Applications of Genetics

Learning Objectives:1. Human genetics• Pedigree Analysis• Genetic Screening• Genetic Counseling• Gene Therapy• Human Genome Project

2. Plant and animal breeding• Artificial Selection• Cloning

3. Recombinant DNA Technology and its applications

4. DNA fingerprinting and its applications5. Implications of genetic manipulation: the

potential benefits, hazards and ethical issues.

Human Genetics1. Pedigree Analysis

Pedigrees = Family Trees• One of the central tasks of the human

geneticist • Pedigree analysis is the construction of

family trees • Family history information is often

collected at major family gatherings • A pedigree is used to trace inheritance of

a trait over several generations.

Symbols used in pedigree charts:

Inheritance of Haemophilic trait in the Royal Family

Three primary patterns of inheritance:

1.autosomal recessive 2.autosomal dominant 3.sex-linked (X-chromosomal)

Autosomal Recessive Pedigree • Recessive: neither parent has the

characteristic phenotype (disease) displayed by the child

• Autosomal: Gene is on one of the autosomes (Chromosomes 1-22).

• Phenotype only occurs under homozygous condition

Autosomal Dominant Pedigree

• Dominant: Affected individuals can appear in every generation

• Autosomal: Gene is on one of the autosomes (Chromosomes 1-22).

• Phenotype occurs under both homozygous and heterozygous conditions

Sex-Linked Pedigree

• X-linked: The trait is preferentially seen in males, who are homozygous. Females are heterozygous "carriers"

• Most X-linked traits are recessive. • Inheritance of red-green color blindness, an X-linked, recessive trait:

•Color blind male is father of "carrier" daughters and normal sons. •Carrier daughters have 50% chance to have color blind sons. •Color blind male x carrier female can produce color blind daughters •Can you determine a pedigree for Red-Green Colorblindness?

For more information, please refer to the following website:Pedigree analysis problem set available at

http://www.biology.arizona.edu/human_bio/problem_sets/color_blindness/intro.html 

Genetic screening • Diagnosis of inherited diseases before symptoms occur by finding abnormalities in the genes or chromosomes.Some examples: •sickle cell anaemia •cystic fibrosis •phenylketonuria (PKU) •Down’s SyndromeMost of these tests performed on cells removed from the fetus (pre-natal diagnosis) and even from a pre-implantation embryo.

Ultrasound

•One of the simplest and easiest genetics screening tests •Evaluate the physical characteristics of the fetus which may help predict certain abnormalities • Measurements of various body parts can be used to suggest an increased risk of Down Syndrome •look for congenital defects such leaky heart valves, and birth defects such as cleft lip or club foot. •Ultrasound is also used to guide the physician during amniocentesis and chorionic villus sampling (CVS).

An example of ultrasound image

Amniocentesis

Remove fetal cells from amniotic fluid 14 to 22 weeks after pregnancy.Culture the cells and look for•chromosome abnormalities (e.g., the three number 21 chromosomes of Down syndrome); •certain enzymatic defects (e.g., an inability to metabolize galactose, hence milk); •the sex of the fetus.

Risks:

1. To the mother: slight discomfort, adverse reaction to medications used, vaginal bleeding or cramping, and infection.

2. To the foetus: inadvertent puncture is small 3. To the pregnancy: chance to miscarriage very

low (not more than 1/200)

Couples who may wish to consider amniocentesis include:

1. Women 35 years of age and over 2. Parents who have had a child with Down's

syndrome or other chromosome abnormality.

3. Couples who are known carriers of a chromosome rearrangement

4. Couples who have had a child with a malformation of the brain or spinal cord

Chorionic villus sampling (CVS)

Procedure:• Suck out placental cells by a tube inserted

through the abdomen or vagina.Advantages:• No need to perform cell culture• Can be performed earlier in pregnancy (10-

12 weeks)• If an abortion is to be performed, it is a

simpler process early in pregnancy

Drawbacks: • More risky than amniocentesis• Increase the chance of miscarriage (approximately

0.8%) • Minor complications such as vaginal bleeding or

cramping occur more frequently following CVS than amniocentesis

• Infection • Transverse limb defects in infants can be resulted • Involve the absence of the distal structures of the

limb • May result in the vascular system disruption of the

limb • Risk for transverse limb defects following CVS is

approximately 0.03%-0.10%

Couples who may wish to consider CVS include:

