Learning Objectives Week 2

Question 1

Multifactorial Inheritance
(characteristics of diseases and other traits that demonstrate multifactorial inheritance)

Answer 1

Multifactorial inheritance:

• increased risk to relatives, but no consistent pattern of inheritance with families. Many of these diseases have traits that aren’t explained by genotype, and different alleles at same gene can yield different severity. Multiple genes + environmental factors –>disease susceptibility. Made up of complex traits which:
• aggregate in families
• don’t follow simple modes of Mendelian inheritance
• are likely due to variants in multiple genes and non-genetic factors that interact
• don’t have a simple relationship between variant and trait in the population.

Question 2

Multifactorial Inheritance

examples of diseases and other traits that demonstrate multifactorial inheritance

Answer 2

Not sure how important this is..
Examples from text:
1) Digenic Pigmentosa = patients heterozygous for EITHER a missense mutation in one gene or for null allele in another gene don’t develop disease; patients heterozygous for both mutations do develop.
2) Venous thrombosis = mutant allele of factor V (FVL) in which arginine  glutamine more frequent in white people. Mutation in prothrombin gene (GA) also more prevalent in Whites. Use of oral contraceptives increase risk for thrombosis, independent of genotype at FVL and prothrombin. Having two of these factors (2 genetic, 1 environmental) raises risk for idiopathic cerebral vein thrombosis.
3) Hirschsprung Disease = HSCR inherited in Mendelian manner; due to mutations in RET gene (affects a tyrosine kinase receptor). Some families require that individual have both RET and GDNE mutation. HSCR = multifactorial disease that results from additive effects of susceptibility alleles at RET, EDNRB, and other loci.
4) Type 1 DM = MHC locus is a major genetic factor in type 1 diabetes. Association between HLA-DR3 and HLA-DR4, which can be subdivided into a dozen+ more alleles. VNTR polymorphisms in promoter of insulin gene itself and other SNPs can affect.
5) Alzheimer’s disease = age, gender, and family history = most significant risk factors. APOE locus = significant genetic factor. Genotype with at least one E4 allele found 2-3x more frequently among patients; patients with 2 E4 alleles have earlier onset (so E4 is a predisposing factor). E4 variant predisposes to a complex trait, but does not predestine anyone carrying allele to develop disease.
(Also, some cancers, IBD, asthma, schizophrenia, clefting, etc.  demonstrate multifactorial inheritance).

Question 3

Multifactorial Inheritance
(strategies used to determine the relative importance of genetic vs. non-genetic factors in contributing to the variation in a complex trait)
-Twin Studies

Answer 3

Twin studies:
-compare MZ to DZ twins. If it can be assumed that MZ and DZ twins are equally similar with respect to non-inherited factors, we can use this to estimate contribution of genetic vs. environmental variation of trait.
(Uses concordance rates. High CR = genetic variation contributes to variation in risk more than environment).
Can study MZ twins raised apart to study environment.

Question 4

Multifactorial Inheritance
(he potential difficulties associated with quantifying the role of genetic factors in contributing to risk of disease at both the population level and the individual level)

Answer 4

-Risk of disease in relatives:
= risk of disease in affected sibs/risk in general population.
-Heritability (h2):
= proportion of variance in trait due to genetic variation.
High heritability = differences among people with respect to a trait (ex: blood pressure) can be attributed to differences in genetic makeup = more genetic variation.
Low heritability = low genetic variation; differences can be applied to the environment.
High heritability does NOT mean non-genetic factors aren’t important and vice versa.

Question 5

Multifactorial Inheritance
(characteristics of complex traits General)

Answer 5

• Incomplete penetrance
• Variable expressivity
• Heterogeneity
• (Presence of phenocopies)

Question 6

Multifactorial Inheritance
(characteristics of complex traits)
-Incomplete penetrance

Answer 6

Incomplete penetrance:
-not everyone with predisposing variant develops disease

Example: Type I diabetes and MHC

Question 7

Multifactorial Inheritance
(characteristics of complex traits)
-Variable expressivity

Answer 7

Variable expressivity:
-no two individuals with the same genetic variant have
exactly the same disease characteristics

Example: Maturity Onset Diabetes in the Young (MODY)

Question 8

Multifactorial Inheritance
(characteristics of complex traits)
-Heterogeneity

Answer 8

Heterogeneity (2 definitions from powerpoint):

1) Different alleles in the same gene result in same trait
2) Different alleles in the same gene result in different traits

(from handout)
-allele and locus: The “same” disease can be caused by different
alleles at one location or by alleles at different locations in the genome

Example (allele): Cystic Fibrosis
Example (locus): Alzheimer Disease

Question 9

Multifactorial Inheritance
(characteristics of complex traits)
-(Presence of phenocopies)

Answer 9

(Presence of phenocopies):
-Individuals who have the disease or trait for reasons
that are not primarily genetic even though clinical presentation mimics the more genetic version

Example: Thalidomide-induced limb malformation vs. genetically-induced

Question 10

Multifactorial Inheritance
(strategies used to determine the relative importance of genetic vs. non-genetic factors in contributing to the variation in a complex trait)
-Adoption Studies

Answer 10

Adoption studies:
-compares similarity between biological siblings raised apart and adopted sibs.
If biological sibling is more concordant with biological sib than adopted sib –> evidence for genetic variation.
(If opposite = evidence for environment.)

Question 11

Genetic Testing
(Define what constitutes a genetic test)

Answer 11

• Analyzing an individual’s genetic material to determine predisposition to a particular health condition or to confirm a diagnosis of genetic disease.
• Examining a sample of blood or other body fluid or tissue for biochemical, chromosomal, or genetic markers that indicate the presence or absence of genetic disease.

Less Restrictive Definition:
-Many ‘tests’ provide information about genetic status/risk without directly testing DNA.

Question 12

Genetic Testing
(Types of Genetic Tests)
-Genotyping

Answer 12

Genotyping:
Used to determine presence/absence of known genetic variant.
• General Uses and Indications: Used to identify sequence changes (mutations) in specific genes. In general you need the following:
o You must know or suspect a specific genetic diagnosis
o The gene must have been identified, and the disorder should exhibit little or no allelic heterogeneity. Genotyping is cost-effective when there are few variants; as the number of candidate variants, grows, sequencing quickly becomes cheaper.
• Can Diagnose: Previously-described mutations in known genes, polymorphic variants.
• Cannot Diagnose: The technique is very specific, assaying only the specific mutation(s) for which the test has been designed. The mutation(s) identified should represent the majority of causative mutations in the gene of interest, otherwise a negative result is uninformative.

Question 13

Genetic Testing
(Types of Genetic Tests)
-Fragment Analysis

Answer 13

Fragment Analysis:
Sizing of PCR products by capillary electrophoresis or, historically, gel electrophoresis.
• General Uses and Indications: Used to identify mutations that are expected to differ in PCR amplicon size (e.g., insertions / deletions). In general you need the following:
o You must know or suspect a specific genetic diagnosis
o The gene must have been identified
o The expected mutation must be of a type expected to result in a larger or smaller amplicon than wild-type, and there must be no other size polymorphisms within the amplicon.
• Can Diagnose: Small-medium (1 to ~2000 nucleotide) deletion/insertions, repeat expansions.
• Cannot Diagnose: This technique will not detect sequence changes other than insertions/deletions.

