STUDENT’S GUIDE

Case Study
Lactose tolerance Steve Cross,
Bronwyn Terrill
and colleagues
Wellcome Trust Sanger Institute
Version 1.1 Hinxton

Case Stu
Case Stud
lactose tolerance

Lactose tolerance
Most humans lose the ability to digest lactose and become lactose
intolerant by the time they are about ten years old. In some populations,
however, people still produce lactase, the enzyme that digests lactose,
in adulthood. The production of lactase gives an evolutionary advantage
to people who have access to cow’s milk.

At least two genetic changes that allow the lactase gene to be

permanently switched on occurred independently in geographically
isolated populations. Which changes in the human genome allow some
people to digest lactose in adulthood? How often and when have such
changes occurred in human history? Lactase non-persistence (lactose
intolerance) varies worldwide.

Outline of the activity 91–100% 31–40%

The majority of people living today are unable to digest lactose in adulthood. 81–90% 21–30%
In this activity you’ll analyse DNA data from different human populations
71–80% 11–20%
to work out which variations in the human genome have enabled some
adult humans to consume dairy products without problems.
61–70% 1–10%
You will also discover which variants are associated with lactose tolerance
51–60% 0%
(or lactase persistence) in different populations. A statistical test (Chi-
squared) will be used to work out whether specific genetic changes have
41–50% No data
significant effects on lactase persistence.

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lactose tolerance

Lactose tolerance and intolerance

Lactose, the sugar found in dairy products and milk, is metabolised by the
enzyme lactase (full name: lactase-phlorizin hydrolase or LPH). Lactase
splits apart the two sugars (galactose and glucose) that make up lactose. The
gene for lactase is known as LCT, and it is mostly expressed in cells in the
epithelium of the small intestine. Although nearly everyone can produce
the enzyme at birth, the majority of people outside Northern Europe lose
the ability to produce a lot of it at some point during the first years of life.
This can be referred to as lactase nonpersistance, adult hypolactasia or,
more commonly, lactose intolerance.

IMAGE FROM: Wellcome Images.

When a lactose-intolerant adult ingests a large amount of lactose, it passes
into their gut without being broken down. The bacteria in the gut then
react to the raised lactose levels by shifting their metabolisms to run on
this sugar. Unfortunately this process produces a lot of gas, which in turn
causes cramping, pain, flatulence and diarrhoea.

Lactose intolerance actually comes in three forms:

1. Primary lactose intolerance. As children are weaned they lose the

ability to metabolise milk, so they become lactose-intolerant adults.
2. Secondary lactose intolerance. This occurs in adults from lactose
tolerant populations who, due to damage caused by diseases of the
stomach or intestines, can no longer properly produce lactase.
3. Congenital lactose intolerance. This is a condition where children are
born unable to metabolise lactose. Before the 20th century it would
normally have resulted in death, but it can now be diagnosed and
treated by moving the infant to a lactose-free diet.

The first medically-documented cases of lactose intolerance — or ‘deficiency’

as it was known at the time — were considered to be the ‘abnormal’ result
of a single-gene, autosomal recessive condition. However, by the 1970s, it
was recognised that most people in the world had this ‘condition’. In Asia,
Africa and the Americas the majority of the native adult population do not
produce lactase as adults. Therefore, studies began to consider how lactose
tolerance or ‘lactase persistence’ ­— being the unusual condition — had
occurred and how it was inherited. Populations that do produce lactase
into adulthood tend to be those with a long history of drinking fresh milk,
including those in Northern Europe and some isolated parts of Africa.
Lactase persistence behaves as a dominant trait because half levels of
lactase activity still enable a significant amount of lactose to be digested.

LACTASE

Lactose Glucose Galactose

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lactose tolerance

Regulating genes
The difference between most lactose-intolerant and lactose-tolerant adults
isn’t that they have different genes — it’s that their genes are controlled
differently. Lactase-persistent people have a lactase gene that is not
switched off after childhood.

The body has three main ways of regulating the effect of a gene:

1. Regulation at the level of gene expression: turning the gene on and

off or raising or lowering the amount of gene product;
2. Regulation at the level of RNA: controlling levels of protein production
by modifying or removing RNA products so that they can’t be translated
into proteins;
3. Regulation at the level of protein: modifying the protein to make more
or less active versions, or degrading the protein to limit its activity.

In Exercise 1, you’ll be looking at the first type of regulation. Studies of

large families have shown there is no difference between the LCT genes
of people with or without lactase persistence. However, the same studies
focused their attention on a 47 kilobase (a kilobase, or kb, is a thousand
bases) region containing a possible control sequence for lactase production
very close to the LCT gene on chromosome 2.

