Medical Genetics- Grody Flashcards
What are the “pros” and “cons” of using Southern blots and what is the importance of restriction enzyme digestion in molecular diagnostics?
Southern Blotting is VERY SPECIFIC using probes
-The restriction enzyme allows for the cutting of large fragments of DNA at specific points, these can be separated by Gel Electrophoresis & then transferred to Nitrocellulose/Nylon and hybridized with probes
CONS: SLOW, not good for prenatal screening
What are the “pros” and “cons” of PCR in molecular diagnostics?
Pros: VERY FAST, can be done from anywhere in the body - NON-INVASIVE
Cons: NOT SPECIFIC (compared to Southern Blot) and cannot run PCR’s on very long DNA fragments
Why is DNA sequencing the “gold standard” (e.g. most accurate and high yield method) for mutation testing?
DNA sequencing is the gold standard because it actually lists the nucleotide sequence. (able to see Base Pair Changes opposed to inferring them from DNA fragments)
List the four different sampling methods used in prenatal diagnosis. What are some advantages and disadvantages associated with them? Which ones are invasive and which ones are non-invasive?
- Aminocentesis - sampling of the amniotic fluid
- Chorionic Villus Sampling - can be done earlier than Amniocentesis, but has HIGH RISK for miscarriage
- Fetal Blood Sampling (PUBS)
- Fetal Tissue Biopsy
All are considered invasive.
Which of the two sampling methods are rarely used and why?
Fetal Blood Sampling (PUBS) and Fetal Tissue Biopsy
There is a great deal that can be done with the data attained from Amniocentesis &CVS, that the last two are rarely done (they are much more invasive & involve a MUCH HIGHER CHANCE of MISCARRIAGE
What is the importance and utility of pre-implantation genetic diagnosis? Under what conditions might a clinician recommend it be used?
“in vitro” fertilization & diagnosis before implantation allows you to select for UNAFFECTED embryos for implantation (avoiding the need for termination later)
-useful if parents are carriers for a genetic disease
What are the different groups that genetic disorders can be classified at the molecular level(4)?
- Disorders for which both the gene and mutation are known.
- Easiest for molecular techniques. - Disorders for which the gene is known, but not the mutation.
- Most commonly encountered. - Disorders for which neither the gene nor the mutation is known.
- Not really dealt with since human genome project.
- Linkage analysis and needs lots of family members. - Polygenic disorders.
- Can’t do at all.
- Typical diseases of internal medicine.
- Affected by many genes and environment.
What are “polygenic disorders” and why are the genes associated with these polygenic diseases difficult to identify using molecular techniques?
Polygenic Disorders are due to hundreds/thousands of gene interactions = DIFFICULT to identify with present techniques
*most diseases in INTERNAL MEDICINE are due to polygenic disorders
What are some important facts about the Duchenne Muscular Dystrophy gene and the DMD gene mutations associated with the disease?
- Largest gene known: >2.5 million bp
- 79 exons
- Mature mRNA transcript of 14 kb
- Deletions found in 2/3 of patients
How is multiplex PCR used in the detection of mutations in DMD patients? What is the advantage of using multiplex PCR when testing for DMD mutations as compared to other molecular laboratory methods?
It was first used to detect deletions in the Dystrophy gene
- Allows for the running of multiple strands of DNA at once & makes it possible to look for multiple deletions rather than one at a time (normal PCR)
What are the clinical features associated with cystic fibrosis (CF)?
Symptoms: •Abnormal secretions •Chronic pulmonary disease •Exocrine pancreatic insufficiency •Failure to thrive, meconium ileus in infants •Sinusitis, nasal polyps •Infertility in males •Elevated sweat electrolyte levels
What are the different methods used in the diagnosis of and screening for CF?
SWEAT TEST - Imunoreactive Trypsin test - DNA analysis (carriers are recessive) *high rate in NORTH AMERICAN CAUCASIANS Gene: •Complete gene locus spans 250 kb •27 exons •Mature mRNA of 6500 bases •Encodes an ion channel of 1480 amino acids (CFTR)
What is the ΔF508 mutation and what does this abbreviation mean?
Most common: Three-nucleotide deletion of codon 508 (phe) in 70% of CF patients.
•∆F508 (deletion of phenyalanine at codon 508)
What is the current recommendation for CF screening?
SWEAT TEST - for those affected
*NIH panel recommended in 1997 that CF screening be an option for anyone who is pregnant/planning to be pregnant (as well of family of people w/CF)
What criteria were used to select specific mutations for inclusion in the core mutation panel for CF screening? What laboratory method is currently used for screening for the mutations in the CF panel?
