Molecular Genetics

Dr Alison Battersby

Dr Martin Evans

Chromosomes

Karyotype

• 23 derived from each parent• Sex determined by X and Y chromosomes• Males XY, females XXNumber, size and shape of chromosomes

arranged in standard mannerPosition of centromere

Convention/Terms

• Metacentric, centromere is median• Acrocentric centromere close to one end• Telocentric centromere terminal ie 1 arm• Short arm on top (P or petit), long arm Q• Biggest first, sex chromosomes last• Banded karyotype

The FISH technique utilizes DNA probes that are specific to regions of individual chromosomes. The probe attaches to the spread of chromosomes from a cell, then a fluorescein stain is applied. This "paints" the chromosome so that it is visible with the aid of a fluorescent microscope. In the example diagram below the chromosome 21 pair have been

painted.

The FISH technique requires viewing many cells to diminish artefactual problems in counting the painted chromosomes. FISH is good at identifying abnormal numbers of chromosomes such as trisomies and monosomies. FISH is also useful when probes are available for specific regions of chromosomes to determine if deletions, translocations, or duplications are present. In the above diagrams, a trisomy 21 is depicted at the right, while a probe for chromosome 22 has detected a translocation, probably to chromosome 9, on the left.

DNA section showing gene ACT on the descending strand and the gene AGT on the ascending strand

DNA double helix

Replication

Central Dogma

DNA contains codes

DNA vs RNA

DNARNA

• Sugar deoxyribose ribose• Bonds with Adenine thymine uracil• # of Strands two one

Kinds of RNA

• Messenger RNA (mRNA)• Ribosomal RNA (rRNA)• Transfer RNA (tRNA)

Transcription

• DNA unwinds• RNA polymerase recognises promotor• Binds• Termination code in DNA

Processing the mRNA transcript

Translation

Mutation

• Frameshift mutation: • THE BIG RED ANT ATE ONE FAT BUG

THB IGR EDA NTA TEO NEF ATB UG?

• Point mutation:• THE BIG RED ANT ATE ONE FAT BUG

THA BIG RED ANT ATE ONE FAT BUG

• Silent, Missense and Nonsense Mutation

Mendelian Inheritance

• 1850’s showed independent units of heredity• Genome: entire complement of genetic

material in a chromosome set• Genetic polymorphism: naturally occurring

differences between individual members of a population

TERMS

• Genotype: Genetic contribution inherited from parents

• Phenotype: Observed variations in physiology/morphology

• Forward Genetics: individuals of 2 distinct phenotypes then to DNA level

• Reverse Genetics: stretch normal DNA and insert a mutation

Single gene inheritance pattern

• Useful for gene discovery eg Cystic Fibrosis/ Tay-Sachs

• Human is a diploid organism with 2 complete genomes and therefore 2 identical chromosome sets ie 2n = 46

• 2 members of a chromosome pair called homologous chromosomes/homologs

• Each gene a gene pair

Mendel’s First LawThe Law of Equal Segregation

• Garden pea, used pre-exisiting mutants• Seven properties each had 2 contrasting

phenotypes• All lines used were pure lines (produced

identical offspring)• Eg seed colour of green or yellow• Made crosses and observed results

Seed colour crosses

• Female (yellow seed) x Male (green seed) = F1 (first filial generation) F1 peas all yellow

• Female (green seed) x Male (yellow seed)= F1 peas all yellow

Selfing the F1 generation

• F1 pea x F1 pea= F2 generation¾ yellow, ¼ greenGreen phenotype had re-appeared fromyellow parents

Selfing the F1 generation

Individually selfed F2 generation

• F2 green seeded peas selfed only gave green peas

• F2 yellow seeded peas selfed were found to be of two types:

• 1/3 pure breeding for yellow• 2/3 progeny ratio of ¾ yellow seeds and ¼

green seeds

More information!

