with Geimsa Stain.Q Banding:Treat with Quinicrine dye giving rise toFluorescent bands.C Banding:Staining of the Centromere.
47Composition of Nucleosomes DNAHistones2( H2A,H2B,H3,H4)
48The Genetic material-Deoxyribonucleic acid (DNA) -Double strandof polynucleotide.-Coiled around eachother formingdouble helix.Strands are anti-Parallel.Sugar phosphatebackbone is outside& bases are inside.A=T and G=C.A/T=1 andG/C=1(Cargaff Ratio)5’3’3’5’
49Nitrogenous bases in DNA and RNA PyrimidinesPurines
52in cytoplasm on ribosomes Replication-in nucleusTranscription-in nucleusTranslation-in cytoplasm on ribosomes
53DNA Replication Replications occurs before cell division. During S Phase ofcell cycle.Entire DNA contentis doubled.Replication isSemi-conservative.Requires:-DNA polymerases-dNTPs(N=A,T,C,G)-RNA primer-Mg++-DNA ligases- Primase- Helicase- SS DNA binding proteinsDNA Replication
54Major Steps in DNA Replication Lagging strandLeading strand,continuous
57Steps in transcription InitiationBinding of RNA polymerase causes opening of the DNA strand and synthesis of the RNAElongationRNA polymerase continues synthesis of RNA complementary to DNA till termination site
58Steps in transcription (contd)Elongation -contdTerminationRho factor binds to the termination site and when RNA polymerase reaches this site, termination occurs
66Mitochondrial DNAIn the human mitochondria the chromosomes are present as 10 circular double helices of DNA.They are self replicative.Contain: 16,596 bp, genes for 22 tRNAs and 2 types of ribosomal RNA required for mitochondrial protein synthesis.They also have genes for 13 polypeptides, involved in cellular oxidative phosphorylation.Both strands of DNA are transcribed and translated.
67Mitochondrial DNA The genes on mitochondrial DNA have no introns. The codon recognition pattern for several amino acids is different from the nuclear DNA.Mitochondria are transmitted in the egg from a mother to all of her children. Thus mitochondrial DNA is only maternally derived.
69The Cell Cycle The cell cycle consists of 2 phases: Mitosis and Interphase.Mitosis (cell division) is the shortest phase.Interphase The period between successive mitosis.The G1, S and G2 phase constitute interphase.In a typical growing cell this lasts hours and mitosis lasts 1-2 hours.Some cells e.g. neurons and RBCs, do not divide and enter the Go phase. Other cells may enter Go but eventually return to continue through the cell cycle (Contd..)
70The Cell Cycle (Contd..)Immediately after mitosis, the cell enters G1 (Gap 1) phase, where there is no DNA synthesis. Some cells spend a few hours others up to years in this phase. At this phase cells perform metabolic functions.S phase - the phase of DNA synthesis.Each chromosome in G1 phase double, and forms two chromatids joined together. By the end of S phase the DNA content of cells is doubled.
71The Cell Cycle (Contd..)G2 phase - The chromatin condenses and forms chromosomes. Each chromosome consists of two identical sister chromatids. During this period the DNA synthesis is restricted, RNA and protein synthesis occur and cell enlarges, eventually doubling its total mass before next mitosis.
73Cell Division Mitosis: Meiosis: - Occurs in Somatic cells. Cell DivisionMitosis:Meiosis:- Occurs in Somatic cells.- Division by which the bodygrows, differentiates and repairs.- Results in two identical daughterDiploid cells with genes identicalto parent cells.- Chromosomes are first doubled,followed by cell division in whichthe number in each cell is halved(diploid).- Occur in cells of germ line.- Only once in generation.- Results in the formation ofhaploid, reproductive cell(gametes: ova and sperms).- Chromosomes duplicatesfollowed by 2 cell divisionsresulting in cells withhalf the number ofchromosomes (haploid).
74MitosisAt conception the human zygote consists of a single cell. This undergoes rapid cell division leading ultimately to the mature human adult body. Each adult human being has approximately 1x1014 cells in the body.In most organs and tissues e.g. bone marrow, skin etc. cells continue to divide throughout life.This process of somatic cell division during which the nucleus divides to produce two identical daughter cells is known as Mitosis.
75Mitosis (contd..)* Each chromosomes divides into two daughter chromatids, one of which segregates into each daughter cells.- The number of chromosomes per cell remains unchanged.Mitosis lasts 1-2 hours.It occurs in five distinct stages :Prophase, prometaphase, metaphase, anaphase and telophase.
76Phases of Mitosis:Prophase: The chromosome condenses and mitotic spindle begins to form. Two centrioles form in each cell from which microtubules radiate as the centrioles move towards opposite poles of the cell.Prometaphase: The nuclear membrane begins to disintegrate and chromosome spread around the cells. Each chromosome becomes attached at its centromere to a microtubule of the mitotic spindle by a specialised structure called Kinetochores.
77Phases of Mitosis (Contd..): Metaphase: The Chromosomes are maximally contracted and most easily visible. The Chromosomes become oriented along the equatorial plain and each chromosome is attached to the centriole by a microtubule forming the mature spindle.Anaphase: The centromere of each chromosome divides longitudinally and the two daughter chromatids separate to opposite poles of the cell.
78Phases of Mitosis (Contd..): Telophase: The chromatids separate completelyand a new nuclear membrane is formed aroundeach set of chromosomes. The cytoplasmseparates (cytokinesis) to form two daughtercells.
80Meoisis:The type of cell division by which the diploid cells of the germline give rise to haploid gamets, i.e. oocytes and sperms.The process involves two successive meiotic divisions:Meiosis I: This is the reduction division and the chromosome number is reduced from diploid to haploid.Meiosis II - follows Meiosis I without an intervening stage of DNA replication. The chromosomes disjoin, and one chromatid of each chromosome passes to each daughter cell.
81Meoisis:Meiosis I: This stage has: Prophase I, Prometaphase I, Metaphase I, Anaphase I & Telophase I, just like mitosis.Meiosis II: has:Metaphase II and telophase II and results in formation of ova in female and sperms in males.
