BMB exam 3

- Del 17q21.31

characteristic long face with high forehead, pear-shaped nose, everted lowerlip, slender fingers, friendly demeanor

Cancer: describe how environmental and genetic factors together define the overall risk of an individual having the condition and the recurrence risk in a sibling or other family member

numerous cancer genes have been identified (major and minor) as well as environemtnal factors to contribute to cancer development - Breast cancer genetic predisposing factors include BRCA1, BRCA2 and p53 gene mutations and environmental risk factors include never giving birth, early first menses or late menopause - Relatives affected increases risk and being female increases risk - Colorectal cancer genetic causes from mutations in APC, MYH and DNA repair genes and environmental risks of lower-fiber diet and lack of exercise

Diabetes: describe how environmental and genetic factors together define the overall risk of an individual having the condition and the recurrence risk in a sibling or other family member

strong association with several HLZ class II alleles and elevated risks for relatives of affected individuals for Type I and Type II has some genetic components as well as environmental factors such as obesity and lack of exercise - psychiatric disorder - Schizophrenia has a high heritability indicating there is a genetic cause playing a role - Bipolar affective disorder also has a high heritability

Patau syndrome (Trisomy 13) -Dominant and distinguishing clinical features

- *Micropthalmia small eyes and some serious cyclopia (single eye), proboscis (projecting tissue just above the eye) - *Polydactyly - *Cleft lip, cleft palate or both - severe developmental delay (motor and cognitive) and cardiac defects - holoprosencephaly a major brain abnormality

3. Explain how variable expressivity and pleiotropy necessitate the need for establishing and, adhering to, the clinical criteria for diagnosis of NF-1, NF-2, and Marfan syndrome

- Because some individuals with these disorders may present only some of the symptoms to varying degrees effecting multiple different organs

- Neurofibromatosis Type II (NF-2) diagnostic criteria is

- Detection of bilateral acoustic neuroma by imaging procedures such as MRI typically in a young individual because the older individuals in the general population not affected by NF-2 can present with unilateral acoustic neuroma - Or First degree relative with confirmed NF-2 and the occurrence of neurofibroma, meningioma, glioma or schwannoma - Or first degree relative with confirmed NF=2 and the occurrence of juvenile posterior subcapsular cataract

Using Down syndrome as examples, rationalize the statement that a syndrome diagnosis does not necessarily confirm the underlying chromosomal causation

- Down syndrome can arise from having 3 chromosome 21(trisomy 21) mostly caused by non-disjunction of the chromosomes during meiosis I or it can arise from an unbalanced Robertsonian translocation involving chromosome 21 and another acrocentric chromosome or it can arise from a robertsonian translocation or isochromosome involving two 21 long arms (21q21q Translocation)

Marfan syndrome diagnostic criteria :When there is a family history (first degree relative confirmed to have Marfan syndrome

- Ectopia lentis OR a systemic score of 7 or great OR aortic root dilation

- Cri-du-chat syndrome (5p-)

- Etiology (chromosome # and location): - Loss of terminal material from the short arm of chromosome 5 (usually paternal origin) - Dominant and Distinguishing clinical features: - Weak high-pitched "cat-cry" - Micrognathia and microcephaly severe developmental delay and cognitive impairment - Hypertelorism, epicanthal folds, low-set ears, and slow growth

- Velocardiofacial syndrome (also known as DiGeorge/Shprintzen/Sedláčková sydrome) (22q11)

- Etiology (chromosome # and location): - Microdeletion at 22q11 gene which codes for a protein that binds precursors to miRNA - Loss of DGCR8 protein results in aberrant miRNA processing and affects multiple organ systems and resulting in multiple anomalies - 1/4,000 births - Dominant and Distinguishing clinical features: - Dysmorphic facial features cleft lip/palate, broad tubular nose and small chin - *submucosal cleft (hard part of palate has a slit) causes a hypernasal speech - long slender fingers - genital abnormalities - hypocalcemia parathyroid hypoplasia - immune deficiency from thymic aplasia - conotruncal heart defects and developmental delay and increased risk for schizophrenia - abnormalities primarily affect the 3rd and 4th brachial arches of embryonic development - if 3rd brachial arch isn't developed no parathyroid low calcium levels - no thymus immune system defective failure to thrive and repeated infections - if 4th brachial arch doesn't develop problems with development of the pulmonary trunk/arteries and aorta of the heart

- Williams syndrome (7q11.23)

- Etiology (chromosome # and location): - Microdeletion in the gene at 7q11.23 (elastin gene) - Dominant and Distinguishing clinical features: - Connective tissue disorder because of the elastin gene - Microcephaly - Facial features are elfin like stellate iris (lace-like) periorbital puffiness/edema, full lips with drooping lower lip, malar hypoplasia (full cheeks), wide mouth, small mandible - high pitched, hoarse voice - bladder diverticular because of poor connective tissue strength - Hypercalcemia (elevated Ca++) causes stones, bone/joint pains and constipation/GI - Cardiac involvement supravalvular aortic stenosis - Proclivity towards music and/or musical fields - Bar-room type personality

- Wolf-Hirschhorn syndrome (4p-)

- Etiology (chromosome # and location): - loss of terminal material from the short arm of chromosome 4 - Dominant and Distinguishing clinical features: - Profound cognitive impairment severe grand mal and/or minor motor seizures - Ocular hypertelorism (wide separation between the two eyes) - Greek helmet appearance broad or beaked nose and micrognathia (small lower jaw) - Cleft lip or palate