Question 14

Genetic Testing
(Types of Genetic Tests)
-Sanger Sequencing

Answer 14

Sanger Sequencing:
Widely used method of mutation detection based on Sanger-Gilbert method. Able to identify the genetic mutation causing many disease conditions.
• General Uses and Indications: Used to identify sequence changes (mutations) in specific genes. In general you need the following:
o You must know or suspect a specific genetic diagnosis
o The gene must have been identified
o The mutation must be detectable by sequencing (deletions, insertions, rearrangements are not always found by sequencing)
o The mutation must be located in a region of the gene that is actually sequenced (promoter and deep-intronic mutations often missed by commercial tests)
• Can Diagnose: Mutations in known genes (mutation can be previously reported or can be novel), polymorphic variants, small (1 to ~100 nucleotide) deletion/insertions. Ideal for looking at the sequence of a known disease gene
• Cannot Diagnose: The technique is very specific, assaying only the region of the gene(s) for which the test has been designed. Frequently, many clinical genetic tests do NOT routinely sequence all parts of a gene (e.g. promoters, introns). This means that although the approach is often very specific, clinical sensitivity is frequently below 100% (this is an important concept to understand). This technique cannot easily detect larger deletions/insertions, rearrangements, and most chromosomal abnormalities.

Question 15

Genetic Testing
(Types of Genetic Tests)
-Massively-Parallel Sequencing / Next-generation sequencing

Answer 15

Massively-Parallel/Nex Gen
Uses massively-parallel sequencing of individual DNA molecules and is likely to replace majority of Sanger DNA sequencing within a few years. Has been in clinical use since 2012. Powerful tool for identifying genetic etiology in difficult cases.
• General Uses and Indications: Used to identify sequence changes in a number of circumstances, but usually most powerful when there is significant genetic or allelic heterogeneity, or when the clinical diagnosis is uncertain. Used in limited by expanding fashion to identify copy-number changes and large deletions.
o You do not necessarily have to suspect a specific genetic diagnosis!! (although it’s helpful)
o The gene must have been identified
o The mutation must be detectable by analysis algorithm (sequence variants and small-moderate insertions/deletions are easy to detect, cytogenetic abnormalities less so)
o The mutation must be located in a region of the genome that is captured (promoter and deep-intronic regions are often omitted)
• Can Diagnose: Mutations in known genes (mutation can be previously reported or can be novel), polymorphic variants, small (1 to ~100 nucleotide) deletion/insertions. Ideal for looking at the sequence of a known disease genes in highly heterogeneic diseases, or for clinical cases for which the diagnosis is uncertain.
• Cannot Diagnose: The technique is very powerful, but typically does not detect large repeat expansions (e.g., Fragile X, HD). This technique cannot easily detect large deletions/insertions, rearrangements, and most chromosomal abnormalities, although the technologies and algorithms are constantly improving.

Question 16

Genetic Testing

Informative

Answer 16

Informative Genetic Test:
• An informative genetic test result is one where the information from a genetic test definitively diagnoses or excludes the disease in question. Put another way, an informative genetic test result is one that is reliably either a true positive or true negative result (ruling-in or ruling-out disease/risk, respectively).

Question 17

Genetic Testing

Non-informative

Answer 17

Non-informative Genetic Test:

• A non-informative genetic test result usually refers to a situation where the genetic test result is normal, but it is not possible to definitively exclude disease/disease risk. A non-informative genetic test result leaves open the possibility that an underlying pathogenic mutation exists, but was missed/not detected by the test.
• A genetic test which cannot completely account for all possible allelic and genetic heterogeneity in a particular disorder can lead to non-informative results.
(•When considering ‘informativity’ ask yourself, how confident you are in your result (does a ‘positive’ result really mean disease/elevated risk and does a ‘negative’ result really mean no disease/no elevated risk). If the answer is ‘no’ then you may be dealing with a non-informative test result.)

Question 18

Genetic Testing
(how allelic heterogeneity and genetic heterogeneity can affect the performance of genetic tests.)
-Allelic Heterogeneity

Answer 18

Allelic Heterogeneity:
refers to the fact that multiple mutations in a particular gene (or at a particular loci) can cause disease. (Allelic heterogeneity in the research setting can also refer to the present of multiple non-pathogenic polymorphisms within a gene)
o Example: Cystic fibrosis is an autosomal recessive disease caused by mutations in one gene, CFTR. Over 1,500 different mutations have been reported. Cystic fibrosis shows allelic heterogeneity but is genetically homogenous (e.g. NO Genetic Heterogeneity).

Question 19

Genetic Testing
(how allelic heterogeneity and genetic heterogeneity can affect the performance of genetic tests.)
-Genetic Heterogeneity

Answer 19

Genetic Heterogeneity:
Mutations in multiple gene associated with the same phenotype
o Example: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease caused by mutations in at least 10 different genes. HCM shows both allelic and genetic heterogeneity.

Question 20

Genetic Testing
(how allelic heterogeneity and genetic heterogeneity can affect the performance of genetic tests.)
-Pleiotropy

Answer 20

Pleiotropy:
Mutations in a single gene can cause multiple disorders
o Example: Mutations in COL1A1 can cause Ehlers-Danlos syndrome, infantile cortical hyperostosis, or osteogenesis imperfecta

Question 21

Turner Syndrome

Clinical Presentation

Answer 21

Karyotype:
-45, XO.
-CVS issues = bicuspid aortic valve, coarctation of aorta, hypertension, prolonged QTc syndrome, partial anomalous pulmonary venous connection, persistent left SVC.
-Eye = inner canthal folds, ptosis, blue sclera.
Skeletal system: cubitus valgus, short 4th metacarpal/tarsal, short stature, scoliosis.
-Ear = sensorineural hearing loss, conductive hearing loss, chronic otitis media.
-Neck = web neck, low hairline, cystic hygroma.
-Learning = difficulty in math, visual spatial skills, and low non-verbal scores.
Poor breast development, shield-shaped thorax, widely spaced nipples, brown nevi, no menstruation.
-Endocrine = hypothyroidism and gonadal dysgenesis.

Question 22

Turner Syndrome

challenges across life span

Answer 22

Infertility, stature, sexual development, concerns regarding health and aging.

Question 23

Turner Syndrome

Pitfalls of medical culture in dealing with affected patients

Answer 23

Secret keeping and not telling patients full truth  causes patient depression, isolation, mistrust, and handling the diagnosis poorly themselves. Difficulty communicating an infertility diagnosis (insensitive, not at all empathetic), perceived negative experiences with physicians (inappropriate humor, too rushed).

Question 24

Autosomal Recessive
(common characteristics of disorders that are of autosomal recessive inheritance.)

Answer 24

AR disorders have phenotypes expressed only in people with 2 mutant alleles of same gene (males and females equally affected). It displays horizontal inheritance; parents of affected child are obligate carriers. Recurrence risk is ¼ for each unborn child; probability of an unaffected sibling being a carrier is 2/3. Majority of mutant alleles present in carriers (not patients), and certain ethnic groups have higher frequencies of mutant allele (high-risk groups). Parental consanguinity causes increased incidence (but if subgroups tend to marry within their group, chances also increase because there was a common ancestor with the allele).

Question 25

``` Autosomal Recessive (Calculate allele frequency and carrier frequency of a given autosomal recessive disease when provided with the disease frequency, and vice versa) ```

Answer 25

Frequency of disease = q2. Find q, find p  2pq = carrier frequency.

Question 26

Autosomal Recessive | Allelic heterogeneity

Answer 26

Allelic heterogeneity: | – presence of multiple mutant alleles at the same gene

Question 27

Autosomal Recessive | Compound heterozygote

Answer 27

Compound heterozygote: | – one who has two mutant alleles at the same gene.

Question 28

Autosomal Recessive | Parental consanguinity

Answer 28

Parental consanguinity: | -if one’s parents share a common ancestor

Question 29

Autosomal Recessive | High-risk groups

Answer 29

High-risk groups: -– ethnic group in which a mutant allele/autosomal recessive disease occurs with higher frequency. Marrying within ethnic group increases chances of producing kid homozygous for condition.

Question 30

Autosomal Recessive | Phenylketonuria PKU

Answer 30

Phenylketonuria (PKU): - high Phe in blood - high Phe metabolites in urine - Hyperactivity - epilepsy - mental retardation - microcephaly

Question 31

``` Autosomal Recessive (biochemical deficiencies in PKU patients and the appropriate treatments) ```

Answer 31

- Defects in PAH (phenylalanine hydroxylase) =98% of people. - Defects in PAH cofactor, B4 = 1-2% of people (B4 also cofactor for Tyr and Trp hydroxylases). - Generally mutations in PAH due to LOF alleles; PAH has high allelic heterogeneity (means many PKU patients will be compound heterozygotes). - For defects in PAH – low Phe diet, generally throughout life. - For defects in B4 = low Phe diet and meds for NT balance.

