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226 Cards in this Set
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What are the characteristics of intellectual disability?
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•Intelligence-quotient (IQ) <70
•Limitations in adaptive functioning •Present before the age of 18 years |
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What is the difference between the culturofamilial and organic ID?
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culturofamilial - multifactoral , IQ ~ 100
organic - monogenic , IQ ~ 35 * normale destribution tops |
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How many XLID genes there are ?
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heterogeneous desease : 150 genes
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How are you going to identificate the XLID genes? ( 3 steps)
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1. Positional cloninig (linkage analysis/chromosome analysis)
2.Analysis of the candidate genes 3.Defective gene |
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What is positional cloning?
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* a strategy for cloning genes based solely on map position.
*involves the isolation of the marker whos location is known and which is tightly linked to the gene you are trying to find. |
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What is the first step in the positional cloning?
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1. Linkage analysis
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What is linkage analysis?
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the aim of LA is to find a candidate gene relatief to a marker, which location on a chromosome is already known.
1. Look at the family tree, determine which chromosome ( parental or maternal ) has a mutation 2. There is always recombination, and the markers that link the desease genes are recombined togather with the desease gene. 3. When you have determined these markers that are linked to the desease gene, you can say something about the candidate region of the genome, the gene responsible for the desease lies somewhere here. * All genes are in the same order on the parental and maternal chromosomes, only sequence differences. * genetic markers shows polymorphysm and can be determined by PRC. |
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What do you do to define the desease gene in the candidate gene regio?
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•Expression pattern (brain)
•Similarities with known ID genes •ID pathway (chromatin remodelling) |
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Which three steps/subtypes of the position cloning there are?
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1. Linkage analysis
2. Breakpoint determination of translocations 3.Deletion duplication mapping |
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How is breakpoint determination of translocation done?
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* FISH - fluorescent in situ hybridization.
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What is array comparative genomic hybridization?
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DNA from a test sample and normal reference sample are labelled differentially, using different fluorophores, and hybridized to several thousand probes. The probes are derived from most of the known genes and non-coding regions of the genome, printed on a glass slide.
The fluorescence intensity of the test and of the reference DNA is then measured, to calculate the ratio between them and subsequently the copy number changes for a particular location in the genome. |
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What are the silent/missence/nonsence mutation/sunstitutions?
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* silent - same aa
* altered aa, may affect protein function or stability * stop codon - loss of function or expression due to degradation of mRNA |
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How do you determine is it a pathogenic mutation or not?
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* segregation ( look at the family tree)
*controls ( single nucleotide polymorphism) *look at the conservation * analysis mutant mRNA transcript *analysis mutant proteins, activity colacolization *analysis metabolites |
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What are the functions of the proteins involved in the iD?
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1) EHMT1 is a methylase - responsibe for histone methylation
2) ATRX - ATPase / helicase , binding to other proteins of the histone inhibiting machinery. 3) MECP2 - Methyl CpG binding HDAC recruitment , Bridge to histone methylation , co - repressor recruitement 4)ZNFs - corepressor 5) other proteins which are involved in histone, creb phosphorylation |
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What is defenition of genomics?
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The study of genes and their function to understand the genome structure and Examination of the molecular mechanisms and
Interplay of genetic and environmental factors in disease. |
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Which types of genetic variations there are?
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MISTAKES DURING MEIOSIS
* SNPs *Variation in repetitive DNA: CACACA versus CACA *Insertion-deletions *Copy number variation *Structural variation: A large chunk of DNA is inverted or translocated to another chromosome *Aneusomy: Chromosomes are not equally divided to daughter cells, resulting in loss or gain of chromosomes |
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Describe the microarray based on 25 base SNP brobes?
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*Microarray consist of DNA fragments ( oligonucleotides ) specific for one of the SNP variants.
* mathching of patients DNAs - fluorescence * determine the overexpression of some SNPs in some patients, this may be a CLUE to the location of the desease gene, but SPSs that are overexpressed are not a desease itself. |
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What is age related macular degeneration?
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* is the leading cause of blindless in the developing world.
*Factor H polymorphism in age-related macular degeneration *nonsynonymous polymorfism ( alters proteins sequnce) - was associated with AMD |
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Which two parameters you can detect with SNP microarrays?
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* genotype of SNP
* SNP genomic number information |
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Which advantage has SNP microarray over karyotypering?
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* the first one has 1000 timer higher resolution.
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How can you study the genes for mutations with sanger sequencing?
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1. Primer design
2. PCR 3. Sanger sequencing 4. Mutation detection |
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What shortcomings have SNP m.array and sanger sequencing?
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* detects only SNPs not the mutations
* misses balanced chromosome reareggement sanger: - misses copy number variations - limited to the analysis of 1-2 genes at once - sequencing 1 genome takes years & hundreds of millions of euros |
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What is the ideal approach to start the desease gene identification?
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* sequence whole genome (with all variations)
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Describe the next generation sequencing.
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*Sample preparation
DNA digested in short fragments *Library preparation Adaptor ligation Used for purification, amplication and sequencing steps *One fragment = one bead A unique DNA fragment is immobilized to DNA bead *Emulsion PCR Each DNA fragment is amplified in microreactor >1,000,000 copies/bead *Sequencing reaction PicoTiterPlate Nucleotide by Nucleotide Light-emission by build-in *Image processing Signal intensity/read in time One bead = one flowgram = one read |
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What is in liquid exome enrichment with Agilent’s SureSelect?
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Is the hybridization of exonic regions with probes and then separation of hybridized and nonhybridezd probes with paramagnetic beads. Wash beads >> get RNA >> sequence
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How to prioritize pathogenic from benign variants?