83 Genetic Consequence of Meiosis - Reduction of chromosome number from diploid to haploid, the essential step in the formation of gametes.- Segregation of alleles, at either meiosis I or meiosis II, in accordance with Mendel’s First Law.- Shuffling of the genetic material by random assortment.- Additional shuffling of genetic material by crossing-over mechanism substantially increasing genetic variation.
84Gametogenesis: Oogenesis: There are differences in female and males gametogenesisOogenesis:Mature ova develops from oogonia by a complex series of intermediate steps:During the first few months of embryonic life :Oogonia originate from primodial germ cellsby a process involving mitotic divisions.At completion of embryogenesis at 3 months of intra-uterine life:The oogonia mature to primary oocytes which start to undergo meiosis.
85Gametogenesis:At birth, all primary oocytes have entered dictyotene, a phase of maturation arrest at which they remain resuspended until meiosis is completed at the time of ovulation .At the time of ovulation, a single secondary oocyte is formed. Most of the cytoplasm is received by the daughter cell from the 1st meiotic division consists largely of a nucleus known as a polar body.Meiosis II then commences during which fertilization can occur. A second polar body is formed.
86Gametogenesis: 2. Spermatogenesis: Rapid process - average duration of days.At puberty, spermatogonia (which have already undergone 30 mitotic divisions) begin to mature into primary spermatocytes.These enter meiosis 1 and emerge as haploid secondary spermatocytes.These undergo second meiotic division to form spermatids, which change to mature spermatozoa
87Genetic Disorders Genetic Disease Mutations in the: * Genome, * Chromosome or* GeneDecrease or increase in the amount of genetic materialAbnormal genetic maerialIncrease or decrease in the amount of gene products (proteins).Decrease in the amount of one protein.Defective function of the protein.- Increased function.- Decreased or complete loss of function.Genetic Disease
89Classification of Genetic Diseases Single Gene Disorders Acquired SomaticGenetic DiseasesChromosomalDisordersMultifactorialDisordersMitochondrialDisorders
90Single Gene Disorders Caused by mutation in or around a gene. Can lead to critical errors in the genetic information.Exhibit characteristic pedigree pattern ofinheritance (Mendelian Inheritance)Occur at a variable frequency in differentpopulationOver 7,000 single gene disorders have beenidentified.May be: - Autosomal- Sex linked
91Chromosomal Disorders Result from defect in the number (i.e. Numericaldisorders) or structure (i.e. Structural disorders)of chromosomes.The first chromosomal disorder was Trisomy 21(Downs syndrome) and was recognised in 1959.These disorders are quite common and affect about1/800 liveborn infants.Account for almost half of all spontaneousfirst-trimester abortions.Do not follow a Pedigree pattern of inheritance.
92Multifactorial Disorders Result from interaction between environmentaland genetic factors.Often polygenic in nature, no single error in thegenetic information.Environmental factors play a significant rolein precipitating the disorder in geneticallysusceptible individuals.Tend to cluster in families.Do not show characteristic pedigree patternof inheritance.
93Multifactorial Disorders CongenitalmalformationsCommon disordersof adult life.
94Mitochondrial Disorders * The defective gene is present on themitochondrial chromosomes.* Effect generally energy metabolism.* Effect those tissues more which requireconstant supply of energy e.g muscles.* Shows maternal inheritance:-effected mothers transmit the disorderequally to all their children.-affected fathers do not transmit thedisease to their children.
95Acquired Somatic Genetic Diseases Recent advances in Molecular Biologytechniques have shown that mutations occuron a regular basis throughout the life of thesomatic cell.These somatic mutations account for1. A large proportion of malignancy and2. possibly involved in events such as'senescence' and the 'ageing process'.
96Single Gene Disorders May be: - Autosomal - Sex linked: Y- linked , holanderic, hemizygoteX- linked , dominant or recessive
97Modes of Inheritance of Single gene Disorders AutosomalSex LinkedRecessiveDominantY LinkedX LinkedAbnormalhomozygousRecessiveDominantNormalhomozygousHeterozygousNormalAbnormal
98Autosomal Inheritance - This is the inheritance of the gene present on the Autosomes.- Both sexes have equal chance of inheriting the disorder.- Two types:Autosomal dominant inheritance, if the gene is dominant.Autosomal recessive inheritance, if the gene is recessive.AbnormalhomozygousNormalhomozygousHeterozygous
99Autosomal Dominant Inheritance - Autosomal dominant inheritance, if the gene is dominant.- The trait (characteristic, disease) appears in every generation.- The trait is transmitted by an affected person to half the children.- Unaffected persons do not transmit the trait to their children.- The occurrence and transmission of the trait is not affected by sex.Normal maleNormal femaleDisease maleDisease female
101Acute Intermittent Porphyria - AD.- Expressed in heterozygotes and homozygotes.- Uroporphyrinogen synthetase deficiency.- Increased urinary excretion of 5-amino levulinic acid and porphobilinogen (diagnostic ) .- Characterized by neurological symptoms that include severe abdominal pain, peripheral neuropathy and psychosis.
102Punnet Square Mother Father Mother Father Affected Normal D d dD dd
103Autosomal Recessive Inheritance - The trait (characteristic, disease) is recessive.- The trait expresses itself only in homozygous state.- Unaffected persons (heterozygotes) may have affected childrens (if the other parent is heterozygote) .- The parents of the affected child maybe consanguineous.- Males and female are equally affected.
104Punnett square showing autosomal recessive inheritance: (1) Both Parents Heterozygous:25% offspring affected Homozygous”Female % Trait “Heterozygous normal but carrier”25% NormalContd.AaAAAaaa
105(2) One Parent Heterozygous: MaleFemale % Off springs normal but carrier “Heterozygous”50% Normal_________________________________________________________________________ (3) If one Parent Homozygous:100% of springs carriers.FemaleAaAAAaAaAa
106Family tree of an Autosomal recessive disorder Sickle cell disease (SS)A family with sickle cell disease -PhenotypeHb ElectrophoresisAA AS SS
108Cystic fibrosis- Most frequent autosomal recessive (AR) disorder (1 in 200births in Caucasians)- Expressed only in homozygotes.- Heterozygote carriers are normal phenotypically- If both parents are heterozygote to abnormal gene than there is 1 in 4 (25%) chance of having child with cystic fibrosis (homozygous).- If one parent has cystic fibrosis (homo) while the other parent is normal, then all childrens will be carriers of the abnormal gene.
