Genetics and Molecular Biology in Medicine

Assigned Readings: pages 251-272 of course reader pages 1-51 of course reader

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What is Genetics
Genetics is the study of variation in a population. Genetics studies how variation is produced, how it is passed on from parents to offspring, and how patterns of variation change from generation to generation.

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Basic Mechanism of Human Inheritance

Genes are present as segments on 23 pairs of strands of DNA molecules (chromosomes) Two pairs each of 22 autosomal chromosome One pair of sex chromosome (two of the same chromosomes in female; one X and a Y in male) Some genes are expressed if they are present in one functional copy (dominant) Some genes are expressed only if they are present in two functional copies (recessive). Same rule apply to sex chromosomes in female, but not in male (only one copy of X)
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Review: Terms to Know

Chromosome- a linear sequence of genes on a strand of DNA. Alleles a particular variation at any point of a chromosome. Locus- the position of a allele on a chromosome. Trait- see phenotype Homozygous- presence of two identical alleles at a given chromosomal locus. Heterozygous-presence of two different alleles at a given chromosomal locus Genotype- genes associated with a trait Phenotype-observable expression of the genotype Genetic Linkage- when two different alleles are passed on from parents to offspring at a greater frequency than expected from random chance.

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Simple Modes of Inheritance

Autosomal Recessive Autosomal Dominant
sick
Heterozygous carrier

sick

sick

sick

Recessive sex-linked inheritance

sick
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sick sick

More Complex forms of Inheritance

Multigenic traits more than one gene encodes for phenotype (ie. diabetes) Quantitative traits (Expressivity) traits that vary in the extent to which they are expressed in each individual (example: height, weight) Incomplete Penetrance not every individual who has the genotype expresses the phenotype. Multifactorial traits Both environmental and genetic components (Hemochromatosis)
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Some exceptions to Mendelian Inheritance

Mitochondrial inheritance- In addition to 23 chromosomes, there is a mitochondrial chromosome and only mother mitochondrial chromosome is inherited. Genetic Imprinting- genes are only expressed from a chromosome orginating in one of the parents, due to silencing of other chromosomes. Uniparental Disomy both copies of each chromosome come from a single parent Genetic anticipation- the severity of the genotype increases from generation to generation (ie. triplet repeat expansion).
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Genomewide Association Studies

A genome-wide association study is an approach that involves rapidly scanning markers across the complete sets of DNA, or genomes, of many people to find genetic variations associated with a particular phenotype or disease state. http://www.genome.gov/20019523

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Allele Frequency
Allele frequency: For a specific chromosomal locus in a population, the number of occurrences of a specific variant (allele), divided by the total number of alleles at that locus.

A patient population has 10 AA, 20 Aa, and 40 aa individualswhat are the allele frequencies of this population? 10AA = 20 A alleles 20Aa = 20 A alleles + 20 a alleles 40aa = 80 a alleles
Total----

40 A alleles + 100 a alleles = 140 alleles total

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Allele A frequency = 40/ 140 = 0.285714 Allele a frequency = 100/ 140 = 0.714286

Genotype Frequency
Genotype frequency: For a single chromosomal locus, the percent of occurrences of different allele combinations in the population.
Q: For a specific chromosomal locus of a patient population, the allele frequency of A is 0.8 and the allele frequency of a is 0.2 What is the expected genotype frequency of the population assuming random mating?

A a
A: p q AA = p

= .8x.8

= .64

A a
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p q

p2 pq

Aa = 2pq = 2 ( .8x.2) = .32 pq q2 2 aa = q = .2x.2 = .04

Hardy-Weinberg equilibrium

For a stable (non-evolving) population, the allele and genotype frequencies are constant from generation to generation

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Haplotype frequency
The population frequency of two or more allele at different chromosomal loci.
# of individuals 20 15 10 A 5 a

B frequency AB = 20/50 = 0.4 frequency Ab = 10/50 = 0.2 frequency aB = 5/50 = 0.1 frequency ab = 15/50= 0.3

Allele frequency A = 30/50 = 0.6 Allele frequency B= 25/50 = 0.5 Allele frequency a = 20/50 = 0.4 Allele frequency b = 25/50 = 0.5
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Haplotype Haplotype Haplotype Haplotype

Linkage Disequilibrium
Linkage disequilibrium: In a population, the non-random association of two alleles at DIFFERENT CHROMOSOMAL LOCI. # of individuals 20 A 15 a 10 A 5 a