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•Dependent on
- inheritance: recessive or dominant - occurrence: sporadic or familial - amount of genetic heterogeneity - (timing of mutation: germline or somatic) - (location of mutation: genic or nongenic) •Arguably most difficult: Dominant diseases that occur sporadically and show genetic heterogeneity! |
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Which hormons influence the differentiation from genital ridge to bipotential gonad?
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*SF1
*WT-1 *FHX-9 |
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Which hormons have influence on dev. of bipotential gonad to ovary and testis?
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*BP gonad > ovary: WNT4a,DAX1
...............> testis : SRY,SOX9 |
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There two types of cells in the males testis? Which function have transcriptional factor SF1 in it?
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Sertoli cells: SF1 influences AMH (anti mullerian hormone) , this leads to mullerian duct regression, thus not formation of female internal genitalia.
Leydig cells: SF1 induces testosterone production , testosterone induces Wolfman duct >>> Forming of male internal genitalia. Testosterone induces ALSO DHT (byproduduct of testosterone in the body) which induces formation of penis and prostate. |
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What importance have WNT4a and DAX1 in woman?
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>>> ovary >>> >>> >>>> estrogen >>> development of female internale genitalia ( Mullerian duct)
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What is SRY?
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Is a Sex Determininig Region on the Y chromosome.
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What does FSH?
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Hypothalamus maakt FSH aan. FSH >>> testis >>> spermatogenesis
FSH >>> ovarium granulosa cells >>> follicle maturation |
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What does LH?
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LH >>> testis >>> testosteron
LG >>> ovarium theca cells >>> androgens ovulation |
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What is Familial Male-limited Precocious Puberty (testotoxicosis)?
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•Autosomaldominant
•Puberty< 4 jaar •Low LH •High testosteron *mutation in LHR, constitutive active , high cAMP * there are more missence mutations that all affect almoust the same region in GPCR (LHR) |
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Describe Leydig cell hypoplasia
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*A non-functional LH-receptor leads to a defect
in the proliferation and/or maturation of Leydigcells in the testis. •Autosomalrecessive •46XY with female genitals •Low testosteron •Absent/hypoplastisticLeydigcellen *Hypoplasia is underdevelopment or incomplete development of a tissue or organ |
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What are the clinical effects on males of testotoxicosis and LCH ?
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tt: early puberty
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What are the effects on females of testoxicosis and leydeg cells hypoplasia?
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tt: none
TCH: amenorrhoa ( absence of menstrual period) |
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What causes uscher syndrome in general?
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*hering loss
*impaired vision *vestibular impairment *ressive inheritance |
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What cause usher I,II,III types of deseases?
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Usher I:
- congenital deafness - vestibular impairment - retinitis pigmentosa Usher II: - congenital hearing impairment - normal vestibular function - retinitis pigmentosa Usher III: - progressive hearing loss - variable vestibular impairment - retinitis pigmentosa clinical heterogenity |
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Which genes are involved by uscher?
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I : MYO7a
II: USH2A III: USH3 |
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Which protein families are involved by uscher?
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cargo proteins ( myosin) , clarin protein family, scaffold, cell adhesion, transmembrane proteins.
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Deschribe the uscher proteins in the inner ear.
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*haricells transport the soundwave movement to the electric signal
*outer cells, andjust these signals: stereocilia ( compound of actine), they are linked by fibrous filaments. * there are fibrous links, that attach stereocilias to each other *stereocilia bestaat zelf uit actine core , and an shell of scaffold proteins. * there are K+ channels in cilias. * when cilias are changed in position it causes depolymerization of these channels. |
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Describe the function of uscherin (USH2a)?
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has FN3 domain which keeps the spacing between the cilias in the proper way.
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Which function have cadherins in stereocilia?
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The tip of one cilia is connected with cadherin with the other cilia ( not the tip).
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Where the uscher proteins in eye are located?
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*calycal process
* connective cilium *ribbons The proteins that are formed in the outer segment are needed for phototransduction. |
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Where are the cilias also found?
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in kidhey , inner ear, brain ventricel and oviduct.
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What does non motile cilia in the human photoreceptor?
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The outer segment of the rod photoreceptor cell in the human eye is connected to its cell body with a specialized non-motile cilium.
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What is the difference between the motile and nonmotile cilia?
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nonmotule: 3plet of 9 microtubules
motile : motile cilium has two central microtubule singlets in addition to the nine outer doublets (called a 9+2 axoneme) The axonemal cytoskeleton acts as a scaffolding for various protein complexes and provides binding sites for molecular motor proteins such as kinesin II, that help carry proteins up and down the microtubules |
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Where are motile cilias involved?
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adhesion between pericilium and photoreceptors
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Where are all cilia proteins involved?
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adhesion ( perecilia, )
transport ( |
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How is the photoreceptor/primary cilium formed?
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Forming of centrocilia starts with centrosome ( 2 centrioles). This centroosme is then translated to the inner part of memrbane and anchored there so the primary/motile cilia is formed.
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What is finally the uscher syndrome?
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*ciliopathy
•Structural role (fibrous links in hair cells & photoreceptors) •(Vesicle) transport •(Ribbon) synapse formation/maintenance •Signaling? |
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Where is mocula zone in eye responsible for?
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macula is cone rich, responsible for color vision, seeing at the daylight , see the details
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Describe how retine is built up?
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light > ganglion cells > bipolar cells > horizontal cells > rods cones > pigment epithelium
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What happens if the "lower:" part of the photoreceptor is not in the pigment epithelium?
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* no signal transduction
* degradation of the photoreceptor inner segments. |
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Describe the Stargardt disease?
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Most frequent juvenile macular dystrophy (1/10.000 individuals)
•Progressive atrophy of macular RPE •Autosomal recessive inheritance *lipid acumulation *gene ABCA4 is involved in STGD1 |
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For what protein does gene ABCA4 codes for?