• Women 35 years of age and older • Parents who have had a child with Down's

syndrome or other chromosome abnormality • Couples who are known carriers of a

chromosome rearrangement • Couples who have a family history of a

genetic condition for which testing is available

Common genetic diseases diagnosed by CVS

•Chromosomal abnormalities such as Down’s, Klinefelter’s and Turner’s syndromes can be identified by karyotype analysis.•The cells can be cultured for DNA analysis that helps diagnosis of 1. Cystic fibrosis 2. Huntington’s chorea 3. Thalassemia

Genetic CounselingGenetic counselors are trained persons who help individual and family to

• comprehend the medical facts, including the diagnosis, probable course of the disorder, and the available management;

• appreciate the way heredity contributes to the disorder, and the risk of recurrence in specified relatives

• understand the alternatives for dealing with the risk of occurrence

• choose the course of action which seems to them appropriate in view of their risk, their family goals, and their ethical and religious standards, to act in accordance with that decision

• make the best possible adjustment to the disorder in an affected family member and/or the risk of recurrence of that disorder.

Who Should be Referred?

1. Families in which one or more members have a serious birth defect or genetic disease.

2. Families who have a child with multiple congenital anomalies, serious developmental delay, or an unexplained abnormality of growth.

3. Families in which more than one close relative has the same disease, e.g., mental retardation, deafness, blindness, cancer, early heart attacks, or schizophrenia.

4. Couples who have had repeated stillbirths or a stillborn baby with birth defects.

5. Couples who are close blood relatives, e.g., first cousins.

Common issues discussed during genetic counseling

1. Making a diagnosis 2. Investigate the family history for previous cases

of genetic diseases through pedigree analysis3. calculate and explain the risk of having

affected children 4. explain the cause of the disease 5. Quality of life• The quality and likely length of an affected

child’s life will be discussed• Availability of treatment, support groups and

financial help will also be discussed.• Effects on other family members will be

discussed

Options:

• These include contraception, sterilization, adoption, artificial insemination to avoid the husband’s genes being passed on or IVF using a donor egg if the problem lies with the woman.

• Prenatal diagnosis is an option if the couple are willing to consider abortion of an affected fetus.

• CVS, amniocentesis and abortion must be discussed.

• The reliability of tests such as DNA analysis, ultra-sound scanning must be explained.

• Possibility of IVF and gene therapy

Ethical issues in genetic counseling

During postnatal counsel•Risk evaluation, preventive options, gene therapy, testing of all family members, confidentiality within the couple During prenatal counsel•Option of pregnancy termination, selective implantation, sex selection Problems of Eugenics (The birth of designer baby)

Gene Therapy

A new method for treating genetic diseases by replacing faulty genes with normal genes or adding normal genes if they are absent.Two types of gene therapy:Somatic cell gene therapy - the insertion of genes into body cells which function only in the treated person and are not passed on to the offspring. Germline gene therapy - the insertion of genes into cells that are involved in reproduction which can be passed on to the future offspring.

Diagrammatic Illustration of Gene Therapy

General Procedure of Gene Therapy:• Isolate and clone the normal gene. • Introduce it into the chosen human cells

with the help of a safe and efficient vector.• The cells may have to be isolated from the

body first, corrected and then replaced. • Easier for blood diseases such as sickle cell

anaemia because the cells that make blood cells can easily be removed from bone marrow and replaced.

• The final problem is to make sure that the gene is expressed normally.

Common Vectors Used:

•Viruses (e.g. Retrovirus) carrying the required gene•Liposomes containing the cDNA clone•Microinjection and electroporation- Direct injection of donor DNA into the cell

Applications of Gene Therapy•e.g. Cystic fibrosis•Cystic fibrosis affects the epithelial cells of the body, but the life-threatening problems mainly affect the lungs. •Lung and trachea epithelial cells are therefore the initial targets for gene therapy. The aim is to get the gene into the cells so that it can make the normal protein, known as CFTR •The cDNA clone is enclosed in a specially designed vector.•Adenovirus infecting the respiratory tract is currently used as the vector for the CFTR gene•The vector does not insert its DNA into the host DNA. If the cell divides, the new DNA is not replaced at the same time so it eventually becomes diluted.•The treatment may only be effective for a few weeks until the epithelial cells die, but it will be easy to repeat the treatment at regular intervals.

