Session 6: Prenatal Genetics Flashcards

Question

what happens in implantation?

Answer 1

at day 5 an enzyme is released which makes a hole in the ZP releasing the blastocyst- the blastocyst then attaches the the uterine membrane - trophoblast cells rapidly divide to form the cytotrophoblast ans syncytiotrophoblast and invade the connective tissue of the uterus - a fluid filled cavity forms within the ICM generating the amniotic cavity enclosed by the amnion

Answer 2

Gastrulation is the process in which the orientation of the body is established and the 3 germ layers are formed: ectoderm endoderm mesoderm The characteristic gastrulation is the formation of the primitive streak

Answer 3

establishes the antero-posterior body axis forms during gastrulation starts as a primitive streak and then develops into a groove then a pit. cells migrate through the primitive streak to the space between the epiblast and hypoblast to generate the 3 germ layers- 1st endoderm, 2nd mesoderm and 3rd ectoderm

Answer 4

lungs, liver, thymus, endocrine glands

Answer 5

blood vessels, muscle tissue, connective tissue

Answer 6

skin and the CNS

Answer 7

All DNA methylation patterns are erased and the pattern specific to the sex of the fetus is set- when fertilised the imprint is maintained stably in all somatic cells of the fetus and throughout life

Answer 8

interuterine insemination- injection of sperm directly into the womb following stimulated ovulation of the woman

Answer 9

adding sperm samples to eggs (havested from a women following stimulated ovulation) in a petri dish. They are incubated together and monitored for successful fertilisation. Successfully developing embryo is transferred back to womans womb

Answer 10

microscopic injections of a sperm directly into an egg nuclei in a petri dish. Similar to IVF but maximises the chance of fertilisation

Answer 11

Microsurgical Epididymal Sperm Aspiration- surgical sperm extraction method and can be used with IUI, IVF or ICSI for men with azoospermia (no sperm in semen)

Answer 12

additional full set of chromosome 69,XX/XY

Answer 13

1-3% of pregnancies 99.9% abort in 1st or second trimester- those that make it to term die in the neonatal period <1 month advanced maternal age is not a risk factor

Answer 14

60-80% are diandry- most common

Answer 15

2 paternal and 1 maternal set of chromosomes

Answer 16

fertilisation of a normal egg by 2 sperm- failure of cortical reaction- 69,XXX or 69,XXY or 69,XYY 69,YYY not viable so not seen minority are due to fertilastion of a normal egg by diploid sperm by a failure of NDJ in spermatogenesis for a complete set of chromosomes

Answer 17

partial hydatidiform mole which is a trophoblastic hyperplasia of the cystic villi increased hCG levels

Answer 18

IUGR head to chest fusion limb growth abnormalities macrocephaly- digyny neural tube defect hydrocephalus- digyny syndactyly heart/renal defects cleft lip/palate

Answer 19

2 maternal and 1 paternal set of chromosomes non hydropic villi NO MOLE and placenta is generally small

Answer 20

fertilisation of a diploid egg - due to NDJ of a complete set of chromosome during meiosis - retention of a polar body in the fertilised egg- failure of meiosis II - fusion of 2 eggs and fertilisation by a haploid sperm

Answer 21

due to the influence of differing imprinted states

Answer 22

usually sporadic - diandry triploidy - recurrence risk is 1-1.5% - digyny triploidy has been reported as recurrent in several families

Answer 23

a group of rare diseases in which abnormal trophoblast cells grow inside the uterus after conception includings - non invasive: mole and partial mole - malignant- invasive mole, choriocarcinoma and placental site trophoblastic tumours (PSRR; very rare), Epithelioid trophoblastic tumors (ETT; even more rare).

