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MBB345 - Human Reproduction and Fertility > Hodgson - history of clinical cytogenetics, human karyotyping and main tech used to detect disease > Flashcards

Hodgson - history of clinical cytogenetics, human karyotyping and main tech used to detect disease Flashcards

1
Q

What are the 3 main referral reasons for couples to fertility services?

A
  • couples who believe they are infertile as trying and cannot get pregnant (don’t have clinically detectable pregnancies)
  • couples who have had frequent miscarriages
  • patients w/ serious risk of genetic disease likely to affect fetus
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2
Q

How are chroms visualised?

A
  • staining and visualised by G banding

- all from single nucleus captured from metaphase

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3
Q

How are chroms organised by morphology?

A
  • position of centromere
  • large metacentric, large submetacentric, medium submetacentric, large acrocentric, small submetacentric, small metacentric, small acrocentric (groups A-G respectively)
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4
Q

What is the protocol for G banding?

A
  • cells cultured to gen mitotic cells (need dividing cells)
  • arrest cell cycle in metaphase (high mitotic index)
  • swell nuclei w/ hypotonic solution (so no overlapping)
  • kill cells using fixative (3:1 methanol:acetic acid) –> fixed
  • drop fixed sample onto glass slide
  • trypsin digest (protease) –> creates pale bands
  • wash, then Leishman’s stain
  • wash, then image analysis
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5
Q

Why are cells fixed in G banding?

A
  • to stop chromosomes from condensing, otherwise would be so small wouldn’t be able to see bands, and also helps sep chroms
  • also kills everything in sample (ie. minimises risk of staff being infected w/ any infectious disease)
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6
Q

What do the dark and pale band represent in G banding?

A
  • dark bands = AT rich

- pale bands = GC rich

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7
Q

What diff samples can be received in the clinic, and what are the sources of these samples?

A
  • blood sample from parents
  • fetal epithelial cells –> taken from amniotic fluid samples
  • placental material –> chorionic villus sample
  • (also fetal blood samples but signif risk of spont abortion, so gen last resort)
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8
Q

Are most cells received actively growing and diving, what is the consequence of this?

A
  • no (except cancers, usually actively growing and dividing)

- so need to culture in order to grow so can gen metaphases to analyse

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9
Q

Why in light microscopy of chroms does 1 side largely appears pale and other dark?

A
  • wherever dye has stained chroms heavily, conformation of chromatin quite open –> theory is this means dye can access binding pocket
  • chromatin collapsed in on itself where pale staining –> dye cannot access binding pocket as well
  • v likely that structure of chromatin determines type of staining you get
  • trypsin wash determines this step
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10
Q

What is an idiogram?

A
  • diag of chrom
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11
Q

In what order are chrom bands numbered?

A
  • no.s increase further away from centromere
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12
Q

What is ISCN?

A
  • standardised nomenclature system for describing chroms and chrom abnormalities
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13
Q

What can affect banding resolution?

A
  • stage of cell cycle cell in when added mitotic blocking agent
  • type of tissue received from patient
  • G banding protocol
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14
Q

Why are chroms analysed in metaphase?

A
  • as move through cell cycle from interphase to metaphase, chroms become longer and more spaced out
  • if more condensed chrom see fewer bands, 2 homologs don’t necessarily condense in perfect synchronicity
  • want to analyse when chroms long enough, so can see all bands, but not too long otherwise will overlap and need more cells
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15
Q

Why does morphology differ dep on type of tissue (ie. type of sample)?

A
  • not fully understood but does slightly correlate w/ maturity of cells –> long chroms when cells terminally differentiated (ie. leucocytes from peripheral blood), but SCs not terminally differentiated and tend to be a lot shorter
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16
Q

Why does trypsin cause pale bands in G banding?