Advantages and Disadvantages of Gene Therapy

Advantages DisadvantagesAddition of normal allele will alleviate symptoms

Only somatic cell therapy allowed

Avoid need for constant medication

Not a permanent cure as cells shed

Extend life expectancy

Viral vector may give inflammation problems

Possibility of DNA entering cells other than target cells with unknown effects

1. Ethical issues of gene therapy• Germline therapy is controversial because it

opens up the whole field of eugenics.• The same technique can be used to add

genes for desired characteristics. (For example, the American public has demonstrated a desire for enhanced growth of normal children with demands for human growth hormone)

• Germline therapy is controversial because the change can be passed on to the children of the treated person and all subsequent generations.

• Do we have the right to alter the genome of the future generation?

Human Genome Project (HGP)The goals of the project are to:• identify all the approximately 30,000

genes in human DNA • determine the sequences of the 3 billion

chemical bases that make up human DNA

• store this information in databases • develop faster, more efficient

sequencing technologies • develop tools for data analysis, and • address the ethical, legal, and social

issues (ELSI) that may arise from the project.

Applications of HGP

1. Molecular Medicine • Improved diagnosis of disease • Earlier detection of genetic predispositions

to disease • Rational drug design • Gene therapy and control systems for

drugs • Pharmacogenomics "custom drugs"

2. Microbial Genomics

• New energy sources (biofuels) • Environmental monitoring to detect

pollutants • Protection from biological and

chemical warfare • Safe, efficient toxic waste cleanup • Understanding disease vulnerabilities

and revealing drug targets

3. Risk Assessment

•Assess health damage and risks caused by radiation exposure, including low-dose exposures •Assess health damage and risks caused by exposure to mutagenic chemicals and cancer-causing toxins •Reduce the likelihood of heritable mutations

4. Bioarchaeology, Anthropology, Evolution, and Human Migration

•study evolution through germline mutations in lineages •study migration of different population groups based on female genetic inheritance •study mutations on the Y chromosome to trace lineage and migration of males •compare breakpoints in the evolution of mutations with ages of populations and historical events

5. DNA Forensics (Identification)

•Identify potential suspects whose DNA may match evidence left at crime scenes •Exonerate persons wrongly accused of crimes •Identify crime and catastrophe victims •Establish paternity and other family relationships •Identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers) •Detect bacteria and other organisms that may pollute air, water, soil, and food •Match organ donors with recipients in transplant programs •Determine pedigree for seed or livestock breeds •Authenticate consumables such as caviar and wine

6. Agriculture, Livestock Breeding, and Bioprocessing •Disease-, insect-, and drought-resistant crops •Healthier, more productive, disease-resistant farm animals •More nutritious produce •Biopesticides •Edible vaccines incorporated into food products (Biopharming) •New environmental cleanup uses for plants like tobacco

Ethnical problems• Fairness in the use of genetic information by

insurers, employers, courts, schools, adoption agencies, and the military, among others.

• Who should have access to personal genetic information, and how will it be used?

• Privacy and confidentiality of genetic information.

• Who owns and controls genetic information?• Psychological impact and stigmatization due

to an individual's genetic differences. • How does personal genetic information affect

an individual and society's perceptions of that individual?

More information on HGP is available at

Dolan DNA learning center (2002), Gene Almanac, [Online] Available at http://www.dnalc.org/resources/resources.html

http://www.web-and-flow.com/members/efitzger1/genetics/webquest.htm (The Human Genome WebQuest)

Selective Breeding of Animals and Plants

Aims: To select desirable traits and remove

undesirable traits of plants and animals through artificial selection

Two types of breeding involved in artificial selection:

(1)Inbreeding (2)Outbreeding

Inbreeding – mating of closely related individualsAdvantages: Create pure line over time Better adapted to steady environment.For exampleParental genotypes: Ffgg x Ffgg

Gametes: Fg , fg Fg , fgF1: FFgg Ffgg Ffgg ffgg

(pure breeding)

Disadvantages of inbreeding

1. Increasing the chance for recessive genes to be homozygous

2. Most recessive genes are undesirable traits

3. Leads to a high frequency of defects present at birth

4. Reduce the genetic variability vigour and fertility of a population.

Outbreeding - mating of individuals that are unrelated

Advantages:• Progeny (offspring) are heterozygous

and the bad recessive genes are masked by normal dominant alleles.