109Sex – Linked Inheritance - This is the inheritance of a gene present on the sexchromosomes.- The Inheritance Pattern is different from the autosomal inheritance.- Inheritance is different in the males and females.RecessiveX-LinkedSex – linkedinheritanceDominantY- Linked
110Y – Linked Inheritance - The gene is on the Y chromosomes. - Shows Holandric inheritance. i.e.The gene is passed from fathers to sons only.- Daughters are not affected.e.g. Hairy ears in India.- Male are Hemizygous, the condition exhibits itself whether dominant or recessive.maleFemale-XY*XXXY*
111X – Linked Inheritance - The gene is present on the X - chromosome. - The inheritance follows specific pattern.- Males have one X chromosome, and are hemizygous.- Females have 2 X chromosomes, they may be homozygous or heterozygous.- These disorders may be : recessive or dominant.
112X – Linked Recessive Inheritance - The incidence of the X-linked disease is higher in male than in female.- The trait is passed from an affected man through all his daughters to half their sons.- The trait is never transmitted directly from father to sons.- An affected women has affected sons and carrier daughters.(1) Normal female, affected maleOvaAll daughters carriers “not affected, All sons are normalXX*X*XYXY
113(2) Carrier female, normal male: Ova50% sons affected,50% daughters carriers,Sperm(3) Homozygous female, normal male:- All daughters carriers.- All sons affected.X*XXX*XXYX * YXY
115Lesch – Nyhan Syndrome - X – linked recessive disease. - Due to deficiency of hypoxanthine guanine phosphoriboyl transferase - Purine salvage pathway is impaired.- Symptoms include:- Self mutilation tendency.- Mental retardation.- Cerebral palsy.- Uric aciduria.- Gout and kidney stones.
116The Hemophilias - X – linked recessive disease. - Expressed in males, very rare in females (homozygotes) [ 1 in 10,000 male births ].- In this abnormality, the blood fails to clot due to abnormality of antihemophilic globulin.- Clinical features include severe arthritis.
117X-Linked Dominant Disorders The gene is on X Chromosome and is dominant.The trait occurs at the same frequency in both malesand females.Hemizygous male and heterozygous females expressthe disease.
118** Punnett square showing X – linked dominant type of Inheritance: (1) Affected male and normal female:OVAAll daughters affected, all sons normal.Sperm(2) Affected female (heterozygous) and normal male:50% sons and 50% daughters are affected % of either sex normal.Contd.XX*X*XYXYX*XXX*XXYX*YXY
119(3) Affected female (homozygous) and normal male: OVAAll children affected..SpermX*XX*XXX*YX*Y
120Chromosomal disorders - These defects result from defects in the chromosomes.- Two groups:* Structural defects– defects in structure of chromosome.* Numerical defects– Increase or decrease in number of chromosomes- These defects are quite common (7 in 1000 live births).- Chromosomal defects do not obey specific pattern of inheritance.- These defects account for over half of all spontaneous abortions in first trimester.
121Increase or decrease in the Change in the structure Chromosomal DisordersNumericalStructuralIncrease or decrease in thenumber of chromosomesChange in the structureof chromosomesEuploidyAneuploidy
122Aneuploidy Euploidy Increase in the total Increase or decrease in set of chromosomese.g 3N or 4NIncrease or decrease inone or more chromosomes.e.g 2N+1, 2N-1-Triploidy (69 chromosomes)found in cases ofspontaneous abortions-Trisomy (46+1) chromosomes(Down Syndrome)-Monosomy (46-1) chromosomes(Turner Syndrome)
130The Philadelphia Chromosome* * Mutation found in all cases of chronic myeloid leukemia* The ABL & BCR fuse due to translocation and form an oncogene
131Mitochondrial Disorders * Effect generally energy metabolism.* Effect more those tissues which requireconstant supply of energy e.g muscles.* Shows maternal inheritance:-affected mothers transmit the disorderequally to all their children.-affected fathers do not transmit thedisease to their children.
133Mitochondrial Inheritance Affected females transmit the disease to all their children.Affected males have normal children.Males cannot transmit the disease as the cytoplasm is inheritedonly from the mother, and mitochondria are present in thecytoplasm.
134Multifactorial Disorders Result from interaction between environmentaland genetic factors.Often polygenic in nature, no single error in thegenetic information.Environmental factors play a significant rolein precipitating the disorder in geneticallysusceptible individuals.Tend to cluster in families.Do not show characteristic pedigree patternof inheritance.
135Multifactorial Disorders CongenitalmalformationsCommon disordersof adult life.
136Acquired Somatic Genetic Diseases Recent advances in Molecular Biologytechniques have shown that mutations occuron a regular basis throughout the life of thesomatic cell.These somatic mutations account for a largeproportion of malignancy and are possiblyalso involved in events such as 'senescence'and the 'ageing process'.
146- The alleles present at one locus. e.g.. Genotype- The genetic constitution (genes on the pair of homologous chromosomes).- The alleles present at one locus. e.g..(a) TT or Tt or tt i.e genes for height.Where T is the “tall” gene and t is the gene for “short” height(b) A A, A S, or S SWhere A is for HbA and S for HbS.
147PhenotypeThe observed biochemical, physiological and morphological characteristics of an individual as determined by his/her genotype and the environment in which it is expressed. e.g.Genotype PhenotypeTT or Tt Talltt ShortAA HbA (normal)Hetero A S HbASSS HbS (SCA)( Homo = Identical , Hetero = different)Dominant* HeteroRecessive
148Genotype – Phenotype relationship Genotype (i.e. genetic make up) determines phenotype (i.e. appearance etc.), though environmental factors may modify the phenotypic expression:e.g.TT (Proper nutrition) TallTT (Poor nutrition) Stunted growth andpoor development.- The Genotype determines the phenotype, but is affected by presence of Recessive or Dominant Gene, e.g (Conti..)
