Genotype-Phenotype correlations 1 Dr MOHAMED FAKHRY 2015

Objectives  Polymorphisms Vs mutations  Detection and clinical applications  Polymorphisms at protein level  Polymorphisms at DNA level  Genotype and phenotype

Genotype 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  S Where  A is for HbA and  S for HbS. 3 Dr MOHAMED FAKHRY 2015

4Phenotype The 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. GenotypePhenotype TT or Tt Tall tt Short  A  A HbA (normal)  A  S (Hetero ) HbAS (carrier)  S  S HbS (SCA) ( Homo = Identical, Hetero = different) Dominant * Hetero Recessive Dr MOHAMED FAKHRY 2015

Genotype – 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) → Tall TT (Poor nutrition) → Stunted growth and poor development. - The Genotype determines the phenotype, but is affected by presence of Recessive or Dominant Gene, e.g. (Conti..) 5 Dr MOHAMED FAKHRY 2015

e.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 → tall tt → short (ii)  s is recessive, it is expressed only in Homozygotes while Heterozygotes are carriers but normal:  A  A ⇒ HbA (Normal)  A  S ⇒ HbAS (Normal but carrier)  S  S ⇒ HbS (Abnormal “Sickle cell anemia”) 6 ⇒ The Genotype determines the phenotype, but is affected by presence of Recessive or Dominant Gene, e.g. (Conti..)

- Genotype differ in the degree of their expression of: Clinical severity, onset age, or both (Variable expressivity).  Variable expressivity refers to the range of signs and symptoms that can occur in different people with the same genetic condition. variable expressivity is probably caused by a combination of genetic, environmental, and lifestyle factors.  Expression of abnormal genotype maybe modified by: Other genetic loci, environmental factors or both  Reduced Penetrance : If some people with the mutation do not develop features of the disorder, the condition is said to have reduced (or incomplete) penetrance in some heterozygous individuals with a dominant disorder, the presence of the mutation is reduced.  “Pleiotropy”; single basic gene defect → multiple phenotypic effects on multiple organs (genetic heterogeneity) e.g Tuberous sclerosis (AD ); rare genetic disease that causes noncancerous (benign) tumors to grow in many parts of the body) : learning disability, epilepsy, facial rash. 7

8 -New Mutations: A sudden appearance of a dominant disorder in the offspring with normal parents. -Codominance: With codominance, a cross between organisms with two different phenotypes produces offspring with a third phenotype in which both of the parental traits appear together. When two allelic traits are both expressed equally in a heterozygote e.g ABO blood groups. ↦ Genotype differ in the degree of their expression of: Clinical severity, onset age, or both (Variable expressivity) Dr MOHAMED FAKHRY 2015

Genetic Polymorphism 9 Dr MOHAMED FAKHRY 2015

Polymorphism Polymorphism is the natural variations in a gene, DNA sequence, or chromosome that have no adverse effects on the individual and occur with fairly high frequency in the general population Polymorphism is the inheritance of genes in different forms termed alleles Is common (probably all genes show allelic variation) Alleles have different DNA sequences 10

Polymorphisms Vs Mutations Genetic polymorphisms: Common alleles > 1% Mutations: Rare alleles < 1% 11 Dr MOHAMED FAKHRY 2015

Polymorphisms: Sites  Inter-genes or intronic: Detected by DNA sequence analysis  Gene coding sequences: Different protein variants →Distinct phenotypes (may be)  DNA regulatory regions: → may affect phenotypes 12

Mutations Genetic diversity among individuals Over generations, the influx of new nucleotide variations has ensured a high degree of genetic diversity and individuality. Deleterious= harmfull mutations not deleterious mutation Disease May effect phenotype 13

Genetic Variation (polymorphism or muation)* I.Some mutations in the gene (coding sequence) Variant protein Altered structure and Altered properties I.Some mutations in the gene (coding sequence) Variant protein,but not critical for the function II.Some mutations in DNA (non-coding regions) Normal properties No effect on proteins structure * Polymorphisms are common, particularly in non-coding regions of DNA 14

Genetic Polymorphism* Many genetic loci are characterized by a number of relatively common alleles, thus producing many phenotypes in normal population Alleles that occur at a frequency of > 1% are said to be polymorphic variants Alleles that occur at a frequency of < 1% are said to be rare variants If there are two or more alleles (several forms of the same genes occupy the same locus) and the rarest occurs at a frequency of more than 1% then this loci will be considered polymorphic. 15

Gene polymorphism e.g. Gene for hair colour Wild type Alleles 16

Types of Polymorphisms (Defined by the method of detection) Types of Polymorphisms (Defined by the method of detection) DNA Polymorphism - Restriction Fragment Length Polymorphism (RFLPs): - Inherited variations in DNA sequence, - Results in gain or loss of a site recognized by restriction endonuclease - Variable 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 Detected by altered DNA sequences Protein Polymorphism Altered physical features Chromosome heteromorphisms Contd….. 17

↦ Polymorphisms: Detection At DNA level  DNA sequence analysis  Restriction fragment length polymorphisms (RFLPs)  PCR-based Methods: Conventional PCR Real-time PCR 18 Dr MOHAMED FAKHRY 2015

Types of Polymorphisms (Defined by the method of detection) Contd… Types of Polymorphisms (Defined by the method of detection) Contd… - Enzyme variant: altered enzyme activity, electrophoretic mobility, thermostability or other physical properties e.g.G-6-PD deficiency. - Antigenic variants: altered antigenic properties e.g. ABO blood groups. Protein PolymorphismAltered physical features Chromosome heteromorphisms Contd….. Detected by: Electrophoresis Altered activity, Altered physical properties 19

↦ Polymorphisms: Detection At protein level ABO System: A, B & O alleles: Chromosome 9 A & B: 4 nucleotide differences O: One base deletion and frame-shift Phenotypes: O, A, B & AB This blood group may be the most complex genetically of all blood type systems since it involves 45 different antigens on the surface of red cells that are controlled by 2 closely linked genes on chromosome 1. Rh System: Rh-D: RH gene located on short arm of Chromosome 1. There are two alleles, D and d. Individuals who are homozygous dominant (DD) or heterozygous (Dd) are Rh+. Those who are homozygous recessive (dd) are Rh- (i.e., they do not have the key Rh antigens). Phenotypes: Rh-positive and Rh-negative Incompatibility:Hemolytic disease of newborn 20

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. Protein Polymorphism 21 Dr MOHAMED FAKHRY 2015

Not all variant proteins have clinical consequences Not all variant proteins have clinical consequences 22 Dr MOHAMED FAKHRY 2015

Types of Polymorphisms (Defined by the method of detection) Contd… Types of Polymorphisms (Defined by the method of detection) Contd… Altered physical features e.g. polydactyly, gigantism, dwarfs, hair on ears, baldness. Altered physical features Chromosome heteromorphisms Detected by: Physical appearance 23

Types of Polymorphisms (Defined by the method of detection) Contd… Types of Polymorphisms (Defined by the method of detection) Contd…  Heritable 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. Chromosome heteromorphisms Detected by: Cytogenetic studies FISH (Fluorescence in situ hybridization) 24

Genetic diversity among individuals Chromosome heteromorphisms Generally, 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) In long arm of chromosome. In chromosomes 1, 9, 16. In short arm of acrocentric chromosomes Protein variations e.g 25

As genetic “Markers”  To distinguish inherited forms of a gene in a family.  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. Uses of Polymorphism 26

Summary Polymorphisms is :  ⇒ High degree of biochemical individuality (Every individual is unique)  Due to Different responses of individuals to environment, diet and drugs  Used as genetic markers; powerful tools in clinical genetics