In Exercise 2, you will study variants, known as Single Nucleotide

Polymorphisms (or SNPs, pronounced ‘Snips’) from this area (called 2q21)
on chromosome 2.

Lactose-reduced milk is produced

by treating milk with lactase.
In this product, an immobilised
enzyme has been used to treat the
milk before packing, hence the label
‘no added enzymes’.

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lactose tolerance

Exercise 1
Genotypes associated with lactose
tolerance and intolerance

A key research paper, published in 2002, identified genetic variants in

northern European populations associated with lactase persistence and
intolerance (Enattah et al, 2002). The extract from the original paper below
reports the results of two variants: one at –13910 bases ‘upstream’ of the LCT
gene and one at –22018 bases ‘upstream’ of the LCT gene.

We analyzed... variants in Finnish DNA samples isolated from a total of

196 biopsy specimens with biochemically determined disaccharidase (LPH)
[lactase] activity.

All 59 samples showing primary lactase deficiency were homozygous with

respect to the C allele of C/T–13910, 6 were heterozygous with respect to the
G/A–22018 variant and the remaining 53 were homozygous with respect to
the G allele.

Of the 137 cases showing lactase persistence, none were homozygous with
respect to the alleles C and G, at C/T–13910 and G/A–22018 respectively; 74
were homozygous for alleles T and A, with 63 being heterozygous at both
positions.

Write the data described in this paragraph in the tables below:

C/T –13910

Condition vs genotypes CC CT TT Totals

Lactase deficiency / lactose

intolerant

Lactase persistence

Total samples

G/A –22018

Condition vs genotypes GG GA AA Totals

Lactase deficiency / lactose

intolerant

Lactase persistence

Total samples

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lactose tolerance

Questions
a. Which of the positions (C/T–13910 or G/A–22018) is 100% associated
with lactase persistence?
b. From this data at this position, which genotype(s) are associated with
lactose tolerance/lactase persistence?
c. From the data at this position, which genotype(s) are associated with
lactose intolerance/lactase non-persistence?
d. How associated is the other variant (C/T–13910 or G/A–22018) with
lactase persistence? (number of non-consistent findings divided by the
total number of samples x 100, subtracted from 100%).

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lactose tolerance

Exercise 2
Other lactose persistence mutations
Although the 100% associated variant you identified in Exercise 1 is thought
to cause lactase persistence in Europeans, this variant is only present at
low levels in other lactase-persistent populations.

A Nature Genetics paper (Tishkoff et al, 2007) sought DNA variations that
could help to explain lactase persistence in East African populations. There,
lactase persistence was most common in Sudanese populations which
raised livestock, and least common in Tanzanian hunter-gatherers. The
researchers sequenced 3.3 kb and 1.8 kb in the same area on chromosome 2
(2q21) that yielded the European variant.

Below are the results from three different African populations at two
different positions in the genome: at 5 bases and 100 bases further
upstream from the LCT gene than the European variant. In this study, a
lactose tolerance blood test (a method of indirectly measuring levels of LCT
actiivty) was used to determine whether a person was LCT persistent, LCT
intermediate or LCT non-persistent.

The Chi-Squared test

The Chi-Squared test can be used here to test the following Null hypothesis.

Null hypothesis: There is no significant effect of genotype (at this position) on

lactase persistence.

Alternative hypothesis: The genotype (at this position) has a significant effect
on lactase persistence.

Kenyans Position Phenotype observed CC CG GG

–14010 LCT persistence 12 48 38
LCT non-persistence 2 10 40
LCT intermediate 3 12 25

Tanzanians Position Phenotype observed CC CG GG

–14010 LCT persistence 17 44 36
LCT non-persistence 5 18 57
LCT intermediate 5 21 28

Afro-Asiatic Kenyans Position Phenotype observed GG GT TT

–13951 LCT persistence 2 7 25
LCT non-persistence 0 1 12
LCT intermediate 0 0 14

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lactose tolerance

Population name: Kenyan Position of allele: –14010

Category Observed (O) Expected* (E) O–E (O–E)2 (O–E)2

(see below) E
Genotype 1 (CC)
LCT persistent
Genotype 2 (CG)
LCT persistent
Genotype 3 (GG)
LCT persistent
Genotype 1 (CC)
LCT non-persistent
Genotype 2 (CG)
LCT non-persistent
Genotype 3 (GG)
LCT non-persistent
Genotype 1 (CC)
Intermediate
Genotype 2 (CG)
Intermediate
Genotype 3 (GG)
Intermediate

TOTALS

* To calculate the Expected values, you will need to count the number of number of samples with a single genotype
found across the entire population. This figure, divided by the total sample size will give you the prevalence of the
genotype. Multiplying the prevalence of each form of the allele by the number of samples in each condition group,
will give you the Expected values.