5. Justify the statement "High concordance rates do not necessarily mean high heritability." and provide one example

- Example Measles there is a high concordance rate for BOTH MZ and DZ twins and thus the heritability is low (0.16) meaning it is less likely genetic cause and more of an environmental cause

Edwards syndrome (Trisomy 18) - Dominant and distinguishing clinical features

- Feeble activity not moving much (don't try to pull or cry) - Hypertonia flexed tight (opposite of down syndrome) and causes fists to clench with the second and fifth digits overlapping the third and forth - Microcephaly (small head) and severe cognitive impairments no developmental mile stones - Often severe malformation of the heart - Low set, malformed ears; micrognathia (small chin); short stenum; small nipples - Rocker-bottom feet (due to abnormal movement in utero) and 2,3 toe syndactyly - Death usually caused by loss of respiratory drive due to brain malformation or dysgenesis

Down Syndrome (Trisomy 21, translocation Down and mosaic Down)- Dominant and distinguishing clinical features

- Flat facial profile nasal bridge is flat, epicanthal folds, and upslanting palpebral fissures, head wider than length (brachycephaly) with a flat occiput - Brushfiel's spots white dots on the pupil - ears are low-set and have a characteristic folded appearance - mouth is usually open and small resulting in a protruding normal size tongue - short neck with loose skin and short stature - broad hands with a simian crease along with incurved 5th digits (clinodactyly) - hypotonia newborn is very weak and floppy - all have some form of developmental delay - and congenital heart disease is present in ~40% so every down syndrome infant requires an echocardiogram

For each of the following probes, compare the information yield and the limitations of FISH

- Gene-specific probes - centromere-specific probes used to identify individual chromosomes by the centromere alone - telomere-specific probes just bind to the ends of chromosomes - allows for identification of submicroscopic telomeric rearrangements of chromosomes that account for 6-7% of these idiopathic cases of cognitive impairment - idiopathic cognitive impairment represents the collection of cases that are not ascribable to gross chromosome anomaly, unusual chromosomal alterations or environmental causes - whole chromosome paint probes spans the whole chromosome so the whole thing lights up - useful in determining if foreign material is located on a specific chromosome because the foreign material will fail to hybridize with the probe an abnormal chromosome wont fluoresce along its entire length

4. For NF-1, NF-2, and Marfan syndrome, explain how gene testing may or may not assist in determining whether an individual has the condition if, on presentation, there is/are: (a) no physical features or symptoms of the particular syndrome (b) only one to several features or symptoms of the particular syndrome

- If an individual has no physical features or symptoms of a particular syndrome but a gene test comes back positive, you will monitor that individual and watch for signs of symptom development because there is 100% penetrance in these disorders - If the individual already have several features or symptoms or a particular syndrome, the genetic test will not help because it will not change your diagnosis or actions, even if it were to come back negative

Illustrate the application of FISH for the detection of a micro-deletion on a chromosome and explain the consequence of detecting an aberration in the proband

- If only one of the probe signals is present, it indicates a deletion of that specific gene - If you detect a chromosome aberration in the proband, you need to check the parents to determine if either of them are carriers of either the same deletion and/or predisposing translocations - Allows for future recurrence risk assessments - Parents may or may not show some signs of this syndrome even though they carry the same deletion due to variable expressivity and pleiotropy

- Patau syndrome (Trisomy 13) incidence and etiology

- Incidence: 1/10,000 live births - 50% die within the first month of life and only 18% survive during the first year - Etiology: three chromosome 13

- Down Syndrome (Trisomy 21, translocation Down and mosaic Down) Incidence

- Incidence: occurs in 1/800 births - Approximately 95% of these cases are a result from trisomy 21 - Approximately 3-4% of these cases are caused by carriers of unbalanced Robertsonian translocations (involving chromosome 21 and another acrocentric chromosome) - Approximately 2% of these cases are due to "mosaic" Down

- Neurofibromatosis Type 1 (NF-1) aka Von Recklinghausen disease incidence and etiology

- Incidence: ~1/4,000 - Etiology: world wide distribution with no particular racial, geographic or ethnic distribution and effects both genders equally - Half of all cases are due to new mutations in the NF-1 gene at 17q11.2 - The mutation rate of NF-1 is 1/10,000 (highest known for any human mutation) - The other 50% of the time is through inheritance autosomal dominant condition

- Neurofibromatosis Type II (NF-2) incidence and etiology

- Incidence: ~1/40,000 (much more rare than NF-1) - Etiology: - Half of all the NF-2 cases are due to a new mutation - Autosomal dominant inheritance

- Marfan Syndrome

- Incidence: ~1/5,000 (may be even more frequent but just not being diagnosed - Etiology: more than 600 mutations in the FBN-1 gene have been described, mostly missense mutations but there have been some nonsense/frameshift mutations - Physiological activity: chromosomal and molecular identification that the condition is due to mutations in the fibrillin (FBN-1 gene) located at 15q21.1 - Fibrillin is an extracellular matrix protein that forms insoluble microfibrils, providing a scaffold for the deposition of elastin in connective tissue and allows for the strength of connective tissues in certain areas of the body

Rationalize the basis for the spectrum of phenotypes that encompass a diagnosis of the Down, Edwards, and/or Patau syndromes and consider how being mosaic for a chromosomal disorder further affects these clinical phenotypes.

- Lots of minor anomalies add up to make a syndrome - No single one of the features/ phenotypes is absolute and needs to be present for a syndrome diagnosis - Some may have all the features, some may not there is variation from one individual to another - Mosaic for a chromosomal disorder means that less than 100% of the cells are abnormal and contain the wrong chromosomes so phenotypes may be milder - Basically any system can be involved because there are 45 other chromosomes impacting the overall phenotype and 30,000 genes that you have inherited from your parents - So one individual may be more likely to develop a heart defect whereas an individual with the exact same chromosome complement may have a different set of features because of the different genes expressed results in a spectrum of phenotypes

Using the region 7q11 as an example, compare and contrast the effects of a micro-deletion versus that of a micro-duplication.