149e.g:(i) As T is dominant, it is expressed in Homozygotes and Heterozygotes, but t is recessive and is expressed only in Homozygotes.TT and Tt talltt short(ii) s is recessive, it is expressed only in Homozygotes while Heterozygotes are carriers but normal:A A HbA – NormalA S HbAS – NormalS S HbS – Abnormal “Sickle cell anemia”
150- Genotype differ in the degree of their expression of: Clinical severity, onset age, or both.(Variable expressivity).Expression of abnormal genotype maybe modified by: Other genetic loci, environmental factors or bothReduced Penetrance: in some heterozygous individuals with a dominant disorder, the presence of the mutation is reduced.Non-Penetrance: when a heterozygous individuals with a dominant disorder has no features of the disorder.“Pleiotropy” – multiple phenotypic effects of a single basic gene defect on multiple organs (genetic heterogeneity) e.g Tuberous sclerosis(AD) : learning disability, epilepsy, facial rash.New Mutations: A sudden appearance of a dominant disorder in the offspring with normal parents.Codominance: When two allelic traits are both expressed equally in a heterozygote e.g ABO blood groups.Pseudodominance: If a homozygous for AR mutation marries a carrier for the same mutation, their children have 1 in 2 chance of being affected (homo). This pattern is like dominant inheritance.
152Genetic diversity among individuals MutationsGenetic diversity among individualsnot deleterious mutationDeleterious mutationsDiseaseMay effect phenotypeOver generations, the influx of new nucleotide variations has ensured a high degree of genetic diversity and individuality.
153Genetic Variation* Some mutations in the gene(coding sequence) Variant proteinAltered structure andAltered propertiesSome mutation in the gene DNA (coding sequence)Variant protein ,but not critical for the functionNormal propertiesSome mutations in DNA (non-coding regions)No effect on proteins structure*Polymorphisms are common, particularly in non-coding regions of DNA
154Genetic Polymorphism* Many genetic loci are characterised by a number of relatively common alleles, thus producing many phenotypes in normal populationAlleles that occur at a frequency of > 1% are said to be polymorphic variantsAlleles that occur at a frequency of < 1% are said to be rare variantsIf there are two or more alleles(several forms of the same genes occupy the same locus) andthe rarest occurs at a frequency of more than 1%then this loci will be considered polymorphic.
155Gene polymorphism e.g. Gene for hair colour Wild type Alleles If there are two or more alleles(several forms of the same genes occupy the same locus) andthe rarest occurs at a frequency of more than 1%then this loci will be considered polymorphic.
156Types of Polymorphisms (Defined by the method of detection) DNAPolymorphismProtein PolymorphismAltered physical featuresChromosome heteromorphismsDetected by altered DNA sequencesContd…..Restriction Fragment Length Polymorphism (RFLPs):Inherited variations in DNA sequence,Results in gain or loss of a site recognised by restriction endonucleaseVariable number of tandem repeats (VNTRs):- Variations in the number of short, repeated nucleotide sequences (eg GC) between restriction sites- VNTRs are extremely polymorphic- Valuable in forensic medicine
157Types of Polymorphisms (Defined by the method of detection) Contd… Protein PolymorphismAltered physical featuresChromosome heteromorphismsContd…..Detected by:ElectrophoresisAltered activity,Altered physical propertiesEnzyme variant: altered enzyme activity, electrophoreticmobility, thermostability or other physical propertiese.g.G-6-PD deficiency.Antigenic variants: altered antigenic propertiese.g. ABO blood groups.
158Protein Polymorphism- Several proteins exist in two or more relatively common,genetically distinct , structurally different & functionally identical.- The causes of polymorphic forms:Mutation in or around gene- Examples :ABO Blood groups, Transferrin, Hb, 1 antitrypsin.
159Not all variant proteins have clinical consequences
160Types of Polymorphisms (Defined by the method of detection) Contd… Altered physical featuresChromosome heteromorphismsDetected by:Physical appearenceAltered physical features e.g. polydacytyly, gagantism, dwarfs, hair on ears, baldness.
161Types of Polymorphisms (Defined by the method of detection) Contd… Detected by:Cytogenetic studiesFISHChromosome heteromorphismsHeritable differences in chromosomal appearances from one person to another, e.g.Variations in the size of the Y chromosome long arm.Variation in the size of the centromere .Variation in satellite size and structure.The occurrence of fragile sites.
162Genetic diversity among individuals Chromosome heteromorphisms Protein variationsGenerally, the karyotype of normal persons of the same sex are quite similar.Occasional variants are seen on staining. These are called heteromorphisms.These reflect difference in amount or type of DNA sequence at a particular location along a chromosome.Almost 25% are silent mutation with no effect on protein structure.Most mutations alter amino acid sequence but do not have phenotypic effect (e.g. ABO blood groups).Rare mutations produce severe phenotype effect or influence survival (e.g. phenylketonuria)e.gIn long arm of chromosome.In chromosomes 1, 9, 16.In short arm of acrocentric chromosomes
163Uses of Polymorphism As genetic “Markers” - To distinguish inherited forms of a gene in a family.- Mapping gene to individual chromosomes by likage analysis.- Presymptomatic and prenatal diagnosis of genetic disease.- Evaluation of high and low – risk persons.- Paternity testing and forensic applications.- Matching of donor-recipient pairs of tissue and organ transplantation.
164Advantages of Polymorphism - Polymorphic forms are produced as result of mutation in the genetic loci.- The advantages are possibly:- Production of more stable forms.- Production of such forms that give resistanceagainst disease:e.g. Hb S Trait are resistance to malarial plasmodia.- Natural selection for survival of the fittest.
165Area of Significance of Polymorphism - Blood transfusion.- Tissue typing.- Organ Transplantation.- Treatment of Haemolytic disease of new born.
166ABO System- First identified by Landsteiner in 1900.- Human blood can be assigned to one of four types according to presence of two antigens, A and B, on the surface of Red Blood Cell andthe presence of two corresponding antibodies, Anti A and Anti B in the plasma.* RBC Antigen Polymorphism:- Useful marker for:- Family and population studies.- Linkage analysis.- Different frequencies in different population.Contd.