A
  • controls how much of chromatin is collapsing
  • longer add trypsin get paler band as cleaving peptide bonds, causing partial collapse of chromatin, so ends up collapsed and smooth, preventing Leishman’s dye from getting to binding pocket
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17
Q

What happens if leave trypsin on too long in G banding?

A
  • eventually chroms fall to pieces
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18
Q

Why is it important that chroms are optimally stained?

A
  • amount of time to analyse chroms signif quicker if optimally stained, so get patient quicker result which is v important
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19
Q

Apart from trypsin digest, what other factors can influence quality of slide?

A
  • ageing –> let slides dry out, longer = better banding quality
  • staining time w/ Leishman’s, too long can blur dark bands together = bad
  • chromosome spread –> too overlapping then can’t see bandin structure properly
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20
Q

What are the 2 types of FISH?

A
  • direct labelling = DNA/RNA probes directly mod by fluorescent nts
  • indirect labelling = fluorescent dyes added after probe hybridised to patient sample
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21
Q

Which type of FISH is used in the NHS, why?

A
  • direct

- much quicker and brighter as labels already manufactured

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22
Q

How is FISH carried out?

A
  • take microscope slide –> DNA in metaphase chrom spread or interphase nucleus (both same protocol)
  • 1st step is like PCR, make patient DNA ss so can hybridise another NA to it
  • -> but lower temps (75-78°), so denatures but doesn’t destroy DNA structures
  • anneal probe
  • series of stringent washes to remove any probe bound to non-complementary regions of sample
  • DAPI is intercalating agent (blue background colour in images), used as counter-stain for both methods
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23
Q

What are the 3 main probes used for FISH in clinical setting?

A

1) FISH probe itself is specific clinical service, ie. if abnormality in fetus suspected go straight to FISH not G-banding, as usually due to gains and losses of chroms (aneuploidy), so need to check chrom no.
2) when querying specific section of chrom that is poss abnormal, after seeing something specific from G-banding
3) whole chrom painting, when unsure about origin of certain piece of DNA

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24
Q

How big are probes?

A
  • tend to be v large (sometimes 100s of kb)
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25
Q

Why is a bright signal of a probe in FISH beneficial?

A
  • allows rapid hybridisation times and rapid, less ambiguous analysis
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26
Q

What are the most common aneuploidies seen in samples?

A
  • ChX –> fragile X (XXX), Turner syndrome (XO)
  • ChY –> Klinefelter (XXY)
  • Ch21 –> DS
  • Ch18 –> Edwards syndrome (trisomy)
  • Ch13 –> Patau syndrome (trisomy)
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27
Q

Can Down’s Syndrome occur w/o full trisomy?

A
  • yes, there is critical region which can be amplified
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28
Q

What are micro deletion probes used for?

A
  • usually for unbalanced foetal karyotype due to ‘abnormal’ inheritance of chroms from a parent w/ balanced rearrangement
  • eg. Cri du Chat, SOTOS
  • often combine multiple diagnostic probes to save money –> 1 probe used as +ve control for other
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29
Q

What is the effect of having a balanced rearrangement?

A
  • gen no other phenotypes, apart from fertility problems
  • can cause fertility problems because fetal karyotype unbalanced, may be so unbalanced that aborts before even realise pregnant, or if slightly imbalanced and can mature part way through dev before aborting
  • if breakpoints near ends of chroms, then fetus can be viable, and dev w/ serious genetic disease
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30
Q

When is whole chrom painting used?

A
  • when unsure where breakpoints are or unsure about origin of certain part of DNA
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31
Q

What is an asynchronous culture?

A
  • contain dividing cells at all stages of mitosis and G0 (quiescence)
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32
Q

Why do patient samples need to be asynchronous cultures?

A
  • human cells take approx 24 hrs to complete 1 cell cycle (and often longer)
  • mitosis lasts for approx 1-2 hrs (4-8% cells in mitosis at any 1 point, and even lower prop in metaphase)
  • therefore need to somewhat synchronise cells
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33
Q

How can DNA synthesis be reg for cell synchronisation?

