W12LECT - Genomics in dental medicine

Question 1

Amelogenesis imperfecta
1. What are the features of Amelogenesis imperfecta?

Answer 1

1/ X – Dominant
2/ Tooth enamel formation problem

Question 2

Amelogenesis imperfecta
1. What are the features of Amelogenesis imperfecta?

Answer 2

1/ X – Dominant
2/ Tooth enamel formation problem
3/ Genes affected”
- AMELX; Xp22.3-1; amelogenin
- ENAM; 4q13.3; enamelin (5%)

Question 3

How do we find mutation is in the patient?

Answer 3

• collect DNA of the patients
  – isolate it from blood
• collect medical data of the patients
• analyze the pedigree

Question 4

What is the use of Sequencing?

Answer 4

• find a new mutation in a specific area
• search for SNPs, small insertions, deletions, duplications

Question 5

How do we perform Genotyping by sequencing?

Answer 5

1/ The sequence of nucleotides and the corresponding signal strength is analyzed.
2/ Color-coded chromatograms facilitate evaluation.
3/ on the chromatogram only one nucleotide specific signal is shown in homozygotes (for a nucleotide).
4/ on the chromatogram two nucleotides specific signals (which are a little decreased) can be seen in heterozygotes
homozygous (C/C)

Question 6

What is the result of Genotyping by sequencing?

Answer 6

1. New SNV in a specific area
2. A deletion was found in some patients

Question 7

What are the features of Genotyping by Sanger sequencing

Answer 7

Question 8

What are the 2 Methods for the genomic backgrounds of diseases?

Answer 8

1. Wet laboratory methods (classical methods, lab experiments)
2. in silico methods (analyze data, bioinformatics)

Question 9

What are the 2 Methods for the genomic backgrounds of diseases?

Answer 9

1. Wet laboratory methods (classical methods, lab experiments)
2. in silico methods (analyze data, bioinformatics)

Question 10

What are the features of In silico methods?

Answer 10

• (Re) analysis of data from on-line databases
• Lots of online software
• Discovering new genes (APOA5)
• Meta-analyzes (joint analysis of several previous studies)

Question 11

What are the 2 Genomics methods for studying the genetic background of disease?

Answer 11

1. Hypothesis-driven approaches
2. Hypothesis-free approaches

Question 12

How do we perform In silico methods?

Answer 12

• alleles or genotypes of markers (e.g.: SNPs, microsatellites), are determined in patients vs. in controls

Question 13

Association studies
1. Association is a statistical statement
=> What is this statement?

Answer 13

1. The specific allele significantly more likely to occur WITH a specific phenotype
2. This means:
   – case-control method, we examine the presence of the selected (candidate) genevariant
   – whether it is more common in patients, than in healthy controls

Question 14

Genetic markers
1. What is a genetic marker?

Answer 14

A genetic marker is usually a sequence variation with a known location on a chromosome that can be used to identify individuals, with a relative high chance to differentiate between different alleles on homologous chromosomes.

Question 15

Genetic markers
2. What are examples of genetic markers?

Answer 15

Question 16

How should Microsatellite detection be?

Answer 16

Question 17

Examples of using STR as markers
1. How do we use STR as markers in Prenatal diagnosis?

Answer 17

5-6 STR markers/chromosome

Question 18

How do we Use SNP/SNV as markers

Answer 18

They are only biallelic, but much more than STR >300 million vs. ≈ 30,000
Easier to detect

Question 19

Give an example of Using SNP/SNV as markers?

Answer 19

• Genes predisposing for numerical differences in teeth are searched
• 21 SNPs were genotyped in 13 genes by PCR-RFLP Patients: 159 congenital hypodontia or oligodontia Controls: 185 fully healthy, wisdom tooth also
• frequency of SNPs were compared in patients and controls
  => result: CHDH gene deficiency affects the number of teeth

Question 20

What is the method that measures Whole genome gene expression?

Answer 20

microarray
e.g.: 44k chip: measures the expression of 44,000 mRNAs simultaneously.

Question 21

What is the role of DNA microarray?