• Mendel crossed an F1 plant with a green-seeded plant

• ½ yellow and ½ green

What this means in modern terms

• Gene: a hereditary factor is necessary for producing pea colour

• Gene has 2 forms or allelesEg Y (yellow phenotype) y (green phenotype)A plant can be Y/Y, Y/y or y/yThe ‘/’ denotes a pair of alleles

Dominant/Recessive

• In Y/y plants the Y allele dominates and phenotype will be yellow

• Therefore Y allele is dominant and y allele is recessive

• In meiosis members of a gene pair segregate equally into the gametes ie Mendel’s 1st Law of equal segregation

Terminology

• A plant with a pair of identical alleles is called a homozygote

• A plant where pair of alleles differ is called a heterozygote

• A heterozygote for one gene is also sometimes called a monohybrid

Which means

• Individual can be:• Homozygous dominant (Y/Y)• Heterozygous (Y/y)• Homozygous recessive (y/y)• Allelic combinations underlying phenotypes

are called genotypes eg Y/Y, Y/y and y/y

Testcross

• A cross of an individual organism of unknown genotype or a heterozygote with a tester

Message

• All 1:1, 3:1, and 1:2:1 ratios are diagnostic of single-gene inheritance and are based on equal segregation in a heterozygote

Sex-Linked Inheritance

• Humans 22 homologous pairs of chromosomes and 2 sex chromosomes

• Females 2 X chromosomes• Males X and Y (non-identical pair)• Homolgous and differential regions• Y chromosome SRY genes (male sexual

function)

Sex Linkage

• Genes in the differential regions show inheritance patterns called sex linkage

• X linkage: mutant alleles in the differential region of the X chromosome show this

• Phenotypic rations can be different in each sex

• Pseudoautosomal region

Human Pedigree Analysis

Human Pedigree Analysis

• No controlled matings• Study medical history of family to see if

consistent with single gene inheritance• Propositus is subject who brought the case to

attention• Draws a family tree using standard symbols• Difficult to use ratios as small numbers

Autosomal recessive

• Eg Phenylketonuria (or cystic fibrosis) p allele recessive

• Sufferers p/p• Others P/P or P/p• Disorder usually appears in progeny of

unaffected parents• Affected progeny includes males and females

Human pedigree of autosomal recessive disorder

Simple Monohybrid Cross

Autosomal dominant disorders

• Defective allele is dominant• So a rare disorder can be dominant• Eg pseudoachondroplasia, Huntington’s disease• Pedigrees show affected males and females in each

generation: they also show affected men and women transmitting the condition to equal proportions of their sons and daughters

X-linked recessive disorders

• Pedigrees have more males than females showing the phenotype under study

• Female would need her mother AND her father to carry the defective allele

• None of female offspring of affected male show the condition but are carriers

• None of sons of affected male show the female as they inherited his Y chromsome

X-linked recessive disorders

• Red-green colour blindness• Hemophilia (Factor VIII)• Duchenne muscular dystrophy• Androgen insensitivity syndrome

X-linked dominant rareeg hypophosphatemia

Calculating risks in pedigree analysis

• Tay-sachs disease

History

• Newly-married husband and wife find they both had an uncle with Tay-Sachs disease (autosomal recessive disease)

• Calculate the probability of the children’s first child having the disease

Calculation

• Neither of the couple has the disease so each could be normal homozygote or heterozygote

• If both are heterozygotes, they could each pass a recessive allele to their child who would be affected

More maths

• We calculate the probability of the couple both being heterozygotes and then, if so, the probability of passing the deleterious allele onto a child

• The husband’s grandparents must have both been heterozygotes (T/t) because they had a t/t child (like a monohybrid cross)

• The husband’s father could be T/T or T/t, but we know the relative probabilities of these genotypes must be ¼ and ½. Therefore 2/3 probability that the father is a heterozygote

And again

• Husband’s mother is assumed to be T/T as allele is rare. Thus if the father is T/t then the mating was T/t x T/T

• Expected proportions of progeny are ½ T/T and ½ T/t

Product Rule

• Overall probability of the husband’s being a heterozygote is calculated using product rule

• “The probability of two independent events both occurring is the product of the individual probabilties”

• Gene transmission is an independent event• Therefore probability of husband being a

heterozygote is 2/3 x ½ = 1/3

Now the wife

• Likewise the probability of her being a heterozygote is 1/3

• If they are both heterozygotes (T/t) their mating is again a standard monhybrid cross so probability of their having a t/t child is 1/4

