Medical Genetics Exam 2 - Glenn Goodwin

Cri Du Chat

"Cry of the Cat", deletion at chrome 5 short arm.

Imatinib

"Gleevac", used to inhibit tyrosine kinase activity in treating CML.

Chronic Myelogenous Leukemia (CML)

"Philadelphia chromosome", reciprocal translocation between chrome 9 and 22. Results in a *constitutively active tyrosine kinase*

Break point in chromosome 9

"abl" whereas in 22, it's "bcr". bcr and abl combine to form the Philadelphia chromosome (chrome 22 becomes the philadelphia chrome)

Tandem repeats

*Only occurs in noncoding* 15% of human genome. Made up of either Minisatelites (10-60 VNTR) or microsatelites (1-6 STR). Used for forensics cases and diagnosis/risk of diseases Assoc. diseases= Huntington's and Fragile X

Types of cuts by restriction enzymes (2)

1. "Blunt"- Same position on both strands- *Hae3* and *Sma1* 2. "sticky"- Cut in a staggered postion- *EcoR1* and *Not1*

Metacentric

A centromere located in the middle of the chromosome--> chromes 1-3. If located at tip--> acrocentric. Between middle and tip= submetacentric--> 4-12

SKY (and applicable disease)

A colored version of FISH used to discover difficult cytogenetic abnormalities. Used in *Acute Promyelocitic Leukemia* (APL) because the color codes the piece of translocated fragment.

BCR-ABL encodes for

A fusion protein (oncoprotein) that ends up having tyrosine kinase turned on indefinitely, resulting in cell proliferation.

Sickle cell Southern blot

A line at 1.35= Sickle cell disease Line at 1.15= Normal Line at both= heterozygous (carrier) Sickle cell will be bigger because of a mutation that results in a loss of the restriction enzyme recognition site

Robertsonian translocation

A long arm of one chrome attaches to a long arm of another. Same shit happens with short arms so you end up with a big ass chromosome and a smaller one (instead of the typical balanced translocation of 2 chromes of same size). *Still considered a balanced translocation* The short chrome is usually degraded because it doesn't have a centromere. *Not dependant on maternal age*, so a young mom with a down's kid usually is due to Robertsonian.

SNP variant affecting response to albuterol

ADRB2

APL treatment

ATRA, "All trans retinoic acid", converts PML-RARa into a co-activator and restores the differentiation of promyelocytes into mature leakocytes

Oncogenes

Abnormal version of proto oncogenes. Proto oncogene structural or genetic change can result in an oncogene. Oncogenes can also come about through DNA/RNA viruses. *Cause growth-signaling pathways to be hyperactive* It's the faulty growth-signaling *proteins* encoded by the oncogenes that cause the cancer.

Assessment question

Answer on following card

Good PCR question

Answer on following card

BCL-2

Antiapoptotic Oncogene overexpressed in Follicular Lymphoma. Translocation from chrome 18--> 14. It lands next to the *Ig heavy chain enhancer* region on 14. Increased BCl-2 leads to increased B cell survival

dideoxy sequencing with capillary electrophoresis

Assigns colors to the nucleotides and you can sequence it accordingly. Done in one tube instead of 4 (as in Sanger).

BCR-ABL

Assoc. with CML, Philadelphia chromosome, reciprocal translocation between chromes 9 and 22 leads to the BCR-ABL hybrid oncogene which codes for a constitutively active *cytosolic tyrosine kinase*

HTLV

Causes adult t cell lymphoma/leukemia

HPV

Causes cervical cancer via viral replications of *E6 and E7* which interfere with normal function of host p53 and Rb proteins (retinoblastoma tumor suppressor), respectively

PML-RARalpha

Chromes 15 and 17; when fused, acts as a corepressor which prevents differentiation of promyelocytes--> Acute Promyelocytic Leukemia (APL).

GDNF

Glial Derived Neurotrophic Factor, activates RET. In cancer, RET is activated independent of GDNF

Example dideoxy mutation case.

Hosni has the mutation whereas the mother is heterozygous for it.