167* Blood Group Substances: - Blood group substances are encoded by allelic genes A and B.- Blood group substances exhibit polymorphism.Polymorphic SystemChromosomal LocationCommon AllelesABOMNSsXg9 q344q28 – 31Xp 22.3A, B and OM and N;S and sXga and Xg.
168ABO Blood groups and Reaction with Antibodies GenoTypeAnti AAnti BCellular AntigenSerum AntiFrequenciesOABABO/OA/A, O/AB/B, O/BA/B-+NOA + BAnti A+BNeither45%42%10%4%
169Clinical Importance of Polymorphism Some diseasegenes occur with polymorphic frequenciesGenetic polymorphisms may produce diseaseSome polymorphisms determine antigenic differencese.g.- HbS in African, Saudi Arabia- Thalassaemia inMediterranean regionSaudi Arabia- Cystic fibrosis in Europeanse.g.On exposure to drugs or environmental factorG-6-PD deficiencyMalignanthyperthermia.e.g.Blood groupHLA antigen fortissue typing.
170Clinical Importance of Polymorphism Contd…..Forensic MedicineAs genetic markerse.g.DNA fingerprint of each individual differs due to polymorphic sites in many non-coding sequencese.g.Predisposing to a disease within families or populations
171Genetic LinkageThe occurrence of two or more genetic loci in such close physical proximity on a chromosome that they are more likely to assort (linked) togetherCrossing over does not take place between closely situated loci – So they are said to be linkedCcCcNoCrossingDuringmeiosisAabbBBbBLinkedNot linked
172Concept of Genetic Linkage Linkage refers to loci, not to alleles (which occupy different chromosomesMeasurement of genetic linkage can only take place in family studiesStatistical method of measuring linkage is by calculation oflod scoreContd….Closeness of a genetic linkage is expressed in Cente Morgans (cM) or percent recombinationUnlinked loci are separated by a genetic distance of 50 cM at a given allele at one locus has a 50% of being transmitted with either allele at an unlinked loci.Loci separated by crossing over in 1% of gametes are 1 cM apartLoci close to each other, so they never separate are linked at a genetic distance of zero cM
173Concept of Genetic Linkage Contd…..Lod ScoreLod score is a acronym for “Logarithm of the Odds”( Logrithm of the likelihood ratio).Lod score of +3 or greater at recombination distance ofless than 50 cM between two loci is considered to be astrong evidence of linkage (1000 : 1 odds for linkage.Lod score of 2 or less is taken as a strong evidencethere is no linkage (100: 1 odds against linkage).
174Concept of Genetic Linkage Contd…..Linkage disequilibriumMeasure in populations, not in familiesThis is the tendency for certain alleles at two linked loci to occur together more often than expected by chance. e.g.If the mutant allele at D occurs on thesame chromosome as Mb more oftenthan expected within a certain populationlinkage disequilibrium is said to exist.MbDDisease locus = DMarker = MAlleles of Marker Ma and Mb.Distance=5cM
175centi Morgan Defines the distance between two gene loci If two loci are IcM apart, there is a 1% change of recombination between these loci as the chromosome is passed from parent to childIt gives a rough unit of distance along the chromosomeDifferent chromosomes have different sizes.Average chromosomes contain about 150 cM.There are about 3300 cM in the whole human genome.This corresponds to 3x109 bp.On average IcM is about 1 million bp (1000 kb).
176Markers tightly linked to a disease The marker linked to a diseasegene, must be on the sameregion on the chromosome(within < 1 cM distance).Markers that are a long distance away on the same chromosome may not appear to be linked, because recombination between the two loci is high
177Clinical Applications of Linkage Linkage is clinically useful as it may permitUsed inPrenatal diagnosisCarrier detectionPresymptomatic diagnosisElucidation of genetic factors in multifactorial disordersMore precise determination of the genotype at an unidentified gene locus on the basis of readily identified linked markersDetermination of the pattern of inheritance or specific for disease that exhibits genetic heterogeneityGene mapping by determining the recombination distance between two genes on a chromosome
178Gene MappingThis is the assignment of genes to specific chromosomal locations.Mapping is done by:Family studies to demonstrate linkage between lociSomatic cell genetic method to show that two loci are not linked (demonstrate synteny) or that an unmapped loci resides on a chromosomeGene dosage studiesCytogenetic techniques e.g. in situ hybridizationIndirect means of identifying location of a gene
179Importance of Gene Mapping To develop optimal strategy for gene therapy by improved knowledge of genomic organizationThe gene map is the anantomy of the human genomeAnalysis of heterogeneity and segregation of human genetic diseasesProvides information about linkage
181Haemoglobinopathies and Thalassaemias GeneticDisordersofHaemoglobin
182Haemoglobin- A conjugated protein consisting of iron-containing heme and protein (globin).- Globin chains are of different types:-chains and non -chains- Each molecule is a tetramer of two - and non - chains.- Each globin binds a haem in a haem binding site.Haemoglobin binds and transports oxygen fromlungs to the tissues, while it transports CO2 fromtissues to the lungs.
183Types of Hemoglobin in adults Globin genes Gene product Tetramers Name of Conc. inChromosome (globin) in RBCs haemoglobin adult11 , -chain 2 2 Hb A , -chain 2 2 Hb A ,-chain 2 2 Hb F <1.0Emberyonic Hb: , -chain 2 2 Hb-Gower II 0 , -chain 2 2 Hb-Gower I 0 , -chain 2 2 Hb-Portland 0
184Structure of each Globin gene Chromosome 11GA5’3’Chromosome 1621215’3’Structure of each Globin gene5’3’Exon 1Intron 1Exon 2Intron 2Exon 3
185Disorders of Haemoglobin Thalassaemias(Biosyntheticdisorderof Hb)Co-existingstructural /biosyntheticdisordersHaemoglobinopathies(Structural disorderof Hb)Constitute a major health problem in severalpopulations of the world(particularly those residing in malariaendemic region)
186Haemoglobinopathies Genetic structural disorder. Due to mutation in the globin gene of haemoglobin.Mostly autosomal recessive inheritance.Result in haemoglobin variants with altered structure and function.Altered functions include:Reduced solubilityReduced stabilityAltered oxygen affinity- increased or decreasedMethaemoglobin formation
187*Types of Mutations in Haemoglobin Point mutation: a change of a single nucleotide base in a DNA giving rise to altered amino acids in the polypeptide chains(e.g. Hb S , Hb Riyadh, Hb C)Deletions and additions: Addition and deletion of one or more bases in the globin genes(e.g. Hb-constant spring which is associated with mild -thalassaemia).Unequal crossing over: as in Hb-lepore and Hb-antilepore associated with -thalassaemias.________________________________________________________*Most abnormal Hbs are produced by mutations in the structural genes which determine the amino acid sequence of the globin chains of the Hb molecule.