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*The STGD1 gene ABCA4 encodes the retina-specific ATP binding cassette transporter type A4 (ABCA4)
*this ABCT has 2 Nucleotide binding domains and two TM domains. |
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Where is The STGD1 ABCA4 protein located?
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located at the rim of rod outer segments
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What is the Normal ABCA4 function?
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Flippase for N - retinylidene – Phosphatidylethanolamine (N-RPE)
from disc lumen to the cytoplasm of the rod photoreceptor outer segment. |
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How N-RPE can be processed?
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N-RPE can be converted into A2E, that is the lipid that finally accumulates in pigment epithelium.
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Where do N-RPE accumulates?
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in the lumen of the rod photoreceptor's disc.
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Describe ABCA4-associated pathology?
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RPE is converted in the disc in A2-PE, which is then somehow(phagocytosis?) transported to the cytosol of the photoreceptor cells.Lysosomal degradation of A2-PE yields > A2E which is when exposed with light induces formation of free radicals > apoptosis > cell degeneration. Due toe the degeneration of the RPE cell ,
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Describe Harry-weinberg principe?
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•Relative proportions of different genotypes remain constant from one generation to another.
•Allele A (e.g. wild-type): frequency p •Allele a (e.g. mutation): frequency q Hardy-Weinberg equation 1: p + q = 1 Hardy-Weinberg equation 2: p2 + 2pq + q2 = 1 |
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What is hypomorphic mutation?
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mild
recessive mutation, mutation that causes a partial loss in gen function. |
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Why you would not get nutrients in the retina with ABCA4 mutation?
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because accumulated A2E blocks nutrient transport
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How do splice donor(5')/ acceptor (3') sites looks like in vertebrates?
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3' ag - splice - g
5' ag - splice gu 3' ag's zijn 100% conserved 5' gu - 100% conserved |
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What causes ABCA4 c.2588G>C mutation?
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Mutation of G in C, just the first exon nucleotide on the splice acceptor site.
This shifts the splice site 3 nucleotides further. So in stead of ProGlyAsp , we get ProAsp, thus we get a deletion of Glycine. OR you will get ProAlaAsp, when the splice site stays the same but the G>C mutation occurs (so Gly>Ala) SO this mutation is a splice site mutation and a missence mutation. |
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What are the diffireces between cone rood dystrophy and retinitis pigmentosa?
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CRD:
• no night blindness • vision loss at 12-15 yrs • normal vessels • macular lesions • cones are more absent than rods RP: • night blindness • vision loss at 6 yrs • thin vessels • midperipheral lesions • rods are more absent that cones |
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Describe consensus primate spilice site?
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AG(exon) | g1 t2 a/g a g(5) t
g1 - 100% conserved t2 - 99.5% conserved, in rare cases C c.4539+1g>t severe c.5714+5g>a moderate |
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Which symptoms are there by Cystis fibrosis?
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*Neonatal darmobstruction
*Pancreas insufficiency *Lung disease No „sperm tube *DF508 = deletion phenylalanine at position 508 |
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Which distribution of mutations/alleles gives normal/desease phenotype by cystis fibrosis?
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*DF508/T5 or T9 >>> normal
*DF508/DF508 >>> desease *5T / 9T normal |
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How can you inhibit Stargardt disease?
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* reduce light exposure
* eat more vitamine A |
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What is mitochonrial heteroplasmy?
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Heteroplasmy is the presence of a mixture of more than one type of an organellar genome (mitochondrial DNA (mtDNA) or plastid DNA) within a cell or individual
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What is X-inactivation?
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Random inactivation of X chromosome during development.
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What are the examples of imprinting deseases?
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Angelman Syndrome 15q11-q13 deletion from mother.
Prader-Willi Syndrome 15q11-q13 deletion from father |
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What is anticipation?
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anticipation is a phenomenon whereby the symptoms of a genetic disorder become apparent at an earlier age as it is passed on to the next generation.
During meiosis, unstable repeats can undergo triplet expansion in this case, the germ cells produced have a greater number of repeats than are found in the somatic tissues. |
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What are the examples of anticipation?
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Huntighton desease
Fragile X syndrome |
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What is mosaicism?
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The presence with same type of cells with different genotype ( different colored skin cells)
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Which dystroglycanopathyes are there?
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* Muscular dystrophy
* Eye abnormalities * Severe brain malformations Is also a example of qualitative effects of mutation. |
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Which effects cause clinical variability?
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*Quantitative effects ( type of mutation)
*Qualitative effects ( type of mutatio) *Genetic context: modifier genes *Surroundings *Coincidence |
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What is Fukuyama congenital muscular dystrophy ?
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*weakness and breakdown of muscular tissue
*wo copies of the defective gene *autosomal recessive disorder *hypomorph mutation ( partially loss of gene function) one gene - no defect ., 2 genes - defect >>> quantitaive |
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What is Walker warburg syndrome?
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*It is associated with brain (lissencephaly, hydrocephalus, cerebellar malformations) and eye abnormalities.
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What are the qualititive effects of mutation?
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Gain-of-function mutation
Loss-of-function mutation Change-of-function mutation |
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Give the example of desease with gain of function mutation?
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* mutation in FGFR2/3
*Constitutive ligand binding Constitutive dimerisation Constitutive kinase activity *Craniosynostosis syndromes *is a condition in which one or more of the fibrous sutures in an infant skull prematurely fuses by ossification |
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Give the example of desease with loss of function mutation?
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*LADD syndrome
*malformations in the network of structures of the eye that secrete tears and drain them from the eyes (lacrimal apparatus) and abnormalities of the forearms and fingers. |
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Give the example of a desease of change of function mutation?
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ECTODERMAL DYSPLASIA
OROFACIAL CLEFTING LIMB MALFORMATIONS p63 Syndrome Family |
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by which mutations are p63 isoforms affected?