- Micro-deletion of 7q11 results in william syndrome with the characteristic phenotype of full lips, good verbal skills (compared to math skills) and outgoing personality - Micro-duplication of 7q11 results in non-specific facial appearance but the individual will have thin lips, poor verbal skills (compared to ma th skills) and will have a withdrawn personality (more autistic-like behavior)

Compare the frequencies of paternal versus maternal origins of aneuploidy for the common chromosomal disorders.

- Most Parental origins of aneuploidy are traced back to maternally derived the majority of the time - The only exception is Turner Syndrome (Monosomy X) which is mostly caused by a sperm that doesn't have an X or Y chromosome in it when it fertilizes a normal egg

1. Define multifactorial inheritance and differentiate the impact that genetic traits have in multifactorial conditions versus conditions due to single gene or chromosomal defect.

- Multifactorial inheritance clinical phenotypes are a result of shared genetic and environmental factors and may involve many different genes or just one gene in combination with environmental variables - Teratogens are one type of environmental factor that can contribute to congenital malformations that are multifactorial include certain medications, maternal conditions or alcohol - In multifactorial inheritance, many conditions are noted to recur in families at an incidence higher than the predicted chance alone and do not display typical mendelian inheritance patterns - Multifactorial traits when environmental influences and combined effects of multiple genes impact the variations noted in a phenotype of a respective trait - Mendelian disorders often have the largest impact on specific families (these are rarer) - Multifactorial disorders have largest impact on population as a whole (more common)

- Neurofibromatosis Type II (NF-2) physiological activity

- Physiological activity: chromosomal and molecular identification that NF-2 is due to a mutation in the Merlin gene located at 22q12 which codes for a tumor suppressor protein in Schwann cells - This lack of tumor suppressor protein leads to many tumor types of the CNS but the major issue is the presentation of an acoustic neuroma bilateral 8th nerve (sound transmission) vestibular schwannomas confirms diagnosis - Symptoms of tinnitus (ringing in the ears), hearing loss and balance dysfunction - 2/3rds of NF-2 patients will develop spinal tumors and ½ will develop meningiomas because it's a predisposing cancer diagnosis

Describe pseudo-autosomal regions of the X and Y chromosomes and how they impact syndromes due to sex chromosome abnormalities.

- Psuedoautosomal regions critical regions on both the X and Y chromosome that have important genetic information that has to be present to impart a normal phenotype - Especially located at the distal ends of Xp and Yp this region pairs during meiosis when crossing over takes place - When these regions are missing such as in Turner syndrome it causes problems

On the basis of your knowledge of crossing-over and DNA recombination, explain the most likely mechanism giving rise to chromosomal deletions and duplications.

- Recombination error where a region that is not quite homologous is recombined and leading to duplication or deletion of parts of the chromosome one of the chromosomes ends up with extra material while the other is miss some - We have evolved from this process of deletion and duplications regions in our genome we have two copies of a region and one of them can evolve into new functions while still retaining the base sort of function that was in the original genomic component

Marfan syndrome diagnostic criteria: - Minor anomalies on the systemic score that are symptoms of Marfan syndrome

- Skeletal: Arachnodactyly (spider fingers), increased arm span, abnormal upper to lower segment ratio (tall stature), hypermobility, pectus excavatum (caved in breastbone), skin includes striae (stretch marks), acetabulo protrusion (penetration of femoral head through the acetabulum of the hip joint - Cardiovascular: major concern is aortic dilation leading to an aneurysm - Optic: acutely dislocated lens that is upwardly shifted and propensity to retinal tear that can lead to vision loss - Dural ectasia: increased diameter of the dural sac surrounding the spinal cord

Explain the ramifications of a diagnosis of a common chromosomal disorder for: (a) the affected individual; (b) a parent of a diagnosed individual who is considering having more children; and (c) diagnosis in an unborn child (e.g. via amniocentesis).

- The affected individual - Parent of a diagnosed individual who is considering having more children - Diagnosis in an unborn child (via amniocentesis)

Explain the fundamental basis for the advanced cytogenetics technique FISH

- The dsDNA can be denatured by alkaline pH or formaide treatment to yield ssDNA and then a ssDNA probe can hybridize (anneal) to its complementary sequence - you can fluorescently label a specific segment of DNA so that it can be used as a probe to detect whether a given sample contains that specific DNA

Explain why certain chromosomal aneuploidies are more likely to be present early in gestation rather than later in gestation and vice versa

- The earlier in gestation the spontaneous abortion occurs, the more likely you're going to find a chromosome abnormality - SAB occurring at less than 8 weeks of gestation are 66% of the time caused by chromosome abnormalities - Turner syndrome is the greatest cause of these earliest spontaneous abortions (20%) - Only 1/5,000 are liveborn most conceptuses that have this chromosome compliment don't even make it past the first few weeks of conception

7. Apply the threshold model of multifactorial diseases and describe how it influences the risk that a relative will develop the same condition.