Question 32

Autosomal Recessive | maternal PKU and its treatment

Answer 32

Pregnant PKU women must remain on low-Phe diet while pregnant because otherwise have higher risk of miscarriage or giving birth to children with malformations and mental retardation, regardless of their genotypes.

Question 33

``` Autosomal Recessive (newborn screening procedures for PKU and importance of the timing of the test) ```

Answer 33

Newborn screening by mass spectrometry (can also use Guthrie test, a bacterial inhibition assay). Detection must be within first few days of life to prevent irreversible brain damage, but not within first 2 days because some children can be missed. Sometime within first 2 weeks.

Question 34

``` Autosomal Recessive (alpha 1-Antitrypsin Deficiency (ATD)) -clinical features of α1-antitrypsin deficiency and the influence of environmental factors on the expression and severity of the disease (ecogenetics) ```

Answer 34

Generally presents with later onset (goes under-diagnosed), common in N. Europeans (1/2500). Increased risk of developing emphysema, liver cirrhosis/cancer (due to accumulation of misfolded α1AT protein in liver). Ecogenetics = earlier and more severe symptoms in smokers. Smoking accelerates onset of emphysema because smoke damages lung, prompting body to send more neutrophils  increased elastase release.

Question 35

``` Autosomal Recessive (Which enzyme is the primary target of α1-antitrypsin) ```

Answer 35

α1AT (aka SERPINA1, a serine protease inhibitor) inhibits the elastase enzyme whose main target is elastin. Elastase binds elastin in connective tissue and digests it. In ATD, there is a defect in SERPINA1 so there’s an increase in elastase and thus an increase in tissue damage in the lungs (alveolar wall damage and emphysema).

Question 36

``` Autosomal Recessive (two most common mutant alleles that cause ATD and the severity of different allelic combinations. Why do some ATD patients have liver failure?) ```

Answer 36

- M alleles encode functional proteins. - Z allele (Glu--> Lys) most common mutant allele--> Z/Z genotype = 15% normal SERPINA1 level. Z allele makes protein that isn’t folded properly and accumulates in ER of liver cells (= liver damage). -S allele (Glu-->Val) makes an unstable SERPINA1 protein, so S/S genotype has 50-60% normal protein level. -Z/S genotype = compound heterozygote = 30-35% normal SERPINA1 activity and may develop emphysema. Treat by either delivering protein via intravenous infusion or aerosol inhalation.

Question 37

Autosomal Recessive | Tay-Sachs Disease (T-S)

Answer 37

T-S: | fatal genetic disorder that causes progressive destruction of CNS.

Question 38

``` Autosomal Recessive (biochemical defects in Tay-Sachs disease and why the brain is the major target) ```

Answer 38

TS = lysosomal storage disorder with accumulation of GM2 ganglioside, which is synthesized primarily in the brain  accumulates in lysosomes of neurons. Patients can’t degrade GM2 because of defective Hexosaminidase A enzyme (which has an alpha and beta subunit encoded by HEXA and HEXB genes  TS patients have mutation in HEXA on C15).

Question 39

``` Autosomal Recessive (Compare similarities and differences between Tay-Sachs disease, Sandhoff disease and the AB variant of Tay-Sachs disease) ```

Answer 39

TS = Type I GM2 gangliosidosis. Mutation in HEXA gene on C15  defect in alpha subunit protein in HexA enzyme (which is made of an alpha and beta unit). (HexB enzyme is normal.) Sandhoff = Type II = same clinical presentation but occurs due to mutation in HEXB gene on C5  causes defects in HexA and HexB enzymes. AP variant of TS = rare form of TS. HexA and HexB enzymes both normal, but still get GM2 accumulation because of defect in GM2-AP (activator protein) which is responsible for facilitating interaction between lipid and HexA enzyme.

Question 40

Autosomal Recessive (Know the high-risk group for Tay-Sachs disease and the available methods for carrier screening and prenatal screening in the high-risk population)

Answer 40

Ashkenazi Jews = 100-fold higher risk for TS (1/3600), whereas general is 1/360000. (Certain French Canadian communities, Amish in PA, and Cajuns of LO, too.) Can determine enzyme activity by running enzymatic activity assay (distinguish between HexA and HexB because HexA inactivated by heat). Carrier screening has 97% accuracy among Jewish population because they have lower HexA enzymes levels in blood. Can perform enzyme test on amniotic fluid cells to perform prenatal screening. Can also do DNA testing, which is able to detect 95% of Ashkenazi Jew carriers and 50% of carriers otherwise. DNA test will miss some carriers.

Question 41

Hemoglobinopathies (Describe the layout of the α- and β-globin gene clusters and the switch between different forms of hemoglobin (Hb) during development. Explain the function of the locus control region (LCR).))

Answer 41

HbA = adult Hb = α2β2 tetramer with 2 alpha and 2 beta chains. . Alpha and alpha-like genes on C16; beta and beta-like genes on C11. 2 copies of alpha, but one copy of beta. alpha cluster = zeta-aplha2-alpha1 (zeta only expressed embryonically) beta cluster = epsilon-gammaG-gammaA-delta-beta Embryonic Hb’s = Hb Gower 1 (zeta2-epsilon2); Hb Gower 2 (alpha2-epsilon2); Hb Portland (zeta2-gamma2) Fetal Hb’s = HbF (alpha2-gamma2) Adult Hb’s = HbA (alpha2-beta2; 95%); HbA2 (alpha2-delta2; 3.5%) (and ~1% HbF) Turn off zeta and epsilon, turn on alpha and gamma during embryogenesis. Turn off gamma and turn on beta and delta around time of birth. HbF is better to bind O2 at placenta because has higher affinity for O2 and low pO2 than HbA. This switches after birth. Homotetramers are poor O2 carriers and precipitate inside RBCs. LCR = located at most upstream region of each cluster; makes physical contact of promoter and regulatory regions of globin genes to influence expression. Deletion of LCR of beta cluster  beta-thalassemia

Question 42

Hemoglobinopathies (Describe the mutations that cause sickle cell anemia and hemoglobin C disease and their consequences. Know the DNA diagnosis method of the sickle cell disease mutant allele)

Answer 42

These are structural variants (qualitative hemoglobinopathies) that affect globin polypeptide properties without affecting its synthesis. Sickle cell anemia (HbSS) more common in African descent (10% = carrier frequency). Glu6Val mutation  HbS less soluble in low-O2 environment  polymerizes into long fibers  sickle shaped RBC. HbCC = milder form of anemia caused by Gly6Lys  less soluble, Hb forms crystals both are auto recessive. Compound heterozygotes have milder anemia than sickle cell. Diagnose sickle cell using PCR, southern blot, and RFLP (MstII restriction enzyme). Cut site is mutated in sickle cell = get different sized products between normal and HbS. Can also diagnose by electrophoresis of Hb protein (separate protein based on charge since different AA substitutions will affect that).

Question 43

Hemoglobinopathies (Know the six possible genotypes of α-globin locus, their clinical phenotypes, and the geographical distributions of α-thal-1 (--) and α-thal-2 (α-) alleles))

Answer 43

*Alpha-thalassemia = low alpha-globin, beta and gamma globin present in excess and precipitate. Usually caused by deletion of alpha-globin genes. ``` *αα/αα = 100% alpha-globin level = normal αα/α- = 75% alpha-globin level = silent carrier ``` * αα/-- = α-thal-1 = 50% alpha globin level = α-thalassemia 1 trait = common in SE Asia; caused by deletion of both copies of alpha globin genes. Heterozygotes have mild anemia. * α-/α- = α-thal-2 = 50% alpha globin level = α-thalassemia 2 trait = common in Africa, Mediterranean, and Asia; due to deletion of one of alpha globin genes. Mild anemia. Heterozygote = αα/α- = silent carrier. * α-/-- = 25% alpha globin level = severe anemia = HbH disease (5-30% of hemoglobin is HbH, β4, which precipitates in blood) = a-thal-1/a-thal-2. SE Asia. * --/-- = 0% alpha-globin level = fetal death/hydrops fetalis (ϒ4) – SE Asia.

