HUMAN MOLECULAR GENETICS

HUMAN MOLECULAR GENETICS
Examples of genetic diseases in Humans Meiosis & Recombination Mendelian Genetics Modes of Heredity Genetic Linkage Analysis

Role of Genes in Human Disease
Genetic Diseases in Humans Role of Genes in Human Disease Most diseases -> phenotypes result from the interaction between genes and the environment Some phenotypes are primarily genetically determined Achondroplasia (-> dwarfism) Other phenotypes require genetic and environmental factors Mental retardation in persons with PKU (polyketonuria) Some phenotypes result primarily from the environment or chance Lead poisoning Down syndrome, achondroplasia 100% Environmental Struck by lightning Infection Weight Cancer Diabetes Height Genetic

Types of Genetic Disorders:
Genetic Diseases in Humans Types of Genetic Disorders: -> Chromosomes and chromosome abnormalities (Down Syndrome) -> Single gene disorders (Haemophilia, sickle cell anaemia) -> Polygenic Disorders (Cancer)

Chromosomal disorders
Genetic Diseases in Humans Chromosomal disorders Addition or deletion of entire chromosomes or parts of chromosomes Typically more than 1 gene involved 1% of paediatric admissions and 2.5% of childhood deaths Classic example is trisomy 21 - Down syndrome KARYOTYPE

Genetic Diseases in Humans
Single gene disorders Single mutant gene has a large effect on the patient Transmitted in a Mendelian fashion Autosomal dominant, autosomal recessive, X-linked, Y-linked Osteogenesis imperfecta - autosomal dominant Sickle cell anaemia - autosomal recessive Haemophilia - X-linked

Single gene disorders Neonatal fractures typical of osteogenesis imperfecta, an autosomal dominant disease caused by rare mutations in the type I collagen genes COL1A1 and COL1A2 A famous carrier of haemophilia A, an X-linked disease caused by mutation in the factor VIII gene Sickle cell anaemia, an autosomal recessive disease caused by mutation in the β-globin gene

Polygenic disorders The most common yet still the least understood of human genetic diseases Result from an interaction of multiple genes, each with a minor effect The susceptibility alleles are common Type I and type II diabetes, autism, osteoarthritis, cancer

Single gene disorders - Polygenic disorders
Genetic Diseases in Humans Single gene disorders - Polygenic disorders Polygenic disease pedigree Autosomal dominant pedigree Male, affected Female, unaffected

Meiosis & Genetic Recombination
Chromosomes & Genes Are long stable DNA strands with many genes. Occur in pairs in diploid organisms. The two chromosomes in a pair are called “homologs” Homologs usually contain the same genes, arranged in the same order Homologs often have different alleles of specific genes that differ in part of their DNA sequence. DNA a b c genes unreplicated pair of homologs Genes and chromosomes

Chromosomes & Genes Genes and chromosomes

Chromosome structure unreplicated chromosome telomeres centromere replicated sister chromatids Each chromatid consists of a very long strand of DNA. The DNA is roughly colinear with the chromosome but is highly structured around histones and other proteins which serve to condense its length and control the activity of genes. Telomeres: Specialized structures at chromosome ends that are important for chromosome stability. Centromere: A region within chromosomes that is required for proper segregation during meiosis and mitosis. Genes and chromosomes

Chromosome structure - Homologs
Meiosis & Genetic Recombination Chromosome structure - Homologs Sister chromatids unreplicated homologs replicated Genes and chromosomes Sister chromatids are almost always IDENTICAL (prior to recombination). Homologues may carry different alleles of any given gene.

Cell Devision Mitosis -> 2n -> 2x 2n (diploid) Goal is to produce two cells that are genetically identical to the parental cell. (somatic cells) Meiosis -> 2n -> 4x 1n (haploid) Goal is to produce haploid gametes from a diploid parental cell. Gametes are genetically different from parent and each other. Genes and chromosomes

Cell Devision -> Mitosis - Meiosis
Meiosis & Genetic Recombination Cell Devision -> Mitosis - Meiosis Mitosis Cross-over 2n 4n 1n I II 2n 4n Genes and chromosomes In mitosis the homologs do not pair up. Rather they behave independently. Each resultant cell receives one copy of each homolog. In meiosis the products are haploid gametes so two divisions are necessary. Prior to the first division, the homologs pair up (synapse -> cross-over) and segregate from each other. In the second meiotic division sister chromatids segregate. Each cell receives a single chromatid from only one of the two homologs. -> contributes to evolutionary variations