A
  • DIAG*
  • have precursor nts (dATP/dCTP/dGTP/dTTP), used by DNA pol and added to new strand as synthesise it
  • rest are some of the precursors to dCTP and dTTP
  • excess dTTP inhibits reduction of CDP to dCDP (precursor for dCTP) by ribonucleotide reductase, this reduces availability of dCTP for DNA synthesis
  • dCTP cellular conc becomes rate limiting –> lymphocytes remain in S-phase for extended periods
  • prop of cells in S-phase increases w/ dTTP block reaction time
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34
Q

How is thymidine block released (part of cell synchronisation)

A
  • by washing –> centrifugation and subsequent suspension of lymphocytes in fresh growth media
  • or addition of dCTP, bypassing need for ribonucleotide reductase (used in healthcare, as centrifuging has health and safety issue)
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35
Q

What happens to cells after released from dTTP block?

A
  • arrested cells process through mitosis in synchronous manner
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36
Q

What is an alt to cell cycle manip for cell synchronisation?

A
  • fluorodeoxyuridylate (FdU) synchronisation
  • excess FdU inhibits the synthesis of dTMP (a precursor of dTTP) –> reduces the availability dTTP for DNA synthesis
  • dTTP cellular concs become rate limiting –> lymphocytes remain in S-phase for extended periods
  • prop of cells in S-Phase increases w/ FdU block reaction time
  • FdU block released by addition of dTTP (or wash and centrifugation, but not preferred)
  • once released, arrested cells proceed through mitosis in synchronous manner
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37
Q

What occurs normally in meiosis?

A
  • in early metaphase spindle fibres polymerised and bind kinetochores
  • once mts connected to kinetochores get tension across spindle
  • sister chromatid cohesion broken down by separase (req tension)
  • immed enter anaphase and chroms condense at opp poles
  • telophase, then cytokinesis
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38
Q

How does colcemid synchronisation work?

A
  • mech of action at mol level not completely understood, but following gen agreed:
  • colcemid binds soluble tubulin, colcemid-tubulin complex may still polymerise, but w/ signif reduced efficiency
  • colcemid-tubulin complex also slows microtubule depolymerisation
  • overall microtubule stability reduced, preventing functional spindle from forming, so no tension and no breakdown of sister chromatid cohesion
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39
Q

Why do we care about mitotic index or banding resolution?

A
  • affects quality
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40
Q

What are the diff aspects of quality, and how is each of these defined?

A
  • accuracy and precision = measure tests general reliability
  • -> test accurate when true abnormality identified
  • -> test precise when repeated analyses yield same result
  • specificity and sensitivity = likelihood of FPs (false +ves) and FNs (false -ves)
  • -> test specific when false +ve rate low, so correctly excludes ‘normal’ patients
  • -> test sensitive when false -ve rate low, so correctly identifies people w/ given disorder
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41
Q

How is QA measured?

A
  • look at 4 diff chroms and see clearly bands indicated for that particular QA in both homologs for 3/4 chroms
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42
Q

What is QA3 used for?

A
  • oncology only –> too low for any fertility services
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43
Q

When is QA4 used?

A
  • for exclusion of aneuploidy and large structural rearrangements –> eg. if prenatal diagnosis, suspected Down Syndrome
  • if can get QA5 rather than QA4 then would, even if only req QA4
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44
Q

When is QA5 used?

A
  • if concerned about fetal dev, for exclusion of aneuploidy and large or more subtle structural rearrangements –> eg. if prenatal diagnosis, ultrasound scan (16-20w gestation) detects morphological abnormalities
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45
Q

When is QA6 (highest) used, and what samples are needed?

A
  • for parental blood samples (as achievable, but cant get QA6 from chorionic villus etc.)
  • for exclusion of subtle structural rearrangements and many microdeletion syndromes –> eg. if recurrent miscarriage (>3)
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46
Q

What holds bivalents together in meiosis?

