Mendelian Genetics II

Data Analysis: Deviation from Expectations

Interpreting Pedigrees

QueeLim Ch’ng
Developmental Neurobiology
King’s College London
queelim@kcl.ac.uk
 Mendelian Genetics II
Part 1: Data Analysis: Deviation from Expectations

QueeLim Ch’ng
Developmental Neurobiology
King’s College London
queelim@kcl.ac.uk
 Previously in Genetics…
Pp x Pp SsYy x SsYy

3:1 9:3:3:1
Segregation Independent Assortment

• Genetic concepts and terms

• Now: Analysis and Interpretation of Genetic Observations
 Previously in Genetics…
Pp x Pp SsYy x SsYy

3:1 9:3:3:1
Segregation Independent Assortment

Mendelian inheritance predicts certain ratios

of phenotypes
Deviations from Mendelian Inheritance

• Non-independent assortment
– e.g., linkage (genes on same chromosome)
• Non-Mendelian segregation of phenotypes
– e.g., complex gene interactions
• These scenarios will result in ratios that differ
from the Mendelian cases

How to identify such deviations?

 Identifying Deviations
Out of 100-coin tosses
Coin Toss Expect: 50 heads, 50 tails
Coin 1 Coin 2
52 heads : 48 tails 68 heads : 32 tails
High probability it Low probability it
Head happened by chance happened by chance
Tail
Is there something
fishy with Coin 2?

• Are these different from what is expected?

– Are the differences big? What is considered big?
– What is the probability that these differences could happen
by chance?
• Calculate answers to these questions with the c2 Test
 The c2 Test: What it does
• Compare observed versus expected results. Are they
different and is that difference by chance?
• The smaller the difference between observed and
expected, the more likely that it happened by chance.
• The c2 Test quantifies the probability that one would
get such a difference by chance.

• By scientific convention, a probability value of less

than 5% indicates that the difference did not occur by
chance; i.e., it’s a significant difference.
 The c2 Test: What it means
c2 represents the magnitude of difference between expected and observed

For a coin toss, there would be 2 categories:

heads and tails, each with expected and
observed numbers

Measures the difference

Square the difference to make it
positive for easy comparison.
e.g., a difference of -3 will be
worth the same as a difference of
3 because (-3)2 = 32 = 9

Measures how big the difference is, by

comparing against the expected value.
Big is relative.
 Is Coin 2 fishy?
Quantify the Difference from Expected
Category Observed, O Expected, Difference,
E O-E
Heads 68 50 18 324 6.48
Tails 32 50 -18 324 6.48
Total = 12.96

Use a chart that convert a c2 value to the probability that the

difference occurred by chance, for the degrees of freedom (dF) c2 = 12.96
of the data. dF = 1
dF = number of values that can vary
independently
= number of data categories – 1
=2-1=1

This is consistent with the fact that only 1 value can vary
independently, as the number of heads could be calculated from
 Is Coin 2 fishy?
Get the Probability
The probability that 68
Conversion Chart heads and 32 tails occurs
Curve for
by chance is ~0.0003.
dF =1
This means that the
chances of getting this
result with a normal coin
is 0.03%, which is quite
unlikely. This is less than
5% which is the cutoff by
convention.
In this case we would
Corresponding conclude that there is
probability something fishy about
= 0.0003 Coin 2 because it doesn’t
behave like a normal coin.
c2 = 12.96
 Talk the Talk: Statistical Terms
• Null hypothesis - the expected result based on an assumption (e.g., 50
heads 50 tails - coin is not biased).
• p-value - probability that difference between observed and expected result
happened by chance (e.g., probability of 0.0003 for 68 heads / 32 tails in
100-coin tosses if the coin was not fishy).
• If observed and expected results are very different, the p-value will be very
low, and the null hypothesis is rejected. This outcome means that the
observed results do not fit the underlying assumption (e.g., reject the
assumption that the coin is unbiased).
• If observed and expected results are similar, the p-value will be high, and
the null hypothesis is not rejected but it doesn’t prove the null hypothesis.
This outcome means that observations do indicate that the coin is fishy.
Two possibilities remain, which we cannot tell apart:
– The coin is fishy, but we did not have enough observations.
– The coin is not fishy.
 Applying the c Test 2

• Testing Independent Assortment

• Experiment done by Mendel’s assistant, Igor
– Did it deviate from Independent Assortment?