Overall

• Overall, the probability of the couple’s having an affected child is the probability of them both being heterozygotes and then both transmitting the recessive allele to the child which again is an independent event

• Therefore probability is:• 1/3 x 1/3 x ¼ = 1/36• Ie a 1 in 36 chance of having a child with Tay-Sachs

disease

Mendel’s Second Law: The Law of Independent Assortment• “Gene pairs on different chromosomes assort

independently at meiosis”• Dihybrid crosses or 9:3:3:1• Punnett square or 4 x 4 grid

The sum rule

• “the probability of either of two mutually exclusive events occurring is the sum of their individual probabilities”

The chi-square test

• A statistical test used to determine the probability of obtaining observed proportions by chance, under a specific hypothesis

• Or “how close to an expected result is close enough”

Polygenic inheritance

• Most variation in natural populations take on the form of continuous variation eg height, weight, colour (bell-shaped distribution)

• Environment plus genes• Interacting genes underlying hereditary continuous

variation are called polygenes or quantitative trait loci (QTL’s)

• Often distributed throughout the genome

Recombination

• “Mapping”• Physical maps shows the genes as segments

arranged along the DNA molecule of the chromosome

• Recombination maps map the loci of genes that have been identified by mutant phenotypes showing single-gene inheritance

• Uses Linkage analysis

Recombinant frequency to recognise linkage

• When geneticists say two genes are linked they mean that the loci of the two genes are on the same chromosome hence any alleles on any one homolog are physically joined

• When 2 genes are close together on the same chromosome pair ie linked they do not assort independently but produce a recombinant frequency of less than 50% ie a recombinant frequency of less than 50% is diagnostic for linkage

How crossovers produce recombinants for linked genes

• How are any recombinants produced?• When homologous chromosomes pair at

meiosis the chromosomes occasionally break and exchange parts in a process called “crossing over”

• Two new products are crossover products

Mapping by recombinant frequency

• Recombinant frequencies for linked genes vary between 0 to 50% depending on closeness providing the basis for a genetic map

• One genetic map unit is the distance between genes for which one product of meiosis in 100 is recombinant

• A recombinant frequency of 10.7% is sometimes called a centimorgan (cM)

Using Lod scores to assess linkage in human pedigrees

• “log of odds” useful in humans with small numbers• Calculates 2 different probabilities• 1. calculate probability of occurring with independent

assortment• 2. calculate probability of occurring with the assumption of a

specific degree of linkage• Ratio of probabilities calculated, the logarithm of the number

taken which is the Lod value• Can add the Lod value to those accumulated from other

researchers• Quite a rigorous test

Gene Interaction

• How do genes in a set interact?• Often several known mutants of a gene• Incomplete dominance- phenotype based on

2 alleles of a single gene but the heterozygote is of intermediate phenotype

Codominance

• Expression of both alleles in a heterozygote• Eg human ABO groups• Genotype Blood type• IA/IA, IA/I A• IB/IB, IB/I B• IA/IB AB• i/i O

Penetrance

• Penetrance: the percentage of individuals with a given allele who exhibit the phenotype associated with the allele

• Why wouldn’t this be 100%?• Environment• Other interacting genes• Subtlety of the mutant phenotype

Expressivity

• The degree to which a given allele is expressed at the phenotypic level ie the intensity of the phenotype eg brown hair

• May be due to variation in the allelic constitution of the rest of the genome or environment

Is a trait heritable?

• Correlations between relatives are only evidence for genetic variation if the relatives do not share common environments familiality or heritability

• Twin/adoption studies: need to have no correlation between adopting families to rule out environment. Very difficult to meet!