Answer 24

46,XX/XY but only paternal chromosomes present

Answer 25

high levels of hCG bunch of grapes appearance NO formation of a fetus

Answer 26

fertilisation of a enucleated egg with a haploid sperm and duplication of the haploid genome (46,XX) remaining are fertilisation of an enucelated egg with 2 different sperm 46,XY or 46,XX 46,YY are not seen as unviable

Answer 27

high hCG uterus may be enlarged more vomiting vaginal bleeding

Answer 28

1 in 100 after 1st 1 in 5 after 2nd

Answer 29

maternal affect AR results in recurrent molar pregnancies the moles that are present are usually **diploid** and genetically **bi parental** which is different to sporadic moles which are diandric

Answer 30

- malignant transformation of a mole - also known as a persistent mole - usually arise form a complete mole - patients present with elevated hCG after a previous molar pregnancy also presents with abormal bleeding abdominal pain or swelling

Answer 31

malignant choriocarcinoma- highly invasive and can cause symptoms locally e.g. uterine bleeding and from distant metastasis in the lungs, CNs and liver (most common) diagnosed by raised hCG and reproductive history

Answer 32

suction evacuation recommended to remove complete and partial molar pregnancies. - after molar pregnancy hCG levels should be monitored until they return to normal. usually ~6 weeks and not adviced to get pregnant during this period if hCG does not return to normal, choriocarcinoma or metastasis is present chemotherapy with methotrexate is recommend

Answer 33

fraternal 2 different eggs fertilised by 2 different sperm more common than identical twins

Answer 34

- increased maternal age - mutations in genes in the TGF9 pathway a.e.g BMP - can be familial

Answer 35

formed from complete cleavage of single embryo

Answer 36

dichorionic diamniotic (DCDA) twins -division at ~ day 4, at morula stage monochorionic diamniotic (MCDA) twins - division at day 4-7 at the blastocyst stage monochorionic monoamniotic (MCMA) twins - embryos that divide after the amnion is formed at day 9

Answer 37

have identical genes but rarely identical due to epigenetic and prenatal environmental post-zygotic mechanisms

Answer 38

- 6 fold increase in perinatal mortality - preterm delivery - IUGR MZ twins have poorer outcomes than DZ MCMA have highest perinatal mortality rate

Answer 39

vascular connections between the feto-placental circulation in each twin - risk on MCDA - monochorionic diamniotic (MCDA) twins

Answer 40

Twin reversed arterial perfusion (TRAP sequence) is a rare condition of monochorionic twin pregnancies. It arises when the cardiac system of one twin does the work of supplying blood for both twins. The twin supplying the blood is known as the "pump twin" and develops normally in the womb. However, the increased pumping of the heart puts this twin at risk for cardiac failure. The other twin — known as the "acardiac twin" — lacks a heart or has one that is not fully formed. It usually has a poorly developed body and may also be missing a head, limbs and torso

Answer 41

MC twins artery to artery and vein-vein anastomoses

Answer 42

one twin is acardiac and perfused in a reverse direction by the pump twin - acardiac twin has abn development of head, thorax and or limb - 'normal' pump twin has poor prognosis and 50% chance of survival due to cardiac overload some are chromosomally abn

Answer 43

twin twin transfusion syndrome

Answer 44

Affects **monochorionic** twins there is an imbalance in the blood exchange between the twins and the placenta. One twin — the donor twin — gives away more blood than it receives in return and runs the risk of malnourishment and organ failure. The recipient twin receives too much blood and is susceptible to overwork of the heart and other cardiac complications.

Answer 45

dihydramnios and IUGR in donor twin polyhydramnios and enlarged bladder and larger size in recipient twin can result in preterm labour due to severe polyhydramnios

Answer 46

- amnio reduction by serial amniocentesis from recipient twin to reduce pressure - laser ablation of connecting anastomoses (vessels) amnio may be sent for testing but array is not indicated

Answer 47

when 1 twin suffers early fetal death (1st trimester) and is not aborted

Answer 48

- areas of degenerate villi or amorphous debris- but often difficult to detect - may be identified by the presence of an empty sac on USS - can explain otherwise inexplicable anomalous maternal serum and alpha fetoprotein and CVS chromosome result

Answer 49

when a twin suffers death and isn't aborted but unlike in a vanishing twin the dead fetus persist (fetal death occurs beyond the first trimester)

Answer 50

determine the degree of identity in the genome of twins

Answer 51

generally requested when one twin has developed a clinical phenotype that is thought to be genetic in origin also used to in transplantation where a twin is a HLA match- if they are identical there is less likely to be GVHD in the recipient

Answer 52

Use QF-PCR to detect microsatellie markers the likelihood of zygosity is then calculated using bayes. the prio risk of monozygosity is 1/3 and dizygosity is 2/3 -if sex is know this can be added (1/2) - the probability of being dizygotic but with the same alleles by chance is calculated from the possible combination of parental alleles.