Question 44

Hemoglobinopathies (Understand the following concepts about β-thalassemias) -thalassemia majors

Answer 44

Show high allelic heterogeneity = lots of compound heterozygotes. -thalassemia majors – severe anemia, thinning bone cortex, enlarged liver and spleen. Consist of β0 (no Hb detected) and β+ (some β expression) homozygotes?

Question 45

Hemoglobinopathies (Understand the following concepts about β-thalassemias) -thalassemia minor

Answer 45

clinically normal, carriers of one beta-thalassemia allele. Consist of β0 and β+ heterozygotes (slightly more expression)

Question 46

Hemoglobinopathies (Understand the following concepts about β-thalassemias) -β+-thalassemia

Answer 46

most common form of beta thalassemia. Some beta-globin is made, so some HbA is present. Decreased beta-globin synthesis caused by mutations affecting transcription, RNA processing, or protein stability

Question 47

Hemoglobinopathies (Understand the following concepts about β-thalassemias) -β0-thalassemia

Answer 47

– zero beta-globin synthesis so no HbA is present --> caused by deletion of beta-globin gene or mutations in 5’ coding region that lead to early stop codon or mutations that result in no RNA synthesis. Hb concentration is 5% of normal level. Rely on HbF and HbA2. β0-thal allele -β+-thal allele

Question 48

Hemoglobinopathies (Understand the following concepts about β-thalassemias) -simple β-thalassemias

Answer 48

caused by mutations or deletions that impair production of beta-globin chain alone; other genes in beta-globin cluster unaffected.

Question 49

Hemoglobinopathies (Understand the following concepts about β-thalassemias) -complex thalassemias

Answer 49

caused by large deletions that remove beta-globin gene plus other genes in that cluster, or the LCR  can cause HPFH.

Question 50

Hemoglobinopathies | Explain hereditary persistence of fetal hemoglobin (HPFH) and its clinical implications

Answer 50

HPFH caused by different deletions that retain gamma so that they keep making HbF. There is no delta or beta synthesis due to deletions of both genes. People with HPFH are disease free since they have adequate levels of gamma chains. 100% of their hemoglobin is HbF. If we can figure out how to express HbF at high levels postnatally  we can maybe treat beta-thalassemia patients and sickle cell anemia patients.

Question 51

Hemoglobinopathies | Give examples of two types of mutations that are known to cause HPFH

Answer 51

Gamma expressed by an extended deletion of additional downstream sequences, bringing a cis-acting enhancer closer to gamma-globin gene. OR can be caused by mutations in the promoter region for one of the gamma-globin genes that destroy the binding site of a repressor, relieving post-natal repression of gamma.

Question 52

``` Thalassemia Vignette (Recognize quantitative and qualitative changes in global chain) ```

Answer 52

In embryonic development, use of Hb Gower 1 (zeta2epsilon2), Hb Gower 2 (alpha2epsilon2), and Hb Portland (zeta2gamma2). In fetal development, primarily rely on HbF (alpha2gamma2)  high affinity for O2. In adulthood, rely on HbA (alpha2beta2) and HbA2 (alpha2delta2). Prior to birth, hematopoiesis occurs in yolk sac, then liver/spleen. Post-birth, occurs in bone marrow.

Question 53

``` Thalassemia Vignette (Know the geographic distribution of the common hemoglobin variants) ```

Answer 53

SE Asia = alpha thalassemia, beta thalassemia, and Hb E. Africa = Hb S, Hb C, alpha and beta thalassemias. West Pacific = Hb E, alpha and beta thalassemias East Mediterranean = Hb S and beta thalassemia Hb S involved with sickle cell (homozygous = anemia, heterozygous = sickle trait). Hb C and Hb E are other qualitative hemoglobinopathies. African = more likely to have deletion in one gene in each parent so offspring has one deletion on each chromosome. Asian = more common to have 2 gene-deletion on each parent such that offspring has no genes = hydrops fatalis.

Question 54

Thalassemia Vignette | Identify the β thalassemia syndromes

Answer 54

Thalassemia = disorder in which globin chain synthesis is reduced = imbalanced globin chain production and defective hemoglobin. 1) β thalassemia major (Cooley’s anemia) = 2 severely abnormal gene. Dependent on transfusion. Presents with dense skull, osteopenia, enlarged spleen, and iron overload. Can treat with transfusion, iron chelators, bone marrow transplant, and splenectomy. 2) β thalassemia intermediate = 2 mildly abnormal genes 3) β thalassemia trait = β-thal minor = 1 normal and 1 abnormal gene. 4) SB0 thalassemia 5) SB+ thalassemia

Question 55

Thalassemia Vignette | Identify the α thalassemia syndromes

Answer 55

α thalassemia types: 1) α thalassemia major 2) α thalassemia 3 gene deletion (HbH disease) = --/- α 3) α thalassemia 2 gene deletion (α thalassemia trait) = --/ αα or –α/-α 4) α thalassemia 1 gene deletion – clinically insignificant 5) Hydrops fatalis = --/-- 6) α thalassemia trait (silent carrier) = - α/ αα

Question 56

Mutational Mechanisms and Disease (Describe, distinguish between, and give examples of the four major mechanisms that genetic mutations lead to disease) 1) loss of function of the protein (most common)

Answer 56

1) loss of function of the protein (most common): - caused by genetic mutations that eliminate/reduce protein function (no gene, no RNA, no protein, or non-functional protein). Most common mutational mechanism. 1. Duchenne Muscular Dystrophy: -Caused by large deletions (multiple exons) resulting from nonsense and frameshift mutations (in-frame deletions -->Becker MD, milder). -X-linked. -Boys with abnormal gait (walking on toes), calf pseudohypertrophy, involvement of respiratory muscles, death ~18 years. Duchenne = complete absence of dystrophin protein. (gower maneuver, walk up thighs, used to get up) 2. Hereditary Neuropathy with Liability to Pressure Palsies: -deletion of PMP22 gene (encodes peripheral myelin protein). Nerves are sensitive to any sort of pressure; repeated focal pressure -autosomal dominant -Deletion occurs due to unequal crossing over between highly homologous repeats on C17 -->left with only 1 copy of PMP22 gene. ( be able to distinguish deletion vs duplication, look at slides) 3. Osteogenesis Imperfecta Type I: - brittle bones, blue sclerae, normal stature -->easily fractured bones. - Type 1 collagen requires 2 proα1 chains and one proα2 chain. - Autosomal dominant. - In OI Type I--> premature termination codons (from nonsense or frameshift) in COL1A1 --> unstable mRNA --> reduction of normal COL1A1 protein. Not making enough protein to make the proper collagen, collagen looks normal; there is simply not enough. 4. turners: chromosome deletion 5. alpha-thalaesmmia: deletion of alpha globin gene

Question 57

Mutational Mechanisms and Disease (Describe, distinguish between, and give examples of the four major mechanisms that genetic mutations lead to disease) 2) gain of function of the protein

Answer 57

2) gain of function of the protein: - caused by genetic mutations that enhance normal functions of a protein, not a novel change, just enhancement like we said. 1. Hemoglobin Kempsey: (point mutation) - Asp99Asn missense mutation in beta hemoglobin gene --> higher oxygen affinity and Hb locked in the relaxed state (high affinity state) --> less O2 distribution to tissues --> body makes more RBCs (polycythemia) 2.Charcot-Marie-Tooth Syndrome (1a): = duplication of PMP22 gene --> demyelinating motor and sensory neuropathy, lower extremity weakness and atrophy. -Autosomal dominant. -people complain of weakness in the feet, lots of foot slapping, could end up in wheelchair 3. Achondroplasia = FGFR3 glyarg mutation increasing signaling via tyrosine kinase (receptor is always ‘on’) 4. Alzheimer disease in Tri21 = patients have 3 copies of APP gene = increased production of APP protein = early onset.

