eRobbins Ch 6 - Genetic Diseases (No Peds)

Question 1

Chromosome classification terminology in Clinical Genetics

Answer 1

1-22 numbered by length1 = longest2 = shortestCentromere = centerTelomeres = endsp arm = petit arm = short armq arm = long armInternationally accepted system of chromosome classification: -Chromosome number -Arm -Region -Band -Sub-band**Capital letters & italicizedSpectrum of testing modalities:DNA sequencing (coding)Cytogenetics: - the study of the structure and function of the cell, especially the chromosomes - can detect both numerical and structural abnormalities in chromosomes - specimens must be fresh, so the cells can be cultured

Question 2

Down Syndrome

Answer 2

Trisomy 21 -> 3x chromosome 21Mental retardationAbundant Neck skinCongenital heart defects - Majority of childhood deaths - Endocardial cushion defects (cells that make up spetum & divide A/V & L/Rintestinal stensosisEpicanthic folds (upper eyelid folds)Flat facial profileSimian crease (only 1 crease in palms)Umbilical herniaHypotoniaIncreased leukemia risk +10-20% Acute megakaryoblastic leukemia

Question 3

Nucleic Acid Hybridization

Answer 3

• Detects the presence of a specific sequence• Based on the principle of complementary base pairing between a target nucleic acid and a labeled probeProcess: - Label probe with fluorescent dye - Denature DNA - Hybridize - Look for fluorscence***Always limitations

Question 4

Patau Syndrome

Answer 4

Trisomy 13Microphthalmia - small eyesPolydactyly - extra fingersCardiac defectsUmbilical herniasRenal defectsMicrocephaly - small headMental retardationCleft lip & PalateOcker bottom feet - rounded arches**Most often fatal- Some have Robertsonian translocations

Question 5

Robertsonian Translocation

Answer 5

Fusion long arms of Chromosome 13 & Chromosome 14 between acrocentric (long arms have all information)Not detectable with a centromeric FISH probe

Question 6

Comparative Genomic Hybridization (CGH) Array

Answer 6

A hybridization technique that can assess the entire genome for copy number changesLabel all patient’s DNA with 1 color (green) & control DNA (red)Control color (red) appears where there are gains/losses in genetic material-> any difference = extra/lack of genetic material

Question 7

Edwards Syndrome

Answer 7

Trisomy 18Low set earsShort NeckCongenital Heart defects - Vent Septal DefectRenal malformations - Horseshoe kidneyLimited HIp AbductionOcker bottom feetMental retardationMicrognathia - small jawProminent OcciputClenched Fist with overlapping fingers

Question 8

Single Nucleotide Polymorphism (SNP) Array

Answer 8

Detect copy number variations like CGH, but they can also detect zygosityCopy Number - 0 is normal - below = lost - above = gainedZygosity- mixtures are in the middle- only 1 parental contribution will be gap in midline -> will be at poles (indicates lack of mutual contribution)

Question 9

Angelman SyndromePrader Willi Syndrome

Answer 9

Angelman = MaternalPrader Wili = Paternal Need SNP Array to tell who contributed what

Question 10

Polymerase Chain Reaction (PCR)

Answer 10

Method for selectively amplifying target nucleic acids up to a million foldReagents:– Template DNA– Two oligonucleotide primers (15-22 bp long)– Thermostable polymerase– Mixture of four deoxynucleotides dATP, dCTP, dGTP, dTTP– Buffers containing magnesiumMechanism:1. Denaturation (95°)2. Hybridization/annealing (55°-72°)3. Synthesis/extension (72°)

Question 11

Amplicon Length Analysis

Answer 11

Gel electrophoresis: - DNA is separated in a gel matrix using an electrical fieldDiagnose triplet repeat disorders - Fragile X syndrome - Huntington disease

Question 12

Restriction fragment length analysis or Restriction Endonucleases

Answer 12

Used to identify point mutations in PCR productsRestriction enzymes are bacterially-derived enzymes that cut DNA at specific recognition sequencesDesign Assays to take advantage of this & target mutant sequences that are differentially recognized by the enzymesRestriction fragment length analysis in the diagnosis of hereditary hemochromatosis

Question 13

Direct DNA sequencing

Answer 13

Sanger sequencing - Gold standard1) All 4 building blocs of deoxynucleotides in same tube & fluorescently labeled2) Capillary electrophoreisis - creates peaks that correlate to where nucleotide is3) Transpose from graph to DNA sequencePyrosequencing - PCR with Luciferase (firefly tail light enzyme)-Probe is specific sequence searching forUseful when testing specific sequence variants - missense mutation of Sickle Cell