Answer 53

- amplifies microsatellite markers on target chromosomes for semi-quantitative detemination of ploidy - multiplexed and uses conditional to allow multiple PCR reactions to be run together - products are differentiated by using different colour dyes and product sizes using capilliary electrophoresis

Answer 54

reaction is stopped in the exponential phase- PCR reagents are not depleted In the exponential phase the level of product is proportional to the amount of starting material and can be used to determine the relative CN of the chromosomes tested BPG recommend 24-26 cycles

Answer 55

Need 2 informative markers- remaining can be inconclusive but not contradictory to the result being reported. recommended to have at least 4 markers per chromosome to increase the chance of an informative result when choosing markers need? - high heterozygosity in the general population - SNP checked - majority are 4bp repeats as these have fewer stutter peaks than dinucleotide repeats

Answer 56

sexing may or may not be included depending on local policy- risk of sex selection of fetus AMEL- present on X and Y and gives peaks 4bp apart. cannot detect sex chr no. but can detect their ration e.g. 1:1 XY or 2:1 XXY SRY- Y chromosome specific non-polymorphic marker to confirm presence or Y TAF9- present on chr3p and Xq used to determine X chromosome copy number based on the assumption that 3p will have a CN of 2 - can be used to distinguish XO from XX with all homozygous markers

Answer 57

peak height/area of smaller peak divided by the peak height/area of the larger peak

Answer 58

1:1 alleles with a range from 0.8-1.4 for alleles >24bp apart a ratio of 1.5 is acceptable because of preferential amplification of the smaller allele

Answer 59

1:1:1 - triallelic and confirms meiosis 1 NDJ 2:1 ratio 1.8-2.4 1:2 ratio 0.46-0.65

Answer 60

a biallelic pattern could indicate meiosis II NDJ or mitotic NDJ and CPM should be considered for CVS - report cautiously - meiotic NDJ not confirmed - follow-up karyotype on cultured cells which contain the mesenchyme and are more representative of the fetus - suggest detailed USS and AF sampling

Answer 61

0.6-0.8 and 1.4-1.8 - can be resolved by running extra single chromosome markers - cannot report a sample as trisomic if there is a single inconclusive marker or single normal marker for that chromosome - may be due to preferential amplification of the smaller allele and can result in inconclusive ratios the greater the distance between the 2 markers.

Answer 62

2018 BPG trisomic results do not need confirmation by another method but sample ID must be confirmed before reporting- usually by repeating the original sample normal result do not need to be repeated

Answer 63

occasionally if normal but this should be stressed on the report- ideally another method e.g. karyotype of FISH should then be used as follow-up to confirm the result for the chromosome

Answer 64

- skewed/inconclusive results for all chromsomes - normal female results - need to request maternal blood if there is a mixed genotype there will be 3 alleles: - 1 fetal (pat) specific, - 1 mat and fetal shared and - 1 mat specific. In a normal sample: the sum of the peak of the maternal and fetal specif alleles should add up to the fetal+mat shared allele and give a normal ratio AMEL can be used to quantify the fetal genotype in a male- if a normal result with non skewed XY is obtained a normal result can be issued

Answer 65

BPG - low level MCC - majority fetal genotype with no inconclusive markers- report - single fetal genotype with no inconclusive alleles- report - inconclusive alleles with mixed genotype- do not report QF-PCR is sensitive to MCC to ~10%

Answer 66

AF-MCC may be obvious due to bloodstaining wither before or after centrifuging - if normal female result obtained need to request mat sample before reporting - could have a tissue plug resulting in MCC with no obvious bloodstaining - culturing will remove MCC from blood as the culture will favour growth of the amniocytes, but contamination from mat tissue may grow CVS- carfeul dissection to remove maternal decidua required to prevent MCC risk - analysis of cultured cells may help but need to be cautious as maternal tissue may still be present and could predominate

Answer 67

presence of extra peaks, skewerd markers for just one chromsome markers may be subtle with alleles still in the normal or inconclusive range diploid/triploid mosaicism can be difficult to distinguish from MCC

Answer 68

- mitotic rescue of a trisomic cell line - mitotic NDJ and generation of a trisomic cell line in a normal conceptus Need to be wary of CPM in CVS samples