188Geographical distribution of common Hb variants Variant Occurrence predominantly in:Hb S (6GluVal) Africa, Arabia, Black AmericansHb C (6Glulys) West Africa, ChinaHb E (26Glulys) South East AsiaHb D (121GluGln) AsiaHb O (121GluVal) Turkey and Bulgury
189Other examples of Haemoglobin variants His Lys Tyr HisCAC AAG UAU CAC NormalShorter chainHis Lys MutationCAC AAG UAA3’3’
194Major abnormalities & problems in SCA Sickling of the red cell during deoxygenation, as the HbShas low solubility at low O2 partial pressure and precipitates.Chronic haemolytic anaemia due to repeated sickling in tissuesand unsickling in the lungs.Plugging of microcapillaries by rigid sickled cells leading to sicklecell crises i.e severe pain and edema. This causes significant damageto internal organs, such as heart, kidney, lungs and endocrine glands.Repeated infections.Frequent cerebrovascular accidents.Hand-foot syndrome (in small,i.e.around age of 3y)Bone deformation – bossing of the forehead.Hepato-spleenomegaly.Growth retardation.Frequent blood transfusion requirements.Psychosocial problems.
195Thalassaemias Genetic disorders resulting from decreased biosynthesis of globin chainsof haemoglobin.
196Thalassaemias A group( not single identity) of Genetic defects. Due to mutations in and around the globin genes.Decreased production of one or more of the globin chains.Result in an imbalance in the relative amounts of the - and non -chains. Altered /non- ratio.A few rare Hb variants are effectively synthesized but are highly unstable, and thus cause thalassaemias as the : chain ratio is altered.As a consequences of thalassaemias there is excess production of the other chains, and a decreased over all haemoglobin synthesis.
197Types of Thalassaemias * Most common- Thalassaemia
198- Thalassaemia Hb - Decreased / ratio In - Thalassaemia productionof - chainsNormal = - ThalassaemiaAccumulation of
199Point Mutation producing - Thalassaemia Less FrequentIntronsChromosome 165’exon1exon2exon33’Base Substitution2bp delChain TerminationDefect5bp delPoly A signalMutation
201-thalassaemia -2 One -gene deletion. -chain production is only about 75% of normal.May be homo- (- /- ) or heterozygous (- / )The patient usually shows a normal phenotypic appearance but there might be mild thalassaemia symptoms.Hypochromic-microcytic RBC’s due to partial reduction of -chain.
202-thalassaemia- 1 Two -genes deletion- (o )thal. The patient synthesizes -chain but it is decreased to about 50% of normal.Anaemic symptoms- hypochromic microcytic anaemia.May be homozygous (- -/- -) or heterozygous(--/ ). If the patient is homozygous than there is no -chain synthesis, and if heterozygous then there is decreased synthesis of the -chain to half normal level.
203Hb H Disease Three -gene deletion. The Hb present during foetal life is “Hb Bart’s” (4), while during adulthood the Hb present is “Hb H” (4).Some of the symptoms include:hepatosplenomegally, impairment of erythropoisis, and hypochromoc-microcytic haemolytic anaemia.
204Hydrops foetalis Homozygous o-thalassaemia. There is a complete absence of -chain (all -genes are deleted).The Hb produced at birth is Hb Barts (4).Hydrops foetalis is lethal and the baby is born dead.Symptoms include: Hepatosplenomegaly, severe hypochromic- microcytic anaemia.
205Hb - Thalassaemia - Thalassaemia Increased / ratio In - ThalassaemiaDecreasedproductionof - chainsNormal = - ThalassaemiaAccumulation of
206-ThalassaemiaIt is characterized by either no -chain synthesis (i.e. o) or decreased synthesis of -chain (+).Excess -chains precipitate in RBC’s causing severe ineffective erythropoiesis and haemolysis.The greater the -chains, the more severe the anaemia.Production of -chains helps to remove excess -chains and to improve the -thalassaemia. Often HbFlevel is increased.Majority of -thalassaemia is due to point mutation.
207o-Thalassaemia The -chain is totally absent. There is increase in HbF with absence of HbA.This is combined with ineffective erythropoisis.In majority of the cases, -gene is present but there is complete absence of mRNA.Characteristics of this disorder are:Skeletal deformities (e.g. enlargement of upper jaw, bossing of skull and tendency of bone fractures).Severe hypochromic- microcytic anaemia.Survival depends on regular blood transfusion.This leads to iron overload (iron accumulates in the blood and tissues, causing tissue damage).Death usually occurs in the 2nd decade of life (i.e. at age of about 20 years) if measures are not taken to avoid iron overload by chelation therapy.
208+-Thalassaemia There is a variable amount of -chain production. There is decreased HbA level, and increased Hb A2, level with normal or increased Hb F level (and there is an increased number of -chains in the free form).The -chain is present but there is decreased numbers of mRNA or there is an abnormality in the mRNA.
210Clinical Classification of Thalassaemias Thalassaemia major:The patient depends on blood transfusions especially if he is homozygous.Thalassaemia intermediate:Homozygous mild +-thalassaemia.Co-inheritance of -thalassaemia.Heterozygous -thalassaemia.Co-inheritance of additional -globin genes. -thalassaemia and hereditary persistence of foetal HbHomozygous Hb leporeHb H disease.3. Thalassaemia minor (trait):o-thalassaemia trait.+-thalassaemia trait.Hereditary persistence of foetal Hb only.-thalassaemia trait.o- and +-thalassaemia trait.