- The risk that a relative will develop the same multifactorial disease (recurrence risk) will increase if: - The affected family member is a close relative (share more genes and environmental factors) - More family members are affected - The disease has a more severe expression within that family - The affected individual is a member of the less commonly affected sex

Explain the nature of the Xist and Tsix genes and how they function in X-chromosome inactivation

- There is a region on the X chromosomes called the X inactivation center (Xic) - Xist (X-inactivate specific transcript) is a gene in this region in band Xq13 and expresses an RNA transcript that binds to and inactivates the X chromosome thus inhibiting gene expression from most but not all the genes on the inactivated X chromosome - Several regions (the pseudo-autosomal regions) on the X chromosome escape X-inactivation - the lone remaining active X chromosome does not express Xist - Tsix gene is the anti-sense transcript of Xist (expressed in the opposite direction across from the Xist locus) - It antagonized Xist function and is only expressed on the active X chromosome it shuts off all other inactivated X chromosomes

Outline procedures for selecting and screening colonies for which have taken up the vector and which of those are recombinant, respectively

- To select the colonies for which have taken up the plasmid and to screen for those that are recombinant (not just an empty vector), individual colonies are spotted in an identical position on a plate with just tetracyclin (the control) and a plate with both tetracyclin and ampicillin - The cells that grow on the plate containing both tetracyclin and the antibiotic will contain empty vectors (did not recombine so dont have foreign DNA) - The cells that grow on the tetracycline but not on the tetracycline and ampicillin contain recombinant plasmids with disrupted ampicillin resistance - Hybridization with a molecular probe is an additional method that is used to screen colonies - In this method, an agar plate with the bacterial colonies is transferred to a filter paper where the cells are treated with alkali to lyse the cells and denature the DNA within it → then a labeled DNA/RNA probe is allowed to hybridize to the cellular DNA and the paper is imaged to identify the colonies containing the DNA of interest

Down Syndrome (Trisomy 21, translocation Down and mosaic Down) Etiology

- Trisomy 21 mostly due to non-disjunction at Meiosis I (~88% of the time maternal and ~8% of the time paternal) - Risk of Down syndrome is about 1 in 100 younger mothers while in older mothers (45yo) risk is 1 in 30 - Translocation Down an unbalanced translocation is usually a de novo event (not inherited) meaning the father and mother aren't the carrier of a balanced translocation - But needs to be checked because sometimes the mother or a father is a carrier and then the mother would have a 10-15% risk of another down syndrome progeny and the father would have a 5% risk of another down syndrome progeny - If the carrier is a 21q21q translocation then all the gametes derived will be unbalanced and ALL surviving progeny will have trisomy 21 - Mosaic Down a post-zygotic non-disjunction event (after the egg has been fertilized) and thus occurs in less than 100% of the cells

Using Turner syndrome as examples, rationalize the statement that a syndrome diagnosis does not necessarily confirm the underlying chromosomal causation

- Turner syndrome is a phenotype, NOT a karyotype there are various possible Turner syndrome karyotypes - In 55% of cases will be caused by monosomy X (45, X) - In 10% of cases it's a mosaic cause (something happened after fertilization) meaning some of the cells will have 45, X karyotype but some cells will have 46, XX karyotype - 20% of cases caused by Isochromosome (46, X, i(X)) - 5% = Ring chromosome (46, X, r(X)) - 5% = deletion portion (46, X, del(Xp) - the last 5% are from other causes

2. Using examples provided in class, explain how twin studies help in distinguishing the effects that genes versus environment have on a given phenotype or disease.

- Twin studies compare monozygotic twins who share 100% of their genes (same in every cell) to dizygotic twins who share 50% of their genes (like any other sibling) - But determining this heritability only tells you if traits are due to a genetic cause or not does not tell you how many genes are involved or how much each gene contributes to the phenotype or the mode of inheritance

Explain the consequences of having X-chromosomal material translocated onto autosomes and why X-chromosome inactivation is important is these translocations.

- When there is a translocation between the X-chromosome and an autosome, non-random X-inactivation occurs because preserving genetic balance is crucial - The normal X chromosome will be preferentially inactivated because the derivative X (containing the translocation) carries autosomal genes on it and inactivation of the derivative X chromosome would cause a loss of autosomal genes - Cells with inactivated normal X-chromosome will survive (but will still probably have a diseased state) - If the abnormal X (derivative chromosome) is preferentially inactivated, cells will fail to survive due to lack of some autosomal gene products - The autosomal material will be lost and that information is critical for cell life

- Marfan syndrome diagnostic criteria with no family of history

- When there is no family history of the disease - Presence of Aortic Root dilatation AND ectopia lentis allows for unequivocal diagnosis of Marfan Syndrome regardless of the absence ofsystemic features - Aortic root dilation and systemic score of 7 or greater the systemic score adds up many different minor anomolys (ex. arm span) - Aortic Root dilation and FBN1 positive gene mutation test - Ectopia lentis and FBN1 mutation

6. Explain how genetic and environmental factors can both contribute to the threshold liability for a multifactorial condition

- a disease such as cleft lip or omphalocele is a multifactorial disease in terms of an individual's liability (risk) to develop the disease the risk of developing the disease lies on a bell curve but once the threshold of liability has been exceeded, the phenotype will be present (either present or not) - The number of predisposing factors (both genetic and environmental) in an individual follow a bell curve but the disease manifests itself in an individual only if a certain number of these predisposing genetic and environmental factors are present must exceed the threshold of liability to present the disease - The more predisposing factors present, the greater the likely hood of exceeding the threshold and presenting the disease

Essential features of a cloning vector are

- an insertion site → where a specific restriction enzyme cleaves to open the vector - an origin of replication → allows the vector to be replicated independently from the host genome - a selectable marker → allows the identification of cells that have taken up the vector

coronary artery disease: describe how environmental and genetic factors together define the overall risk of an individual having the condition and the recurrence risk in a sibling or other family member