Size of population
Category Prevalence of allele Expected
(ignoring alleles)
Genotype 1 (CC)
LCT persistent
Genotype 2 (CG)
LCT persistent
Genotype 3 (GG)
LCT persistent
Genotype 1 (CC)
LCT non-persistent
Genotype 2 (CG)
LCT non-persistent
Genotype 3 (GG)
LCT non-persistent
Genotype 1 (CC)
Intermediate
Genotype 2 (CG)
Intermediate
Genotype 3 (GG)
Intermediate

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lactose tolerance

Population name: Tanzanian Position of allele: –14010

Category Observed (O) Expected* (E) O–E (O–E)2 (O–E)2

(see below) E
Genotype 1 (CC)
LCT persistent
Genotype 2 (CG)
LCT persistent
Genotype 3 (GG)
LCT persistent
Genotype 1 (CC)
LCT non-persistent
Genotype 2 (CG)
LCT non-persistent
Genotype 3 (GG)
LCT non-persistent
Genotype 1 (CC)
Intermediate
Genotype 2 (CG)
Intermediate
Genotype 3 (GG)
Intermediate

TOTALS

* To calculate the Expected values, you will need to count the number of number of samples with a single genotype
found across the entire population. This figure, divided by the total sample size will give you the prevalence of the
genotype. Multiplying the prevalence of each form of the allele by the number of samples in each condition group,
will give you the Expected values.

Size of population
Category Prevalence of allele Expected
(ignoring alleles)
Genotype 1 (CC)
LCT persistent
Genotype 2 (CG)
LCT persistent
Genotype 3 (GG)
LCT persistent
Genotype 1 (CC)
LCT non-persistent
Genotype 2 (CG)
LCT non-persistent
Genotype 3 (GG)
LCT non-persistent
Genotype 1 (CC)
Intermediate
Genotype 2 (CG)
Intermediate
Genotype 3 (GG)
Intermediate

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lactose tolerance

Population name: Afro-Asiatic Kenyans Position of allele: –13915

Category Observed (O) Expected* (E) O–E (O–E)2 (O–E)2

(see below) E
Genotype 1 (GG)
LCT persistent
Genotype 2 (GT)
LCT persistent
Genotype 3 (TT)
LCT persistent
Genotype 1 (GG)
LCT non-persistent
Genotype 2 (GT)
LCT non-persistent
Genotype 3 (TT)
LCT non-persistent
Genotype 1 (GG)
Intermediate
Genotype 2 (GT)
Intermediate
Genotype 3 (TT)
Intermediate

TOTALS

* To calculate the Expected values, you will need to count the number of number of samples with a single genotype
found across the entire population. This figure, divided by the total sample size will give you the prevalence of the
genotype. Multiplying the prevalence of each form of the allele by the number of samples in each condition group,
will give you the Expected values.

Size of population
Category Prevalence of allele Expected
(ignoring alleles)
Genotype 1 (GG)
LCT persistent
Genotype 2 (GT)
LCT persistent
Genotype 3 (TT)
LCT persistent
Genotype 1 (GG)
LCT non-persistent
Genotype 2 (GT)
LCT non-persistent
Genotype 3 (TT)
LCT non-persistent
Genotype 1 (GG)
Intermediate
Genotype 2 (GT)
Intermediate
Genotype 3 (TT)
Intermediate

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lactose tolerance

(O–E)2
The total of E across all four groups is the chi-squared value ( χ 2).
Using this information, calculate the probability of the Null hypothesis
being true using this lookup table, where p is the probability.

p 0.9 0.75 0.25 0.1 0.05 0.025 0.01 0.005

χ 2 1.06 1.92 5.39 7.78 9.49 11.14 13.27 14.86

Questions
For each of the three populations:

e. What is the χ 2 value?

f. What is the probablity that the Null hypothesis is true?
g. Does this mean that the result is statistically significant?
h. Were any of the results for these positions statistically significant?
If so, which one(s)?
i. If all of the lactase-persistent individuals in the world have the same
genetic change that allows them to drink milk, there is a possibility
that the change has only happened once, and that all these people share
a common, milk-drinking ancestor. If, however, there are a number of
changes that give rise to milk-drinking, it is likely that it has evolved
more than once. What can you conclude from these results about the
origins of lactase persistence?
j. What other data would you need to explore lactase persistence further?

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