Question 14

Huntington disease

Answer 14

Autosomal dominantProgressive movement disorders and dementia Caused by CAG repeats in coding region of the the first exon of HTT (4p16.3) -> toxic gain of function in proteinPolyglutamine expansionCharacterized by: - Dementia - Jerky movements/motor neuron- Toxic protein kills brain

Question 15

Fragile X syndrome

Answer 15

Most common cause of mental retardationCGG repeats in promoter region of FMR1 genePCR primers on either side of promoter region & amplifyNormal = baselinePremutation = elevatedFull Mutation = nothing - because too long to amplify**limitation of test - but also diagnositc to be CONCLUSIVELarge, protruding ears (one or both)Long face (vertical maxillary excess)High-arched palate (related to the above)Hyperextensible finger jointsHyperextensible (‘Double-jointed’) thumbsFlat feetSoft skinPostpubescent macroorchidism (Large testes in men after puberty)Hypotonia (low muscle tone)

Question 16

Real-Time PCR

Answer 16

• DNA template amplification and detection steps occur simultaneously, allowing quantification of the template• A fluorescent reporter emits light in direct proportion to the amount of the PCR product.• Requires a special thermal cycler that can measure fluorescence.Several techniques:TaqMan probe- labeled with quencher & reporter molecules when cleaved - reporter emits lightUse a LOT to quantify - Viral - Hep B - Hep C - Minimal residual disease amount

Question 17

Sickle Cell Anemia

Answer 17

Misssense Mutation6th Codon in Beta HbDNA = T-A -> A-TRNA = A-U-> U-AAA = Glutamic Acid -> ValineSymptomatic: Stoke

Question 18

Next-Generation sequencing

Answer 18

Involves high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at onceMassive parallel sequencing- Can produce upwards of 1 billion sequences per instrument on 1 run- Multiple genes in one assay- Same genes multiple times Cost/nucleotide sequence is FAR CHEAPERTremendously powerful

Question 19

Marfan Syndrom

Answer 19

Autosomal dominantDefect in extracellular glycoprotein fibrillin-1 due to mutations in FBN1 (15q21.1) or FBN2 (5q23.31)Characterized by:Skeletal abnormalities Very tall & Thin Long arms/legs & Feet/Fingers *Think Michael Phelps bodyChest pectus excavatum - indentation pectus carinatum - protrusionScoliosisEctopia lentis - eye problemsCardiovascular lesions mitral valve prolapse, dilated ascending aorta due to cystic medionecrosis)Cystic medionecrosis in Aorta:- Typically regular dense CT- Elastic is disrupted & CT becomes irregular**Next Generation sequencing - because mutations are interspersed through the span the FBN1 and FBN2 genes - need broad net

Question 20

Non-invasive prenatal testing

Answer 20

Fetal cell free DNA in Maternal blood - Sequence with Next generation sequencing from that

Question 21

Turner syndrome

Answer 21

Monosomy X or 45, XShort statusLower posterior hairlineWebbing of neckLymphangioma in posterior neckPigmented neviCoarctation of aortaShield chest - broad chest with wide nipplesStreak ovaries, infertility, amenorrheaPeripheral edema at birthCubitus valgus - deformity where forearm is angled more angled away from the body when fully extended

Question 22

Congenital

Answer 22

Simply implies “present at birth.” Of note, some congenital diseases are not genetic (e.g., congenital syphilis)

not all genetic disorders manifest in infancy and childhood, andconversely, many pediatric diseases are not of genetic origin

Question 23

Mutations

Answer 23

permanent changes in the DNA

Point mutations - 1 nucleotide substituted
(SIckle Cell - B chain of Hb)

Missense mutations = alters meaning of genetic code

Nonsense - mutated into stop codon

Frame shift = insertion/deletion of 1+ base pairs (not multiple of 3) and alters the reading of the strand’s codons

Trinucleotide repeat mutation - amplification of a sequence of 3 nucleotides; 2 of which are G & C
i.e. Huntinton’s diseas or Fragile X

Question 24

Polymorphisms

Answer 24

DNA variations

SNP’s (single nucleotide polymorphisms)

• variation at single isolated nucleotide positions and are almost always biallelic
• less than 1% of genome = variances between humans

CNV (Copy Number Variations)

• form of genetic variation consisting of different numbers of large contiguous stretches of DNA from 1000 base pairs to millions of base pairs
• a LARGE portion of human phenotypic diversity

Question 25

Epigenetics

Answer 25

Modulation of gene or protein expression in the absence of alterations in DNA sequence
i.e., mutation) or structure of the encoding gene

above genes -> not in the genes

Methylation of cytosine @ promoters
- Heavily methylated promoter become inaccessible to RNA polymerase, leading totranscriptional silencing

Histone Proteins with DNA wrapped around = Nucleosome