A
  • COs
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47
Q

What % of clinically recognisable conceptions spontaneously abort, and how many of these are due to abnormal chrom complement?

A
  • 15% of all clinically recognisable conceptions will spont abort
  • approx 50% of all spont abortions have an abnormal chromosome complement
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48
Q

What is the most common cause of spontaneous abortion?

A
  • polyploidy most common aborted (and triploidy most common of this)
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49
Q

What is the most common trisomy, and what does it result in if detected?

A
  • trisomy 16
  • seen in approx 1-1.5% of pregnancies
  • when detected in fetus always results in spont abortion, but if confined to placenta then can come to term (not all cells in embryo are destined to be in fetus, some diverge to form placenta)
50
Q

What is the most common aneuploidy?

A
  • 45X
51
Q

What % of cytogenetically abnormal pregnancies are due to gains and losses of whole chroms or chrom sets?

A
  • 89%
52
Q

What are the commonly viable autosomal aneuploidies, and what % spont abort?

A
  • trisomy 13: Patau Syndrome, 99.5% spont abort
  • trisomy 18: Edwards Syndrome, 95% spont abort
  • trisomy 21: Down Syndrome, 78% spont abort
53
Q

What are the common sex chrom aneuploidies, and what % spont abort?

A
  • 21% spont abort
  • 47, XXX
  • 47, XXY
  • 47, XYY
  • 45, X
54
Q

Why are sex chrom aneuploidies gen not as big of a problem?

A
  • as Y chrom is gene poor so has little effect and extra X copies inactivated
55
Q

How are these common aneuploidies detected in PND service?

A
  • rapid service (gen w/in 24hrs) = FISH or cell-free fetal DNA qPCR
  • karyotype analysis (gold standard) –> provides full report after rapid test carried out
56
Q

Why are FISH and cell-free fetal DNA qPCR so rapid?

A
  • don’t need to grow sample, can do on interphase samples
57
Q

How does advanced parental age contrib to infertility?

A
  • MI or MII NDJ results in abnormal gametes
  • advanced maternal age is signif risk contributor, errors in MI more common than MII
  • paternal errors do happen, but much more unlikely
  • can get mitotic NDJ –> v early in dev of embryo, chrom NDJ occurs and 3 copies ended up in cells destined to dev into fetus
58
Q

Is bias towards maternal errors unique to chrom 21, why?

A
  • no, also evident for many other aneuploidy syndromes, eg. 18, 13
  • 100% maternal errors in 16
  • reason for this relates to no. COs
59
Q

How does NDJ events affect the no. of COs?

A
  • reduced CO obs when NDJ originated from MI

- increased CO obs when NDJ originated from MII

60
Q

What is cM a measure of?

A
  • unit of measure of genetic recomb freq
  • equal to 1% chance that marker at 1 genetic locus will be sep from marker at 2nd locus due to CO (which occurs in MI)
  • a single cM will equal diff no. of bps between genomic loci, due to differing rates of COs
61
Q

What is the role of sister chromatid cohesion in COs and meiotic chrom NDJ?

A
  • homologous chroms covalently joined via CO
  • so faithful disjunction dep on distal sister chromatid cohesion (relative to centromere)
  • after DNA rep, cohesin (clamping prot) laid down behind pol, holds sister chromatids together
  • ring closed by another prot = Scc1 in mitosis and Rec8 in meiosis
  • early in MI, Spo11 (a tpm) loaded onto DNA and creates ds breaks
  • later in MI ds breaks repaired by HR machinery –> can be repaired to form CO or non CO
  • position and no. of COs highly reg
  • if only 1 CO, then position v important, as when meiotic spindle grabs chroms, what holds them together important as preventing NDJ
  • only sister chromatid which is mechanically holding these homologous chroms together is between CO and telomere –> so the further down CO is located, the less cohesin is holding them together
62
Q

What is cohesin, ie. what is it made up of?