HER2

Human EGF Receptor, encoded by *ERBB2*, located in long arm of chromosome *17*. Regulates growth, differentiation, and adhesion. AKA Neu. *200 fold gene amplification* Assoc. with breast cancer

Turner syndrome

Missing sex chromosome (45 "X0"), will have *high FSH*. Ovaries are unresponsive. Widely spaced nipples

Gain of function mutation

Tend to be *Dominant* mutations. Usually activation of an oncogene

Stringency

The degree to which non complementary sequences are able to bind. Higher stringency= fewer mismatches tolerated. Condtions producing high stringency= Increasing heat, lowering salt, higher concentration of denaturing agents.

Burkitt Lymphoma

Translocation between 8 and 14 results in lymphoma. C-Myc (a TF upregulatory gene) goes from 8 to 14. 14 encodes for Ig heavy chain so you end up with an increased activity of Ig/lymph synthesis.

Allele Specific Oligonucleotide (ASO)

Type of probe that recognizes either normal or mutant DNA sequences. Very high stringency--> can detect a single nucleic acid difference. Will present as a "dot plot". High temp and low salt Used to confirm *Sickle cell*

PCR youtube video

https://www.youtube.com/watch?v=iQsu3Kz9NYo

SNP variant affecting response to Plavix

CYP2C19, and other CYPs

MYC gene

TF upregulatory gene on chrome *8*--> Reciprocal translocation between 8 and 14--> Burkitt's Lymphoma Also assoc. with *EBV*

Fragile X repeated sequence

>200 repeats of CGG When answering Southern Blot questions: A line at or above 5.7Kb= full mutation for Fragile X A line in between 5.2-5.7= "premutation" A line under 5.2= "normal"

PCR uses (5)

1. Bacterial/viral ID 2. Diagnosis of single gene disorders 3. Paternity testing 4. Mutation/translocation breakdowns- "RFLPs" *Multiplex PCR* 5. Quantitation of DNA/RNA in a sample

Diseases detected by PCR (5) ("CS-PHD")

1. CF- Deletion of CTT--> Deletion of Phe at 508 2. Sickle Cell 3. PKU 4. Huntingotn's 5. DMD

Rearrangements of RET genes lead to (2)

1. Papillary thyroid carcinoma 2. Lung adenocarcinoma

Applications for genome sequecing

1. Pre-implantation diagnosis of embryos (single cell WGS) 2. Diagnosis of diseases and single celled variants that cause them. 3. Disease susceptibility 4. Genotype-based therapy 5. Monitor disease progression

Signaling pathways in BCR-ABL (4)

1. RAS- MAPK pathway--> Cell proliferation 2. inhibition of apoptosis--> PI3-K/PKB 3. CRKL-CAS--> Interferes with cell adhesion (such as *paxillin*) which allows the cancer to spread 4. Also utilizes JAK-STAT pathway--> Initiator of transcription

Steps of DNA sequencing (4)

1. Separate the strands 2. Add radiolabeled primer 3. Elongate 4. Incorporate a chain terminating "ddNTP"- either ddGTP, ddATP, ddTTP, or ddCTP 5. Each dideoxynucleotide is in a separate tube *Those are used for chain termination because they lack the 3'-OH*

"ingredients" used in PCR (5)

1. Taq- "Thermus Aquaticus", fast and no proofreading 2. Pfu- "pyrococcus Furiosus", slow proofreading 3. 2 primers, forward and reverse, 18-25bp (Forward primer is identical to coding strand and reverse primer is complementary to coding strand). 4. dNTPs 5. Mg used as a buffer 20 cycles will yield *1E^6 copies*

Exome

180K exons, 1% of total genome but 85% of diseases. Can have the exome sequenced for about $1000 and it takes a day. Limited though because it won't determine diseases/abnormalities in mitochondria, regulatory regions (promoters/enhancers), or structural variants (translocations)

Number of chromosomes in human

22 pairs of autosomes and 1 pair of sex

Human genome numbers

3E^9 base pairs, 39K functional genes

APL chromosome

46 XY t(15, 17), translocated between 15 and 17 (PML and RAR fusion respectively)

Klinefelter

47 XXY, will have *increased GnRH* levels (because testes don't respond).

Sporadic cancers

75-80% of cancers, come about through random mutations and are *acquired*. Hereditary is about 5-10% of cancers. Mutation is present at birth and usually has an early onset of cencer Familial cancers- present among members of same family but are not necessarily hereditary. *Genetic and environmental*