Meiosis/perfect linkage
Meiosis & Genetic Recombination Meiosis/perfect linkage P L p l only parental-type gametes Genes and chromosomes

Meiosis/with recombination
Meiosis & Genetic Recombination Meiosis/with recombination P L p l Genes and chromosomes In some meiotic divisions these recombination events between the genes will occur resulting in recombinant gametes -> contributes to variation (evolution) Meiotic recombination in a grasshopper Meiosis is not conservative, rather it promotes variation through segregation of chromosomes and recombination

Mendelian Genetics The laws of heridity
Gregor Mendel ( ): “Father of Genetics” Augustinian Monk at Brno Monastery in Austria (now Czech Republic) -> well trained in math, statistics, probability, physics, and interested in plants and heredity. Genes and chromosomes Mountains with short, cool growing season meant pea (Pisum sativum) was an ideal crop plant. Work lost in journals for 50 years! Rediscovered in 1900s independently by 3 scientists Recognized as landmark work!

P F1 F2 One Example of Mendel’s Work D d Tall Dwarf x All Tall
Phenotype Clearly Tall is Inherited… What happened to Dwarf? F1 x F1 = F2 F2 3/4 Tall 1/4 Dwarf -> Phenotype: 3:1 Dwarf is not missing…just masked as “recessive” in a diploid state Tall is dominant to Dwarf Use D/d rather than T/t for symbolic logic DD dd Dd Genotype Homozygous Dominant Recessive Heterozygous d D Punnett Square: possible gametes

Mendelian Genetics The laws of heridity The Law of Segregation:
Genes exist in pairs and alleles segregate from each other during gamete formation, into equal numbers of gametes. Progeny obtain one determinant from each parent. -> Alternative versions of genes account for variations in inherited characteristics (alleles) -> For each characteristic, an organism inherits two alleles, one from each parent. (-> homozygote/heterozygote) -> If the two alleles differ, then one, the allele that encodes the dominant trait, is fully expressed in the organism's appearance; the other, the allele encoding the recessive trait, has no noticeable effect on the organism's appearance (dominant trait -> phenotype) -> The two alleles for each characteristic segregate during gamete production. Genes and chromosomes

2. The Law of Independent Assortment Members of one pair of genes (alleles) segregate independently of members of other pairs. -> The emergence of one trait will not affect the emergence of another. -> mixing one trait always resulted in a 3:1 ratio between dominant and recessive phenotypes -> mixing two traits (dihybrid cross) showed 9:3:3:1 ratios -> only true for genes that are not linked to each other 3:1 Genes and chromosomes 9:3:3:1

After rediscovery of Mendel’s principles, an early task was to show that they were true for animals And especially in humans Genes and chromosomes

Problems with doing human genetics: -> Can’t make controlled crosses! -> Long generation time -> Small number of offspring per cross So, human genetics uses different methods!! Genes and chromosomes

Major method used in human genetics is -> pedigree analysis (method for determining the pattern of inheritance of any trait) Pedigrees give information on: -> Dominance or recessiveness of alleles -> Risks (probabilities) of having affected offspring Genes and chromosomes

Standard symbols used in pedigrees:
Mendelian Genetics The laws of heridity Standard symbols used in pedigrees: carrier Genes and chromosomes ”inbreeding”

Modes of Heredity Autosomal Dominant
Most dominant traits of clinical significance are rare So, most matings that produce affected individuals are of the form: Aa x aa Genes and chromosomes -> Affected person can be heterozygote (Aa) or homozygote (AA) -> Every affected person must have at least 1 affected parent -> expected that 50% are affected /50% are uneffected -> No skipping of generations -> Both males and females are affected and capable of transmitting the trait -> No alternation of sexes: we see father to son, father to daughter, mother to son, and mother to daughter

Modes of Heredity Autosomal Dominant Examples:
Tuberous sclerosis (tumor-like growth in multiple organs, clinical manifestations include epilepsy, learning difficulties, behavioral problems, and skin lesions) and many other cancer causing mutations such as retinoblastoma Brachydactyly Genes and chromosomes