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*EEC syndrome mutations cluster in the DNA binding domain
EEC mutations affect all p63 isoforms impaired DNA binding alter transactivation activity mutations exert dominant effects |
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What includes HayWell syndrome?
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• Ankyloblepharon
• Ectodermal dysplasia • Cleft palate or cleft lip • No/mild limb defects |
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Where are mutations located in p63 protein with HayWell syndrome.
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*SAM domain
*AEC mutations disrupt protein-protein interactions |
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What is the mutation in EEC?
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R280H
*EEC - ectodermal dysplasia |
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What is SHFM?
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Split-hand-split-foot malformations
*•milder phenotype •reduced penetrance than by EEC |
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Whuch mutation causes SHFM?
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R280H
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SHFM has milder phenotype than EEC? Why is that possible in context of modifier genes? What is modifier genes, give the defenition.
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Modifier Genes. Instead of masking the effects of another gene, a gene can modify the expression of a second gene.
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What causes spinal muscular atrophy?
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Spinal Muscular Atrophy (SMA): recessive mutation of SMN1
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How can SMA become less severe? In the context of modifier genes.
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* Level of functional SMN protein determines phenotype
* rescue by increased SMN2 copy number1 * But also other genetic factors modulate the SMA phenotype *Plastin 3 is also a genetic modifier *Unaffected individuals have increased levels of Plastin 3 RNA and protein |
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Give an example of a desease where the surroundingsfactors are involved?
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hypercholesterolemia
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Give the examples of deseases where coincidence play a major rol?
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*BRCA1 en Borstkanker
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What do you see with the karyotiping of ID patient?
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del(16)(q13q22)
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Which chromosome abberations are belong by which deseases?
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Downsyndrome - trisomy 21
Cri-du-Chat Syndrome - Deletion 5p DiGeorge syndrome - Deletion 22q11 |
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Which desease you would not see with karyotiping?
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Wolf Hirschhorn syndroom (deletion 4p)
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What if u dont know where to look with karyotyping/ FISH?
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microarray
A microarray is a glass slide with a large quantity of (FISH) probes |
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Which colors of interpretation are there in microarray?
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Geel = Equal amounts green and red DNA
Meaning: Chomosomal locus is present in normal amount/copies Green = More green than red DNA Meaning: Chromosomal locus is present in a larger amount/copies in person 1 met disease A Red = Less green than red DNA Meaning : Chromosomal locus is present in less copies/ammounts in person 1 with disease A |
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What is uniparental disomy?
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Uniparental disomy (UPD) occurs when a person receives two copies of a chromosome, or part of a chromosome, from one parent and no copies from the other parent.[1].
UPD could involve isodisomy (meiosis II error) or heterodisomy (meiosis I error) For the determination and confirmation of heterodisomies TRIO data is needed, |
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Describe the pathology of the duchene dystrophy.
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•Start between 2-5 jaar
•Delayed motor development •Progressive •Death around 20th years levensjaar Gower sign Wheelchair bound from 12 yr Heart disease Scoliosis pneumonia |
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What does dystrophyn protein?
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connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane.
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Describe the genetics of DMD?
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•X-linked, recessive
•1:3500 boys •1/3 new mutations •Majority deletions: nonsense mediated decay |
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What is nonsence mediated decay?
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Nonsence mediated decay – results in the rapid degradation of mRNA with stop codons that occur before the last splice junction in the mRNA since in most cases, such mRNA arise from errors in RNA splicing.
Ribosome normally remove all exon junction complexes from the mRNA but if there is a stop codon, the ribosom will stop and will not remove EJC. EJC will interact with other factors which will lead to the degradation of mRNA. |
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How is diagnosis of duchenne occurs?
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•Majority deletions: problem with carrier detection
•Southern blot analysis •Multiplex PCR Several exons per reaction Several different reacties •MLPA (Multiplex ligation-dependent probe amplification (MLPA)[1] is a variation of the multiplex polymerase chain reaction that permits multiple targets to be amplified with only a single primer pair[) |
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What is genetics/pathology of becker muscular dystrophy?
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•Allelic with Duchenne
•Clinically less severe •Deletions, but in-frame: still protein production |
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What is the therapy for duchenne?
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•Exon-internal primers
•Open secondary mRNA structure •Steric hindrance of splicing |
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In which exon there is a deletion?
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exon 50 so exon49+51 are spliced out togather >>> nonfunctional out of frame mRNA
if you block exon 51 splicing, exon 49+52 will be spliced out, so the partly functional protein will be formed. |
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What are the other mutation in the dystrophyn complex?
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•-dystroglycan: (glycosylation) MEB, Walker Warburg, Fukuyama
•Sarcoglycans: Limb-girdle muscular dystrophies |
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What is Walker-Walkburg, describe pathology.
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Muscle
- Congenital muscular dystrophy - High creatin kinase Eye -Retina ablation -hypoplasia nervus opticus -Microphthalmia Brain - Agyria or pachygyria - Major white matter disorganisation - Hydrocephalus -Occipital encephalocele -Cerebellar hypoplasia Enlarged ventricles |
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Which types of CMD there, discribe each? And which proteins are involved by each type?
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•CMD I: hypotonia, muscle weakness and contractures
FKRP (Fukutin related protein) •CMD II: muscle- and brain abnormalities Fukutin •CMD III: Muscle, eye and brain abnormalities (mild) (MEB) POMGnT1 •CMD IV: Muscle, eye and brain abnormalities (severe) (WW) POMT1 |
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Which O linked mannosylation is distrurbed in Walker-Walkburg desease and in muscle-eye-brain desease?
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beta-1,2-N-acetylglucosaminylation
in MEBD and O-mannosylation in WWD. In Brain, peripheral nerve, skeletal muscle a-dystroglycan |
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Why is O-linked mannosylation importand for the desease??