- dozens of genes involved in lipid metabolism (LDL receptor mutations or apolipoprotein B mutations) have been identified to play a role in this disease - but other factors such as obesity, hypertension, smoking, positive family history also contribute and increase the risk of disease

Pyloric stenosis: describe how environmental and genetic factors together define the overall risk of an individual having the condition and the recurrence risk in a sibling or other family member

- males are more commonly affected (1/200) than females (1/1000) so males have lower threshold - so brothers of an affected sibling has a higher risk than a sister

neural tube defect: describe how environmental and genetic factors together define the overall risk of an individual having the condition and the recurrence risk in a sibling or other family member

- neural tube defect spina bifida and anencephaly (failure of neural tube to close completely at ~4 weeks gestation) - gen population risk is 1/500 to 1/1000 but recurrence risk is 3% (1/33) for a first degree relative - the chance of occurrence may be lowered (50 to70%) with folic acid supplementation an environmental factor

Neurofibromatosis Type 1 (NF-1) physiological activity

- product of the NF-1 gene is Neurofibromin 1 a tumor suppressor protein that downregulates Ras in the MAP Kinase pathway - Ras protein is active in lots of tissues such as skin, nerves etc - causes an overgrowth syndrome with increased risk of cancer optic nerve gliomas; brain tumors; juvenile chronic myelogenous leukemia myelodysplastic syndrome; and malignant peripheral nerve sheath tumors in adults - diagnosis of this disease should cause you to closely investigate symptoms possibly indicative of a mass - very high likelihood of learning disability (commonly associated with macrocephaly)

8. Describe sex-based differences in thresholds and explain how they influence the recurrence risk for members of different sexes in the same family

- the threshold of liability may differ depending on sex differences the less commonly affected sex would have a higher threshold of liability than the more commonly affected sex - if the affected individual belongs to the less commonly affected sex (higher threshold of liability), the recurrence risk is higher overall than if the affected individual belongs to the more commonly affected sex (lower threshold) - because if the less commonly affected sex is already present in the family, that means more factors were present in the family for that individual to be affected than if the individual was the opposite sex

General procedure of cloning

1) foreign DNA and vector DNA are digested by an appropriate restriction enzyme - 2) utilizing the matching sticky ends, the insert DNA is ligated into the vector using the enzyme DNA ligase - 3) bacteria cells are then forced to take up the recombinant plasmid by transformation - 4) the cells are grown on agar containing the appropriate antibiotic so that there can be selection for bacteria that have taken up the plasmid

Nomenclature examples Pericentric inversion

46,XX,inv(9)(p12;q13) - involves both p AND q arm of the chromosome

Nomenclature examples Paracentric

46,XY,inv(10)(q11.23;q26.3) - involves ONE arm of the chromosome

Paternal %

80% monosomy X

% Maternal

87% Trisomy/monosomy 13-15 95% in trisomy/monosomy 18 93% trisomy/monosomy 21 95% trisomy/monosomy XXX

Describe a derivative chromosome and provide an example in terms of a karyotype

A derivative chromosome is a structurally rearranged chromosome generated by a strcutural change between two or more chromosomes. It is named after the chromosome that has an intact centromere. I think for an example, we will say chromosome B is short, and chromosome A is long. Chromosome A will have part of it added to chromosome B. Now, chromosome B is longer, and A is shorter than before. The karyotype is noted with der. There is no karyotype notation in the course packet that I could find According to our friend Wikipedia: derivative chromosome 46,XY,der(4)t(4;8)(p16;q22)t(4;9)(q31;q31) would refer to a derivative chromosome 4 which is the result of a translocation between the short arm of chromosome 4 at region 1, band 6 and the long arm of chromosome 8 at region 2, band 2, and a translocation between the long arm of chromosome 4 at region 3, band 1 and the long arm of chromosome 9 at region 3, band 1.

Using the conventions of nomenclature, identify a specific location on a chromosome (e.g. 3p21.31) in terms of chromosome number, arm, region, band, and sub-band

Chromosome 3 Small arm Region 2 Band 1 Subband 31

Describe the concept of clone libraries and how such libraries are constructed. Compare the information content of genomic libraries and cDNA libraries.

Clone libraries → a collection of clones made simultaneously from a given sample - Genomic libraries → a set of clones that together contain all of the DNA sequences of a given genome - cDNA library → a set of clones that together contain all the DNA sequences produced by reverse transcription of the mRNA isolated from a tissue or cell (represents all the genes being transcribed at a given time)

Describe the features of DNA sequences recognized by restriction endonucleases. Given a DNA sequence and a list of recognition and cleavage sites for restriction enzymes, identify the positions and number of DNA fragments that would be produced.

DNA sequencing by restriction endonucleases → restriction enzymes are used to cut DNA into smaller fragments in order to study and identify genes - It also allows for recombining (joining) the DNA from different genomes to study the gene's function, expression and regulation or to create recombinant gene products for treatment of disease (such as insulin and HGH) - Restriction sites are typically 4 to 6 bases long and the sequence is an inverted repeat (single stranded sequence of nucleotides followed downstream by its reverse complement) - All restriction endonucleases will cut at a restriction site → this produces DNA fragments with either "sticky" or "blunt" ends that can be resealed using the enzyme DNA ligase - Note: restriction enzymes will not recognize the sequence going 3' to 5' (only 5' to 3')

Recall the types of foreign DNA that can be used as an insert. Compare the informational content of genomic and cDNA and synthetic DNA clones.