Modes of Heredity Autosomal Dominant Examples: Achondroplasia
-> short limbs, a normal-sized head and body, normal intelligence -> Caused by mutation (Gly380Arg mutation in transmembrane domain) in the FGFR3 gene -> Fibroblast growth factor receptor 3 (Inhibits endochondral bone growth by inhibiting chondrocyte proliferation and differentiation Mutation causes the receptor to signal even in absence of ligand -> inhibiting bone growth Genes and chromosomes

Modes of Heredity Autosomal Recessive
These are likely to be more deleterious than dominant disorders, and so are usually very rare The usual mating is: Aa x Aa Genes and chromosomes -> Affected person must be homozygote (aa) for disease allele -> Both parents are normal, but may see multiple affected individuals in the sibship, even though the disease is very rare in the population -> Usually see “skipped” generations. Because most matings are with homozygous normal individuals and no offspring are affected -> inbreeding increases probablility that offspring are affected -> unlikely that affected homozygotes will live to reproduce

Modes of Heredity Autosomal Recessive Examples:
Sickle-Cell Anaemia (sickling occurs because of a mutation in the hemoglobin gene -> affects O2 transport; occurs more commonly in people (or their descendants) from parts of tropical and sub-tropical regions where malaria is common -> people with only one of the two alleles of the sickle-cell disease are more resistant to malaria) Cystic fibrosis (also known as CF, mucovoidosis, or mucoviscidosis; disease of the secretory glands, including the glands that make mucus and sweat; excess mucus production -> causing multiple chest infections and coughing/shortness of breath; especially Pseudomonas infections are difficult to treat -> resistance to antibiotica) Genes and chromosomes

Modes of Heredity Dominant vs. Recessive
Is it a dominant pedigree or a recessive pedigree? 1. If two affected people have an unaffected child, it must be a dominant pedigree: A is the dominant mutant allele and a is the recessive wild type allele. Both parents are Aa and the normal child is aa. 2. If two unaffected people have an affected child, it is a recessive pedigree: A is the dominant wild type allele and a is the recessive mutant allele. Both parents are Aa and the affected child is aa. 3. If every affected person has an affected parent it is a dominant pedigree. Genes and chromosomes

Modes of Heredity X-Linked Recessive
-> Act as recessive traits in females (XX) -> females express it only if they get a copy from both parents) -> dominant traits in males (XY) -> An affected male cannot pass the trait on to his sons, but passes the allele on to all his daughters, who are unaffected carriers -> A carrier female passes the trait on to 50% of her sons Examples: About 70 pathological traits known in humans -> Hemophilia A, Duchenne muscular dystrophy, color blindness,….. Genes and chromosomes

Other sex-linked disease
Modes of Heredity Other sex-linked disease X-linked dominant: -> caused by mutations in genes on the X chromosome -> very rare cases -> Males and females are both affected in these disorders, with males typically being more severely affected than females. -> Some X-linked dominant conditions such as Rett syndrome, Incontinentia Pigmenti type 2 and Aicardi Syndrome are usually fatal in males Y-linked (dominant): -> mutations on the Y chromosome. -> very rare cases -> Y chromosme is small -> Because males inherit a Y chromosome from their fathers -> every son of an affected father will be affected. -> Because females inherit an X chromosome from their fathers -> female offspring of affected fathers are never affected. -> diseases often include symptoms like infertility Genes and chromosomes

Exceptions to Mendelian Inheritance
Modes of Heredity Exceptions to Mendelian Inheritance Mitochondrial inheridance: Mitochondrial DNA is inherited only through the egg, sperm mitochondria never contribute to the zygote population of mitochondria. There are relatively few human genetic diseases caused by mitochondrial mutations. -> All the children of an affected female but none of the children of an affected male will inherit the disease. -> Note that only 1 allele is present in each individual, so dominance is not an issue Genes and chromosomes

Summary of mutations which can cause a disease
Three principal types of mutation Single-base changes Deletions/Insertions Unstable repeat units Two main effects Loss of function Gain of function

Mapping a disease Locus
Genetic Linkage Mapping a disease Locus Linkage Although Mendel's Law of Independent Assortment applies well to genes that are on different chromosomes. It does not apply well to two genes that are close to each other on the same chromosome. Such genes are said to be “linked” and tend to segregate together in crosses.