Answer 21

• It measures DNA or uses DNA as a part of its detection system.
• In each of the spots in this array, a known DNA sequence or gene is orderly arranged.

Question 22

What are the features of sequencing 2nd generation (New generation, ie NGS = next generation sequencing or short-read sequencing, massively parallel sequencing)?

Answer 22

• NGS (next generation sequencing)
• Advantage: The majority of genetic variations can be detected
• Disadvantage: difficult to evaluate (too many data)

Question 23

What are the examples of sequencing 2nd generation (New generation, ie NGS = next generation sequencing or short-read sequencing, massively parallel sequencing)?

Answer 23

1. Exome sequencing: for monogenic diseases
2. RNA-seq: gene expression measurement
3. Bisulfite sequencing: methylation pattern
4. Sequencing of pathogens
5. Metagenomics: determination of the microbiome 55

Question 24

Explain WES (whole exome sequencing) vs. WGS (Whole genome sequencing)

Answer 24

1. Genome > 3 billion bp; >15 million bp difference vs reference
   genome
2. Exome: 1.2 % of the genome; ≈180.000 exons
3. Majority of monogenic diseases are caused by mutations in protein coding regions (exons)

Question 25

What are Advantages of Exome Sequencing?

Answer 25

1. Majority of monogenic diseases are caused by mutations in protein coding regions (exons) 2. Provides a cost-effective alternative to whole-genome sequencing (4–5 Gb of sequencing per exome compared to ≈90 Gb per whole human genome) 3. Produces a smaller, more manageable data set for faster, easier analysis compared to whole-genome approaches 4. Cheaper, easier to interpret

Question 26

What are weaknesses of Exome sequences?

Answer 26

In ≈70% of patients in whom there was a high degree of pre-test suspicion for a monogenic rare diseases, exome sequencing provides no molecular diagnosis.

Question 27

What are the reasons of Exome sequences?

Answer 27

1. Mutations in non-coding regions: RNA genes (underrepresented); regulatory regions (enhancers, promoters, introns) 2. Mosaicism 3. Short read sequencing is not good in detecting indels (small and large insertion-deletions), copy-number variations (CNVs), and chromosomal rearrangements 4. Epigenetics

Question 28

What are the 2 Methods for identification of larger deletions or inversions?

Answer 28

1. MLPA: Multiplex ligation-dependent probe amplification (see in practice) 2. Long range sequencing (third generation sequencing) creating ten thousand to a hundred thousand bases) length DNA sequences. - Nanopore sequencing - PacBio sequencing

Question 29

What are the Advantages of third generation, long read sequencing?

Answer 29

“long-read sequencing harbors the potential to deliver near-perfect genome analysis in the future and long-read optical mapping may replace almost all karyotyping, FISH, and CNV- microarray assays in routine diagnostics.”

Question 30

What is the Possible role of epigenetic regulation in diseases?

Answer 30

1. MZ twins with identical pathogenic mutation in WAS gene 2. Discordant phenotypes: - X-linked thrombocytopenia (XLT) - More severe Wiskott-Aldrich syndrome-like (WAS; OMIM 301000) phenotype 3. Researchers found that the sibling with a more aggressive phenotype showed a DNA hypermethylation compared to the brother with XLT.

Question 31

What is the mechanism of Detection of methylation pattern: whole-genome bisulfite sequencing (WGBS)?

Answer 31

1. In bisulfite conversion cytosine is converted to uracil using sodium bisulfite, while 5-methylcytosine (5-mC) remains intact. 2. After PCR uracil becomes thymine 3. Comparison of the bisulfite-treated and non-treated by sequencing. Where cytosine remained cytosine, it was methylated.

Question 32

What are Shortcomings of animal models?

Answer 32

1. Similar diseases can have different pathomechanisms 2. At genomic levels the differences are larger (junk regions). 3. Homologous genes with different functions

Question 33

What are features of Genetically modified animals?

Answer 33

1. „KO” animals: – Disease models can be developed with inactivating genes – Study of gene functions 2. Transgenic animals: disease models, gene functions 3. In vivo gene modification, see CRISPR/Cas9

Question 34

Give the Summary: Methodology

Answer 34