Foreign DNA can be genomic DNA, cDNA or synthetic DNA - Genomic DNA → simply DNA derived directly from a genome - genomic DNA would incorporate some sequences such as introns or regulatory sequences that would not be found in cDNAs - cDNA clones (reverse transcription)→ DNA that is synthesized from mRNA using reverse transcriptase - Will contain the open reading frame, 5' and 3' untranslated regions and polyA tail but will not contain introns, promoters, enhancers or intergenic DNA - Allows for the ability to express a eukaryotic gene in a prokaryotic host - Synthetic DNA clones→ chemically synthesized oligonucleotides that are used as primers for in vitro DNA synthesis and probes in hybridization assays - Disadvantage = only up to ~100 nts can be chemically synthesized

Describe the process of gel electrophoresis and what information is obtained

Gel electrophoresis is used to identify the size and shape of DNA and RNA sequences - Smaller fragments move farther down the gel towards the positive electrode than large fragments - Fragments of nucleic acids if known size are run adjacently to serve as a molecular weight marker

Vector type: Bacterial artificial chromosomes (BACs)

Host: E.Coli Insert range size: 50-200kb Special features: Very large fragment for gene mapping, genome organization

Vector type: Cosmids

Host: E.Coli Insert range: 25-40 kb Special features: Clone even longer fragments

Vector type: gamma phage (y)

Host: E.Coli Insert size range: 10-15kbp Special feature: Double stranded linear Efficient cloning of larger fragments

Vector type: Yeast artificial chromosomes (YACs)

Host: yeast Insert size range: 100-1000 kbp Special feature: very large fragment for gene mapping, genome organization. replication originm centromere, telomeres

Vector type: Plasmid

Hot organism: E. Coli, yeast Insert size range: up to 10 kbp Special features: Double stranded circular, antibiotic resistance

Explain the physical basis of hybridization assays. Recall the composition of probes and the factors that determine their stringency.

Hybridization/ annealing is the process of base pair formation between a probe and its target DNA and relies on the ability of two complementary single stranded nucleic acid oligomers to form a double stranded structure - Probes are typically composed of synthetic DNA or RNA but can sometimes be composed of cDNA or genomic DNA fragments - The stringency is the degree to which a probe will tolerate mismatches and still anneal and can be manipulated by changing the probe length and the experimental conditions (temperature and salt) → can be as accurate as a single mismatch will prevent annealing - Probes are labeled in order to detect the degree to which hybridization occurs → can be labeled using a radioactive isotope, fluorescent tag or affinity tag - Common hybridization assays include southern, northern and western blots, allele specific oligonucleotides (ASO), fluorescence in situ hybridization (FISH) and microarrays

General information

If more than 2% of genome is missing, the condition is lethal But trisomies of the larger metacentric chromosomes is also lethal, because there is just too much extra genetic material Deletions or duplications that are visible using routine karyotyping (~550 bands) are usually large, termed macrodeletions or macroduplications Smaller ones not seen on karyogram but can be seen by FISH or chromosome microarray, are called microdeletions or microduplications CNV = copy number variation/variants = the quantitative difference in the amount of DNA due to a deletion or duplication Depending on size and location of CNV, the condition can be pathogenic or benign

Describe the synthesis of cDNA, including the role of reverse transcriptase. Be able to distinguish between cDNA and genomic DNA based on the components present in the sequence.

It is possible to obtain DNA that does not contain introns (cDNA) by using the enzyme reverse transcriptase → this enzyme will synthesize DNA that is complementary (cDNA) to the mRNA - Since the template is spliced mRNA the product contains the Open reading frame, the 5' and 3' untranslated regions and the poly A tail but it does not contain introns, promoters, enhancers or intergenic DNA - This approach allows us to express eukaryotic genes inside a prokaryotic host

Describe the process and products of the polymerase chain reaction (PCR).

PCR is an in vitro amplification of a specific fragment of DNA without an origin of replication → it allows for obtaining large amounts of DNA of interest so that it can manipulated in other lab techniques - Steps: - Denature the double stranded target DNA by heating - add two synthetic DNA primers that are complementary to the DNA sequences flanking the target region - Decrease the temperature to allow the primers to anneal to the target DAN - Temperature is increased to ideal temperature for extension of the primer by a special heat stable DNA polymerase called Taq DNA polymerase - These steps are cycled 25-30 times and each step doubles the amount of target DNA in the previous step → get an exponential increase in copy number of the fragment of interest - The products of PCR can be used for many things such as sequencing, cloning, digestion with restriction enzymes or as probe

Schematically diagram and write the karyotype for a reciprocal translocation.

Reciprocal translocation- Exchange of material between two or more distinct chromosomes. There are two kinds. Balanced is when there is no apparent gain or loss of genetic material. When it is balanced, there is usually no phenotypical change. However, the offspring of the parents affected can result in the offspring being off balanced. Unbalanced is when there IS a gain or loss of genetic material. Cell is either missing or contains an extra chromosome material. Karyotype: Written as 46,XX,t(4;20)(q22.1;q31.2) That means: 46 chromosomes, females, t= translocation, ( the smaller chromosome listed first, followed by the larger one), (q is the long arm, 2 region,2 band, 1 subband; long arm,3 region,1 band, 2 suband) The portion that is (q22.1;q31.2) are the areas that are switched between the chromosomes.

Describe in words or diagrams the general concept of recombinant DNA. Describe the role of DNA ligase

Recombinant DNA → is DNA that has been formed artificially by combining constituents from different organisms - The process of cloning involved combining a specific gene or DNA segment with a small carrier DNA called a vector and then replicating it - Replication of the recombinant DNA occurs in two ways: increasing the cell number and by creating multiple copies of the DNA per cell - DNA ligase → this enzyme is used to ligate the insert DNA into the vector

Schematically diagram and write the karyotype for a Robertsonian translocation

Robertsonian translocation is when there is a fusion of two acrocentric chromosomes. Acrocentric chromosomes are chromosomes where the centromere is located very close to the end of the chromosome. Acrocentric chromosomes are chromosomes 13,14,15,21 and 22. There is no loss of genetic material of the long arms of the acrocentrics. There is loss of satellites on the short arms, but the genes lost are redundant information, and are on other satellites.

