16. Molecular genetic methods and applications in human genetics III

Question 1

Applications of genetic tests

Answer 1

1) Diagnostic/predictive
- Direct detection of mutation, confirm/excude mutation or qualify prognosis
2) Population wide screening
3) Prenatal diagnosis

Question 2

Prenatal diagnosis

Answer 2

Invasive

• CV=chorionic villus sampling (10-12 wks gestation)
• Amniocentesis (14-16 wks gestation)

Non-invasive

• Maternal serum screening at 16 wks (AFP)
• US
• Study of fetal cells from maternal blood

Preimplantation diagnosis

Question 3

Non-invasive studies to test Down syndrome

Answer 3

1) US - nuchal translucency increased + nasal bone absent
2) Maternal age
3) Maternal serum markers - hCG increased

Question 4

Maternal serum markers

Answer 4

• PAPP-A (pregnancy associated plasma protein A
• AFP (alphafetoprotein)
• Estriol (UE3)
• Inhibin-A (inhibin)
• hCG (human chorion gonadotrophin)

Question 5

Microsatellites

Answer 5

Microsatellites are di-, tri-, or tetra nucleotide tandem repeats in DNA sequences. The number of repeats is variable in populations of DNA and within the alleles of an individual - kind of a “fingerprint”.

• PCR of chromosome specific markers
• Capillary electrophoresis

Question 6

PrenaTest

Answer 6

Non-invasive prenatal testing

- Can identify 7 chromosomal disorders

Question 7

Comparative genome hybridization (CGH)

Answer 7

Whole genome of patient and control as samples

• Nick translation
• Fluorescent dyes - red for patient, green for control
• Chromosomes are fixed in metaphase + hybridized to slide

Results:

• Where patient and control are equally hybridized - rate of fluorescent signal is also equal (=1 - yellow)
• More intense patient signal - genome has extra copies
• Less intense patient signal - genome has a deficit

Question 8

CGH advantages compared to karyotyping and FISH + disadvantage

Answer 8

1) Advantages over karyotyping
- Easily noticable differences
- Higher resolution (100 kb vs 5-10Mb)
2) Advantages over FISH
- Can examine more than only specific, pre-selected regions
3) Disadvantage
- Contamination (in tumor samples are also healthy cells)

Question 9

Microarray technology

Answer 9

High resolution nucleotide chain hybridization

1) Nucleotide probes bound to solid surface (glass chip, bead)
2) Labelled samples are hybridized to probes
3) Measure signal intensity

Question 10

Microarray applications

Answer 10

• aCGH/CNV/SNP
• Gene expression
• microRNA
• Methylation array

Question 11

Clinical use of aCGH

Answer 11

• Disease focused arrays (DMD array)
• Hematological and cancer arrays
• Prenatal arrays
• Preimplantation arrays

Question 12

Gene expression microarray

Answer 12

• Mainly in research
• Compare mRNA expression in tested samples
• Complex bioinformatical analysis (GSEA)

Question 13

Gene expression microarray applications

Answer 13

• Microbiology-pathogen and host interaction studies
• Drug development, drug efficiency testing
• Biomarker studies (tumor diagnostic)

Question 14

MammaPrint

Answer 14

Check breast cancer - risk of recurrence within 10 years after diagnosis (early stage breast cancer)

• Low risk vs high risk
• Expression pattern of 70 genes
• Low risk: don’t need chemo after surgery

Question 15

microRNA microarray

Answer 15

microRNA profile is distinct in healthy and cancer tissues, or between tumors

Question 16

Methylation detection by PCR

Answer 16

Bisulfite PCR, MS-PCR (methylation sensitive)

• Treatment of DNA with bisulfite converts cytosine residues to uracil in CpG sites - but leaves 5-methylcytosine residues unaffected
• During PCR unmethylated cytosines are displayed as thymines in the resulting amplified sequence
• Can be verified by sequencing

Question 17

Methylation microarray

Answer 17

• Used to test DNA methylation

- Used for epigenetic studies

Question 18

Huntington disease detection

Answer 18

PCR

• Detection of a length polymorphism (VNTR)
• CAG repeats
• Molecular marker including a range of CAG repeats should be used in each analysis

Question 19

Huntington normal vs mutant

Answer 19

Normal: 11-34 CAG repeats

Mutant 42 -> CAG repeats

Question 20

Triplet repeat mutation diseases

Answer 20

• Fragile X
• Freidrich’s ataxia
• Muscular dystrophy
• Myotonic dystrophy
• Spinobulbar muscular dystrophy
• Spinocerebellar ataxia
• Huntington disease

Question 21

Detection Hb S steps

Answer 21

1) DNA amplification
2) Digestion with restriction endonuclease
3) Electrophoresis
4) Staining with ethidium-bromide

Question 22

Indentification of a disease causing gene sequence

Answer 22

1) Linkage
2) Gene localization
3) Gene identification
4) Gene structure and sequence analysis
5) Gene product
* This gives better predictive analysis, better diagnosis and better treatment

Question 23

Identification sequence of CFTR

Answer 23

1) Linkage:
- Salt transport involvement
- Find a genetic disease feature with known location: DOCRI-917
2) Gene localization
- Tight linkage with RFLP-markers on long arm of chr 7
3) Gene identification
- 920bp long cDNA found from sweat gland
4) Gene structure and sequence analysis
- Sequence show that gene codes for a protein with a transmembrane region
- Expressed in every affected tissue!
- Samples from patients showed triplet deletion (phenylalanine)
5) Gene product
- Chloride ion channel - gene is CFTR

Question 24

CFTR mutations

Answer 24

• More than 1000 mutations of CFTR gene are known

- Most frequent mutation: del-F508

Question 25

Method for identification of delF508

Answer 25

Most frequent mutation in CF

1) PCR
2) Allele specific PCR
3) Capillary electrophoresis: Separation of PCR products

Question 26

NGS

Answer 26

Next generation sequencing assay

• Detection of rare mutations
• MiSeqDx for CF was first FDA-cleared in vitro diagnostic NGS assay

Question 27

Detection of DNA polymorphism (SNP, VNTR)

Answer 27

• Blot-based indentification: by specific probes (!) (RFLP)

- PCR-based identification: by specific primers (!) - forward and reverse primers