B frequency AB = 20/50 = 0.4 frequency Ab = 10/50 = 0.2 frequency aB = 5/50 = 0.1 frequency ab = 15/50= 0.3

Allele frequency A = 30/50 = 0.6 Allele frequency B= 25/50 = 0.5 Allele frequency a = 20/50 = 0.4 Allele frequency b = 25/50 = 0.5

Haplotype Haplotype Haplotype Haplotype

Expected Haplotype frequency AB = 0.6 x 0.5 = 0.3 Expected Haplotype frequency Ab = 0.6 x 0.5 = 0.3 Expected Haplotype frequency aB = 0.4 x 0.5 = 0.20 Expected Haplotype frequency ab = 0.4 x 0.5 = 0.20 D = observed haplotype freq expected haplotype freq = 0.4 0.3 = 0.1 2009 Gus Rosania

Gene association study data analysis

Genetics studies in human population are complicated by variations in age, height, weight, gender, ethnicity, environmental exposures, prior drug use,etc. These are not issues in controlled animal studies. In human populations, linkage disequilibrium for variant loci that lie in close proximity to each other tend to be the rule, so the number of haplotypes are much less than expected based on the total number of genetic variants. Gene variations and environmental exposures are often linked to ethnic background of population.
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Gene association study designs

Case control- uses patients who already have a phenotype and look back to see if there are genotype characteristics of these patients that differ from those who dont have the outcome. Cohort- uses patients who have a purported genotype and look forward to see if there are resulting phenotypic characteristics that differ from those who dont have the genotype. Trio- relates the phenotype and genotype characteristics of the parents to the phenotype and genotype characteristics of the offspring to detect specific phenotypes that occur more frequently in offspring with a specific genotype.
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Linear Regression
A mathematical method to assess the relationship between a measured dependent variable, on one or more independent variables.

For example: y = A + Bx + Cx + Dx + Ex + 1 2 3 4

Where y and x are some measurable, quantitative phenotypic feature (like y is rate of drug clearance; x1 is patient weight, x2 is age, x3 is 1 if a patient carries the major allele and 0 if the minor allel, and x4 is 1 if the patient carries the minor allele and 1 if the major allele, and is an error term that is calculated) For a patient population, y,x1, x2, x3 and x4 can be measured. Linear regression calculates a single value for A, B, C, and D that minimizes the sum of the errors . These calculated values for A, B, C, D and E capture the contribution of each independent variable x on the dependent variable y.

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Logistic regression
a related way to assess the relationship of several different factors to a probabilistic outcome, as modeled by the logistic equation:

y=

1 1 + e -z

z = A + Bx + Cx + Dx + Ex + 1 2 3 4 Where y is the probability of something occuring (say, the probability that the drug will cure the patient from 0 to 1) and z is the total contribution of all measured (and error) values to the probability y (for example, x1 can be patient weight, x2 is age, x3 is 1 if a patient carries the major allele and 0 if the minor allel, and x4 is 1 if the patient carries the minor allele and 1 if the major allele, and is an error term that is calculated)
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Homework No. 1
Due next class: Answer questions 1,3,5,7,9 in course reader pages 271-272 In addition, answer the following five questions:
S and s are two alleles of the same chromosomal locus. Given a sample population of 50 SS, 25Ss, and 25 ss individuals, answer the following :
1) What is the allele frequency of the sample population? 2) What is the allele frequency of the first generation, assuming random mating amongst the sample population? 3) What is the allele frequency of the second generation, assuming random mating amongst the first generation? 4) Was the original sample representatitive of a population in Hardy-Weinberg equilibrium? 5) Inspection of a neighboring locus reveals that every chromosome that has the S allele has a B allele, while every chromosome that has an s allele has a b allele. Are alleles S and B in linkage equilibrium?

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Molecular Biology
Molecular biology is the study of how genes encode the structure and function of living things, from the level of the individual molecules to that of organisms.