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•Alpha-dystroglycan only in
Brain, muscle, peripheral nerves •O-linked mannosylation needed for proper function •all types CMD effect on O-linked mannosylation •There may be a correlation between length of the (residual) sugar chain and severity of disease |
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Describe the gene and protein dystroglycan.
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•One gene, two proteins: a- en b-dystroglycan
•a-dystroglycan is an extracellular proteint b-dystroglycan is a transmembrane protein •Mutations in dystroglycan are embryonal lethal in mice, in humans not found |
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what is Limb-girdle muscular dystrophy?
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•Autosomal recessive
•Starts in hip region, then shoulders, (or vice versa) juvenile onset •Progressive, variable, wheelchair 20-30 years after first symptoms •Death in middle age, variable •genetically heterogeneous |
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What is there to conclude about dystrophin complex?
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•One protein complex, many genes
•Different syndroms, same protein complex •Mutations in the protein-coding genes, but also in the genes coding for modification-enzymes •Often secundary loss of -dystroglycan |
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How can you see chronical myeloide leukemia?
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poorly differentiated cells, seen only in the bone maarrow not in blood.
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Which deletions are frequent in acute lymphoblastic leukemia?
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CDKN2A deletions are frequent in ALL
Also (homozygous) deletions of the RB1 gene |
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Descreibe functions CDK,CYCL,RB1?
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CyclD\CDK6 inhibits Rb1
CyclE\CDK2 inhibits Rb1 |
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Which genes are involved in B cell differentiation?
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Ikaros,E2A,CREG
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Which heriditary forms of colorectal cancer there are? And which mutations are involved there?
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1. Familial adenomatous polyposis (FAP) APC gene mutations
2. Hereditary nonpolyposis colo-rectal cancer (HNPCC - Lynch syndrome) mutations in DNA repair genes |
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Which genes are associated with heretary nonpolyposis colo-rectal cancer (NHPCC)????
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*MSH6,2
*PMS1,2 *MLH1 genes of the MISMATCH repair proteins( A ~ C is a mismatch ) |
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What is microsatellite instability?
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The repeats (CA repeat) may become longer or shorter and would not be repaired.
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HOW DOES THE CELL DEAL WITH
DNA DAMAGE? |
• induction DNA repair
• induction cell cycle arrest • induction apoptosis |
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What are the functions of p53?
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*cell divisions stops and p53 trigger the enzymes which repair the genes.
*p53 allows cell with repaired DNA to divide *p53 allows destruction of cells damaged beyond repair |
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What is the role of ROLE OF TRANSCRIPTION MACHINERY?
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• DNA damage detection
• DNA repair dosimeter • Apoptosis induction |
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What causes blockade of transcription?
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*p53 modification/accomulation - apoptosis
*trapping of NES containing proteins in the nucleus, preferantial inhibition of large survival genes. |
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|
Where to lead altered DNA metabolism?
|
chromosome aberrations >
(point)mutations > CANCER cell cycle defect > apoptosis > AGING |
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|
What is xeroderma pigmentosum?
|
XERODERMA PIGMENTOSUM
- sunlight sensitive - pigmentation anomalies - dry skin - neurological anomalies - skin cancer (>2000x) Global genome repair deficient Nucleotide excision repair, DNA deformation due to base modifications. defect |
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What is COCKAYNE SYNDROME?
|
COCKAYNE SYNDROME
- sunlight sensitive - growth retardation - neurological anomalies - retina degeneration - rapid aging - no skin cancer Transcription coupled repair deficient |
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Wat is WERNER SYNDROME?
|
transcription coupled defect, premature aging
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Wat is HUTCHINSON-GILFORD SYNDROME?
|
mutation is lamin A , disruption of nuclear lamina, activation of proteins that accelerate aging.
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What is the first line of the innate immunity defence?
|
defensins:
Antimicrobial peptides *present on epithelial surfaces as a first line of defense *ancient defense system |
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What does IgD antibody?
|
activation of basophils and mast cells.
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What is somatic recombination?
|
light / heavy gene segments of Ig and a/b chains of TCR have V,D,J,C segments. These are recombined on the DNA level voor each light/heavy chain of a/b chain. So the diversity of different chains is created.
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How is only of the chains k or labda are produced?
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Signals of the antigen receptor shut off the production of the recombinase enzyme amd further recombination of unrecombined light chain loci. as a result each B cell produces either one K or labda light chain from one of the inherited parental alleles.
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How is the diversity in receptors further increased?
|
* somatic hypermutatation ( affinity maturation)
* addition and substraction of bases during somatic recombination |
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How is the B cells activated?
|
* by T helper cell MHC peptide presentation + antigen presentation on the IgG receptors on the B cell surface
* somethimer there is a strong B cells activation only through the abtigen- IgGR interactions. |
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How is differentiation of lymphocytes goes?
|
double negative T lymp. >>> double positive T lymph ( 95% undergoes apoptosis, death by neglect) >>> the rest will be either 4+ or 8+ CD cells.
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What is postitive / megative selection?
|
* +selection >>> weak self antigen recognition >>> maturation
* negative selection >>> strong antigen recognition >>> cell death |
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Where MHCI either MHCII are present in the body?
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* MHC I all cells
* MHC II APCs |
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What is the structure of MHCI/II?
|
* MHC I a1,2,3 b2 microglobbulin , closed binding cleft
*MHC II a1,2 , b1,2 , open binding cleft. |
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How many MHC lasses molecules has every human?
|
3 MHC class I and 3 MHC class II molecules are
encoded by the MHC of each parent! every human has maximally six different MHC class I and class II molecules |
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What is immunproteasome?
|
immunoproteasome: exchange of certain
subunits by MHC-coded ones – induced through cytokines – change of peptide repertoire |
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What is MHCII processing pathway?
|
extracellular material is obsorbed and moved to the endosomal compartiment with with acid proteases
CLIP occupies the cleft of newly senthysezed MHCII molecules. When exocytic vesicle with CLIP fuses with endosomes with peptides the CLIP dissociates and a peptide binds to MHCII. |
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What is cross presentation?
|
• The presentation of antigens taken up by endocytosis on MHC class I molecules
•processing pathway required for CD8+ T cell activation by dendritic cells •in this way, also non virus-infected dendritic cells can presented virusderived peptides |
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How do different APCs take antigen up?