A
  • heterodimer between Smc1 and Smc3
63
Q

Why do cells not want too many COs to form close to each other, and how is this prevented?

A
  • would increase risk of non disjoining chroms in MII

- think when 1 CO occurs, mech to inhibit others forming too close by

64
Q

Why such a bias to maternal NDJ?

A
  • female gametogenesis: oocytes formed, chrom pairing and CO all occur during fetal dev, held in this stage till puberty (w/ CO), single ovary released, enters 1st division, 2nd only completed upon fertilisation
  • -> if only held together by single CO, and the closer is to bottom then fewer cohesin mols, then more likely to non disjoin, esp if damaged
  • male gametogenesis: spermatogonial cells differentiate to spermatocytes, which divide by meiosis to form spermatids, this is continuous process
65
Q

What can mosaicism result from?

A
  • mitotic chrom nondisjunction event resulting in trisomic and monosomic daughter cells
66
Q

What can mosaicism affect?

A
  • fetus, placenta or both
67
Q

What is confined placental mosaicism (CPM)?

A
  • when mosaicism only in placenta
68
Q

If do chorionic villus and detect mosaicism in placenta by FISH, then how is it found out if in placenta or both?

A
  • do amniotic fluid sample
  • good for genetic analysis as contains epithelial cells from fetus from lots of places, eg. skin, epithelial lining of internal origins etc.
69
Q

Why are nearly all cases of T16 CPM of low birth weight?

A
  • can disrupt dev of placenta, which can impact fetus
70
Q

What causes T16 CPM?

A
  • nearly all caused by trisomic conception resulting from maternal MI nondisjunction, means CPM for T16 almost always formed by trisomic rescue (= mitotic NDJ of abnormal cell, which gen normal diploid cell)
  • or start w/ 2 normal gametes, fetus disomic for all chroms and 2 normal sex chroms, then undergoes mitotic divisions, can have mitotic NDJ event, if goes on to dev into placenta then have normal gamete and abnormal placenta
71
Q

What is confinement of trisomic cell line to placenta dep on?

A
  • stage of embryonic dev when rescue event occurred and in which cell
72
Q

In cases of T16 CPM what must also be considered?

A
  • UPD of Ch16 –> 16 is an imprinted chrom w/ phenotypic consequences
73
Q

Why are males more susceptible to structural rearrangements?

A
  • can inhibit gametogenesis –> can cause azoospermia or oligospermia
  • oogenesis more robust
74
Q

What are the 4 types of large structural rearrangements?

A
  • balanced (no loss of genetic material) reciprocal translocations (= exchange of material between 2 or more chroms)
  • Robertsonian translocation (unbalanced)
  • insertion
  • inversion: pericentric or paracentric
75
Q

What structural rearrangements are most common?

A
  • reciprocal and Robertsonian translocations far more common than insertions and inversions (v rare in fertility setting)
  • insertions more common in cancer
76
Q

What is a Robertsonian translocation?

A
  • unbalanced rearrangement between acrocentric chroms
  • occur between 2 acrocentric chroms, resulting in fusion of 2 long arms
  • breakpoints in both chroms located in short arms, so RTs are dicentric and unbalanced –> as some of p arms of both chroms involved lost
  • loss of acrocentric p arms not linked to any known adverse clinical phenotypes, as contain repetitive seqs
  • RTs either homologous (both q arms derived from same chrom) or non-homologous (diff chroms) –> 10 poss non-homologous and 5 homologous translocations
  • homologous RTs are a type of isodicentric chrom
77
Q

Apart from homologous RTs are there any other isodicentric chroms?

A
  • yes, others been obs in humans
78
Q

Are isodicentric chroms of non-acrocentrics viable?

A
  • not viable when part of a constitutional karyotype and are therefore not observed in fertility investigations
79
Q

How is ISCN written?