Question 58

Mutational Mechanisms and Disease (Describe, distinguish between, and give examples of the four major mechanisms that genetic mutations lead to disease) 3) acquisition of a novel property by the mutant protein

Answer 58

3) acquisition of a novel property by the mutant protein: - caused by genetic mutations that confer a NEW property on protein without altering normal function. 1. Sickle cell anemia: - glu-->val in beta globin gene. Transports oxygen normally, but in low-oxygen states, valine leads to polymerization of Hb into long fibers that cause RBCs to take sickle shape and then get caught in capillaries. 2. Osteogenesis Imperfecta Type II, III, IV: - mutation in COL1A2 -->abnormal protein ( no longer the same protein) with a different, severe function. Half of collagen trimers are normal, but the other half are abnormal and lead to severe phenotype. (So better to have OI Type I and just have half the amount of normal collagen). Other types besides I, fuck shit Upppp. 3. Huntington disease: - CAG repeat in gene increases number of glutamine residues --> causes huntingtin protein to become toxic.

Question 59

Mutational Mechanisms and Disease (Describe, distinguish between, and give examples of the four major mechanisms that genetic mutations lead to disease) 4) perturbed expression of a gene at the wrong time (heterochronic expression) or in the wrong place (ectopic expression), or both

Answer 59

4) perturbed expression of a gene at the wrong time (heterochronic expression) or in the wrong place (ectopic expression), or both: caused by mutations that alter regulatory regions of a gene, causing it to be expressed at wrong time (heterochronic) or location (ectopic). 1. Cancer = gene that is normally silent is abnormally expressed = abnormal proliferation. 2) .Hereditary persistence of fetal Hb = normal switch from fetal to adult Hb doesn’t occur and HbF remains expressed. Due to deletions of genes in beta hemoglobin locus, but retention of fetal (gamma) gene such that gamma-globin continues to be expressed since beta-globin gene is missing

Question 60

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) General:

Answer 60

1) Transcription: 2) Translation 3) Polypeptide folding 4) Post-translational modification 5) Assembly of monomers into holomeric protein 6) Subcellular localization of polypeptide 7) Cofactor or prosthetic group binding to peptide 8) Function of a correctly folded, assembled, and localized protein in normal amounts

Question 61

Mutational Mechanisms and Disease (Explain the mechanism of genetic anticipation in tri/tetra-nucleotide repeat disorder and recognize the phenotypes of these disorders.)

Answer 61

Generally neurodegenerative Mutations in 5’ UTR: 1) Fragile X = CGG (>200) = transcriptional silencing and LOF = loss of RNA binding (impaired translational repression of target RNAs) 2) Fragile X tremor/ataxia (60-200) = CGG = increase in FMR1 mRNA = GOF = neuronal inclusions. Novel properties. Mutations in Introns: 1) Friedreich ataxia = GAA (>200) = impaired transcriptional elongation =LOF in frataxin = reduced heme synthesis, increased Fe in mitochondria 2) Myotonic dystrophy 2 = CCTG (>75) = same effects as MD1? Novel properties. Mutations in Exons: 1) Huntington Disease = CAG (>40) 36-39 might get it; 27-35 your okay, but could expand in offspring= expanded polyglutamine tracts in huntingtin protein (makes it toxic) --> increased protein-protein interactions with TFs? LOF. Risk of expansion from paternal side Pedigree stuff: the expansions happen when male make sperm; girls will not make expansion, children wont have it. but if your a guy in the same boat 32 copies, you will have expansions. your children will have more expansions 2) Spinocerebellar ataxia Mutations in 3’ UTR: 1) Myotonic dystrophy 1 = CTG (>50) = expanded CUG repeats in RNA confer novel properties on RNA = increased amounts of RNA-binding proteins --> impaired RNA splicing. Maternal expansion more likely. Cataracts, myotonia, weakness. Congenital = >2000 repeats. (problem when women make eggs, so opposite of HD, so males will be fine no expansion; females not the case all their offspring will be affected) ( cant undo hand shake, fails grip test release, thenar eminence stimulation leads to a reaction; you get stuck to doors and shit) Genetic anticipation = risk of expansion such that disease gets worse in each subsequent generation and has earlier age of onset.

Question 62

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) 1) Transcription:

Answer 62

1) Transcription: - Thalassemias due to reduced production of globin mRNA because of deletions/mutations in regulatory/splice sites - hereditary persistence of HbF = results from increased postnatal transcription of gamma-globin genes

Question 63

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) 2) Translation

Answer 63

2) Translation | - thalassemias due to non-functional mRNAs with nonsense/frameshift mutations

Question 64

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) 3) Polypeptide folding

Answer 64

3) Polypeptide folding | - many hemoglobinopathies due to abnormal Hb with AA substitutions/deletions  unstable globins

Question 65

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) 4) Post-translational modification

Answer 65

4) Post-translational modification | - I-cell disease = LSD due to failure to add phosphate group to mannose residues (which target enzymes to lysosomes)

Question 66

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) 5) Assembly of monomers into holomeric protein

Answer 66

5) Assembly of monomers into holomeric protein | - osteogenesis imperfecta – AA substitution in procollagen chain impairs assembly of normal collagen triplex

Question 67

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) 6) Subcellular localization of polypeptide

Answer 67

6) Subcellular localization of polypeptide - familial hypercholesterolemia mutations in C-terminus of LDL receptor = impaired localization of receptor to clathrin-coated pits  prevents internalization of receptor and recycling to cell surface

Question 68

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) 7) Cofactor or prosthetic group binding to peptide

Answer 68

7) Cofactor or prosthetic group binding to peptide | - types of homocystinuria due to poor binding of cofactor (pyridoxal phosphate) to cystathionine synthase apoenzyme

Question 69

Mutational Mechanisms and Disease (Discuss and cite examples* of the eight steps at which mutations can disrupt the production of a normal protein) 8) Function of a correctly folded, assembled, and localized protein in normal amounts

Answer 69

8) Function of a correctly folded, assembled, and localized protein in normal amounts - diseases in which protein is normal except for one critical function altered by AA substitution (ex: Hb Kempsey)

Question 70

``` Autosomal Dominant (Know the characteristic pattern in a pedigree showing autosomal dominant inheritance) ```

Answer 70

- Manifests in homozygotes and heterozygotes: - equal in males and females. - Can be passed by either parent. - Vertical transmission = shown in every generation. HH = rare when dominant mutation causes the disease.

Question 71

``` Autosomal Dominant (Understand features that may complicate the assessment of an autosomal dominant pedigree) ```

Answer 71

Penetrance: -measurement of individuals with a disease genotype that actually express the phonotype. (Reduced penetrance in retinoblastoma, BRCA mutations, and Huntington’s). Expressivity: -the degree to which a genotype is phenotypically expressed. Pure dominance: -homozygotes and heterozygotes equally affected; incompletely dominant = homozygotes are affected more severely (ex: Achondroplasia).

Question 72

Autosomal Dominant (clinical features and molecular basis) -Achondroplasia

Answer 72

Achondroplasia: -autosomal dominant, skeletal dysplasia. 80% new mutations (dwarf born to totally normal parents). Small stature, limb shortening (more so of proximal limbs = rhizomelic), short fingers, bow-legged, large head, midfacial retrusion, trident hands, small foramen magnum (yields craniocervical instability). Due to AA substitution mutation of FGFR3 (fibroblast growth factor receptor), a tyrosine kinase receptor--> inhibits chondrocyte proliferation/differentiation. AA is lethal so only see presentation in Aa form (aa is normal).

