eRobbins Ch 6 - Genetic Diseases (No Peds) Flashcards
Chromosome classification terminology in Clinical Genetics
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
Down Syndrome
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
Nucleic Acid Hybridization
• 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
Patau Syndrome
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
Robertsonian Translocation
Fusion long arms of Chromosome 13 & Chromosome 14 between acrocentric (long arms have all information)Not detectable with a centromeric FISH probe
Comparative Genomic Hybridization (CGH) Array
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
Edwards Syndrome
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
Single Nucleotide Polymorphism (SNP) Array
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)
Angelman SyndromePrader Willi Syndrome
Angelman = MaternalPrader Wili = Paternal Need SNP Array to tell who contributed what
Polymerase Chain Reaction (PCR)
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°)
Amplicon Length Analysis
Gel electrophoresis: - DNA is separated in a gel matrix using an electrical fieldDiagnose triplet repeat disorders - Fragile X syndrome - Huntington disease
Restriction fragment length analysis or Restriction Endonucleases
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
Direct DNA sequencing
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
Huntington disease
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
Fragile X syndrome
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)
Real-Time PCR
• 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
Sickle Cell Anemia
Misssense Mutation6th Codon in Beta HbDNA = T-A -> A-TRNA = A-U-> U-AAA = Glutamic Acid -> ValineSymptomatic: Stoke
Next-Generation sequencing
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
Marfan Syndrom
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
Non-invasive prenatal testing
Fetal cell free DNA in Maternal blood - Sequence with Next generation sequencing from that
Turner syndrome
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
Congenital
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
Mutations
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
Polymorphisms
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
Epigenetics
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
