Genetic variation exists between individuals and populations. This document discusses genetic variation and polymorphisms, including single nucleotide polymorphisms (SNPs). It also discusses allele frequencies, the odds ratio, and Hardy-Weinberg equilibrium. The document summarizes a research article that developed a multiplex assay using SNaPshot minisequencing to simultaneously screen for SNPs associated with fetal hemoglobin levels in genes like BCL11A and HBS1L-MYB.

1. Genetic Variation In Individual And Population
Polymorphism and consequences, Genotype, Phenotype, and
Gene frequencies, Odd ratio, and Hardy-Weinberg Equilibrium
Miss May Soe Thu
5836362 MTMT/M
1

2. Outline
• Genetics
• Genetic Variation
• Polymorphism
• Allele frequencies
• Odd ratio
• Hardy-Weinberg equilibrium
• A Research Article
2

3. Genetics
• The study of genes, heredity, and genetic variation in living organisms
http://ghr.nlm.nih.gov
Human Genome Project: 20,000 – 25,000 genes
www.pinterest.com
3

4. Why genetics is important?
Genetics
Physical
features
Personality
Susceptibility
to diseases
Intelligence
4

5. How is it work?
www.caribbeanbiopharma.com
Genotype Phenotype
DNA RNA Protein
5

6. Basic Genetics Terminology
• Chromosome : A thread-like structure located in the nucleus
• Locus : The position of genes on the chromosome
• Allele : A particular characteristics of the gene at the locus
• Gene : A basic physical and functional unit of heredity
• Genotype : The genetic constitution of an individual organism
• Phenotype : A morphological, physiological, biochemical, or
behavioral characteristics of an individual organism
www.ck12.org
6

7. Genetic variation
• Phenotypic and genotypic differences among individuals or in a population
Identical twins ???
7
www.bio.vtn2.com Popular-archaeology.com

8. Effects of genetic variation
• Can affect how humans develop diseases and respond to pathogens,
chemicals, medication, vaccines, and other agents
P. vivax
Duffy Antigen (-) HLA-B35 Antigen (+)
P. falciparum
8
www.africans-abroad.org

9. Polymorphism
• Variation in parts of the DNA sequence among individuals, no distinction a
person’s appearance.
• In at least 1% of a population
What is the effect on our health?
• No adverse effects on the individual and occur
with fairly high frequency in the general population
http://www.kawarada-lab.com/research/biosensing/DNA_sensor_SNPs.html
9

10. Polymorphisms
Kelly A. et al. Human genetic variation and its contribution to complex traits: vol10, 2009.
Single nucleotide variation
Insertion-deletion variation
Block substitution
Inversion variation
Copy number variation
Structuralvariation
10

11. Single Nucleotide Polymorphisms (SNPs)
• Change in base consequence at a single point
• About 10 millions position in the genome
• Distributed uniformly in genomic DNA
• Used to track the inheritance of disease gene
within families
Coding sequence of gene
Non-Coding regions of gene
Intergenic regions between gene
http://learn.genetics.utah.edu/content/pharma/snips/
11

12. SNP Genotyping Methods
 Next generation sequencing
 SNP microarrays
 Allele-specific hybridization
 Molecular beacons
 RFLP
 PCR-based methods
 Oligonucleotide ligase assay
http://www.nature.com/nbt/journal/v26/n10/fig_tab/nbt1486_F1.html
12

13. Mutation
1) Missense Mutation 2) Nonsense Mutation
http://ghr.nlm.nih.gov/
13

14. Mutation
3) Insertion Mutation 4) Deletion Mutation
http://ghr.nlm.nih.gov/
14

15. Mutation
5) Frameshift Mutation 6) Repeat Expansion Mutation
http://ghr.nlm.nih.gov/
15

16. Gene Frequencies
• How often a particular gene variant occurs in a particular population
Alleles
frequencies
Phenotype
frequencies
Genotype
frequencies
Proportion of all alleles at a given locus in a population
Proportion of how common a condition or trait is
Proportion of all genotypes in a population
16

17. Importance of Knowing Allele frequencies
Shifting the allele
frequencies
Microevolution
• Genetic drift
• Individual
migration
Macroevolution
• Formation of
new species
Underlying rule to track
Constant state of allele frequencies
Hardy-Weinberg
Equilibrium 17

18. Hardy-Weinberg Equilibrium
• To understand evolution, it is necessary to understand how a
non-evolving population works.
www.quora.com
p2
= frequency of homozygous dominant genotype
pq = frequency of heterozygous genotype
q2
= frequency of homozygous recessive genotype
18

19. Hardy-Weinberg Equilibrium
http://www.gloucesterhs.ocdsb.ca/UserFiles/File/Biology.html
19

20. How to calculate Genotype frequencies
AA
AA
AA
AA
AA
AA
AA
AA
aa
Aa
Aa
Aa
Aa
Aa
aa
aa
aa
aa
Aa
20Benjamin A. Pierce. Genetics: A Conceptual Approach. 4th Ed.
𝑓(𝐴𝐴) =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐴𝐴 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠
𝑁
𝑓(𝐴𝑎) =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐴𝑎 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠
𝑁
𝑓(𝑎𝑎) =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑎 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠
𝑁