Question 73

Autosomal Dominant (clinical features and molecular basis) -Neurofibromatosis Type 1

Answer 73

Neurofibromatosis Type 1: autosomal dominant; 50% new mutation rate. 6+ café au lait spots, neurofibromas, plexiform neurfibroma, freckling in axillary/inguinal area, optic glioma, Lisch nodules (in eye), osseous lesions, affected 1st DR. Mutation in NF1 (a TSG) = loss of function mutation. Really shows variable expressivity. (Ch 17)

Question 74

Autosomal Dominant (clinical features and molecular basis) -Marfan Syndrome

Answer 74

Marfan Syndrome: autosomal dominant; 25% new mutation rate. Disorder of connective tissue (ocular, skeletal and cardiovascular)  aortic root enlargement, ectopia lentis. Due to mutation in FBN1 gene that messes up fibrillin protein (extracellular matrix protein). Causes severe reduction in number of microfibrils.

Question 75

Autosomal Dominant (clinical features and molecular basis) -Autosomal Dominant Polycystic Kidney Disease

Answer 75

Autosomal Dominant Polycystic Kidney Disease: -bilateral renal cysts, cysts in other organs, vascular abnormalities. Due to mutation either in PKD1 or PKD2 gene (example of locus heterogeneity  how same disease can be called by alleles at different loci), yielding truncated Polycystin 1 or Polycystin 2 protein.

Question 76

Autosomal Dominant (clinical features and molecular basis) -Familial Hypercholesterolemia

Answer 76

Familial hypercholesterolemia: -autosomal dominant. High cholesterol and LDL levels, xanthomas, and premature coronary artery disease. Mutation in LDLR gene and LDL receptor  body doesn’t clear LDL like it should.

Question 77

Autosomal Dominant | Know unique features of Trinucleotide-Repeat disorders

Answer 77

Expansion of a DNA segment, generally due to slipped mis-pairing in which mis-pairing of bases in regions of repetitive DNA replication causes bases to slip out--> polymerase will correct for the entire repeat. Anticipation = severity of disease tends to increase in the next generation (due to expansion of repeats) and have earlier onset. Parental transmission bias = trinucleotide expansion is more prone to occur in gametogenesis of either a male or female (so maternal vs. paternal expansion). Can be AD, AR, and x-linked.

Question 78

Autosomal Dominant | Understand clinical features and molecular basis of Huntington Disease

Answer 78

HD = autosomal dominant, CAG repeat (>39-40 = bad). Certain populations have predisposition to instability (Venezuelans). Exhibits progressive neuronal degeneration causing motor, cognitive, and psychiatric disturbances. Age of onset = 35-44; death approximately 15 years post-onset. Exhibits chorea. Due to mutation in HTT gene = abnormal huntingtin protein  expansion of glutamine may cause altered structure or biochemical property. > 60 repeats = juvenile onset. <27 = normal.

Question 79

X-linked and Mitochondrial Inheritance | Know the differences between X-linked dominant and X-linked recessive inheritance

Answer 79

X linked disorders in general are more common in males and see no male-to-male transmission. X-linked recessive = phenotype expressed in all males to carry affected genotype; only expressed in homozygous females. Heterozygous females are carriers. X-linked dominant expressed in male hemizygotes and female heterozygotes.

Question 80

X-linked and Mitochondrial Inheritance | Know the characteristics of an X-linked pedigree

Answer 80

Affected fathers pass to all daughters. Carrier mothers have 50% chance of passing to children.

Question 81

X-linked and Mitochondrial Inheritance (clinical features and molecular basis) -Hemophilia A

Answer 81

X-linked recessive. Generally in males but 10% of carrier females are affected (probably due to weird pattern of X inactivation; generally see after injury or incision). Spontaneous bleeds into joints, muscles, or intracranial; excessive bruising, and prolong bleeding after cut; delayed would healing. Due to mutation in F8 gene that causes a deficiency in Factor VIII protein (generally by 22A inversion).

Question 82

X-linked and Mitochondrial Inheritance (clinical features and molecular basis) -Duchenne Muscular Dystrophy

Answer 82

Dystrophinopathies = x-linked recessive. Spectrum of muscle disease; Duchenne MD is more severe, Becker MD is less severe. Due to mutation in DMD gene that causes defects in dystrophin protein. In Duchenne’s, progressive muscular weakness, calf hypertrophy, and dilated cardiomyopathy; onset < age 5, and death in 30’s – due to complete absence of dystrophin protein. Becker MD = similar symptoms, but later onset and death in 40s  due to abnormal quantity or quality of dystrophin protein. DMD-associated dilated cardiomyopathy due to no dystrophin in myocardium; skeletal muscle not involved; early death.

Question 83

X-linked and Mitochondrial Inheritance (clinical features and molecular basis) -Fragile X Syndrome

Answer 83

Fragile X Syndrome: due to CGG repeat; most common cause of male mental retardation. 1/3 females affected; disease shows pattern of maternal anticipation. Generally have intellectual disabilities, dysmorphic features (large ears, long face, macroorchidism), autistic behavior, social anxiety, hand flapping/biting, aggression. Due to a mutation in FMR1 that causes failure to express FMRP protein. 56-200 repeats = permutation zone. >200 = full mutation.

Question 84

X-linked and Mitochondrial Inheritance | What are the unique features of mitochondrial inheritance and the clinical manifestations of these mutations

Answer 84

mtDNA shows maternal inheritance  majority of mtDNA encodes genes involved in respiratory chain. Exhibits replicative segregation = at cell division, multiple copies of mtDNA replicate and sort randomly among newly synthesized mitochondria  can be normal or mutated DNA. Threshold effect = when you have more mutated mtDNA than normal  caused mitochondria to become mutant. Heteroplasmy = when the mitochondria has normal and mutated mtDNA inside it. Mom always passes her mtDNA to her offspring; never see transmission from dad. All daughters will pass on, no sons will. Disorders of mitochondria generally caused by dysfunction of the respiratory chain and oxidative phosphorylation; occurs in tissues with high energy requirements (brain, eyes, muscle, heart, kidneys, liver).

Question 85

Epigenetics | Understand the four main characteristics of epigenetic phenomena

Answer 85

1) Epigenetics allows for different gene expression patterns/phenotypes, even with identical genome (more or less). 2) These epigenetic characteristics can be inherited through cell division, even through generations. 3) Epigenetics are like an on/off switch (ex: gene can be silenced or not). Epigenetic characteristics are reversible (pathway to disease). 4) Erase-able. (Again, can be turned on or off  both therapeutic potential or can cause disease).

Question 86

Epigenetics | Explain the basic principle of Waddington’s epigenetic landscape

Answer 86

Cell fates established in the way a marble rolls down to point of lowest elevation. Ball has capacity to roll down hill via multiple paths  cells can differentiate into different cell states. Marbles come to rest at lowest point on hill  cell eventually is fated to be something specific (their ‘low energy’ state). Also like flipping switches. Undifferentiated cell has all switched flipped on and certain ones flipped off as it differentiates to a specific purpose. We can reprogram adult cells to all ‘switch on’ = induced pluripotent stem cell.

Question 87

Epigenetics | List three specific examples of epigenetic phenomena

Answer 87

Erasure and resetting of methylation patterns of imprinted genes during gametogenesis  cells maintain inherited pattern until gametogenesis. In meiosis, undergo DNA de-methylation and then sex-specific gene silencing (DNA methylation) so there’s no overlap at fertilization. (Methylation occurs on cytosines  5-meC  gene silences by solidifying ‘repressed’ state). Packaging of DNA into chromatin  modification of histones can be inherited as well. H3 modifications affect gene expression. Genes in heterochromatin are repressed. Turned off = methylation; turned on = acetylation. Epigenetics also impacts X-inactivation. Chromatin-mediated gene silencing (heterochromatin domains, x-inactivation, imprinting). Centromere marking by CENP-A (attachment to spindle during mitosis). Reinforcing feedback loops. Bacteriophage lambda repressor mechanism. Feedback loops in cancer that operate through cytosolic signaling proteins.