Genetic Linkage Mapping a disease Locus Basic rules of linkage Loci on different chromosomes will not be co-inherited i.e. locus A on chromosome 1 will not be co-inherited with locus B on chromosome 2 Loci on the same chromosome may be co-inherited The closer two loci are on the same chromosome the greater the probability that they will be co-inherited i.e the likelyhood of recombination is small

Genetic Linkage Mapping a disease Locus Linkage analysis The mapping of a trait on the basis of its tendency to be co-inherited with polymorphic markers Why map and characterize disease genes? Can lead to an understanding of the molecular basis of the disease May suggest new therapies Allows development of DNA-based diagnosis - including pre-symptomatic and pre-natal diagnosis

Genetic Linkage Mapping a disease Locus First question to ask in a mapping exercise: -> Are there functional or cytogenetic clues? Functional Clues Osteogenesis imperfecta (OI) Collagen I Haemophilia A Factor VIII Haemophilia B Factor IX Cytogenetic Clues (structure and function of chromosomes) Duchenne muscular dystrophy Translocation at Xp21 Polyposis coli Deletions in 5q -> If there are clues, then one can target a particular gene or a particular chromosomal region -> If there are no clues, then one needs to conduct a genome-wide linkage scan

Genetic Linkage Mapping a disease Locus Example: Sweat Pea Purple & Long Consider the following pair of genes from the sweet pea that are located on the same chromosome -> linked: Trait affected Alleles Phenotype Purple Flower color p P purple red Long Pollen length L l short Gene

Genetic Linkage Mapping a disease Locus Example: Sweat Pea Purple & Long Mating type - more clearly reveals what gametes (and how many) were contributed by the F1 generation. P/P L/L X p/p l/l > homozygote F P/p L/l X p/p l/l "tester" F ? -> result can give indication if loci are linked or not

Genetic Linkage Mapping a disease Locus Calculation of Recombination Frequency Recombination frequency is a direct measure of the distance between genes. The higher the frequency of recombination (assortment) between two genes the more distant the genes are from each other. A map distance can be calculated using the formula: # recombinant progeny /total progeny X 100 = map distance (% recombination) 1 map unit = 1% recombination = 1 centimorgan (cM) 1 cM (Thomas Hunt Morgan) is the unit of genetic distance Loci 1cM apart have a 1% probability of recombination during meiosis Loci 50cM apart are unlinked -> LOD Score - a method to calculate linkage distances (to determine the distance between genes)

Genetic Linkage Mapping a disease Locus Example: Sweat Pea Purple & Long -> Calculation of map distance between the P and L genes gametes zygote phenotype observed P L P/p L/l Purple long parental type P l P/p l/l purple short recombinant p L p/p L/l red long recombinant p l p/p l/l red short parental type TOTAL # recombinant progeny /total progeny X 100 = map distance 305 were recombinants (154 P l p L) 305/2840 X 100 = 10.7 map units or 10.7% recombination frequency

Genetic Linkage Mapping a disease Locus Build a map Recombination frequencies for a third gene (X) were determined using the same type of cross as that used for P and L. P to L map units P to X map units X to L 2.8 map units . Map 13.1 units P L X 10.7 units units We can deduce from this that L is between P and X and is closer to L than it is to P. Thus it is possible to generate a recombination map for an entire chromosomes.

Genetic Linkage Mapping a disease Locus Chromosomes and Linkage The maximum frequency of observed recombinants between two genes is 50%. At this frequency the genes are assorting independently (as if they were on two different chromosomes). 50% parental gametes (AB, ab) A a B b 50% non-parental gametes (aB, Ab) If on the same chromosome, but greater than 50 map units apart, crossovers will actually occur > 50% of the time but multiples will cancel each other out. A a B b parental gametes (AB, ab) -> Two genes can be on the SAME chromosome but will behave as if they are unlinked in a test cross. non-parental gametes (aB, Ab)

Mapping using molecular markers
Genetic Linkage Mapping a disease Locus Mapping using molecular markers Molecular markers are most often variations in DNA sequence that do not manifest a phenotype in the organism. However they can be used to map genes in the same way that markers affecting visible phenotypes are. An example of this would be a restriction fragment length polymorphism (RFLP) Gene of interest restriction sites -> markers

Genetic Linkage Human linkage map

Genetic Linkage Mapping a disease Locus Polymorphic markers -> A marker that is frequently heterozygous in the population -> One can therefore distinguish the two copies of a gene that an individual inherits -> They are not themselves pathological - they simply mark specific points in the genome Technique used for mapping with markers: Primers are made to the unique DNA sequence to each side of a given repeat, and these primers are used to amplify the repeat using the polymerase chain reaction (PCR). -> copies of the repeat are either radioactively or fluorescently labeled and then run on a gel to separate the different sizes from one another. -> The size of each sequence, which correlates with the number of repetitive sequences within it, can then be assessed.