21. How to calculate Allele frequencies
p = all dominant alleles
q = all recessive alleles
21Benjamin A. Pierce. Genetics: A Conceptual Approach. 4th Ed.
𝑝 = 𝑓 𝐴 =
2𝑛𝐴𝐴 + 𝑛𝐴𝑎
2𝑁
𝑞 = 𝑓 𝑎 =
2𝑛𝑎𝑎 + 𝑛𝐴𝑎
2𝑁
𝑝 = 𝑓 𝐴 = 𝑓 𝐴𝐴 +
1
2
𝑓(𝐴𝑎)
𝑞 = 𝑓 𝑎 = 𝑓 𝑎𝑎 +
1
2
𝑓(𝐴𝑎)

22. Odd Ratio (OR)
• A measure of association between a genetic variation and a disease
• Case-control study
www.scalelive.com
Geneticvariation
Disease
Result Interpretation
OR > 1 Increased risk of disease
OR < 1 Decreased risk of disease
OR = 1 No change in frequency
OR =
Odds of genetic variation with disease
Odds of genetic variation without disease
=
𝐴/𝐶
𝐵/𝐷
22

23. Genotyping of BCL11A and HBS1L-MYB SNPs
associated with fetal haemoglobin levels:
a SNaPshot minisequencing approach
Pavlos Fanis, Ioanna Kousiappa, Marios Phylactides and Marina Kleanthous
Fanis et al. BMC Genomics 2014, 15:108
http://www.biomedcentral.com/1471-2164/15/108 23

24. Globin Genes
http://www.jci.org/articles/view/25398/
24

25. Beta-thalassaemia
• It is characterized by a genetic deficiency in the synthesis of beta-globin
chains, leading to excess alpha chains.
Absent β-globin
Decreased β-globin
Inadequate ϒ-globin
Excess α-globin
No HbA
Decreased HbA
Severe Anaemia
α-globin precipitates
25

26. Sickle cell disease
http://faculty.etsu.edu/millerh/ learn.genetics.utah.edu
Point Mutation in Beta-globin gene
26

27. BCL11A gene
• Encodes a zinc-finger transcription factor
• Repress HbF synthesis (ϒ-globin expression)
• Occupy sites within the locus control region (LCR) and intergenic regions
of the β-globin locus
27
http://140.226.65.22/MOLB7800/Mechanisms%20of%20Gene%20Expression/

28. HBS1L-MYB intergenic region
• Associated with elevated fetal hemoglobin (HbF) levels and alterations
of other clinically important human erythroid traits
28Swee Lay Thein et al. Intergenic variants of HBS1L-MYB are responsible for a major quantitative trait locus
on chromosome 6q23 influencing fetal hemoglobin levels in adults. 2007
Fig: Overview of the 6q23 region and the HMIP locus
Proto-oncogene
Encode c-MYB transcription factor
Erythroid differentiation
MYB

29. Objective
• To develop the multiplex assays based on the SNaPshot-minisequencing
approach for simultaneous screening of known SNPs
www.biomerieux-diagnostics.comLifesequecing.blogspot.com
29

30. Overview
Biological samples
SNP Selection
Design & Optimization of PCR and minisequencing primers
Detection of SNPs
Genotypic profile verification
30

31. Biological samples
Genomic DNA
samples
• 25 confirmed β-
thalassaemia
Extraction • Gentra Puregene Kit
(Qiagen)
DNA
concentration
& purity
• Nandrop ND-1000
spectrophotometer
31

32. SNPs Selection
12 BCL11A + 16 HBS1L-MYB
Updated Hemoglobinopathy sequence variation database
Polymorphisms with variable HbF levels in different ethnic populations with HPFH,
beta-thalassaemia, sickle cell anaemia, beta-cell heterozygotes, and HbE heterozygotes
32

33. Design & Optimization of mPCR primers
Identify the genetic regions of SNPs
Design 44 F & R primers
Screen & Check the primer database
Predict 2’ structure & primer-primer interactions
Amplicons for BCL11A mPCRs
(69-649 bp)
Amplicons for HBS1L-MYB mPCRs
(60-221 bp)
• BLAST
• Biomaths calculator program
• “Human genomic plus transcript
(Human G+T)” database
• OligoAnalyzer 3.1 tool
Purified by standard desalting 33

34. Design & Optimization of minisequencing primers
Identify the genetic regions of SNPs
Design 28 F & R primers with 3’ base
corresponding to the base of the SNPs
Screen & Check the primer database
Predict 2’ structure, primer-dimer interactions, etc.
Primers for BCL11A
(20-57 bp)
Primers for HBS1L-MYB
(16-68 bp)
• BLAST
• Biomaths calculator program
• “Human genomic plus transcript
(Human G+T)” database
• OligoAnalyzer 3.0 tool
Purified by HPLC 34