Answer 69

SMM PSP SMD partial chromosome imbalance CNV

Answer 70

No - stutter peaks may be incorporated into the peak for the shorter allele resulting in misinterpretation of allele ratios

Answer 71

somatic microsatellite mutation - single marker with 1:1:1 ratio and unequl peak heights (2 will add up to the larger one) - due to mutation of an allele and mosaicism for the de novo and orginal allele - caused by defective proofreading during DNA rep in early embryogenesis cant use as an informative marker for reporting, lower annealing temp to distinguish from a PSP

Answer 72

polymorphsim to primer binding site resulting in efficient amplifcation of an allele and a single inconclusive result 0.6-0.8 or 1.4-1.8 repeating with a lowered anealing temp should normalise the ratio

Answer 73

submicroscopic micrsatellite dup - trisomic result for a single marker- all other consistent with normal - some are inherited and benign- list on known inherited SMDs available - if flanked by 2 normal markers assumed to represent an SMD and not reported - parents may be requested to check if SMD is inherited and not associate with abn phenotype distinguish from PSP be repeating at lower annealing temp detail findings on report if not considered benign and extra testing with flanking markers may be required

Answer 74

normal and abnormal results for the same chromosome- 2 or more consecutive markers showing the same result follow up with karyotype, array, parental bloods

Answer 75

normal and abnormal results for the same chromosome- 2 or more consecutive markers showing the same result follow up with karyotype, array, parental bloods

Answer 76

twins can be identified as dizygotic or probably monozygotic which may be clinically useful - likelihood of monozygosity is determined by the number of informative markers and the maternal/paternal relatedness sampling the same twin twice cant be excluded which twin the report is for should be clearly identified e.g. twin 1 upper left

Answer 77

cheaper needs less sample less labour intensive can detect MCC in all pregnancies not just male can detect UPD may detect some unbalanced rearrangements

Answer 78

- assumption that fetal material was tested - result is consistent with trisomy as only specifc markers tested - include interpretative sentence e.g. consistent with a diagnosis of down syndrome 2 alleles results- risk that it may represent CPM- follow up karyotype, AF and USS - should perform karyotype follow-up of abnormal results to determine the structure and recurrence risk

Answer 79

- does not give structure - higher resolution that G-banding - cant detect MCC in female pregnancies but may be suspected if there is mosaicism - more expensive ans time consuming that PCR - higher failure rate that PCR - need to scan lots of cells are there can be a range of artefactural staining e.g. monomsomy from co-localisation of 2 signals or trisomy from 2 cells being on top of each other. need to scan even more if referral indicates mosaicism

Answer 80

repetitive sequence probes are targeted to the alpha satellite sequences found at the centromere - composed on array on tandomly repeated monomers the are sufficiently different to allow distinction of all chromosome except chr13 and 21 and chr14 and chr22 so need to look for 5 signals for a trisomy and follow-up testing required to confirm the chromosome involved. target specific probes targeted to specific sequences on the q arms of chromosomes are therefore preferred

Answer 81

1-1.5% of pregnancies have major congenital abnormalities 20-25% of these are chromosomal

Answer 82

higher resolution than karyotype increases diagnostic yeild - BPG recommend minimum resolution of 400Kb - 2.4% increase in diagnostic rate for all and 7% for abscan referrals - carriers of familial rearrangements not detected - culturing generally not required (except small samples or MCC cases

Answer 83

- increased resolution can increase the pick up of VUS which can be difficult to interpret- may need MDT. - should only report variants that are class 4 or 5 in prenatals, may also need to request parents to aid interpretation - may miss low level mosaisicism - may not detect triploidy - may detect IFs- need reporting policy (report IFs that are clinically actionable to the family) - culturing may be required for follow-up as array does not give structure

Answer 84

Evaluation of ArrayCGH in prenatal diagnosis of fetal anomalies - multi centre study comparing aCGH to karyotype in fetus with 1+ USS abn + NT>3.5mm - 500 recrutied - QF-PCR followed by aCGH for those with a -ve result - ISCA arrays - variants categorised as benign, VUS or pathogenic (arrays are now using a 5 clas system as with SNV interpretation) - only reported variants associated with a known clinical syndrome of significant chromosomal imbalance

