Genetic testing and medical diagnostics

Question 1

More than _____ human gene tests are in diagnostic use.

Answer 1

900

Question 2

It is becoming increasingly prevalent to

Answer 2

directly examine an individual’s DNA for mutations associated with disease.

Question 3

Genetic tests are used for

Answer 3

prenatal, childhood, and adult prognosis and diagnosis of genetic diseases.

Question 4

A prognostic test

Answer 4

predicts a person’s likelihood of developing a particular genetic disorder.

Question 5

A diagnostic test

Answer 5

for a genetic condition identifies a particular mutation or genetic change that causes the disease or condition.

Question 6

Can also test for

Answer 6

‘carriers’.

Question 7

These tests usually detect

Answer 7

gene alterations associated with single-gene disorders inherited in a Mendelian fashion.

Question 8

Examples include

Answer 8

sickle-cell anemia, cystic fibrosis, Huntington disease, hemophilia, and muscular dystrophy.
Also for more complex disorders like cancer.
Sampling for DNA testing are from white blood cells and cheek (buccal) swabs, hair cells or from gametes.
Results using either method are exactly the same (genomic DNA).

Question 9

Prenatal genetic tests are used for

Answer 9

certain disorders in which waiting until birth is not desirable

Question 10

For prenatal testing, fetal cells are obtained by

Answer 10

amniocentesis (fetal cells from amnionic fluid is harvested) or chorionic villus sampling (CVS – cells from the fetal part of the plasenta wall are collected by suction).

Question 11

Captured fetal cells can then be

Answer 11

subjected to genetic analysis by techniques that involve PCR (such as allele-specific oligonucleotide testing, or sequencing.

Question 12

More genetic testing has been used to detect genetic conditions in babies than in adults.

Answer 12

About 60 conditions that can be detected, although not all use DNA-or RNA-based genetic tests – some test for proteins or other metabolites.

Question 13

Question 14

Approx. _____ of the DNA in a pregnant mother’s blood belongs to the fetus.

Answer 14

3-6 %

Question 15

Approx. 3-6 % of the DNA in a pregnant mother’s blood belongs to the fetus.
These are called cell-free DNA (cfDNA) and…

Answer 15

can be extracted and analyzed for genetic conditions such as Down syndrome (MarteniT® 21PLUS).

Question 16

WGS of maternal blood-plasma cfDNA can be used to accurately sequence the entire exome of a fetus.

Question 17

Deducing fetal genome sequences from maternal blood – haplotype analysis (contiguous DNA fragments that do not undergo recombination during gamete formation).

Question 18

For any given chromosome, a fetus inherits one copy of a haplotype from the mother (maternal copies, M1 or M2) and another from the father (paternal copies, P1 or P2). Here the fetus inherited haplotypes M2 and P2 from the mother and father, respectively. DNA from the blood of a pregnant woman would contain paternal haplotypes inherited by the fetus (P2), maternal haplotypes that are not passed to the fetus (M1), and maternal haplotypes that are inherited by the fetus (M2). The maternal haplotype inherited by the fetus (M2) would be present in excess amounts relative to the maternal haplotype that is not inherited (M1).

Question 19

Restriction fragment length polymorphism (RFLP) analysis

Answer 19

Variation in the length of DNA fragments generated by restriction endonucleases.

Question 20

Variation in the length of DNA fragments generated by restriction endonucleases.

Answer 20

These variations are caused by mutations that create or abolish cutting sites for restriction enzymes.

Question 21

RFLPs are inherited in a
_____________ fashion and are useful as genetic markers

Answer 21

codominant

Question 22

Example of RFLPs

Answer 22

Sickle cell anemia

Question 23

Only about________ of all point mutations can be detected by RFLP analysis
______ revealed many more disease-associated mutations
Now use _________ to detect these mutations.

Answer 23

5-10 %
HGP
PCR and synthetic oligonucleotides (ASOs)

Question 24

Allele-specific oligonucleotides

Answer 24

Short, single-stranded fragment of DNA designed to hybridise to a complementary specific allele in the genome.

Question 25

Under proper conditions, an ASO will hybridise only with

Answer 25

its perfect complementary DNA sequence and not with other sequences, even those that vary by as little as a single nucleotide.

Question 26

How does Allele-specific oligonucleotides

Answer 26

Region of interest is PCR-amplified and spotted on DNA-binding membrane. Hybridised with labelled ASO and visualised by fluorescence. Can detect single nucleotide polymorphisms (SNPs). Rapid and relatively cheap – routine tests.