• Hybrid vigour – Progeny are tougher, more fertile and have a greater chance of survival

• Produces variation/heterozygosity; on which natural selection can act.

Disadvantage:No more pure line exists

ExamplesThe cross of larger, non-sweet-tasted tomato with small, sweet-tasted tomato. The cross of high-resistant to pest and disease crop with higher-yield crop.

Parental genotypes: FFgghhIIjj x FFGGHHiiJJ Gametes: FghIj FGHiJF1: FfGgHhIiJj

Higher chance of heterozygosity in F1Result in hybrid vigour

hybrid maleparent

femaleparent

The hybrids are stronger and bigger in size than their parents

Artificial Insemination for Domestic Animals

Placing semen within the female reproductive organs through any means other than sexual intercourse. Particularly useful with: •Conditions preventing vaginal ejaculation •Sperm with low motility (movement) •Cervical mucus that is hostile to sperm •Presence of sperm antibodies

Procedure:Intracervical insemination (ICI) OR Intrauterine insemination (IUI) ICI - Fresh ejaculate (semen) is placed directly into the cervix (opening to the uterus) by using a syringe and cannula (long, slender tube).

IUI - "Washed" semen is placed directly into the uterus via a catheter through

the cervix.

Success Rates

ICI - 2% or higher chance of pregnancy per cycle

IUI - Higher probability of pregnancy than ICI. Generally, 5 - 20% chance of pregnancy per cycle Rates are higher when AI is accompanied by superovulation using fertility drugs.

Typical Procedure Protocol:

Pre-procedure: • Thorough evaluation of both male and female for

suspected causes of infertility • Determination of drugs to be used (or not) and timing

of treatment cycle • Monitoring by ultrasound and blood work to evaluate

ovarian response to drugs and reduce potential side effects

• If drugs result in ovarian hyperstimulation syndrome, treatment cycle will be discontinued immediately.

• In case of no drug use, patient may be required to monitor own cycle with ovulation predictor kits and other methods induction of ovulation by HCG , if necessary

During procedure:

• Male partner must produce a semen sample, usually in the clinic (may be collected at home in some cases), or donor semen must be available

• Female partner is inseminated while lying down, feet in stirrups, on examination table

• Female may be advised to refrain from strenuous physical activity for 24 to 48 hours after insemination

Advantages of using artificial insemination

1. Sperm from one male can be used for many females;

2. One ejaculate can be used for several inseminations;

3. Speeds up selective breeding; 4. Saves cost of keeping male and travelling

for mating5. Less stress than mating;6. Allows genetic testing

Disadvantages of using artificial insemination

• Danger of inseminating too many females with one male causing inbreeding;

• With consequent genetic problem;• Undetected genetic weakness• Requires expertise;• Cost of vet.;• Sperm damage in storage;• Not successful in some species ;

Diagram showing how sperms are collected

Selective breeding of animals – sperm bank (for domestic animals and human)

1. Sperms are collected and frozen in liquid nitrogen for further use.

2. Endangered animal species, e.g. giant pandas can be bred

3. Sperm from one male (with good quality) can be used for many females

4. Infertility treatment in human

Selective breeding of animals –embryo transfer breeding (for domestic animals and human)

Procedure:• Injections of hormone to stimulate and

ovulation in the animals that you want to get the embryos from.

• The donor is inseminated at normal time • Seven days later, rinsing out the uterus to

extract the embryos and ova (unfertilised, fertilised or degenerate)

• Isolation of the good embryos using a microscope and then transfer into the recipient animals e.g. surrogate.

Applications:• Domestic animal e.g. cow1. Produce up to ten or more progeny per year

from their best cows.2. Profit from the increased sale of quality

genetics without losing the bloodlines.3. Extend the productive life of some older cows,

incapable of carrying another calf by producing further progeny through the use of embryo transfer.

4. Conserve the genetics in their herd through the uses of, embryo freezing for, export, domestic sale or future transfers on their own farm.

• Infertility treatment in human