Answer 85

- to optimise prenatal DNA extraction techniques - incoporate aCGH into standard lab service - all CNVs recorded in a standard way and in a central database

Answer 86

CMA is a robust, cost effective, acceptable diagnostic technique for prenatals with a NT >3.5mm and US abn and should replace karyotype 1-3% increased diagnosis compared to karyotype

Answer 87

2015 working group on behalf of the joint committee on genomics in medicine - gudiance on reporting in prenatal samples e.g. whether come low penetrances susceptibility loci should be reported recommends that: NIPA1 dels and dups are not reported 15q13 dups not reported STS dups not reported 16q13 dups not reported het del of gene that cant be linked to phenotype i.e. carrier status should not be reported

Answer 88

at the morula to blastocyst stage many abnormal embryos arrest ~ 30% of pregnancies fail to implant and another 30% implant transiently - this results in little disruption to the menstrual cycles cause is commonly polyploidy or monosomy and frequency increases with advancing maternal age

Answer 89

fetus is incapable of surviving independently < 24 weeks, therefore below this is classed as miscarriage and above this is a still birth

Answer 90

50% are chromosomally abnormal majority are trisomy, triploidy and monosomy X 60% single trisomy 12-15% monosomy X 12% triploidy 3% tetraploidy ~5% structural rearrangement ~5% multiple aneuploidy

Answer 91

~10-15% usually towards the end where the placenta takes over the nourishment of the fetus so the body recognises abnormality

Answer 92

T16 T22 T21 T15 T13/18 only T21,18 and 13 are seen in liveborns non-mosaically the frequency of trisomy in pregnancy is greater than in live birth as many are lethal and will result in miscarriage

Answer 93

T16 is seen in 1% of pregnancies and accounts for 10% of pregnancy loss those that survive are mosaic or CPM and only in placenta- this can result in placental insufficiency and IUGR

Answer 94

Majority are from an error (NDJ) in maternal meiosis I - T18 associated with NDJ in meiosis II significantly associated with maternal age

Answer 95

2-5% with 20% chromosome abn rate

Answer 96

20% chromosomally abnormal - more likely to be viable trisomies (21, 18, 13) as others lost in 1st trimester) - increased proportion of structural rearrangments

Answer 97

reduced proportion of aneuploidy (T21, T18, T13 and 45,X) increased proportion if sex chr abn and structural rearrangements and there is less genetic imbalance than in a trisomy

Answer 98

T16- 100% unless mosaic T21 - 80% T18 + T13 - 95% 45,X- 95-99% tertraploid- almost 100% triploid- almost 100% balanced rearrangement- 15% unbalanced rearrangement- 95% (depending on size of imbalance) normal karyotype- 8%

Answer 99

4 copies- usually due to failed early mitotic division resulting in duplicated genome and 92,XXX or 92,XXYY 2-3% of spontaneous miscarriage

Answer 100

10% of miscarriages 2/3 are diandric due to dispermy - partial hyadatiform mole and IUGR - most miscarry in 1st trimester a few make it to second trimester 1/3 digyny due to diploid egg from NDJ of complete chromosome set in oogenesis or failure to expel polar body - misacarry in 1st trimester - severe IUGR, macrocephaly and small placenta (non molar)

Answer 101

95-99% die in utero hypothesised that complete 45,X is lethal and that all TS patients have some level of mosaicism

Answer 102

Only seen for chr 21 ~1 in 1000 karyotyped abortions

Answer 103

diandric diploidy- complete mole due to 2 sperm fertilising and empty egg - increased risk of choriocarcinoma digyny diploidy- ovarian teratoma. fatal. may contain fully differentiated tissue e.g. hair and nails

Answer 104

likelihood of miscarriage of abnormal offspring is dependent on the size and genetic content of the imbalanced region (if unbalanced); balanced rearrangements seen in the same frequency in liveborns

Answer 105

CPM with the abnormality only in the placenta can still result in pregnancy loss due to abnormal development of the placenta which can have a physiological effect on the fetus

Answer 106

3rd consecutive recurrent miscarriage (<24weeks)-n RCOG = test the POC and follow- up in parents if an abnormality is detected. Only test parents by karyotype in exceptional circumstances pregnancy loss with significant fetal abnormalities or termination (irrespective of gestation) still birth (pregnancy loss >24 weeks)

