W12LECT - Genomics in dental medicine Flashcards
Amelogenesis imperfecta
1. What are the features of Amelogenesis imperfecta?
1/ X – Dominant
2/ Tooth enamel formation problem
Amelogenesis imperfecta
1. What are the features of Amelogenesis imperfecta?
1/ X – Dominant
2/ Tooth enamel formation problem
3/ Genes affected”
- AMELX; Xp22.3-1; amelogenin
- ENAM; 4q13.3; enamelin (5%)
How do we find mutation is in the patient?
- collect DNA of the patients
– isolate it from blood - collect medical data of the patients
- analyze the pedigree
What is the use of Sequencing?
- find a new mutation in a specific area
- search for SNPs, small insertions, deletions, duplications
How do we perform Genotyping by sequencing?
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)
What is the result of Genotyping by sequencing?
- New SNV in a specific area
- A deletion was found in some patients
What are the features of Genotyping by Sanger sequencing
What are the 2 Methods for the genomic backgrounds of diseases?
- Wet laboratory methods (classical methods, lab experiments)
- in silico methods (analyze data, bioinformatics)
What are the 2 Methods for the genomic backgrounds of diseases?
- Wet laboratory methods (classical methods, lab experiments)
- in silico methods (analyze data, bioinformatics)
What are the features of In silico methods?
- (Re) analysis of data from on-line databases
- Lots of online software
- Discovering new genes (APOA5)
- Meta-analyzes (joint analysis of several previous studies)
What are the 2 Genomics methods for studying the genetic background of disease?
- Hypothesis-driven approaches
- Hypothesis-free approaches
How do we perform In silico methods?
- alleles or genotypes of markers (e.g.: SNPs, microsatellites), are determined in patients vs. in controls
Association studies
1. Association is a statistical statement
=> What is this statement?
- The specific allele significantly more likely to occur WITH a specific phenotype
- 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
Genetic markers
1. What is a genetic marker?
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.
Genetic markers
2. What are examples of genetic markers?
How should Microsatellite detection be?
Examples of using STR as markers
1. How do we use STR as markers in Prenatal diagnosis?
5-6 STR markers/chromosome
How do we Use SNP/SNV as markers
They are only biallelic, but much more than STR >300 million vs. ≈ 30,000
Easier to detect
Give an example of Using SNP/SNV as markers?
- 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
What is the method that measures Whole genome gene expression?
microarray
e.g.: 44k chip: measures the expression of 44,000 mRNAs simultaneously.
What is the role of DNA microarray?
- 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.
What are the features of sequencing 2nd generation (New generation, ie NGS = next generation sequencing or short-read sequencing, massively parallel sequencing)?
- NGS (next generation sequencing)
- Advantage: The majority of genetic variations can be detected
- Disadvantage: difficult to evaluate (too many data)
What are the examples of sequencing 2nd generation (New generation, ie NGS = next generation sequencing or short-read sequencing, massively parallel sequencing)?
- Exome sequencing: for monogenic diseases
- RNA-seq: gene expression measurement
- Bisulfite sequencing: methylation pattern
- Sequencing of pathogens
- Metagenomics: determination of the microbiome 55
Explain WES (whole exome sequencing) vs. WGS (Whole genome sequencing)
- Genome > 3 billion bp; >15 million bp difference vs reference
genome - Exome: 1.2 % of the genome; ≈180.000 exons
- Majority of monogenic diseases are caused by mutations in protein coding regions (exons)
What are Advantages of Exome
Sequencing?
- Majority of monogenic diseases are caused by mutations in protein coding regions (exons)
- Provides a cost-effective alternative to whole-genome sequencing (4–5 Gb of sequencing per exome compared to ≈90 Gb per whole human genome)
- Produces a smaller, more manageable data set for faster, easier analysis compared to whole-genome approaches
- Cheaper, easier to interpret
What are weaknesses of Exome sequences?
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.
What are the reasons of Exome sequences?
- Mutations in non-coding regions: RNA genes (underrepresented); regulatory regions (enhancers, promoters, introns)
- Mosaicism
- Short read sequencing is not good in detecting indels (small and large insertion-deletions), copy-number variations (CNVs), and chromosomal rearrangements
- Epigenetics
What are the 2 Methods for identification of larger deletions or inversions?
- MLPA: Multiplex ligation-dependent probe amplification (see in practice)
- Long range sequencing (third generation sequencing) creating ten thousand to a hundred thousand bases) length DNA
sequences.
- Nanopore sequencing
- PacBio sequencing
What are the Advantages of third generation, long read sequencing?
“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.”
What is the Possible role of epigenetic regulation in diseases?
- MZ twins with identical pathogenic mutation in WAS gene
- Discordant phenotypes:
- X-linked thrombocytopenia (XLT)
- More severe Wiskott-Aldrich syndrome-like (WAS; OMIM 301000) phenotype - Researchers found that the sibling with a more aggressive phenotype showed a DNA hypermethylation compared to the brother with XLT.
What is the mechanism of Detection of methylation pattern: whole-genome bisulfite sequencing (WGBS)?
- In bisulfite conversion cytosine is converted to uracil using sodium bisulfite, while 5-methylcytosine (5-mC) remains intact.
- After PCR uracil becomes thymine
- Comparison of the bisulfite-treated and non-treated by sequencing. Where cytosine remained cytosine, it was methylated.
What are Shortcomings of animal models?
- Similar diseases can have different pathomechanisms
- At genomic levels the differences are larger (junk regions).
- Homologous genes with different functions
What are features of Genetically modified animals?
- „KO” animals:
– Disease models can be developed with inactivating genes
– Study of gene functions - Transgenic animals: disease models, gene functions
- In vivo gene modification, see CRISPR/Cas9
Give the Summary: Methodology