Question 27

ASOs are ideal for pre-implantation genetic diagnosis (PGD)

Answer 27

PCR amplification of small quantity of DNA allows for genetic analysis of single cells from embryos created by in vitro fertilisation (IVF). Any alleles that can be detected by ASO testing can be used during PGD. Sickle-cell anemia, cystic fibrosis, and dwarfism are often tested for by PGD, but alleles for many other conditions are also often analysed. PGD should improve embryo implantation success rates and reduce miscarriages for couples.

Question 28

pre-implantation genetic diagnosis (PGD)

Answer 28

Question 29

Demand for genetic tests that detect complex mutation patterns or previously unknown mutations in a single gene associated with genetic diseases and cancers.

Answer 29

Microarrays can generate, simultaneously, an immense amount of information from a single array (50K spots).

Question 30

For example, the CFTR gene (responsible for cystic fibrosis) is 250 Kb in size and has 27 exons and contains approx. 1000 known mutations (about half are point mutations, insertions, and deletions - and they are widely distributed throughout the gene).

Question 31

Eg of microarrays:

Answer 31

Gene expression analysis Disease diagnosis and drug development. Nutrigenomics Genome scanning DNA genotyping

Question 32

DNA genotyping

Answer 32

– test for gene mutations. Human genome microarrays containing probes for most human genes are available, including the p53 gene and the BRCA1 gene.

Question 33

Genome scanning

Answer 33

– test for SNPs. Uses SNP sequences as probes on a DNA microarray allowing to simultaneously screen thousands of mutations that might be involved in single-gene diseases as well as those involved in disorders exhibiting multifactorial inheritance. Possible to analyze a person’s DNA for dozens or even hundreds of disease alleles .

Question 34

Gene expression analysis

Answer 34

–compare differential expression under defined conditions.

Question 35

Disease diagnosis and drug development.

Question 36

Nutrigenomics

Answer 36

– gene expression profiles linked to the metabolism of specific nutrients.

Question 37

Question 38

Gene expression analysis –compare differential expression under defined conditions.(Transcriptomics)

Answer 38

mRNA is isolated from two different cell or tissue types – e.g. normal cells and cancer cells arising from the same cell type. The mRNA samples contain transcripts from each gene that is expressed in that cell type. The expression level of each mRNA can be used to develop a gene-expression profile that is characteristic of the cell type under the specific conditions.

Question 39

Certain cancers have distinct patterns of gene expression, correlated to factors such as the cancer stage and response to drugs.

Answer 39

Gene expression analysed in normal white blood cells and in cells from a white blood cell cancer known as diffuse large B-cell lymphoma (DLBCL). Assayed the expression profiles of 18,000 genes and discovered that there were two types of DLBCL, with almost inverse patterns of gene expression!

Question 40

Pathogen Identification – use of Microarrays and NGS

Answer 40

Whole-genome analysis to identify genes associated with infections

Question 41

Hosts are infected in vitro with bacteria, yeast, protist, or viral pathogens, and then gene-expression microarrays are used to analyze pathogen gene-expression profiles.

Answer 41

Patterns of pathogen gene activity during infection and replication are useful for identifying pathogens and understanding mechanisms of infection.

Question 42

NGS and third-generation sequencing (TGS) methods are being used for pathogen identification.

Answer 42

Whole-genome sequencing (WGS) is a quick way to identify pathogenic bacteria, viruses, or other microorganisms, and also valuable for tracking genetic variations in microorganisms. CERI @SU: NGS provided a large body of data on evolving strains of SARS-Cov-2, identified delta and omicron variants.

Question 43

A primary goal of infectious disease research is to prevent infection. Gene-expression profiling can identify important pathogen genes and the proteins they encode. The latter may prove to be useful targets for subunit vaccine development or for drug treatment strategies to prevent or control infectious disease.

Question 44

Genome screening for “good” genes/mutations Natural or “healthy knockouts” - the fortunate few individuals who may lack a specific gene or have a mutation in a disease-causing gene that provides a health benefit

Answer 44

A rare mutation (1 person in 10 million) in the human gene ISG15 protects individuals with this mutation against many, if not most viruses. ISG15 mutations knock out a function that helps to dampen inflammation, so individuals with this mutation have a heighted inflammatory system, which helps fight off viruses. CCR5 delta-32 mutation makes one immune to HIV infection (1% of Northern European population).