Answer 107

epidemiological factors parental/ embryonic factors infections acquired thrombophilic defects anatomical abnormalities (uterus, cervix) maternal endocrine and immune anomalies (thyroid dysfunction) UNEXPLAINED

Answer 108

QF-PCR for common aneuploidies and sex - some centres also include other trisomies common in miscarriage 16, 22, 14, 15 ArrayCGH Karyotype no longer performed as tissue can be hard to culture and prone to infection or failure plus array provides a higher resolution and expecting to find imbalance. Donaghue et al 2017- PCR and array = lower failure rate and higher yeild that karyotype or MLPA strategies

Answer 109

11-14 weeks - pregnancy dating - ID multiple pregnancies - placental location - ID fetal anomalies (50%) can be detected at 1st trimester scan

Answer 110

subcutaneous fluid under the skin at the nape of the neck - most powerful measure for detecting aneuploidy - included in the combined score (with mat age, PAPP-A and B-hCG) - requires a highly trained sonographer to be measured and variation in measurement has been reported

Answer 111

increasing size is correlated with increased risk of chromosomal abn >3.5mm is significant and may be due to chromosomal abnormality (T21, 18, 13, XO), cardiac defect/failure or delayed development of the lymphatic system can either regress or evolve into cystic hygroma, oedema, fetal hydrops in second trimester

Answer 112

USS markers that may be associated with anuploidy but are also seen in normal pregnancies - absent nasal bone (seen in 2% of normal fetus and 9% in normal afro-Caribbean pregnancies) - dilated cisterna magna - echogenic foci in the heart - 2 vessel cord - echogenic bowel with density lower than bone

Answer 113

DS screening programme has increased the detection of aneuploidy at first trimester therefore if normal variants are detected at the second trimester and the mother had a normal combined screen score they are not referred for genetic testing

Answer 114

ventriculomegaly >10mm echogenic bowel with density greater than bone nuchal fold >6mm renal pelvic dilation

Answer 115

holoprocencephaly- failure of brain to divide into 2 lobes (T13, 18, SHH gene) Exomphalos- herniation of fetal abdomen 9t18, 13 or BWS) megacystis - enlarged fetal bladder (T13 and 18) skeletal abn e.f. T21, 18 and X) associated with shortened long bones

Answer 116

diaphragmatic hernia short limbs abnormal hands and feet

Answer 117

IUGR microcephaly cleft palate heart defect

Answer 118

heart defect cleft lip/palate

Answer 119

lissencephaly (LISS1)

Answer 120

cardiac abn (TOF) renal abn polyhydramnios cleft lip and palate

Answer 121

IUGR, cardiac defects

Answer 122

overgrowth, exomphalos

Answer 123

asymmetrical IUGR

Answer 124

short long bones frontal bosing

Answer 125

cardiac defects-VSD duodenal atresia IUGR hydrops echogenic bowel renal anomalies syndactyly

Answer 126

rocker bottom feet talipes clenched hands exomphalos VSD oesopageal atresia IUGR renal abn neural tube defect

Answer 127

holoprosencephaly diaphragmatic hernia cleft lip/palate IUGR renal abn

Answer 128

cystic hygroma hydrops increased NT and nuchal fold fetal oedema IUGR renal abn 2 vessel cord

Answer 129

IUGR holoprosencephaly 2 vessel cord partial mole in diandric IUGR, macrocephaly and small placental in digyny

Answer 130

PIGD aims to select embryos that are unaffected for transfer to the uterus to prevent the birth of a baby with a disease causing genetic mutation.

Answer 131

1. biopsy of polar body just prior to conception (1st polar body) or just after fertilisation (2nd polar body) 2. biopsy of day 3 cleavage stage embryo (5-cells) 3. biopsy of trophoectoderm from bastocyst (day 5-6 embryos)

Answer 132

used to be FISH but now being replaced by array (structural) and PCR (molecular diagnosis by linkage) can also do direct mutations analysis by sanger

Answer 133

CF, HD, NF1, DMD/BMD, chromosomal rearrangments

Answer 134

Preimplantation genetic screening-screens embryos for aneuploidies with the aim of selecting euploid embryos for transfer the use for PGS is controversial, at least 11 randomised control trials have found no clinical benefit and 1 study has shown that PGS significantly lowered the birth rate from IVF when applied to women of advanced maternal age Not currently supported by or offered by the NHS