SsYy x SsYy
Smooth Smooth Wrinkled Wrinkled
Yellow Green Yellow Green Total
Observed 2834 920 951 287 4992
Expected Ratio 9 3 3 1 16
Expected 2808 936 936 312 4992
Expect 9:3:3:1 if How to calculate Expected?
there is Expect 9 out of 16 of the total peas to
independent be smooth and yellow
assortment So, 9/16 x 4992 = 2808
 Calculations for Igor
Category Observed, Expected, Difference,
O E O-E
Smooth Yellow 2834 2808 26 676 0.24
Smooth Green 920 936 -16 256 0.27
Wrinkled Yellow 951 936 15 225 0.24
Wrinkled Green 287 312 -25 625 2.00
Total 4992 4992 c2 = 2.75

4 Categories c2 = 2.75
Therefore, Degrees of Freedom, dF = 4 – 1 = 3 dF = 3
 Did Igor’s results deviate from Independent
Assortment?
The p-value/probability that
Conversion Chart Igor got such data if we
Curve for assume independent
dF =3 assortment is 0.45 or 45%.
Corresponding This means that one would
probability accept that these results do
(p-value) not seem to deviate from the
= 0.45 9:3:3:1 ratio. The null
hypothesis is not rejected,
because the p-value is
greater than 5%.
In this case, we would
conclude that Igor’s results
did not deviate from
independent assortment.
c2 = 2.75
 Summary: The c Test 2

means sum across all

categories

• Purpose: to test if difference between observed

and expected is significant
• The terms in the equation have meaning
• Calculate the c2 value and the degrees of freedom
and look up the corresponding probability (p-
value) of getting the observation given the
expected values
• Draw conclusions from the p-value
 Mendelian Genetics II
Part 2: Interpreting Pedigrees

QueeLim Ch’ng
Developmental Neurobiology
King’s College London
queelim@kcl.ac.uk
 How to Study Inheritance in
Humans?
• Mendel’s peas – can do controlled matings
X X

• Cannot control matings in humans

• Can only look at available families
– Pedigree analysis
• Not many offspring
– Few observations
 Pedigree Analysis

• Look at family trees

• Determine pattern of inheritance (disease)
– Dominant or recessive?
– Chances of being carriers or affected
 Human Chromosomes
Humans
• 2 matching (homologous)
sets of chromosomes*
• Females have two X
chromosomes
• Males have X and Y
chromosomes

• Non-sex chromosomes are

called autosomes
• Today: focus on autosomal Sex
inheritance Autosomes Chromosomes
 Pedigree Analysis
What the symbols mean

Generation
I Parents
1 2

II Offspring in birth order

1 2 3

Female Male Female Male

Normal Affected
 Case study: Neonatal Diabetes
Genotype Phenotype
INSA/INSA normal
INSA/INSB normal INSB is recessive to
INSB/INSB Neonatal Diabetes INSA
INSA/INSD Neonatal Diabetes
INSD is dominant
INSD/INSD Neonatal Diabetes
over INSA

• Neonatal diabetes are caused by mutations in the INS

gene that encodes insulin
• INSA, INSB, and INSD are alleles of the INS gene
• Disease-causing alleles can be dominant or recessive
 Case study: Neonatal Diabetes
Genotype Phenotype
INSA/INSA normal
INSA/INSB normal INSB is recessive to
INSB/INSB Neonatal Diabetes INSA
INSA/INSD Neonatal Diabetes
INSD is dominant
INSD/INSD Neonatal Diabetes
over INSA

• Neonatal diabetes are caused by mutations in the INS

gene that encodes insulin
• INSA, INSB, and INSD are alleles of the INS gene
• Disease-causing alleles can be dominant or recessive
 Autosomal Recessive Inheritance
Consanguineous

Hallmarks:
• Disease appears in progeny of unaffected parents (skip
generations)
• Affected progeny can be male or female (autosomal, not
sex-linked)
• Consanguineous (parents who are related) matings are more
likely to produce progeny who are homozygous recessive
 Inferring Genotypes:
Autosomal Recessive Inheritance
Consanguineous

