Biotechnology / Pharmacy

Nutrigenomics And Pharmacogenomics

Protocol. 2

PROTOCOL 2. NUTRIGENETICS
PERSONALIZED NUTRITION
1. INTRODUCTION

In the first practical session we have carried out a nutrigenomic analysis. That is, we have
studied the effect of one nutrient (cholesterol) on gene expression using microarrays.

In this second practical session, we will focus on nutrigenetics and personalized nutrition.

2. GENOTYPING METHODS

As part of a nutrigenetic analysis, we must define the allelic variants that an individual carry
and then to study how these variants affect to disease predisposition and how they interact
with nutrients to modify such predisposition. This can be done using different approaches.

Once again, the development of new omics techniques has allowed the broad study of millions
of variants simultaneously. This is called Genome Wide Association Studies (GWAS). Two
different methodologies can be used for GWAS studies:

a. Microarrays

We have learnt before how microarray studies are used to define the effect of nutrients on
gene expression. Genotyping microarrays are similar in terms of probes immobilization,
labeling and hybridization. However, there are some differences:

1. The starting material is genomic DNA.

2. Two probes are used for each SNP, one for each allele.
3. These probes are usually labeled with FAM and VIC.
4. The hybridization is allele specific. This means that only one allele hybridizes in one
probe.

Due to these characteristics genotyping microarrays do not give intensity levels of both
fluorophores. Instead of that, we are detecting FAM, VIC or both at the same intensity
depending on if the individual is homozygous or heterozygous (Figures 1 y 2).
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Figure 1. Schematic representation of the genotyping method using FAM and VIC probes.

Figure 2. TaqMan probes mechanism of action.

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The result of the genotyping is a spots cloud with three components according to three signal
possibilities: FAM/FAM, VIC/VIC, or FAM/VIC (Figure 3).

Figure 3. Genotyping spots cloud.

This kind of analysis does not require normalization. The procedure is called “variant calling”
and is the determination of the genotype of everyone. However, like with expression
microarrays, some quality parameters must be bearded in mind to determine confident
signals. There are different quality parameters that can be used (Figure 4).

Figure 4. Quality parameters

All determinations that do not fit these parameters must be discarded.

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b. Next Generation Sequencing

NGS applied to genotyping purposes is like that applied to gene expression analyses. In this
case, the most common method used is DNA-seq that uses the whole genome as starting
material. The first step is the fragmentation of the DNA. Then, adaptors must be ligated to
make the DNA library and samples are hybridized and amplified.

Similarly, to gene expression, the result is a data matrix of sequences and runs (how many
times that sequence has been read). Then, sequences must be defined with an alignment
algorithm and, finally the variant calling must be done (Figure 5).

Figure 5. Schematic representation of the sequence alignment method.

To reach a confident allelic determination, some quality controls must be made according to:

- The number of runs

- The quality of the run
- The chain bias (when the allelic variant is detected only in one chain)

c. Functional analysis

Data obtained from both, microarray and NGS, give rise to a list of SNPs. A statistical and
functional analysis is needed to determine the clinical value. The statistical analysis refers aims
to find significant associations between the SNP and the risk of developing a health condition.
That means, we must determine if the SNPs is more frequent in individuals with or without the
condition. The association test depends on the type of variables (quantitative/qualitative) and
the type of experimental design (case/control, prospective, interventional trials, etc.).
Whatever the test used, when we analyze thousands or millions of SNPs, a correction for
multiple testing needs to be applied. The most common correction method is Bonferroni.
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Once we have found associations between selected SNPs and health parameters, we must
define the biological meaning of those associations. To do this, we will use SNPs data bases:

Genecards: https://www.genecards.org/

SNPedia: https://www.snpedia.com/index.php/SNPedia dbSNP:

https://www.ncbi.nlm.nih.gov/

OMIM: https://www.omim.org/

Genecards

It is an integrative database that provides information on all annotated and predicted human
genes. It automatically integrates gene-centric data from ~150 web sources, including
genomic, transcriptomic, proteomic, genetic, clinical, and functional information. In the
searching bar, the name of the gene must be introduced (i.e., FTO). Among the information it
provides is:

- Other gene aliases

- Links to other databases like Ensmbl, OMIM or UniProt
- A summary of the function
- Regulatory elements indicating the transcription factors that bind them
- Genetic context
- Protein details including size, domains, 3D structure and modifications
- Animal models, antibodies, and other molecular tools for its study
- Phenotypes associated to gene mutations
- GO terms
- Cellular location
- Metabolic pathways from BioSystems (NCBI) and Reactome
- Interacting proteins including a STRING network
- Expression levels in different tissues
- Gene variants
- Publications

SNPedia

It is a database of polymorphisms. To search for a polymorphism, you must type the “rs” code
in the searching bar (i.e., rs9939609). This database provides a brief summary of each paper
that mentions the polymorphism and includes a link to the original paper. There is also a right
vertical menu where the following information is provided:

- The alleles and the risk with a color code where red means higher risk and green
means lower risk.
- Links to different databases, including dbSNP
- Allele frequencies
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- Boxes where GWAS studies are summarized with the associated disease, the risk allele,
the OR, and a link to the original study
- Boxes where related diseases are summarizes, including the corresponding links to
OMIM. dbSNP

It is also a database of gene variants that are searched for using the “rs” code. By clicking in
the correct link, the information card of this SNP will appear. This information includes:

- The allelic change

- The corresponding aminoacid change
- The orientation (FWD/REV)
- The minor allele frequency
- The experimental validation of the SNPs
- A link to OMIM and ClinVar databases of disease associations
- A link to Pubmed
- The genetic contexts where the SNP is located
- The allelic frequency in different populations

OMIM

It is a catalogue of human diseases and disorders. To search for information, the name of the
gene must be typed in the searching bar. The information it provides includes:

- Chromosomic location and related diseases

- Cloning, mapping, structure, and function
- Molecular function
- Animal models
- Gene variants
- References

3. EXPERIMENTAL DESIGN

Then, we will develop a genetic profile and the corresponding personalized nutritional advice.
We have a list of SNPs from the 24Genetic test. These SNPs allow the characterization of:

- Personalized consumption of nutrients

- Response to different dietary patterns
- Specific requirements of vitamins and minerals
- Predisposition to dyslipidemia
- Taste perception
- Regulation of appetite and emotional eating
- Predisposition to obesity
- Metabolism of caffeine
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- Predisposition to T2DM

The final aim is the elaboration of a nutrigenetic report with some individualized nutritional
advises.

4. EXERCISES

4.1 Exercise 1

First, we will explore the described databases using FTO gene and its SNP rs9939609. DESEASE,
RISK ALLELE, FREQUENCY, GENE ,INTERACTION WITH DIET

Gene function: This gene is a nuclear protein of the AlkB related non-haem iron and 2-
oxoglutarate-dependent oxygenase superfamily but the exact physiological function of this
gene is not known. Other non-heme iron enzymes function to reverse alkylated DNA and RNA
damage by oxidative demethylation. Studies in mice and humans indicate a role in nervous and
cardiovascular systems and a strong association with body mass index, obesity risk, and type 2
diabetes. [provided by RefSeq, Jul 2011]

Diseases asociated: FTO (FTO Alpha-Ketoglutarate Dependent Dioxygenase) is a Protein Coding

gene. Diseases associated with FTO include Growth Retardation, Developmental Delay, And
Facial Dysmorphism and Body Mass Index Quantitative Trait Locus 14. Among its related
pathways are DNA Damage Reversal and Homology Directed Repair. Gene Ontology (GO)
annotations related to this gene include ferrous iron binding and oxidative RNA demethylase
activity.