(Grody’s study)
Test that could be done early on in pregnancy, and used the cytobrush method of gathering mouth mucosal cells for PCR test.
-Grody tested the GENERAL POPULATION (not exclusively families that had a known CF history)
Screening for 6 of the more widely known mutations increased the specificity of the test (GREATER ACCURACY)
- led to the development of the Probe Testing Unit for CF
Why is the residual risk of testing negative after a negative CF genetic test not 0?
A negative test implies only 92% risk of NOT being a carrier this is based on the six mutations that they screen for.
Certain mutations were removed from the panel since they occurred much less than expected (1078delT) and others turned out to be polymorphisms rather than mutations responsible for CF, but were present when there were other deletion somewhere else that caused CF (I148T)
What is the significance of the BRCA 1 and 2 genes in familial breast and ovarian cancer?
compared with normal population, those with BRCA 1,2 more LIKELY TO HAVE BREAST & OVARIAN CANCER
Linked to Ovarian cancer, although rates of ovarian cancer are less compared with breast cancer, it is still significantly higher than normal population (1% for population; hereditary 44% BRCA1 and 27% BRCA2)
What is the mode of inheritance of the BRCA 1 and BRCA 2 disease-causing mutations?
BRCA is a Dominant disease
How do mutations in BRCA 1 and BRCA 2 increase the hereditary risk for early onset breast and for early onset ovarian cancer as compared to the general population risk?
WT risk: 10% likelihood by age 90
BRCA1 and BRCA 2: Over 87% likelihood by 90
How is genetic testing for BRCA 1 and BRCA 2 mutations performed?
Sequence Analysis:
BRCA1: 22 coding exons, > 5,500 bp
BRCA2: 26 coding exons, > 11,000 bp
What are the various possible results of genetic testing for BRCA 1 and BRCA 2 mutations? What is the significance of the following possible test results?
1. When a patient tests positive for a deleterious mutation 2. When there is no mutation detected 3. When the identified mutation has uncertain clinical significance (6 steps)
Test Results:
-Positive for a deleterious mutation
-No mutation detected
• Mutation previously identified in the family
• No known mutation in the family
-Uncertain clinical significance
1. When a patient tests positive for a deleterious mutation: you can proceed with surgery or whatever treatment necessary for the pt
2. When there is no mutation detected: you still must have further testing since you can’t rule out the possibility of new mutations
*If there is no one in the family then you can utilize population risk for the disease to acquire at least some measurability
3. When the identified mutation has uncertain clinical significance:
i. Examine genetic code, nature of AA substitution
ii. Position of affected residue in 3-D protein structure
iii. Study of additional family members
iv. General population studies
v. Evolutionary conservation
vi. Correlation with phenotype: family hx, histology, molecular/biochemical defects
What are some approaches for classifying novel missense variants in the BRCA1 and BRCA2 genes identified by the genetic test? Which one of these approaches provides the most convincing evidence that a specific novel missense variant is pathologic?
- Examine genetic code, nature of AA substitution
- Position of affected residue in 3-D protein structure
- Study of additional family members
- General population studies
- Evolutionary conservation
- Correlation with phenotype: family history, histology, molecular/biochemical defects (e.g., MSI, ER/PR)
- Functional studies
- Family hx is most convincing
What is the “economy class syndrome” and what is its relationship to the factor V Leiden mutation?
Economy Class Syndrome: predisposition to clotting more than normal (formation of embolysim during flight with not enough movement)
Factor V- Leiden:
•Clotting factor 5 has a mutation that does not allow it be to cut/deactivated.
•Get clots.
•Not recommended for mass population. Pretty rare.
Penetrance anywhere from 10-90% that if you have it you still might not get it
What are the mutations seen in fragile X patients and why is Southern blotting used to detect these mutations? What is the full mutation range of the CGG repeat in fragile X syndrome and what occurs to alleles with over 200 repeats?
•Affects mostly boys.
•Mental retardation and dysmorphic facial features.
•Looks like the end of the chromosome is about to break off where the mutation is located on the X chromosome.
•CGG repeat:
•Normal range: 6-54 repeats
•Premutation range: 52-200 repeats
•Full mutation range: 200-> 1000 repeats
oHard for PCR to detect because it can be so big.
•Alleles with >200 repeats are hypermethylated, transcriptionally repressed (the DNA pol “fell off”)