Polymorphic markers -Variable number tandem repeats (VNTRs)
Genetic Linkage Mapping a disease Locus Polymorphic markers -Variable number tandem repeats (VNTRs) Changes in the numbers of repeated DNA sequences arranged in tandem arrays ACGTGTACTC 3-repeat allele 4-repeat allele Polymorphic markers - Microsatellites Particular class of VNTR with repeat units of 1-6bp in length Also known as short tandem repeats (STRs) and sometimes as simple sequence repeats (SSRs) The most widely used are the CAn microsatellites CACACACACACA CACACACACACACACA 6 (CA) allele 8 (CA) allele Polymorphic markers - Single nucleotide polymorphisms (SNPs) a polymorphism due to a base substitution or insertion or deletion of a single base

Genetic Linkage Mapping a disease Locus
Practicalities of Linkage Analysis The genotype for a microsatellite marker on chromosome 1 Chrom. 1 6 (CA) allele 8 (CA) allele Paternal copy Maternal copy * Determine the genotype of each family member for polymorphic markers across the genome -> The individuals genotype for this location is (6 8)

Genetic Linkage Mapping a disease Locus Uninformative and informative meioses 6 6 6 8 9 10 8 9 6 10 Uninformative Completely informative A lab technique used to determine whether two genetic markers are linked to each other and how closely linked they are. It uses sexual reproduction which produces offspring in which the two markers may have crossed over during DNA recombination. Informative -> if repetitive sequences (markers) are different at the same location

Genetic Linkage Mapping a disease Locus An autosomal dominant disease for which the gene resides on chromosome 1 But you don’t know that! 1 Disease gene

Genetic Linkage Mapping a disease Locus Marker Studies Disease gene 5 6 4 7 2 3 Marker studied 2 3 1 5 4 4 2 4 2 5 2 7 1 5 3 5 6 7

Genetic Linkage Mapping a disease Locus Genotype data for the whole family (24) (25) (27) (23) (16) (14) (26) (46) (34) (13) (33) (58) (12) (18) (78) (47) (67)

Genetic Linkage Mapping a disease Locus The next step - define the maximal region of linkage Disease gene Gene resides here

Genetic Linkage www.ensembl.org Mapping a disease Locus
And then? -> Make a list of the genes within the interval

Genetic Linkage Mapping a disease Locus Gene content of chromosome 1

And finally-> Find the mutation! Target candidate genes within the interval by DNA sequencing Two important considerations for single-gene disorders: Allelic heterogeneity different mutations at the same locus (or gene) cause the same disorder. -> β-thalassemia may be caused by several different mutations in the β-globin gene Locus heterogeneity Determination of the same disease or phenotype by mutations at different loci (or genes) -> medullary cystic kidney disease (ADMCKD; synonym: medullary cystic disease, MCD); maybe huntington disease

What about mapping polygenic disorders? Gene1 Gene 2 Gene 3 Gene 4 PHENOTYPE Environment Schizophrenia Asthma Hypertension (essential) Osteoarthritis Type II diabetes (NIDDM) Cancer -> Unrelated affected individuals share ancestral risk alleles

Genetic Linkage -> No clear inheritance pattern
A polygenic phenotype Affected individual joining the family, emphasizing the common nature of the disease An affected individual with unaffected parents -> No clear inheritance pattern

Genetic Linkage Summary Mapping single gene disorders
Use clues If none, genome-wide linkage analysis DNA sequence analysis of linked region Mapping polygenic disorders Model-free genome-wide linkage analysis Functional analysis of associated polymorphisms within the refined genomic interval Conclusions For a single gene disease identifying the causal mutation is now relatively straightforward Technological and analytical advances are also making polygenic diseases tractable Genetics is going to play an ever increasing role in medical diagnosis and in the development of improved treatment regimes

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