211Hb-LeporeThis is an abnormal Hb due to unequal crossing-over of the - and -genes to produce a polypeptide chain consisting of the - chain at its amino end and - chain at its carboxyl end.The -fusion(hybrid) chain is synthesized inefficiently and normal and -chain production is abolished.The homozygotes show thalassaemia intermediate and heterozygotes show thalassaemia trait.Unequal crossing-over can be explained as crossing over between similar DNA sequence that are misaligned resulting in sequences with deletions or duplications of DNA segments; a cause of a number of genetic variants.The adjacent and -genes differ at only 10 of their 146 a.a. residues, if mispairing occurs followed by intergenic crossing over, two hybrid genes result: one with a deletion of part of each locus (lepore gene) and one with a corresponding duplication (anti-lepore gene).
212High Persistence of Foetal Hb (HPFH) A group of disorders due to deletions or cross over abnormalities which affect the production of and chains in non-deletion forms to point mutations upstream from the -globin genes.
213Double heterozygous indicates the presence of combinations of the following: Hb S + O-thalassaemia.Hb S + --thalasaemia.Hb S + -thalasaemia.Hb S + HbC diseaseHb S + HbE disease
215Diagnosis of Genetic Diseases Family History*Estimation ofHaematologicalparametersChromosomalAnalysisDetermination ofEnzyme Activityor Specific ProteinRecombinantDNATechnologyClinicalPresentation*Estimation ofBiochemicalParameters* Important for all genetic diseases
2161. Family History Consanguinity of parents. Presence of other siblings with the same disorder.Occurrence of the disorder in other members of the family.Repeated abortions or still births,mother and fathers ages.Drawing punnet square helps to determine the mode of inheritance of the genetic disorders.Autosomal or X-linkedDominant or recessive
2172. Clinical Presentation Certain clinical features are specific for a disease:Chronic anaemia:HaemoglobinopathiesThalassaemiaOther genetic anaemiasAcute anaemia, under certain stressful conditions.G-6-PD deficiencyHypoxia – sickle cell disease.Dependence on blood transfusion - -thalassaemia (major)Severe immune deficiency – ADA deficiency.Emphysema - 1 anti-trypsin deficiency.Hypercholesterolaemia – familial hypercholesterolaemia.Delayed blood coagulation – Haemophilia (decrease in factor VIII or IX).Mental retardation – Fragile syndrome (in X chromosome) or phenylketonuria (PKU).Muscular weakness and degeneration – Duchenne muscular dystrophy.
218Recombinant DNA Technology ( Genetic Engineering)
219Recombinant DNA Technology ( Genetic Engineering) Techniques forcuttingand joining DNA
220Requirements for DNA technology Restriction endonucleases PrimersVectorsNTPsProbesDNAOther enzymese.g ligases,Taq polymerasesSpecial chemicals andequipment
221Restriction Endonuclease Endonucleases.Synthesized by procaryotes. Do not restrict host DNA.Recognize and cut specific base sequence of 4-6 bases in double helical DNA.The sequence of base pairs is palindromic i.e. it has two fold symmetry and the sequence, if read, from 5’ or 3’ end is the same.5’-GAATTC-3’3’-CTTAAG-5’
222Restriction Endonuclease Produce either Blunt Ends or Staggered ends:5’-GAATTC-3’3’-CTTAAG-5’5’-GAA TTC-3’3’-CTT AAG-5’Blunt Endsor5’-GAATTC-3’3’-CTTAAG-5’5’-G AATTC-3’3’-CTTAA G-5’Staggered Ends
223Uses of Restriction Endonuclease Obtaining DNA fragments of interest.Gene mapping.Sequencing of DNA fragments.DNA finger printingRecombinant DNA technologyStudy of gene polymorphism.Diagnosis of disease.Prenatal diagnosis
224Sources of DNA cDNA Genomic DNA Synthesised from Synthesis of DNA mRNA using reversetranscriptaseSynthesis of DNADNA extractedfrom cellsUsing DNA synthesiser
226Vectors Cloning vesicles DNA molecules. Can replicate in a host e.g bacterial cells or yeast.Can be isolated and re-injected in cells.Presence can be detected.Can be introduced into bacterial cells e.g. E. coli.May carry antibiotic resistance genes.
227Types of vectors Type Insert size Plasmid : circular, double stranded cytoplasmic DNA in procaryotic e.g. PBR 3 of Ecoli.Bacteriophage lambda: a bacterial virus infects bacteria.Cosmids: a large circular cytoplasmic double stranded DNA similar to plasmid.Yeast Artificial Chromosomes (YAC)Insert size<5-10 kb.Upto 20kb.Upto 50kb.~ kb.
231Recombinant DNA Technology Amplification of DNAStudy of DNA structureand functionsOthersPolymerase chain reactionDNA cloningARMSDNA sequencingDGGERT PCRDot blot analysis
232Principles of Molecular Cloning Involves:Isolation of DNA sequence of interest.Insertion of this DNA in the DNA of an organism that grows rapidly and over extended period e.g. bacteria.Growing of the bacteria under appropriate condition.Obtaining the pure form of DNA in large quantities for molecular analysis.
234Polymerase Chain Reaction (PCR) Method to amplify a target sequence of DNA or RNA several million folds.Developed by Saiki et al in 1985.Based on Enzymatic amplification of DNA fragment flanked by primers i.e. short oligonucleotides fragments complimentary to DNA. Synthesis of DNA initiates at the primers.DNA5’ ATCAGGAATTCATGCCAAGGTTGATCGATGATCGATCGATCGATTGAT 3’3’AGCTAGCTAGCT 5’Primer
236Application of PCRDiagnosis of genetic disease by amplification of the gene of interest, followed by detection of mutation.Detection of infectious agent e.g. bacteria and viruses.DNA sequencing.In forensic medicine.