A
  • no. chroms, sex chrom complement, descrip of abnormality, (2 chroms in numerical order dep by ;), (cytogenetic breakpoints where think occurred)
  • eg. 45,X-,t(14;21)(q10;q10)
80
Q

What are the most common non homologous RTs?

A
  • RT 13;14 accounts for 76% of non-homologous RTs
  • 14;21 is 10%
  • rest are roughly evenly distrib in prop
81
Q

What is needed for recomb to happen, what does this mean is uncommon?

A
  • need ds break, homology and homologous seqs in same place at same time
  • so rearrangements between non homologous chroms uncommon
82
Q

How are ds breaks in dividing cells usually repaired?

A
  • using homology of sister chromatid
83
Q

How are RTs so common?

A
  • if look at seq of p arms of all acrocentric chroms, they are site of rRNA
  • in G1, nucleolus is site of biosynthesis of ribosomes, some of DNA making up the nucleolus is encoding rRNA mols going to transcribe, so all acrocentric p arms come together in same space at same time –> this is where DNA break likely to occur, have homology as all rRNA genes are there, but even greater homology in repetitive microsatellite DNA
84
Q

Why are there likely 2 diff mech for RTs?

A
  • break site and fusion site homogenous in patients for 13;14 and 14;21 RTs
  • whereas others are heterogeneous
85
Q

How does breakpoint diversity illustrate distinct mechs for RT formation?

A
  • FISH studies identified that majority of breakpoints in RTs are proximal to NOR region (w/ respect to centromere) and therefore result in del of NOR regions
  • non 13;14 and 14;21 RTs
    –> HR between repeats unlikely due to variability of break sites
    –> random breakage and exchange more likely
    → acrocentric;acrocentric predisposed due to close prox of NOR region in nucleoli formation during MI
  • 13;14 and 14;21 RTs
    –> low variability in breakpoints indicate diff mech of formation
    –> most likely based on HR between repetitive seqs w/ opp orientation
86
Q

What microsatellites are present in band p11 of acrocentric chroms, and what is their role?

A
  • I, II, III and IV microsatellites and β satellite

- mediate recomb

87
Q

Why is orientation of microsatellites v important, relating to RTs?

A
  • if microsatellites had same orientation on tips of chrom 14 and 21 then would line up, repair and separate, inc exchange of material of p arms
  • but they are inverted, so when pair up get exchange, then end up w/ dicentric chrom
88
Q

What questions are family likely to have during genetic counselling?

A
  • are they able to have a normal child?
  • are they at risk of further miscarriages?
  • are they at risk of having an abnormal live born child, if so, what is the risk?
  • are there any clinical interventions to help?
89
Q

How must meiosis be considered when deciding outcomes of an RT?

A
  • remember how many functional centromeres there are

- RT has 50% chance of going each way, as do the other chroms

90
Q

When calc risk in RTs what assumptions are made?

A
  • that meiosis I segregation random (may not be true in those w/ RT)
  • clinically when dealing w/ indiv don’t know exact risk (esp for RTs), as other factors need to account for
  • must also account for trisomy or monosomy rescue events, advanced maternal age and assoc increased likelihood of NDJ and abnormalities of other chroms (not just +21) and issues regarding UPD and UPID
91
Q

How can genetic counselling lead to initiation of family studies?

A
  • if proband has siblings may be worth checking parents, as siblings could also be affected –> has ethical considerations
  • if parent carrier, then would recommend siblings attend genetic counseling
  • but if parents both normal then proband classed as de novo
  • but still worth investigating siblings as mother of proband could be mosaic
92
Q

What is UPD/UPID?

A
  • both homologous chroms inherited from single parent, w/ no contrib from other parent
  • UPD = both homologous chroms from 1 parent
  • UPID = 2 copies of same chrom from 1 parent
93
Q

In what chroms can UPD occur, and what conditions result?