Question 88

Epigenetics | Describe how DNA methylation can be inherited through cell division.

Answer 88

DNA replication is semi-conservative. Methyltransferases propagate epigenetic marks by using the hemimethylated state/parent strand to re-establish pattern on new strand. Occurs only on cytosines of CpG repeats  5-meC  solidifies repressed state.

Question 89

Epigenetics | (Name three chemical modifications to DNA or histones that can potentially be inherited.

Answer 89

DNA replication during S phase  chromatin in disrupted. The new histones have to be made with the same epigenetic character? Repressive histone marks = methylation = off. Active histone marks = acetylation = on. Problem for maintaining an epigenetic state = DNA is half old, half new; histones are half old, half new.

Question 90

Epigenetics | Understand that epigenetic mechanisms and inheritance can occur both inside and outside the nucleus

Answer 90

For example, in the nucleus for gene silencing and outside the nucleus for chromatin remodeling. Or, cytosolic epigenetic inheritance in cancer (NFkB?)

Question 91

Epigenetics (Name a specific type of gene that, when aberrantly methylated with 5meC, can lead to cancer and an approach to therapeutic intervention in this case)

Answer 91

If tumor suppressor gene (TSG) is silenced, it can lead to cancer (not actively suppressing it). Therapy focuses on un-methylating them to turn them back on. Other stuff: HDACs can silence genes because they get rid of acetyl groups. New research trying to correct epigenetic patterns and prevent inheritance of aberrant gene silencing by using DNA methyltransferases and HDAC inhibition (both silence tumor suppressors?).

Question 92

Genetic Counseling | Have an appreciation of factors that have impacted the process of genetic counseling and how it is currently practiced

Answer 92

Started as a eugenics movement --> moved to academic counseling and goal of social reform. Now it’s a professional field with goal of education and helping people with genetic disease.

Question 93

Genetic Counseling | Identify present-day goals of genetic counseling

Answer 93

Help individual/family comprehend medical facts (diagnosis, disorder, and management), appreciate the way heredity contributes to disorder and risk of recurrence, understand the alternatives for dealing with risk of recurrence, choosing a course of action that is appropriate for them, and making the best possible adjustment to the disorder. Emphasis on appropriately trained people, communication, and client autonomy in decision-making.

Question 94

Genetic Counseling | Recognize indications for genetic counseling

Answer 94

Hereditary condition in the family, fetus/child with birth defect, child with mental retardation, exposure to carcinogen, consanguinity, advanced maternal age, family history, ethnicity.

Question 95

Genetic Counseling | Identify fundamental ethical principles of genetic counseling as it is practiced today

Answer 95

Respect for patient autonomy in making decisions, beneficence where personal well-being is promoted, non-maleficence (do no harm), and justice (provision of equal care for all). Patient confidentiality. Tenets = counseling should be educational, unconditional, supportive, and nondirective.

Question 96

Genetic Counseling (Know the reproductive options currently available for couples with increased risk for having a child with a genetic disorder, including which options are appropriate or available given the mode of inheritance and/or diagnostic information available)

Answer 96

Take the risk, no reproduction, adoption, prenatal diagnosis with or without interruption of affected pregnancy, sperm/egg donor (autosomal = egg or sperm donor; X-linked = egg donor), pre-implantation genetic diagnosis.