35. Multiplex Minisequencing detection of SNPs
mPCR A + mPCR B (Amplification Reaction)
Amplify with genomic DNA+Buffer+dNTP+
DNApolymerase+primer mix+D/W
Amplified PCR products-Analyze in 2% & 3% w/v EtBr
stained smear agarose gel for BCL11A & HBS1L-MYB
Treat & incubate with ExoI/Antarctic Phosphatase mix at
37 ̊C * 1hr
Inactivate the enzymes at 75 ̊C * 15’’
Template (treated PCR product)
BCL11A
69-333 bp in mPCR-A
411-649 bp in mPCR-B
HBS1L-MYB
66-221 bp in mPCR-A
60-190 bp in mPCR-B
35
mPCR A mPCR B

36. Multiplex Minisequencing detection of SNPs
Treated PCR products
Minisequencing primers
SNaPshot Multiplex Ready Reaction Mix
1X Antarctic
Phosphatase Buffer,
37 ̇̇̊̇̇̊̊C * 1 hour
Enzyme Inactivation,
75 ̇̇̊̇̇̊̊C * 15 minTreated Mini-sequencing Products
36

37. Denaturation
Electrophoresis
GeneMapperTM v4.1
software
Multiplex Minisequencing detection of SNPs
ABI 3130xl genetic analyzer
37

38. Summary of Multiplex Minisequencing
38
Cláudia M.B. Carvalho and Sérgio D.J. Pena. Optimization of a multiplex minisequencing protocol for population studies and medical genetics. 2005
Polymorphic sites Multiplex PCR amplification Multiplex product inspection
PCR product digestion with
exonuclease I & Antartic
phosphatase
PCR product
Minisequencing reaction
using fluorescein-ddATP
Electropherogram obtained in a
monochromatic fluorescent sequencer

39. Multiplex Minisequencing detection of SNPs
BCL11A HBS1L-MYB
SNP ( Detected Genotype): rs11886868(GA),
rs4671393(CC), rs7557939(CT), rs10189857(TC),
rs6545816(GG), rs1427407(CC), rs6706648(GG),
rs10184550(CT), rs7606173(CG), rs6732518(GA),
rs766432(TT), rs7599488(GA).
SNP ( Detected Genotype): rs9389268(AG),
rs9376090(TC), rs9402686(GA), rs4895440(AT),
rs35959442(CG), rs9399137(TC), rs11759553(AT),
rs1320963(AA), rs28384513(TT), rs6929404(CC),
rs9494142(TC), rs6934903(TA), rs6904897(TT),
rs7776054(AG), rs4895441(AG), rs9402685(TC).
Size (nt)
Fluorescentunits
39

40. Genotypic profile verification
Sanger
Amplified
mPCRs (A&B)
Purify
Prepare 9 & 13 cycle
sequencing reactions for
BCL11A & HBS1L-MYB
Sequence by
ABI 3130xl
genetic analyzer
PCR-RFLP
2SNPs at BCL11A
& 5SNPs at
HBS1L-MYB
Singleplex
PCR
reaction
Digest with restriction
enzymes
Visualize in 2%
Et-Br stained
agarose gel
SNaPshot
minisequencing
Agreement in results
40

41. Strengths of the SNaP shot assay
Several selected polymorphisms can be detected simultaneously in a single reaction
The results can be presented in a single electropherogram
It can be easily performed in any labs equipped with a thermocycler and DNA sequencer
Highly accurate and sensitive for genotyping of large numbers of clinical samples
Cost and time efficient compared to other common genotyping techniques
41

42. Opportunities of Analyzing the SNPs
• Can provide the crucial genetic information
• Can help predict the severity of diseases in newborn
• Can help to better understand the molecular mechanisms of action of
modifying factors that influences the production of haemoglobin
Future Expectation
• Can provide more personalized patient care
42

43. 43

44. References
• 1. Geoffrey M.Cooper REH. THE CELL: A Molecular Approach. 4th Ed.
• 2. Kelly A. Frazer SSM, Nicholas J. Schork and Eric J. Topol. Human genetic
variation and its contribution to complex traits: Macmillan Publishers Limited; 2009.
• 3. Benjamin A. Pierce. Genetics: A Conceptual Approach. 4th Ed.
• 4. Menzel S, Garner C, Gut I, Matsuda F, Yamaguchi M, Heath S, et al. A QTL
influencing F cell production maps to a gene encoding a zinc-finger protein on
chromosome 2p15. Nature genetics. 2007;39(10):1197-9.
• 5. Thein SL, Menzel S, Peng X, Best S, Jiang J, Close J, et al. Intergenic variants
of HBS1L-MYB are responsible for a major quantitative trait locus on chromosome
6q23 influencing fetal hemoglobin levels in adults. Proceedings of the National
Academy of Sciences of the United States of America. 2007;104(27):11346-51.
44