237Application of Recombinant DNA Technology1. Clinical Chemistry:Diagnosis of disease e.g. sickle cell anaemia by Mst II.Prenatal diagnosis,Premarital “Presymptomatic “Neonatal screening
241Cytology, Histology and Pathology Synthesis of protein in bacterial 2. Human GeneticsMutations in genes causing hereditary disease e.g. diagnosis of fibrosis Channes disease.Forensic MedicineAnalysis of stains of blood, semin.VirologyDetection of viral diseases e.g. hepatitisMicrobiologyUsing specific gene probes for detection of E.coliCytology, Histology and PathologyUsed in detection of tumor.Synthesis of protein in bacterialInsulinGHSomatostatinInterferonTransgenic animal production
245Genetic Counselling for Mendelian Disorders Genetic disorders:ChromosomalSingle geneMultifactorialMitochondrialAcquired somaticOnly single disorders follow a clearly defined pedigree pattern of inheritance “Mendelian Pattern”.During genetic counselling it is essential to establish whether or not the disorder is Mendelian andto calcualte the precise risk of recurrence.
246Essential Components of Genetic CounsellingRecurrence RiskHistory andpedigreeconstructionFollow-upConfirmatorydiagnosisClinicalExaminationCounselingCalculation ofrecurrence risk- History findings- Clinical examination findings- Radiology findings- Laboratory parameter results- DNA studies results- OthersAvailableoptions5
250Establishment of Mendelian Inheritance Pattern of transmission judged from family tree.For several diseases the family tree may be conclusive even if accurate diagnosis is not made.For some diseases pedigree pattern is not helpful and only clinical diagnosis is usedFor some disorders the pattern looks complicated and the exact diagnosis cannot be made.More common by combination of clinical diagnosis and comparable pedigree pattern.
253Complexities in AD Disorders Late or variable onset of the disease.How old will the family members be, to be certain of not developing the disease, e.g.Huntington’s disease, adult onset polycystic kidney disease, myotonic dystrophy.For some conditions life tables have been prepared.2. Lack of penetrancePenetrance:- Is the index of the proportion of individuals with the affected gene who present the disease.- Some disorders show lack of penetrance I.e. biochemical defect is present, but clinical features are absent, e.g.Huntington disease – Penetrance decreases with age.Retinoblastoma: Lack of penetrance unrelated to age.
254Complexities in AD Disorders Variation in Expression:Several AD disorders show variation in clinical expression and hence the disorders cannot be ruled out unless careful examination is carried out.Mild Moderate Severe expression*Problems in G.C. since those who reproduce are leastseverely affected, but may have severely affected childrene.g. Tuberousclerosis, Myotonic dystrophy, Huntingtonsdisease.*Disease severity may depend on sex of the transmitting parent.“Anticipation: refers to the state that a genetic disease worsens with successive generation.
255Factors underlying variability in AD disorders Factors EffectGenomic imprinting Phenotype varies accordinglyAnticipation due to unstable More severe phenotype inDNA successive generationMosaicism Mild ornon-penetran phenotypeModifying alleles Influence of unaffected parentSomatic mutations also Variable penetrancerequired for presentation(e.g. familial cancers)New mutations Sudden appearance of (AD)disorder in normal parent
256II. Complexities in AR Disorders Difficult to confirm as homozygote born to phenotypically normal (carrier) parents, who may not have an affected relative.Horizontal transmission ( sudden appearance of a disorder in a generation)Diagnosis makes the mode of inheritance certain.LowRiskVery low
257Problems with AR disorders Genetic heterogeneity.Lack of penetrance and variation in expression are much less.
258If consanguinity present the risk is increased: Rare disorder increase in the number of effected childrendue to consanguinity(c) Extensive consanguinity Appear like AD inheritance (pseudo AD)
259Population Risk Can be calculated from: Hardy Weinberg Equilibrium p + q = 1 [p2 + q2 = 2pq = 1]q2 = Abnormal homozygotep2 = Normal2pq = Heterozygotee.g. 2 patients of PKU in screened.q2 = 2; q = =p = 1 – q = 0.986(hetero)2pq =
260Disease Gene Carrier Risk for Risk for Risk of transmitting an AR disorder in relation to disease incidences (the spouse is healthy)Disease Gene Carrier Risk for Risk forfrequency frequency frequency offspring offspring(q2)/10000 (q) (%) =2pq(%) homo. (%) healthy(affected sib) sib
261X-Linked Disorders Occupy a prominent place in genetic counselling. >100 X-linked disorders recognised.Majority XR; some dominant (often lethal in hemizygous male).X-chromosomes inactivation (lyonns phenomenon). applies to almost all human X-chromosomes.
262Recognition of X-Linkage No male-to-male transmission.Affected male All daughters carriers (XR). All daughters affected (XD).Unaffected males never transmit disease to either sex.A definite carrier women risk ½ sons affected.Carrier women ½ daughters carrier (XR) ½ daughters affected (XD).Homozygous affected women are few affected male are much more.These guidelines will cover most genetic counseling problems.
263Mitochondrial Inheritance No transmission in descendents of males, affected or not.Both sexes may be affected.Females may be symptomless carriers.All daughters of an affected or carrier female are at risk of transmitting the disorders or of becoming affected.All sons may become affected, but do not transmit it to their children
264First degree……………………………………. 1/4 Sibs (brothers & sisters) Degree of Relationship to patients Proportion of gene sharedFirst degree……………………………………. 1/4Sibs (brothers & sisters)Dizygotic twinsParentsChildSecond degree …….. ………………………….. 1/4Half sibsUncles, auntsNephew, niecesDouble first cousinsThird degree: ……………………………………. 1/8First cousinsHalf uncles, auntshalf nephew, nieces
265Relation shared of Homo. Gene ChanceDegree ofRelation shared of Homo.Monozygotic twinDizytotic twin st 1/ /4Sibs st 1/ /4Uncle-nephew(aunt-niece) nd 1/ /8Half-sibs nd 1/ /8Double 1st cousin nd 1/ /8First cousin rd 1/ /16
266Consanguinity Consanguinity relevant Not relevant Only relevant to genetic risks if it involves both parental lives not just one.Consanguinity relevant Not relevantThe rarer the disorder the higher the proportion of affected individuals from consanguineous marriages.Consanguinity must be seen in the context of particular community. An apparent relationship of a particular disorder is much less certain if 30% cousin marriages, compared to non-consanguineous mating.Extensive consanguinity (AR) appears like AD.