INSA/INSB INSA/INSB
INSA/INSB INSA/INSB

INSB/INSB INSB/INSB

• Having inferred autosomal recessive inheritance, we can infer the genotypes

of individuals
• Affected individuals
– genotype must be INSB/INSB
– must have inherited one INSB allele from each parent
• Parents unaffected, therefore their genotype must be INSA/INSB
• Those who are unaffected but carry a disease-causing allele are called carriers
 Chances of Getting Disease
Mother’s
INSA/INSB If both parents Gametes
INSA/INSB are
heterozygous
INSA INSB
INSA/ INSA/
INS A

Gametes
Father’s
INSA INSB
INSB/INSB
INSA/ INSB/
INSB
INSB INSB

• For this family, what is the probability that their next child
would have neonatal diabetes?
• Each pregnancy is an independent event. So, the chances that
the next child will have the disease is still 1 in 4 (standard 3:1
Mendelian ratio).
 Chances of Being a Carrier
Mother’s INSA/INSB INSA/INSB
If both parents Gametes
are INSA INSB
heterozygous

INSA/ INSA/
INS
Gametes

A
Father’s

Unaffected Son
INSA INSB
?
INS /
A
INS /B
INSB INSB/INSB
INSB INSB
• For this other family, what is the probability that the unaffected son
is a carrier (INSA/INSB)?
• Since he is unaffected, he is not INSB/INSB
• Possibilities left are either INSA/INSA or INSA/INSB (shaded squares)
• From the Punnett’s Square, there is a 2 out of 3 chance that he is a
carrier (2/3 or 67%)
 Case study: Neonatal Diabetes
Genotype Phenotype
INSA/INSA normal
INSA/INSB normal INSB is recessive to
INSB/INSB Neonatal Diabetes INSA
INSA/INSD Neonatal Diabetes
INSD is dominant
INSD/INSD Neonatal Diabetes
over INSA

• Neonatal diabetes are caused by mutations in the INS

gene that encodes insulin
• INSA, INSB, and INSD are alleles of the INS gene
• Disease-causing alleles can be dominant or recessive
 Autosomal Dominant Inheritance

Hallmarks:
• Disease appears in every generation
• Affected progeny can be male or female (autosomal,
not sex-linked)
• Affected individuals have affected parents
 Inferring Genotypes
Autosomal Dominant Inheritance
Mother’s INSA/INSA
Gametes Affected
Mother
INSA INSD
INSA INSA
INS
Gametes

A
Father’s

/INSA /INSD Affected

Son INSA/INSA
INS A
INS A
INSA
/INSA /INSD
• Unaffected individuals must be INSA/INSA
• The affected mother could be INSD/INSD or INSA/INSD
• The unaffected son must have received one INSA allele from each parent, which
means the mother must be INSA/INSD
• The affected son must be INSA/INSD, because he would have received one INSA
allele from the father
• If the disease is rare, it is highly likely that the dominant disease-causing allele
is also rare. Affected individuals will thus tend to be heterozygous.
 Summary: Pedigree Analysis
• Examine patterns of inheritance:
– Autosomal: both sexes affected equally
– Recessive: affected individual from unaffected parents;
disease skips generations
– Dominant: affected individual have affected parents; disease
occurs every generation
• Once dominance/recessive inheritance is identified, one
can infer the genotypes of individuals in the pedigree
• Infer parent’s genotype from children’s genotype and
vice versa (like Sudoku)
• Using a Punnett’s Square, calculate the chances of an
individual being affected or being a carrier
 Summary: Analysis of Inheritance
• Use statistics (c2 Test) to:
– Evaluate the effects of chance on genetic data
– Determine if the pattern of inheritance fits
expectations based on certain assumptions
• Examine pedigrees to:
– Determine the mode of inheritance
– Infer the genotype of individuals
– Calculate the chances of an individual being a
specific genotype
 References and Image Credits
Key References
• Mendel’s Peas and Mendelian Genetics
– Chapter 3, Essentials of Genetics, Klug et al.

Primary Literature
• Insulin Mutations and Neonatal Diabetes
– Garin et al., Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):3105-10. doi:
10.1073/pnas.0910533107
– Støy et al., Proc Natl Acad Sci U S A. 2007 Sep 18;104(38):15040-4.

Images
• £1 coins - http://richardjstark.wordpress.com/author/richardjstark/
• The Simpsons – www.wikipedia.com