Answer

C

Answer

C, patient 3. More questions at end of lecture 13

Restriction enzymes

Cut up a piece of DNA at a specific point without use of ATP. Recognize 4-6 bases and *palindromes* in DS DNA

Short arm of chromosome

Denoted by "p", long arm is "q"

Burkitt and EBV

EBV induces expression of *LMP-1*. LMP-1 mimics an activated cytokine receptor which results in cell proliferation via: 1. MAPK 2. PI3/PKB 3. NF-kB

Trisomy 18

Edward syndrome, 50% are still born. Of those that live, 90% die within 1st year. Cleft palate, heart/liver/kidney defects/GI

Fragile X

Elongated face and weird ears as well. "Hanging by thread X chromosome" CGG repeat of >200.

RET (2)

Encodes Receptor Tyrosine Kinase which is involved in neural crest development. *Point activating* mutations in the *Cysteine* or *Tyrosine* domains cause constitutive activation and leads to 1. Multiple Endocrine Neoplasia (MEN2A and 2B) 2. Familial Medullary Thyroid Carcinoma (FMTC)

Criminal DNA typing

FBI uses 13 standardized STR markers for DNA typing (CODIS combined DNA typing system)

Robertsonian acrocentric chromosomes

Important for "Robertsonian" 13-15, 21, and 22

EBV (3 pathways)

Induces expression of membrane protein, *LMP-1* LMP-1 mimics *TNFR*, which is a cytokine that causes proliferation of cells. Works via 1. MAPK pathway 2. PI3-k/PKB pathway 3. NF-kB

Tumor suppressors

Job is to arest cell in a certain part of cycle as well as aid in DNA repair. "Loss of function" mutations. 1. Gate keeper- Regulate transition through checkpoints- p53, Rb 2. Caretakers- Protect genome survival/integrity- BRCA 1/2, NF1, Apaf-1

HIV causes this type of cancer:

Kaposi's Sarcoma

Translocations during hematopoiesis leads to___

Lymphomas and leukemias

Paternity testing slide

Male 2= father

Trisomy 13

Patau syndrome

Probe

Radiolabeled DNA sequence complementary to the sequence you're trying to identify.

SNPs

Responsible for 90% of polymorphisms in humans. Occur every 100-300 BPs. Can occur in coding/non coding regions.

RAS

Small GTP-binding proteins, point mutations result in its locked active form leading to indefinite cell-cycle progression. Large number of cancers assoc. with RAS--> Colorectal and pancreatic

Organization of DNA fragments on electrophoresis

Smaller pieces will be towards the "bottom"- positive end. Big pieces on top (negative)

Types of RB

Sporadic affects 1 eye whereas hereditary forms/LOH affects both. May also cause osteosarcoma, soft tissue sarcoma, and melanoma. RB1 gene located on chrome *13*

Simean crease

The single hand crease seen in Down's patients. Can also be present in 1-2% "normal" individuals

Unbalanced translocation

There is a net gain or loss of genetic material to a chromosome.

Multiplex PCR disease

Used to determine presence of DMD (exons 48-51 deleted)

Loss of function

Usually recessive. Usually inactivation of a tumor suppressor

Loss of heterozygosity

When a mutation in one allele causes a mutation in a normal. Can give the appearance of a dominant mutation. Assoc. with Tumor suppressor mutations and first observed in *retinoblastoma* Mutant, *rb*, causes the loss or mutation of normal *RB1* so cancer can occur through hetero or homozygous.

Translocation

When equal sized pieces of 2 chromosomes trade places. Ex: A piece of chromosome A goes to chromosome B. A same size piece goes from Chrome B to A. The trade is known as *reciprocal exchange* and results in a *balanced translocation*

Nondisjunction during meiosis 1

Will result in 50% cells with 1 chromosome 50% 3 chromosomes 2 gametes will have a big chrome and 2 will only have a small chrome.

Nondisjunction during Meiosis 2

Will result in 50% normal (2 chromes) 25% trichrome 25% 1 chrome (only a small chrome, no big ones).