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Basic tenets of molecular biology

Genes are encoded in the sequence of base pairs of DNA. DNA is transcribed to RNA which can be translated to proteins Proteins are largely responsible for structural and functional activities that we associate with life. Some genes encode for functional RNA molecules (ie. tRNA, ribosomal RNA, miRNA) so not all genes code for proteins)
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DNA Replication
histones DNA AT CG CG TA G C chromatin

helicase A polymerase A

Nucleus A A G T G C A T T C chromosome

T T C G AT CG GC TA

A C G T

Cell
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Transcription and Splicing

exons DNA transcription hnRNA introns Non-coding DNA Non-coding DNA Non-coding DNA

NUCLEUS

splicing
mRNA translation

polypeptide

CYTOPLASM
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Translation
Amino AminoAminoAmino acid acid acid acid polypeptide

tRNA

UCA CUATAAAGUUAAUUCCC U AGGGACCC

mRNA

rRNA Ribosome DNA changes that affect aminoacid sequence are said to be non-silent DNA changes that do not affect aminoacid sequence are said to be silent
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The Genetic Code

TTT TTC TTA TTG CTT CTC CTA Phe Phe Leu Leu Leu Leu Leu TCT TCC TCA TCG CCT CCC CCA Ser Ser Ser Ser Pro Pro Pro TAT TAC TAA TAG CAT CAC CAA Tyr Tyr STOP STOP His His Gln TGT TGC TGA TGG CGT CGC CGA Cys Cys STOP Trp Arg Arg Arg

CTG
ATT ATC ATA ATG

Leu
Ile Ile Ile Met*

CCG
ACT ACC ACA ACG

Pro
Thr Thr Thr Thr

CAG
AAT AAC AAA AAG

Gln
Asn Asn Lys Lys

CGG
AGT AGC AGA AGG

Arg
Ser Ser Arg Arg

GTT
GTC GTA GTG

Val
Val Val Val

GCT
GCC GCA GCG

Ala
Ala Ala Ala

GAT
GAC GAA GAG

Asp
Asp Glu Glu

GGT
GGC GGA GGG

Gly
Gly Gly Gly

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The Cell Cycle

cytoplasm cytoplasm cytoplasm Nucleus Nucleus

Nucleus

Chromosome condensation Chromosome segregation Mitosis cytokinesis

Nucleus

Chromosome decondensation Cell growth DNA replication

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Development of Multicellular Organization

Sperm egg zygote Growth factors Hormones Signal Transduction Transcription factors Extracellular matrix Morphogens

embryo

worm

fly

mouse

monkey

human

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Genes affect normal physiology, growth and reproduction

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Genes affect disease and aging

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OMIM: http://www.ncbi.nih.gov
OMIM: Online Mendelian Inheritance in Man

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Some Important MolBiol Tools

Hybridization DNA Amplification DNA Sequencing Recombinant DNA

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Hybridization
Double stranded DNA Single stranded DNA TACGCCTTTTTAAAAATTTTTACG ATGCGGAAAAATTTTTAAAAATGC

TACGCCTTTTT ATGCTTAAAAA

1. heat ATGCTTAAAAA

2. cool

TACGCCTTTTTAAAAATTTTTACG ATGCGGAAAAATTTTTAAAAATGC

TACGCCTTTTTAAAAATTTTTACG ATGCTTAAAAA TACGCCTTTTT Hybridized DNA ATGCTTAAAAA 2009 Gus Rosania

Example: Genechip/Microarrays
Fluorescent labeled probes Derived from patient DNA T A C A T T T T A T A C A C A C A T A T T T T T AT TA GC TA AT AT

A T G T A A

A T G T A A

AT TA GC TA AT AT

A T G T A A

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DNA Amplification
PCR- polymerase chain reaction A, T, G, C nucleotides Heat stable polymerase Primers flanking sequence of interest DNA 1. Denature with heat

3. Repeat 2. Cool down

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DNA sequencing
ATCGA TAGCAAAGCTTA A A T G C ATCGA TTCG A TAGCAAAGCTTAAA ATCGA TTCGA A TAGCAAAGCTTAAA ATCGA T TAGCAAAGCTTAAA ATCGA T T TAGCAAAGCTTAAA ATCGA TTCGAA T TAGCAAAGCTTAAA

ATCGA TAGCAAAGCTTA

A T G C

ATCGA TAGCAAAGCTTA

A T G C A T G C

ATCGA TTC G TAGCAAAGCTTAAA

ATCGA TAGCAAAGCTTA
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ATCGA TT C TAGCAAAGCTTAAA

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Recombinant DNA
TATGC ATACG 1. cut T ATGC ATAC G TATGC ATACG Bacterial plasmid DNA 3. mix 4. ligate TATGC ATACG

2. cut TATGC ATACG TATGC ATACG ATGC G T ATAC 4. Insert In bacterium

DNA to be cloned

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Applications to PGx analysis

Patient Classification Drug Distribution and Metabolism Drug Receptors Interethnic Pharmacogenetics Multifactorial Pharmacogenetics

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