Diagnostic Molecular Genetics- Grody

Question 1

What are the “pros” and “cons” of using Southern blots and what is the importance of restriction enzyme digestion in molecular diagnostics?

Answer 1

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 probesCONS: SLOW, not good for prenatal screening

Question 2

What are the “pros” and “cons” of PCR in molecular diagnostics?

Answer 2

Pros: VERY FAST, can be done from anywhere in the body - NON-INVASIVECons: NOT SPECIFIC (compared to Southern Blot) and cannot run PCR’s on very long DNA fragments

Question 3

Why is DNA sequencing the “gold standard” (e.g. most accurate and high yield method) for mutation testing?

Answer 3

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)

Question 4

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?

Answer 4

1. Aminocentesis - sampling of the amniotic fluid2. Chorionic Villus Sampling - can be done earlier than Amniocentesis, but has HIGH RISK for miscarriage3. Fetal Blood Sampling (PUBS)4. Fetal Tissue BiopsyAll are considered invasive.

Question 5

Which of the two sampling methods are rarely used and why?

Answer 5

Fetal Blood Sampling (PUBS) and Fetal Tissue BiopsyThere 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

Question 6

What is the importance and utility of pre-implantation genetic diagnosis? Under what conditions might a clinician recommend it be used?

Answer 6

“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

Question 7

What are the different groups that genetic disorders can be classified at the molecular level?

Answer 7

1. Disorders for which both the gene and mutation are known.-Easiest for molecular techniques.2. Disorders for which the gene is known, but not the mutation.-Most commonly encountered.3. 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.4. Polygenic disorders.-Can’t do at all.-Typical diseases of internal medicine.-Affected by many genes and environment.

Question 8

What are “polygenic disorders” and why are the genes associated with these polygenic diseases difficult to identify using molecular techniques?

Answer 8

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

Question 9

What are some important facts about the Duchenne Muscular Dystrophy gene and the DMD gene mutations associated with the disease?

Answer 9

-Largest gene known: >2.5 million bp-79 exons-Mature mRNA transcript of 14 kb-Deletions found in 2/3 of patients

Question 10

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?

Answer 10

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)

Question 11

What are the clinical features associated with cystic fibrosis (CF)?

Answer 11

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

Question 12

What are the different methods used in the diagnosis of and screening for CF?

Answer 12

SWEAT TEST- Imunoreactive Trypsin test- DNA analysis (carriers are recessive)*high rate in NORTH AMERICAN CAUCASIANSGene: •Complete gene locus spans 250 kb•27 exons•Mature mRNA of 6500 bases•Encodes an ion channel of 1480 amino acids (CFTR)

Question 13

What is the ΔF508 mutation and what does this abbreviation mean?

Answer 13

Most common: Three-nucleotide deletion of codon 508 (phe) in 70%.•∆F508 (deletion of phenyalanine at codon 508)

Question 14

What is the current recommendation for CF screening?

Answer 14

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)

Question 15

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)

Answer 15

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

Question 16

Why is the residual risk of testing negative after a negative CF genetic test not 0?

Answer 16

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)

Question 17

What is the significance of the BRCA 1 and 2 genes in familial breast and ovarian cancer?

Answer 17

compared with normal population, those with BRCA 1,2 more LIKELY TO HAVE BREAST & OVARIAN CANCERLinked 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)

Question 18

What is the mode of inheritance of the BRCA 1 and BRCA 2 disease-causing mutations?

Answer 18

BRCA is a Dominant disease

Question 19

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?

Answer 19

WT risk: 10% likelihood by age 90BRCA1 and BRCA 2: Over 87% likelihood by 90

Question 20

How is genetic testing for BRCA 1 and BRCA 2 mutations performed?

Answer 20

Sequence Analysis:BRCA1: 22 coding exons, > 5,500 bpBRCA2: 26 coding exons, > 11,000 bp

Question 21

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)

Answer 21

Test Results:-Positive for a deleterious mutation-No mutation detected • Mutation previously identified in the family • No known mutation in the family-Uncertain clinical significance1. When a patient tests positive for a deleterious mutation: you can proceed with surgery or whatever treatment necessary for the pt2. 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 measurability3. When the identified mutation has uncertain clinical significance: i. Examine genetic code, nature of AA substitutionii. Position of affected residue in 3-D protein structureiii. Study of additional family membersiv. General population studiesv. Evolutionary conservationvi. Correlation with phenotype: family hx, histology, molecular/biochemical defects

Question 22

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?

Answer 22

-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

Question 23

What is the “economy class syndrome” and what is its relationship to the factor V Leiden mutation?

Answer 23

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

Question 24

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?

Answer 24

•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 repeatsoHard for PCR to detect because it can be so big.•Alleles with >200 repeats are hypermethylated, transcriptionally repressed (the DNA pol “fell off”)

Question 25

What are some late-onset symptoms/manifestations of the fragile X premutation in male and female carriers?

Answer 25

•Recent reports of premature ovarian failure in female premutation carriers.•Late-onset tremor-ataxia-dementia syndrome in male premutation carriers.•May be due to mRNA interference with expression of the normal FMR1allele or of other genes.

Question 26

Why is asymptomatic and predictive genetic testing of children not usually done for adult onset diseases (e.g. Huntington’s disease). Under what circumstances might predictive genetic testing of children be done?