Question 97

``` Genetic Counseling (Recognize factors that may impact a client’s perception of risk and their selected course of action. ) ```

Answer 97

Pre-existing assumptions about the level of risk, personal experiences with a condition, attitudes about illness/disability, anticipated burden of having disease/raising child with disease, temporal factors (time change how people view their situation), gender.

Question 98

Finding Disease Genes | Understand the rationale for finding disease genes

Answer 98

By finding genes, we can improve diagnostics, preventative medicine, pharmacogenomics, therapy, our understanding of the basic biological defect. Hard to determine environmental risk factors, but we can systematically discover disease genes. (Want to be able to apply personalized medicine, but it’s hard because of the low ORs of most disease susceptibility genes.)Just because there’s a low OR, doesn’t mean Population Attributable Risk is also low  can still have high utility. Functional cloning = knowing the function of the gene to figure out the map. Positional cloning = mapping the gene to figure out it’s function.

Question 99

Finding Disease Genes | 1) Candidate-gene association study (hypothesis-driven approach)

Answer 99

1) Candidate-gene association study (hypothesis-driven approach): = most common type of study; depends on a priori hypothesis. Most powerful for common risk alleles with small to moderate effects/ORs (complex polygenic traits). Most hypotheses are wrong  leads to false positives. Depends on LD  SNPs on same piece of DNA tend not to be separated by recombination so will be co-inherited. Use a genotype marker in the candidate gene in cases and controls, and compare allele frequencies. Looking for mutations in genes of interest. Need like a hundred different samples, and need to include evidence from every study ever, including the unpublished ones.

Question 100

Finding Disease Genes | 2) Genome-wide association study (hypothesis-free approach)

Answer 100

2) Genome-wide association study (hypothesis-free approach) = studies the gene indirectly. Tests MANY SNPs across the genome and searches for significantly different allele frequencies in case versus control; looking for regions of genome associated with disease. Need to match cases and controls ethnically  can detect/correct for population stratification. Significant associations require follow up association studies of specific SNPs; most effective for common alleles with small to moderate effect sizes (ORs). GWAS = only successful approach for complex diseases. Works because self-contained, controls for ethnicity, allows to measure and correct for degree of ethnicity mismatching, allows for use of standardized correction, and demands independent replication studies. Need like a thousand different samples (cases and controls).

Question 101

Finding Disease Genes | 3) Genetic Linkage study (hypothesis-free approach)

Answer 101

3) Genetic Linkage study (hypothesis-free approach): = studies genes indirectly; searching genome for segments disproportionately co-inherited along with disease through multiplex families (assumes relatives share susceptibility genes). Best for Mendelian traits (uncommon alleles with strong effects); less powerful for complex traits. Depends on recombination = loci near each other on a chromosome tend not to be separated by recombination. LOD scores assume locus homogeneity (significance is 3 for Mendelian, 3.3 for polygenic).

Question 102

Finding Disease Genes | 4) Exome sequencing

Answer 102

4) Exome sequencing: = only about 1% of genome; cheaper to sequence only the protein coding regions is its only strength. Problem is that we don’t have tools to recognize which variations are pathologic versus not. Used mostly to search for causal mutations in Mendelian diseases  not good for complex diseases because there’s so much variation that we can’t sort it out. Only association studies for complex traits.

Question 103

Finding Disease Genes | Understand the basic statistical approaches used to test association and to test linkage

Answer 103

To test association = statistical significance (P value by chi squared). To test linkage = use statistical measure of LOD score (log of odds) = log10 (likelihood of data if loci linked/likelihood of data if loci unlinked)  assumes locus homogeneity  significance = 3 for Mendelian, 3.3 for polygenic trait.

Question 104

Finding Disease Genes (Be aware of the three most commonly used types of DNA polymorphisms) -1) Microsatellites

Answer 104

1) Microsatellites: = STRPs = simple sequence repeats that re multi-allelic. Used for linkage and forensics. # of repeats differs on certain alleles for different people and can be inherited. Repeats of 1-6 bps.

Question 105

Finding Disease Genes (Be aware of the three most commonly used types of DNA polymorphisms) -2) SNPs

Answer 105

2) SNPs: = single base changes you can score anywhere in the genome. Allele frequencies differ for different ethnic groups/populations. Used for association. Ex: the 1000 Genomes Project is sequencing SNPs (and other variants) in diff ethnic groups. Sometimes SNP alleles are in linkage disequilibrium blocks where recombination doesn’t happen  tend to be co-inherited. Stable and don’t change much; have something to do with haplotypes (combos of alleles for different genes located closely together and tend to be inherited together).

Question 106

Finding Disease Genes (Be aware of the three most commonly used types of DNA polymorphisms) -3) CNVs

Answer 106

3)CNVs: = recurring parts of the genome, hundreds to thousands nucleotides in size. Occurrence frequencies differ in different ethnic groups; individually rare and collectively common. Can be in genes or out, can include genes. We’re not sure how causal they are for human disease.

Question 107

Treatment of Genetic Disease (Discuss that while curing genetic diseases remains challenging, many genetic diseases are amenable to some level of treatment/management)

Answer 107

Actual genetic cures may be rare because it’s hard to correct the root cause of an abnormality (could be single-gene, could be complex, could be chromosomal  especially hard after you’ve already developed), but we can treat the conditions and manage the disease. Can’t remove or silence extra material, insert missing material, remove/silence/regulate single genes or insert them, and gene-environment interactions not fully understood. You can have general management of a disease, and then primary disease specific therapies (protein, cellular, organ), can counsel, and can offer secondary and primary prevention (for families and population).

Question 108

Treatment of Genetic Disease | Identify genetic conditions that currently can be treated and those for which treatment may soon be available

Answer 108

Trisomy 21 = much better supportive care and cardiac surgery = increased survival. Multiple endocrine neoplasia = autosomal dominant and many have RET mutations that will eventually cause cancer. Genetic testing can identify pre-symptomatic carriers and improve their survival by prophylactic thyroidectomy (take out the thyroid before cancer has chance to even develop). For PKU = can do a newborn screening for early diagnosis and treat to decrease morbidity. Most metabolic disorders manifest autosomal recessive inheritance and there are various ways we can intervene. (Add tables later if needed

Question 109

Treatment of Genetic Disease | Discuss examples of genetic disorders that are treated on the basis of protein/enzyme replacement therapy

Answer 109

Ex: Alpha 1-AT deficiency = auto recessive, elastases go unchecked. Can use recombinant AT1 therapy to supplement the protein. Fabry disease = x-linked, deficiency of alpha-galactosidase A activity (cleaves galactose). Causes microvascular diseases, neuropathy, nephropathy, cardiomyopathy. Can treat with recombinant alpha-galactosidase. Can also treat with intravenous infusion of galactose which ‘chaperones’ protein to help it fold properly. (Add table 2 later if needed)

Question 110

Treatment of Genetic Disease | Identify the principles and theoretical risks of gene therapy

Answer 110

Gene therapy = intro of DNA into human to treat a disease. Can be ex vivo (culture patient cells and insert DNA outside of body, then re-introduce into patient) or in vivo (direct directly into patient). Requires targeting (to appropriate cell and location  want to make sure you don’t end up inserting it in the middle of a crucial gene, oncogene, or tumor suppressor), expression (new DNA must lead to adequate expression and duration  doesn’t necessarily have to be at ‘normal’ physiological levels), and toxicity (must be tolerable). Delivery can be viral (adenoviral and retroviral) or non-viral (liposomal, protein-DNA conjugates, injection of naked DNA, artificial chromosomes). Current gene therapy directed at gene replacement/deficiency where gene/protein is missing or non-functional. Harder for dominant negative or GOF mutations (probably because requires more regulation than just replacing function?) (Add table 3 later if needed) Emerging treatment = replacement of deficient products, compensation with novel drugs, small molecules, manipulation of gene expression, and manipulation of pre-mRNA splicing. Replacement = enzyme replacement (lysosomal storage diseases, alpha-1-AT); protein replacement (factor replacement in hemophilia) Compensation with novel drugs = Farnesyl transferase inhibitors, which can help with lamin A/C mutations in progeria (premature aging). Lamin A/C mutation  mutant progerin protein that is targeted to nuclear membrane via farnesyl groups  pathological effects at the membrane. FTI’s reduce progerin sequestration. Also, in Marfan, FBN1 mutations increase cytokine transforming growth factor (TGF-beta). Angiotensin II receptor blockers (ARBs) can block this. Small molecules = Gleevec and CML, chaperone proteins Gene expression = suppression of nonsense signals Manipulation of pre-mRNA splicing = siRNA’s can selectively degrade mRNA; miRNA can suppress translation, anti-sense oligos can suppress splicing

Question 111

Case Studies | (1) Achondroplasia

Answer 111

Autosomal dominant disorder caused by GOF mutation in FGFR3 (1138G>A or 1138G>C; both cause Gly380Arg). FGFR3 is a trans-membrane tyrosine kinase receptor that binds fibroblasts and inhibits proliferation of chondrocytes  less bone growth. Mutation causes ligand-independent activation of FGFR3  inappropriately inhibiting = shortening of long bones. ~80% patients have de novo mutations from father’s germ-line (increase in mutation frequency with paternal age). Present with rhizomelic shortening, short stature, megalencephaly, spinal cord compression, trident hand, brainstem compression (due to smaller foramen magnum), and lumbar spinal stenosis. Midface hypoplasia. Management = mainly looking for any sorts of compression throughout life, spinal stenosis, otitis media, etc. Full penetrance disorder; can only have heterozygous dominant because homozygous dominant is lethal.

Question 112

Case Studies | (2) Nonsyndromic Deafness

Answer 112

Allelic heterogeneity with dominant and recessive inheritance patterns. Congenital deafness in the recessive form. Progressive childhood deafness in the dominant form (starts later in childhood). 1 in 500-1000 neonates. Conductive = anatomy; nervous = sensorineural. Of genetic deafness, half is congenital. 75% nonsyndromic, 25% syndromic. In nonsyndromic deafness, mutations of GJB2 is most common cause  cause DFNB1 (congenital auto recessive) and DFNA3 (progressive auto dominant). GJB2 encodes connexin26 that forms gap junctions in cochlea  failure to from these = loss of cochlear function. Diagnose with newborn screening (otoacoustic emissions or automated ABR tests) to intervene as soon as possible (hearing aids, cochlear implants) to help improvement in language development. Syndromic Deafness = systems outside ears are involved. Can have intellectual disability, seizures, and dysmorphic syndromes. - With retinitis pigmentosa = Usher (AR) syndrome -with thyroid goiter = Pendred (AR) syndrome -with arrhythmia or sudden death = Jervell and Lange-Nielson (AR) syndrome -with white forelock = Waardenburg (AD) syndrome -with 8th nerve schwannomas = Neurofibromatosis type II

Question 113

Case Studies | (3) Fragile X Syndrome

Answer 113

-X-linked mental retardation disorder caused by mutations in FMR1 gene -FMR1 gene makes FMRP. Mutations due to CGG repeat in 5’UTR. Normal = 6-50. [[Premutation = 59-200. Not hypermethylation  actually get increase in FMRP protein.]] Maternally unstable; larger pre-mutations at greater risk for expansion. Mutation = >200 repeats. Cause hypermethylation of the region and the FMR1 promoter  silences promoter, causing loss of FMRP expression. Maternal transmission with risk of expansion. Can have haplotype predisposition to expansion  presence of a few AGG triplets in the string of CGG repeats inhibits expansion of the repeats; absence = predisposition to expansion. Causes moderate mental retardation in males (mild in females). Hyperactivity, hand flapping/biting, temper, autistic features. Long face, prominent jaw/forehead, large ears, macro-orchidism. Severity of phenotype depends on repeat length mosaicism and methylation (mosaicism can mean higher mental function for both repeat length and methylation because it’s better than every cell being affected). Females = degree of severity dependent on degree of skewing of X-chromosome inactivation. FXTAS = also X-linked, but due to premutation triplet repeat expansion. Onset in adulthood, have ataxia/tremor, eventually memory loss, parkinsonism, and neuropathy. Men > women. Too much FMRP protein. Premature ovarian failure also X-linked, due to permutation repeat expansion. Too much FMRP protein.