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• Dendritic cells: Antigen uptake through macropinocytosis and phagocytosis peptides,
viral antigens, allergens • Macrophages: Antigen uptake through phagocytosis particulate antigens, intracellular and extracellular pathogens •B-cells: Antigen uptake via antigen-specific receptor (Ig) soluble antigens, toxins, viruses |
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Where to leads the synapse formation between APC and T cell?
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Formation of the synapse leads to a reorientation of the microtubuli inside the T cell – vesicles containing cytokines are released into the T-cell/B-cell contact
area. |
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Which molecules are the parts of the synapse beween T cell and APC?
|
APC: ICAM I , CD80, MHC
T cell: LFA 1 , CD28, CD3(TCR),CD8 |
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How do receptor clustering leads to phosphorylation?
|
LCK anchored to the CD4 co-receptor. LCK phorphorylates ZAP-70 and thereby made enzymatically active. ZAP-70 bind on the ITAMs of the CD3 proteins. ZAP-70 then phosphorylates other proteins of the signalling cascade. Such as LAT, LAT is a Linker for
activation of T cells. LAT is a scaffold protein. |
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What rol has LAT in signal transduction?
|
LAT > PLCgamma > IP3 > calcium> calcineurin > target of important immunosuppressants.
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What happens in the case of wiskott-aldrich protein defect?
|
•Blood clotting disorder
• Impaired T cell function • Failure of antibody responses to encapsulated bacteria • Located on X-chromosom defect in WASP a scaffold protein which is needed for actine cytoskeleton rearrgengement. |
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Give the summary of early signalling?
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Clustering of cell surface receptor
Tyrosine kinase activation Phosphorylation of a scaffolding protein Binding of adaptor proteins Activation of down-stream events |
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What are the important molecules on the T cells?
|
* CD28
*CTLA4 - cytotoxic T lymphocyte antigen *ICOS - inducible costimulatory molecule |
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What are the important molecules on the antigen presenting cells?
|
–B7.1 (CD80)/B7.2 (CD86): CD28/CTLA4
ligands –B7H: ICOS ligand 67 |
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What are the functional characteristics of CD28?
|
•CD28
–expressed on naive and activated T cells (6 x 104 molecules/cell) –naive T cell responses are more dependent on this costimulus –lowers the threshold for T cell activation –promotes activation by low-affinity peptides *CD28 – key player in the ‚two-signal hypothesis‘ |
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What is the rol of the CTLA-4 molecule?
|
It's a negative regulator.
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What is the two signal hypothesis connected with CD28 receptor?
|
complete T cell activation
requires Ag-mediated triggering of the TCR (signal 1) in concert with costimulatory signals that can be provided by the CD28 molecule |
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How do you transport the cytokines to the B,T cells?
|
via the kinetochore.
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What does LCK kinase?
|
it gets in the proximity of TCR and phoshorylate TCR. TCR tyrosine which are phosphorylated become the docking sites to the zap-70
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What is function of LAT?
|
LAT is a scaffold protein that has many phosphorylation domains. When its phosphorylated it becomes docking site for the proteins with SH domain.
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What is 2photon microscopy?
|
*imaging of living tissue
*red shifted excitation light *reduced background noice * deep tissue penetration can be used in stead of confocal microscopy due to 2 last advantages. |
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|
Describe TIRF?
|
TIRF maakt gebruik van het verschil in brekingsindex van glas en een waterige oplossing. Hierdoor wordt licht, waarmee onder een bepaalde hoek op het glasoppervlak wordt geschenen, met daarboven de waterige oplossing, bijna volledig gereflecteerd. Een klein gedeelte van het licht penetreert wel in de oplossing, maar slechts tot een paar honderd nanometer. De kleine
volumina zijn nodig, omdat we effectief maar aan één molecuul willen meten. In grotere volumina kan dit niet, omdat we dan teveel last hebben van scattering van licht door het oplosmiddel, de zgn “Background noise”. Hierdoor is de signaal/ruis verhouding van het te meten molecuul te klein voor detectie. * so you excite the molecules that are close to plasma membrane. |
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|
What is AFM?
|
Een AFM heeft een groter bereik in kracht, maar een kleinere resolutie (tot ~ 5 pN). Een
AFM kan ook gebruikt worden voor scannen, nanolithografie (slijpen van gemodificeerde oppervlakken) en nanofabricatie (single molecule cut and paste assembly). De AFM tip en oppervlak kunnen dichterbij het biomolecuul aangehecht worden. |
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What is Near-Field Scanning Optical Microscopy?
|
*the size of the opening determines optical resolution , wavelenghts.
*Excitation only occurs within layer of < 100 nm away from aperture: Low background signal due to small excitation/detection volume |
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|
Which leukocyte migration patterns there are ( neutrophyls, T-lymfocytes, DC)
|
Neutrophyls: bloodvessel > endothelial wall > inflammation > cell death
T lymphocytes > thymus > blood > lymph> blood> tissue DC: blood> tissue > lymphoid organs |
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|
Descrobe the cell adhesion cascade of the lymphocyte.
|
1) Macrophage at the resident site of the infection produces chemo-,cytokines.