Explain the basis of the Sanger (dideoxy) sequencing method. Outline the steps involved. Note the limitations of the method

Sanger (dideoxy) sequencing → a method to determine the DNA sequence of an unknown region by utilizing the idea that DNA polymerases need a 3'-OH group for synthesis to occur - Steps: - a sequencing primer binds to the template of unknown sequence and primes the synthesis of a complementary DNA strand (5' to 3') - Then the addition of four dNTPs and four ddNTPS (dideoxynucleoside triphosphates with no OH on either the 2' or 3' position of the ribose nucleotide) → each of the four ddNTPS is labeled with a different dye - Key: the DNA pol can't extend the chain because no free OH - results in a collection of fragments whose lengths differ by one nt - Each fragment is separated by capillary electrophoresis to generate a pattern of colored peaks indicating the nt - Limitations: the read length for one sequencing reaction is 400-600 nucleotides and the throughput is low

Klinefelter syndrome

Syndrome (47, XXY) males with an extra X chromosome - Dominant phenotypic features: - Tall and thin with relatively long legs disproportionally long arms and legs - Signs of hypogonadism around puberty testes remain small (hypoorchidism) and secondary sexual characteristics remain underdeveloped - Testicular dysgenesis and gynecomastia - Usually infertile - IQ is slightly but significantly reduced on certain intelligence performance tests - 2/3rs have educational problems such as dyslexia - poor psychosocial adjustment - higher frequency of non-hodgkins lymphoma and other lymphoid cancers and higher frequency of certain autoimmune diseases and type I diabetes some immune regulation is not right

Karyogram

Systematic visual picture of chromosomes. Homologous pairs of chromosomes are arranged by size, centromere location, and banding pattern. Each chromosome can be compared band by band with its homolog. Provides the basis for an karyotype.

Explain the main limitation and the uses for chemically synthesized DNA

The main limitation of chemically synthesized DNA is the amount → only ~100 nts can be synthesized - Chemically synthesized DNA is primarily used as primers during in vitro DNA synthesis or as probes in hybridization assays for identifying a specific DNA sequence

Idiogram

This is a drawing/depiction of the chromosomal pattern. Shows the relationship between short and long arms, centromeres, stalks and satellites.

Karyotype:

This is a written description of the chromosomes of the individual. There are some basic elements of karyotype. Number coresponding to the total number of chromosomes in a cell, followed by a comma, then followed by the sex chromosomes, followed by another comma, and then information important for the karyotype individual.

Euploidy

This is when a cell or organism has a multiple of the haploid chromosomes. AKA, a multiple of 23. Has one or more than one set of the same set of chromosomes

Aneuploid

This is when the number of chromosomes is NOT a multiple of the haploid number (23). This can result in phenotypic consequences. Having less genetic material is worse than having extra. In nondisjunction during mitosis, there is a failure of homolog separation in either meiosis I or failure of sister chromatid separation in meiosis II (or mitosis). The most common is a failure in meiosis II, where the gametes contain 2 copies of one homolog of a chromosome. It is as also more common in maternal meiotic divisions, and risk increases with age.

Describe the basic cytogenetic technique of karyotyping and compare the resolution of standard versus high resolution banding.

You take a blood sample, and add phytohemaaglutinin. This chemical will stimulate cell division. Once cultured for 3 days, you then add colchicine and hypotonic solution. This arrests the cell in mitosis. Cells are then fixed to the slide, and then is digested with trypsin and stained with Giemsa. You can now analyze the metaphase spread. Standard resolution: Can see between 400 and 500 bands, and see large rearrangements. High resolution: This uses chemicals that interfere with chromosome condensation (prophase cells), resulting in longer chromosomes for band resolution. See more than 650 bands, and allows for detection of small rearrangements.

- Turner Syndrome

a deficiency in a female who lacks two functioning X chromosomes - Dominant phenotypic features: - Lymphedema localized fluid retention and tissue swelling due to lymphatic obstruction - Webbed neck due to fluid in the neck during gestation and when it drains it leaves a bunch of excess skin - Short stature endocrine problems (hypothyroidism) - Primary ovarian failure due to gonadal dysgenesis - Lack of secondary sexual characteristics shield shaped chest with side spaced nipples and underdeveloped breasts - Elevated frequency of renal and cardiovascular anomalies (aortic stenosis) - Intelligence is usually average or above average - Osteoporosis is common; congenital hip dislocation; scoliosis occurs in 10%

Southern Blot

a hybridization assay used to identify a specific DNA sequence - General steps include: separating fragments of genomic DNA after restriction digest by gel electrophoresis → then blot (transfer) them onto a filter paper and add the sequence specific probe that will anneal to the DNA → image the filter paper to see the hybridized fragments

Northern Blot

a hybridization assay used to identify a specific RNA sequence

Western Blot

a hybridization assay used to identify the size and abundance of a protein (an antibody probe would be used instead of DNA/RNA probe)

- Del 1p36

characteristic straight eyebrows, hypotonia (poor muscle tone), microcephaly, seizures and severe learning difficulties