Risk alelles : Alleles T>A

Frequency: Global 84190 T=0.60143 A=0.39857

The odds ratio associated with heterozygotes is 1.34 (CI 1.17-1.52), and for homozygotes, 1.55
(CI 1.3-1.84).

Effects of common FTO gene variants associated with BMI on dietary intake and physical
activity in Korean

4.2 Exercise 2

You must find information that allow you to determine the biological significance of 32 SNPs
associated with diabetes. Fulfill the table adjusted and apply a weighted and normalized GRS
calculation. The percentage is 35.88%

4.3 Exercise 3

Now, we will work with the provided list of SNPs to define the genetic profile of your patient
for different traits. For these traits:
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LDL
HDL
TG

Use the provided color scale and point, with an arrow, to the approximately estimated risk of
your patient.

For the remaining traits, describe the profile.

All of them are in the normal ranges meaning the patient has a avarege response to TG and
colesterol.

4.4 Exercise 4

Nutrigenetic Report
1. Caffeine Metabolism
 Genetic Profile: Quick metabolizer of caffeine due to a CYP1A2 mutation.
 Implication: Caffeine will have a lower and less lasting effect.
 Advice: The patient can consume caffeine in moderate amounts without concerns for
prolonged effects. However, be mindful of the potential for increased tolerance.
2. Appetite and Diet
 Genetic Profile: Susceptible to increased appetite and low satiety due to mutations in
ANKK+E+A95:H96, ADIPO, ADRB2, FTO genes.
 Advice:
 Include high-fiber foods (e.g., whole grains, legumes, fruits, and vegetables) to
enhance satiety.
 Practice portion control and mindful eating.
 Regular meal timings to manage hunger levels.
3. Emotional Eating and Sugar Cravings
 Genetic Profile: Prone to emotional eating and sugar attacks due to FGF21 gene
mutation.
 Advice:
 Engage in stress management and emotional wellness coaching.
 Keep healthy snacks handy to manage sugar cravings responsibly.
 Gradually reduce sugar intake to lower the threshold for sweet tastes.
4. Taste Preferences
 Genetic Profile: Unable to taste bitter flavors due to mutations in TAS2R38 gene.
 Advice:
 Incorporate bitter-tasting vegetables like cauliflower and broccoli, which are
likely to be more palatable.
 Explore a variety of vegetables to take advantage of this unique taste
preference.
5. Diet and Weight Management
 Genetic Profile: High responsiveness to Mediterranean diet, poor response to low-fat
diets, linked to PPARG and TCF7L2 gene variations.
 Advice:
 Follow a Mediterranean diet rich in fruits, vegetables, whole grains, olive oil,
fish, and lean meats.
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 Avoid low-fat diets; instead, focus on healthy fats like nuts, seeds, and
avocados.
 Regular physical exercise is crucial for effective weight management.
6. Vitamin Deficiencies
 Genetic Profile: Low levels of Vitamin B6, C, and K due to variations in MTHFR, NBPF3
genes.
 Advice:
 Vitamin B6: Increase intake of poultry, fish, potatoes, chickpeas, bananas, and
fortified cereals.
 Vitamin C: Consume citrus fruits, berries, tomatoes, bell peppers, and broccoli.
 Vitamin K: Include green leafy vegetables, Brussels sprouts, cabbage, and
broccoli in the diet.
 Consider supplements under medical supervision if dietary changes are
insufficient.
7. Lipids and Cholesterol
 Genetic Profile: Average lipid and cholesterol levels.
 Advice:
 Maintain a balanced diet.
 Regular check-ups to monitor lipid levels.
Overall Recommendations
 Regular Medical Check-Ups: To monitor the effectiveness of dietary changes and
supplements.
 Lifestyle Modifications: Consistent physical activity, stress management, and adequate
sleep.
 Continuous Monitoring: Adjust diet and lifestyle habits based on regular health
screenings and genetic updates.