2) cytokines stimulate selectin expression and are responsible for leukocyte rolling. 3) chemokines> increase in affinity of the integrin to the endothelium. 4)Chemokines ( in the tissue) stimulate the motility of leukocytes > transmigration. |
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What is the function of chemoattractants?
|
*vasodilation,angiogenesis
*proliferaiton *mast cells degranulation *chemotaxis and activation of integrins |
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Which type of the receptors are chemokines?
|
GPCR
|
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Which adhesion molecules of the immune system there are?
|
Cadhering, integring, Ig-superfamily, selectins
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Which interaction between adhesion molecules are homo/hetero typic?
|
integrins-ICAMs
selectins - selectin ligans. |
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Which slectin endothelium interaction of the leukocyte with the endothelium there are?>
|
*L,P,E selectins
L-selectin (T lymf.) - L selectin Ligand ESL (T lymf.) on E selectin PSGL-1 aan P selectin en aan E selectin |
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|
What happens upoun leukocyte activation by chemokines presented on the endothelium?
|
* L selectin is shed
*cytoskeleton changes *integrin upregulation/activation ICAM + VCAM on endothelium *firm adhesion with integrins IG superfamily. |
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On which molecules is transmigration/frim adhesion dependent on ?
|
integrins, heterodimers, one beta subunit can bind on many different alfa subunits.
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Do integrins expressed on both endotheliun and leukocytes?
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* ja
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Which Integrins and Ig superfamily ligansds are involved in cell migration.
|
T cell: LFA-1 (a1,b2) vs ICAM-I/2: endothelium
T cell: LFA-1 (a1,b2) vs ICAM-I/2: endothelium T cell: LFA-1 (a1,b2) vs ICAM-I/2: endothelium T cell: a4,b7 vs VCAM/MADCAM endothelium |
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Where are the integrins also involved ?
|
phagocytosis
degranulation communication/proliferation signal transduction |
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|
Which integrin is involved in phagocytosis ?
|
AmB2
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|
Which integrines stabilize immunological synapse?
|
LFA1-ICAM-1
|
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|
Clustering (avidity) , activation (affinity)
|
***
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|
Which molecules are responsible for which defect in the leukocyte adhesion deseases?
|
Defects in rolling (selectins)
(firm) adhesion (b2 integrins) coagulation (b3 integrins) Recessive, ‘loss of function’ mutations |
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|
Which immunological adhesion defects there are?
|
defects in chemokine receptors
leukocyte adhesion deficiency I,II,III |
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|
Describe LAD I.
|
point mutations in integrine B2 subunit
high mortality Mild form LAD I – decreased expression of b2 integrins (5 – 10% of total) loss of function mutation |
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|
Describe LAD II?
|
Defect in fucose metabolism; mutation in CD15s=ligand for selectins
infecions, growth / mental retardation |
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|
What are the differences between LAD I and II ?
|
Since LAD I has very bad phagocytosis and T/B cell activity, it is not able to eradicate the infections which are present in the tissues ( since macrophages are already there). This is the crutial difference between these two deseases.
Both deseases have no neutrophil adhesion/migration chemotaxis. |
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|
Describe new adhesion deficiency , a new variant of LAD I >>> LADIII.
|
involves all leukocyte integrins (b1,b2,b3)
no structural mutations in integrins mutations in the kindlin-III, aprotein involved in the signal transduction through integrins. |
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|
When is anti-adhesion therapy usefull?
|
Inhibition of chronic infections ( chronh desease_)
treatment of autoimmune deseases blocking tumor metastasis |
|
|
a) What adhesion molecule would you block to induce tolerance after organ transplantation and which processes do you inhibit using this approach?
|
Since the chronic rejection is induced mainly by CTLs, i would block aLb2 (ICAM)and a4b1(VCAM) on lymfocytes , macrofages, DC. But then the immune system will be also impaired.
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b) Reperfusion damage after organ transplantation is caused by activation of endothelium due to restored blood flow. This leads to local inflammation and chemokine production that attracts large numbers of neutrophils. Upon neutrophil activation and degranulation, the tissue becomes damaged. What adhesion molecule would you block to inhibit this unwanted inflammation
|
aMb2 (ICAM)
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|
Which cytokines produce Th1/Th2 subsets cell and to help which T or B lymfocytes?
|
Th1>>> T cells
Th2 >>> B cells |
|
|
Which different dendritic cells there are?
|
Myeloid DC
PDC Nibbling DC Langerhans cells Dendritic cells cross talk and there efficiency depends on cross talk. |
|
|
Describe the roles of Plasmacytoid DC and conventional DC?
|
*Plasmacytoid DC are the!
main IFN producing cells! in our body. Immune ! modulating DC *Conventional DC are the! professional antigen! presenting DC in our body. Direct Immune responses. |
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|
What is the function of myeloid DC?
|
Stimulating T cells, fighting wound infection
|
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|
How do the maturation of DCs occurs?
|
it needs a danger signal
|
|
|
Which different T helper cells subsets there are and what do they do?
|
Th 1 > Cytokines IFNy (T cytotoxic)
Th2 > Cytokine Il-4 ( B cells, helminic parasites, ab production) Th17 > Cytokine Il-17 (mediators of inflammation) Treg > cytokine TGF-beta prevent immune pathology. |
|
|
Which 3 functions has a mature DC?
|
- co-stimulatory molecules!