A
  • been detected in humans for almost every chrom
  • some result in abnormal phenotypes as these chroms host imprinted genes
  • 5 clinically relevant imprinted chroms:
  • -> 6: PAT = DMTN
  • -> 7: MAT = Russell Silver syndrome
  • -> 11: PAT = Beckwith-Wiedemann syndrome
  • -> 14: MAT = Temple syndrome, PAT = Kagami-Ogata syndrome
  • -> 15: MAT = Prader-Willi, PAT = Angelman
  • -> 2, 16, 20 = poss clinical pathology
94
Q

Is UPD the only way in which imprinted chroms can lead to disease?

A
  • no, may also result in abnormal phenotype through LOH, resulting in recessive disease
95
Q

What are the diff mechs for UPD/UPID to occur?

A
  • trisomic rescue (can result in UPD)
  • monosomic rescue (can result in UPID)
  • gamete complementation (could result in either)
96
Q

How can UPD be determined?

A
  • quantitative PCR of unique microsatellite regions of DNA on these chroms
  • looks for allelic diffs between mat and pat chroms and amp them by diagnostic PCR
97
Q

What can large structural rearrangements result in?

A
  • loss or reduced fertility in males due to failure of spermatogenesis
  • recurrent miscarriages (unbalanced constitutional karyotype of fetus)
  • live born abnormal child (if diff is significantly small and can be tolerated)
98
Q

Why do paternal carriers of structural rearrangements usually not pass them on?

A
  • tend to be infertile
99
Q

Why are not all translocations and other balanced rearrangements not inherited from mother (even though paternal carriers tend to be infertile)?

A
  • paternal errors in meiosis occur de novo to gen a rearrangement in prop of gametes (under these circumstances male is a mosaic, the translocation in the germline and would not be detected following analysis of a peripheral blood sample
  • eg. if karyotype parents and both normal, but child is carrier of balanced structural rearrangement, would call this de novo event as appears not to have been inherited
  • however in vast majority of cases the translocation HAS been inherited as male partner is mosaic
  • thought that vast majority of structural rearrangements do actually happen in latter stages of spermatogenesis –> so these males are mosaics and would not be identified as carrying the mutation
  • MI/II normal and balanced translocations likely occur de novo in haploid cells gen (ie. immature spermatids)
100
Q

Where are the vast majority of translocations in the pop seen?

A
  • cancer patients
101
Q

Are translocations gen inherited?

A
  • vast majority are recurrent –> ie. seen in unrelated indivs
  • but also see familial translocations
102
Q

How are breakpoints identified?

A
  • look at diffs between normal and derivative affected chroms
  • use idiograms to identify which bands are present where
103
Q

What do familial translocation gen cause in males and females?

A
  • in a male patient would likely cause infertility

- in females could explain recurrent miscarriages (normal other fertility problems)

104
Q

What is the consequence if breakpoint in translocation is v close to end of chrom?

A
  • genetic imbalance is small

- so great risk of having live abnormal child w/ genetic disease

105
Q

What is the most common recurrent non-Robertsonian, and what genetic disease is it assoc w/?

A
  • t(11;22)(q23q11)
  • in approx 1 in 7000 indivs (but likely more common, as don’t know if have it)
  • Emanuel Syndrom
106
Q

How are translocation breakpoints characterised?

A
  • by palindromic AT rich repeats (PATRR)
107
Q

What causes Emanuel syndrome?

A
  • 3:1 malsegregation of the abnormal Ch22 and supernumerary inheritance of this derivative chrom
108
Q

What are the symptoms of Emanuel syndrome?

A
  • severe mental retardation and distinctive morphological features
  • kidney and heart abnormalities
109
Q

Are oligospermia and azoospermia usually due to abnormal genetics?

A
  • are most common reasons for infertility, but vast majority of the time not due to abnormal genetics (and even smaller prop due to structural rearrangements)
110
Q

What are about 30% of oligospermia and azoospermia cases caused by?