Answer 26

Genetic testing of children for adult onset diseases should not be undertaken unless direct medical benefit will accrue to the child and this benefit would be lost by waiting until the child has reached adulthood.- No reason to stigmatize a child if he/she is asymptomatic.- If a child tests positive, so does the parent.-When to do predictive testing: If direct medical benefit can be gainedExample: multiple endocrine neoplasia(MEN2) (tumors in endocrines) or FAP (colon cancer with polyps)

Question 27

What is the importance of “40 or above” repeats in Huntington’s disease. What types of repeats are they and what amino acid do they code for?

Answer 27

40 or more CAG repeats in the HTT gene CROSSES THE THRESHOLD & will produce a dsyfunctional protein, causing manifestation of huntington disease.

Question 28

What are the major types of polymorphic markers used in genetic testing? Which type of marker is used in forensic and paternity testing? What are the applications of DNA fingerprinting?

Answer 28

1. Restriction fragment length polymorphisms (RFLP)2. Variable number tandem repeats (VNTR)3. Short tandem repeats (STR)•1-4 repeats in tandem, repeated by PCR.•Most commonly used.4. Single nucleotide polymorphisms (SNP)*Used for paternity testing Applications of DNA fingerprinting:-Paternity testing-Establishing zygosity of twins-Determining bone marrow transplant engraftment-Identifying mislabeled pathology specimens-Pedigree analysis of animals and animal products-Establishing identity of rapists, murderers, etc.

Question 29

What is CODIS? What kind of polymorphic markers are used in CODIS? How are searches in CODIS performed and how are matches called? What are partial matches and are they permitted under the act of Congress that established CODIS?

Answer 29

Combined DNA Index System-Established through DNA Identification Act of 1994-Administered by FBI-Blends forensic DNA technology with computer technology-Arrestee states: VI, TX, LA, NM and CA. (DNA taken upon arrest)-Can use low-stringency DNA searches to look for family members who share similarities. •Has ethical concerns.

Question 30

What is direct to consumer (DTC) genetic testing? What are some objections to DTC testing?

Answer 30

23 and me-Micro array w/ ½ million SNPs.-Mostly for entertainment.-Do test some real stuff like BRCA which can be bad because they don’t provide counselors.-This and other direct-to-consumer genetic tests are not good predictors and most are fraudulent.

Question 31

What is “next generation” (massively parallel) DNA sequencing? What is exome sequencing? How is the high capacity of next generation sequencing achieved? What kinds of novel/unexpected sequence variants can be discovered by next generation sequencing?

Answer 31

Next generation DNA sequencing:-Bust up entire genome into 300 million fragments (about 100 nt each).-Each fragment is amplified.Classes of Novel/Unexpected Sequence Variants Identified by Whole Genome Sequencing:-Missense variants of uncertain significance in known gene-Variants and deleterious mutations in unknown gene(s)-Deleterious mutations in unintended target (e.g., BRCA mutations in a baby)

Question 32

What are “off-target” (incidental) findings? How can we avoid the problem of incidental findings?

Answer 32

-Revelation of “off-target” mutations-Many revealed disorders will have no prevention or treatment-Revelation of nonpaternity, consanguinity, incest-Revelation of intersex disorders: Klinefelter, Turner, XY female, XYY male, etc.-Costs of genetic counseling and follow-up-Possible forensic uses of data-Data storage and privacy-Huge number of novel missense variants

Question 33

What are some of the ethical concerns raised by the use of next generation sequencing and what types of medically actionable information should be given to patients?

Answer 33

Informed consent for whole genome sequencing: patient choices:-Receive all information (CD, DVD?)-Receive relevant/targeted information. • Not actionable for BRCA.-Receive medically actionable information for patient’s age • Not actionable for BRCA.-Receive medically actionable information for future. • Actionable for BRCA.-Receive medically actionable information for relatives

Question 34

What is circulating cell-free fetal (ccff) DNA? How can ccff DNA and next generation DNA sequencing be used in prenatal diagnosis? What are some advantages associated with using free fetal DNA in prenatal diagnostics? What are some ethical issues raised by non-invasive prenatal screening by ccff DNA?

Answer 34

ccff are fetal DNA/RNA circulating through mother’s plasma, can potentially test mother’s blood and diagnose fetus, non-invasive-Uncertain informed consent requirements-Uncertain pre-test genetic counseling requirements-Detection of “off-target” disorders-Ease of specimen collection allows DTC testing-Ease of specimen collection prompts screening for minor disorders, traits, sex, paternity, adult-onset disorders-Potential for “trivialization” of prenatal diagnosis and pregnancy termination

Question 35

What was the main argument of the ACLU attorneys seeking to overturn the Myriad Genetics patent on the BRCA1 & BRCA2 genes?

Answer 35

-Genes are products of nature, not inventions.-It is unconstitutional to patent a person’s individuality.-Patients are prevented from seeking a “second opinion”.-Gene patents are overly broad.-Legal principles bar patenting of laws of nature, products of nature, and abstract ideas.-Gene patents violate the First Amendment by inhibiting free speech and access to information.Ruling: Natural genes and isolated sequences from those genes are not patentablecDNA is still patentable

Question 36

What is the estimated number of nucleotides and genes in the human genome?

Answer 36

3.2 Billion Base Pairs, ~23000 genes