- antigen presentation! - cytokine production |
|
|
Which different stimuli do DC integrate?
|
endogenous danger signals - (PAMPS)
cytokines (IFN-y) endogenous danger signals (tissue damage DAMPS) |
|
|
Do maturation of DC needs T helper cell CD40 ligand interaction?
|
YE$
|
|
|
Which molecules can be induced by the infection beyond the TLRs?
|
Rig Like helicases
NT binding oligomerization-domain like receptors Lectins |
|
|
What are the melanoma associated antigens?
|
gp100, tyr, MAGE
TRP-2 ( in B16 mouse model) |
|
|
What is DC vaccination strategy in melanoma patient?
|
Cytopheresis ( monocytes are separated from the blood of the patient)
addtion of molecules (Ils, GFs ) to monocytes ex-vivo >>> immature DC > adding DC maturating cocktail and tumor derived peptides. The intradermal, intravenous, intranasal giving this cells to the patient. |
|
|
How can be DC vaccines be tracked?
|
by non invasive MR imaging.
|
|
|
What does correlates with goed clinical uitcome of cancer patient transfected with mature DC?
|
Specific DTH infiltrating T cells correlate with a favorable clinical
outcome! |
|
|
What are the characteristics of T reg cells?
|
Treg characteristics:!
Originate in the Thymus! Depletion of Treg results! in auto-immunity! Suppression is cell-contact! dependent and requires! TCR triggering! Multiple subsets! Regulatory T cell! Foxp3+! CD4+CD25+++! GITR+/CTLA-4+! CD103+! CD25 depletion prior to DC vaccination enhances tumor rejection!!!! |
|
|
Wat is het doel van neonatale screening?
|
Het opsporen van aandoeninigen waarbij de behandeling na de geboorte meer voordeel voor het individu hebben dan de interventie in de late stadium, waarbij de aandoeding veel meer schade had aangedaan.
|
|
|
Beschrijf sensitivity versus specificy ?
|
Sensitivity: proportion of affected patients that has a positive test result
Specificity: proportion on non-affected individuals that have a negative test result (can one exclude the disease if test is negative) |
|
|
Bij welke metabole ziektes wordt neonatale screening toegepast?
|
•Galactosemie
• Biotinidase deficiëntie • Tyrosinemie I |
|
|
What is the Classical phenotype of Congenital Disorders of Glycosylation?
|
•hypotonia/epilepsy/cerebellar atrophy/mental retardation/ataxia
•inverted nipples, fat pads, strabismus, feeding problems, hypogonadism, coagulopathy, etc. |
|
|
How can cdg be diagnosed?
|
1) screening: isofocusing of plasma transferrin
2) Enzyme activity 3) Structure analysis (N-glycan or glycolipid analysis) 4) DNA diagnostics |
|
|
What assays will you do in fresh muscle biopsy?
|
•Substrate oxidation rates:
pyruvate, succinate and malate •ATP production rate from pyruvate and succinate. •Single enzymes •NADH:Q1 oxidoreductase (complex I). •Succinate Dehydrogenase (Complex II). •Decylubiquinol: Cyt C Oxidoreductase (Complex III). •Cytochrome C Oxidase (Complex IV). •ATPsynthase (Complex V). •Pyruvate Dehydrogenase complex •Citrate synthase (CS). |
|
|
Which gene has to be investigated by the mitochondrial desease?
|
The mode of inheritance is an indication for the candidate gene which has to be investigated!.
|
|
|
How does DNA sequence analysis occur?
|
•amplification of DNA fragment islinear (1:1) and performed in the presence of one primer
•nucleotide at 3’-end is fluorescent labeled •label is dependent on nucleotide type Based on the retention time and fluorecent label the sequence is determined |
|
|
What should you do if In case the sequence variation you has found has not been described?
|
Check at least 100 control alleles of control population
•On internet (EST’s) |
|
|
What is NDUFS7 mutation?
|
is responsible for complex I deficiency
|
|
|
What is positional candidate gene approach?
|
•Chromosome transfer in combination with linkage analyses and/or candidate gene approach.
* OXPHOS defect is expressed in fibroblasts. * Fibroblast are immortalized. |
|
|
Describe citric acid cycle
|
*glucose is processed by glycolisis in puryvate.
* puryvate is processed by putyvate dehydrogenase to acetyl-COA *the citric acid cycle generates NADH and QH2 *puryvate dehydrogenase is regulated by kinase and phosphatase. *NADH en Acetyl-COA - stimuleren kinase activity *NAD,puruvate inhibit kinase activity |
|
|
Describe complex 1?
|
The reaction that is catalyzed by this (NADH reductase) enzyme is the two electron reduction by NADH of coenzyme Q10 or ubiquinone (represented as Q in the equation below), a lipid-soluble quinone that is found in the mitochondrion membrane:
As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space. |
|
|
Describe Succinate-Q oxidoreductase (complex II)?
|
It oxidizes succinate to fumarate and reduces ubiquinone. As this reaction releases less energy than the oxidation of NADH, complex II does not transport protons across the membrane and does not contribute to the proton gradient.
|
|
|
Cytochrome c oxidase (complex IV)
|
Pumps 4 protons, uses Oxigen, generates water and reduced cytochrome c
|
|
|
What does ATP synthase (complex V)?
|
The enzyme uses the energy stored in a proton gradient across a membrane to drive the synthesis of ATP from ADP and phosphate (Pi)
|
|
|
Congenital disorder of glycosilation
|
nervous system, muscles, and intestines are affected.
|
|
|
What is PPM2?
|
Phosphomannomutase 2 catalyzes the isomerization of mannose 6-phosphate to mannose 1-phosphate. Mannose 1-phosphate is a precursor to GDP-mannose necessary for the synthesis of dolichol-P-oligosaccharides.
In the cytosol of the cell. These mutations change the structure of the PMM enzyme in different ways; however, all of the mutations appear to result in reduced enzyme activity. Decreased activity of the PMM enzyme leads to a shortage of GDP-mannose within cells. As a result, there is not enough activated mannose to form oligosaccharides. Glycosylation cannot proceed normally because incorrect oligosaccharides are produced. The signs and symptoms in CDG-Ia are likely due to the production of abnormally glycosylated proteins in many organs and tissues. |
|
|
What causes CDG-Ib ?
|
phosphomannose isomerase deficiency
|