Molecular Techniques

• Restriction enzymes: An endonuclease (cuts nucleotide chain with straight or ‘sticky’ends) that will recognise specific target nucleotide sequences in DNA and break the DNA chain at those points; a variety of these enzymes are known and they are extensively used in genetic engineering

Southern blot

Agarose gel and X ray film

Restriction Fragment Length Polymorphisms

Some definitions

• Cloning: in recombinant DNA research is the process of creating and amplifying specific DNA segments

• Gene therapy: the correction of a genetic deficiency in a cell by the addition of new DNA and its insertion into the genome. Different techniques have the potential to carry out gene therapy only in somatic tissues or to correct the genetic deficiency in the zygote

Prenatal identification

• Chorionic Villus Sampling used to detect specific known genetic disorders eg Down syndrome. Reliable test. 11-13 weeks of pregnancy. Cells grown in the lab for 2 weeks, detects trisomies 13, 18 and 21 (after 72 hours). Produces a karyotype

• Amniocentesis week 16 onwards. Cells grown and 2 weeks to results

Genetic counselling

• Tests performed on people who do not have the disease themselves, there are a number of issues to discuss before testing. The counsellor discusses pros and cons of testing eg do you want to know? What would you do with the information and how might you react?

Role clinical geneticist• Diagnosis of genetic disorders affecting all ages and all body

systems, birth defects and developmental disorders• Investigation and assessment of genetic risk• Genetic counselling• Follow up, support, co-ordination of health surveillance• Offer genetic services to extended family if appropriate• Liaison with genetic labs• Education and training• research

Organisation of clinical genetic services

• Usually regionally based in a major city• Currently 25, 100 consultants in field• Many centres offer joint clinics with different

departments• The interface between scientists and patients

DNA Banks• Researchers from the UK are taking part in a global study of the link

between genetic variation and diseases. • Scientists from the UK, US and China will work together to create the

largest DNA database in the world.• The 1000 Genomes Project will map the DNA make-up of 1000 people

from different parts of the world to create a detailed catalogue of the most common genome variants.

• It is hoped this will lead to a greater understanding of the relationship between genetic variation and common diseases - benefiting both the medical and human biology sectors.

Now for the test!

Terms to remember

• Congenital abnormalities• Genetic abnormalities• Terratogenic• Heritability, proband• Chromosomal abnormalities, including

numerical• Deletions/ microdeletions

Terms to remember• Autosomal versus• X-linked: females carriers, males show the disease• Heterozygotes/ homozygotes-who is affected Mendelian

patterns• Alelles• Genome• Genotype/fenotype• Genetic studies: family, twin, adoption- concordance rate,

monozygotic, dyzigotic

Terms to remember• Mitosis• Meiosis• Mithocondrial DNA (always maternal, both sexes can suffer)• Linkage• Polygenic trait is one whose phenotype is influenced by more than one

gene. Traits that display a continuous distribution, such as height or skin color. Do not show the phenotypic ratios characteristic of Mendelian inheritance, though each of the genes contributing to the trait is inherited as described by Gregor Mendel.

• Many polygenic traits are also influenced by the environment and are called multifactorial.

Genetics linked with:Schizophrenia: heritability about 82%, life time risks in

relatives of patients with schizophrenia, probably genetically heterogenous, no single locus responsible demonstrated so far; children of mothers with S have 13% chance of S both in adoption and twin studies page 358 Puri Hall

Huntignton Disease• Wilson Disease• Prader Willi- deletion chromozome 15• Learning disability syndromes and autism

Genetic aspects of epidemiology/ what is involved, gender rates, ethnicity, onset

• Schizophrenia- risk if a parent, other sibling or twin brother have schizophrenia

• Mood disorders: 26% biological: 12% adoptive parents, complex genetic heterogeneity, overlapping sets of susceptibility genes ie. COMT encoding for tyrosine hydroxilase, serotonin transporter and BDNF brain derived neurotropic factor

• Depression/ endogenous• Bipolar affective disorder• Anxiety disorders- spectrum of neurotic traits, prevalence is

1in 6 in general population in UK• Personality disorder/ psychopatic traits

Example-Dementia• Neuron loss with age but not necessarily loss of

function • Accumulation of Tau protein resulting in

neurofibrillary tangles• Senile plaques: aggregation of amyloid• APP gene (amyloid precursor gene) in early onset

dementia, runs in families• Amyloid deposition predisposed by apolipoprot E e4

allele on Chr 19• Presenilin 1 PS1 on Chr 14 and PS2 on Chr 1