- Barr bodies

chromatin positive bodies in female cells but not found in normal male cells - Represent more condensed DNA that is not being transcribed the second X chromosome in every cell of a female is being condensed and shut down so only the psudoautosomal regions are being expressed - one of the first cytological tests to distinguish female from male cells - There is much less genetic information (gene content) present on the Y chromosome relative to the extra X chromosome so the female cells need to manage this genetic informational imbalance through X inactivation only one X chromosome remains active while additional X chromosomes (barr bodies) are inactivated (lyonization) thereby maintaining adequate X chromosomes derived gene expression (dosage) levels - Patients with abnormal sex chromosome numbers have one less barr body than the number of chromosomes - Ex. People with Turner syndrome lacked a barr body and males with klinefelter syndrome had a barr body in each cell

, - Neurofibromatosis Type 1 (NF-1): list the clinical criteria for diagnosis

clinical diagnosis is based on a patient having any two of the following symptoms: - six or more cafe au lait macules present in 99% of NF-1 patients - skinfold freckling in the axillary (underarm) or inquinal (groin) regions - two or more neurofibromas (swelling of the nerve sheath that surrounds nerves) of any type (99% present by adulthood) or one plexiformneurofibroma (large coalescence of several neurofibromas) - optic glioma causing tunnel vision and headache - two or more Lisch nodules benign hamartomas of the iris - this is the penetrance of the gene if you're going to have anything you're going to have lisch nodules by the time they're an adult - Osseous lesion (sphenoid dysplasia) or thinning of long bone cortex with or without pseudoarthrosis (false joint) - a first degree relative (parent, sib, or offspring) diagnosed with NF-1 as defined by these criteria

Thrombosis: describe how environmental and genetic factors together define the overall risk of an individual having the condition and the recurrence risk in a sibling or other family member

formation of blood clots - general population risk increases with age 1/100,000 in childhood and 1/100 in elderly - attributed to many genetic factors and environmental factors Genetic factors: Factor V Leiden, Prothrombin 20210 mutation, protein S deficiency, protein C deficiency, antithrombin III deficiency Environmental factors: Surgery/trauma, cancer, cholesterol, smoking, prolonged inactivity

- Concordance rates

how often two individuals share the same trait/condition - Diseases/traits among twins are determined to be concordant (shared) or discordant (not shared)

- Edwards syndrome (Trisomy 18) Incidence and etiology

occurs in about 1/6,000 to 1/8,000 live births (~10 times less frequent than down syndrome) three chromosome 18s in every cell of the body - Also been some incidence of mosaic trisomy 18 due to some random event during cell division early in embryonic development

Deletions

oss of a segment of a chromosome, resulting in unbalanced information Phenotype depends on size and location of deletion Ex. Wolf-Hirschhorn syndrome If a deletion of part of a chromosome is noticeable on the karyogram, it is a large deletion! If too small to be detected on a karyogram, it is considered to be a "microdeletion" The other, homologous chromosome can be normal Can have deletions at the ends of the chromosome ("terminal deletion") or somewhere in the middle ("interstitial deletion") Nomenclature: Terminal deletion: 46,XX,del(4)(p16.3) Interstitial deletion: 46,XY,del(7)(q11.23;q21.2)

Duplication

part of a chromosome has duplicated genetic material A result of unequal crossing-over during meiosis Results in a partial trisomy for the region Clinical consequences depend on size and location, but generally not as severe as losing genetic information

Insertion

segment of a chromosome is inserted into a completely different chromosome (i.e. part of chromosome 4 is inserted into chromosome 20) Genetic information is still balanced The chromosome that receives the extra info does not give any information back to the donor May affect the offspring because the donor chromosome normally would line up with its homolog, but now it wants to line up with the recipient chromosome Nomenclature: 46,XX,ins(20;4)(q13.1;q12q27) Always list the recipient chromosome first!

- Heritability

the measure of the proportion of the total variance of a trait that is caused by genes and is determined by comparing the concordance rates between MZ and DZ twins - Heritability = 2 x (concordance of MZ - concordance of DZ) - The greater the difference between the concordance rate, the higher the heritability =more likely genes are influencing the condition (heritability closer to 1.0) - 100% concordant among MZ twins and 50% concordant among DZ = determined solely by genes - The more similar the concordance rates are, the lower the heritability = environment is predominantly responsible

Inversion

when a single chromosome has two breaks, and when they reform, the segment between the two breaks gets inverted Balanced, because no information is lost

Ring chromosomes

when small deletions on both ends of a chromosome (p- and q-arms), the ends fuse to form a ring Results in an imbalance, because information is lost due to deletions on the ends Usually cause an abnormal phenotype Nomenclature: if chromosome 15 is a ring, written as 46,XX,r(15)(p__;q__) Seen on karyotype and microarray Uncommon 99% are sporadic They are unstable during cell division and are often lost, leads to mosaicism

Pericentric inversion

when the segment between the two breaks does include the centromere Can have offspring with deletion and duplication of that chromosome segment

Paracentric inversion

when the segment between the two breaks does not include the centromere Lethal!

Isochromosomes

when two p arms or two q arms of the same chromosome are joined at the centromere, and the other arm is lost, causing a "mirror image" chromosome Occurs because two sister chromatids are divided incorrectly at the centromere A horizontal split, rather than a vertical split So you get two copies of the same arm and a mirror image at the centromere; deletion of one arm, while the other arm is duplicated This is unbalanced because information is lost when one arm is deleted Nomenclature: if there is an isochromosome of the long arm of the X-chromosome, would be written as i(Xq) I.e. 46,X,i(Xq) Extremely uncommon, but when it does occur, usually affects the X chromosome Usually lethal if it occurs on most autosomes Can be seen on karyotype and microarray