A
  • dels of particular region on y chrom
  • 3 important genes in the region: AZFa, AZFb, AZFc
  • thought mech by which these genes can be del v similar to how form translocations and other rearrangements in genome, governed by HR based mech
  • repeat seqs facilitate repair, which results in del of 1 or more of these genes
111
Q

What are repetitive pieces of DA scattered all over DNA responsible for?

A
  • a lot of genomic abnormalities which can impact on infertility and increased risk of genetic disease
  • having these seqs doesn’t mean will have rearrangement, but does increase chance
  • likelihood of genetic abnormality seems to be dep on length of homology in repetitive seqs and distance between repetitive seqs –> more likely to suffer genetic abnormality when seqs are longer and closer together
112
Q

How is t(11;22)(q23;q11) usually detected?

A
  • can be detected by male referred due to problems w/ infertility
  • but usually following fertility investigation or genetic investigation of dev delayed child (Emanuel syndrome)
113
Q

What is the ICSN karyotype of Emanuel syndrome?

A
  • 47,XY,+der(22)t(11;22)(q23;11)
  • 47 chroms, male, all chroms normal apart from extra derivative c22 (ohas 2 normal 22 and 11s), derived from abnormal translocation events between c11 and 22
114
Q

What is a Pachytene cross?

A
  • when 4 chroms pairing up in MI

- forms as all chroms need to pair up and held together w/ normal no. of COs

115
Q

What are the diff ways of seg chroms in MI, and what are the resulting gametes?

A

1) normal way is alt seg, when you co-seg alt centromeres
- -> get 4 gametes following MII
- -> 2 normal
- -> other 2 would result in normal pregnancy as not genomic imbalance (all of each chrom present), but child would be carrier of rearrangement
- other ways gen abnormal, unbalanced gametes
2) adj seg I = adj centromere (non-homologous) seg together
- -> means all gametes gen are unbalanced, so most likely outcome is unbalanced zygote, most likely spont aborted
3) adj seg II = adj centromere (homologous) seg together
- -> prod 4 v unbalanced gametes
- -> 2 are missing parts Ch22
- -> 2 are almost nullisomic for Ch11

116
Q

What is the result if nondisjoin chroms and get 3:1 MI malsegregation?

A
  • gen 4 unbalanced gametes
  • -> 2 sufficiently unbalanced to result in miscarriage (monosomic)
  • -> but in other 2 have 1 whole copy of both ch11 and ch22 (ie. not missing any genetic material), but does have supernumerary derivative ch22 and if fuse w/ normal gamete get partial trisomy for 11q23-ter and for 22pter-q11.2, BUT is viable (still risk of spont abort) = Emanuel syndrome
117
Q

What is the mech for dev of Emanuel syndrome?

A
  • repetitive region involved, but full mech not understood
  • PATRR located at 22q11 known to be particularly unstable and been identified as being involved in other genomic rearrangements involving Ch22
  • PATRRs thought to be susceptible to mis-pairing, leading to formation of 2° structures which are able to induce genomic instability
  • de novo gen of the 11;22 translocation also appears to be restricted to spermatogenesis
  • formation of 11;22 translocation likely to involve colocalisation of PATRR11 and PATRR22 during late spermatogenesis, DSB formation and subsequent aberrant repair
  • DSB repair likely to occur vis NHEJ, as late spermatids cannot undergo HR
118
Q

How many cells are generally analysed?

A
  • 5 cells

- 3 fully analysed –> to see small abnormalities (unless find something wrong)

119
Q

How common is trisomy 16, and what is the consequence?

A
  • most common chrom abnormality detected at conception and in 1st trimester fetal loss
  • babies w/ full trisomy 16 almost always lost in v early pregnancy and virtually none have been born
120
Q

What is the most likely seg in a pachytene cross?

A
  • generally longest straight line can draw

MBB345 - Human Reproduction and Fertility (3 decks)

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