Answer 135

NHS reference group for medical genetics published a clinical commissioning policy for PGD which outlines the conditions under which PGD can be commissioned

Answer 136

- provide guidance, consistency and clarity in the commissioning of PGD services - improve equality of access to PGD - ensure PGD is offered for diseases where there is sufficient evidence of clinical benefit and cost effectiveness - reduce variation in clinical practice and conditions referred for PGD

Answer 137

NHS will commission up to 3 courses of PGD for a couples - at risk of having a child with a sever genetic disease >10% risk - fertile- different to IVF treatment for infertility - HEFA licence for the disease - genetic counselling - female partner <40yrs and BMI between 19 and 30 - both partners should be non-smokers

Answer 138

not offered for sex selection for family balancing in the UK - PGS is not commissioned in the NHS as there is no evidence that it provides clinical benefit - not to be used to address fertility problems or recurrent miscarriage with unknown aeitiology

Answer 139

The Human Fertilisation and Embryology Authority (HEFA) oversees the use of gametes and embryos in fertility treatment and research human fertility embryology authority - licence and monitor UK fertility clinics and all research involving embryos on the UK - licence that diseases for which PGD can be offered

Answer 140

IVF--> biopsy cells --> genetic test --> transfer unaffected embryos

Answer 141

in IVF a women undergoes ovarian stimulation then oocyte removal. The oocyte can then be fertilised by a sperm in a petri dish. In IVF the sperm and the egg and incubated in the same petri dish and the sperm are left to fertilise the egg. Improved fertilisation can be achieved by ICSI where the sperm is injected into the egg. sperm for ICSI are morphologically selected- sperm with a large vacuole are more likely to result in aneuploidy

Answer 142

- 1st and 2nd polar body - cleavage stage embryo (day 3) - blastocyst (day 5-7) trophoectoderm - balstocyst- blastocoele fluid

Answer 143

longer time to complete genetic testing without requiring crypreservation, can only detect maternal mutations generally only used in countries where testing cleavage stage embryos is illegal - Austria, Switzerland and Germany

Answer 144

well assessed methodology may hamper embryo viability 60% show mosaicism for anueploidy but rectified by the blastocyst stage

Answer 145

more cells to test = more DNA for analysis may not be representative of the inner cell mass which goes on to develop into the embryo

Answer 146

less invasive and safe and contains DNA from ICM and trophoectoderm only ~ 50% of samples contain DNA suitable for analysis and there can be low concordance with cleavage stage embryos

Answer 147

embryoonic DNA is isolcated from the blastocyst culture medium combined with blastocoele fluid can be used for genetic testing. - aspiration of blastocoele fluid is less technically challenging and invasive than TE cell biopsy origin of DNA in the blastocoele fluid is still under investigation and is routinely discarded during hr vitrification process to prevent formation of crystals

Answer 148

Monogenic disorders - 1st PGD cases where directed at the Y chromosome for X-linked disease - HD is the most commonly tested for AD disease - direct and indirect testing is available - CF- due to the high number of mutations a muliplex linkage based approach has been used so that the same design can be used for all mutations.

Answer 149

PGD can be used to select embryos with a low level of the mutatn mtDNA less prone to allele drop our as there is a high copy number of mtDNA when there is a high level of mut mtDNA or homoplasmy PGD is not suitable main issue is even if an embryo is selected with a level of heterpolasmy below that associated with disease they may still be affected due to the mitochondrial bottleneck which could result in higher levels in the fetus

Answer 150

PG haplpotyping used for diagnosis of monogenic disorders by using linkage to determine if the high or low risk allele has been inherited - same test can be used for all families rather than needing to design mutation specific tests for each mutation - can be used for disease where the causative mutation has not been determined - ideally need DNA from parents and an affected proband to ID the high risk allele but an unaffected family member can be used for exclusion testing - useful if mutation is not amenable to PCR detection - need markers either side of gene and should determine that there are enough informative markers before offering test - need to determine the risk of a double recombination resulting in low risk haplotype carrying the causative mutations - where there is an informative marker either side of the gene the risk of a double recombination is much lower than if the informative markers are only on one side.

Answer 151

used bacteriophage for whole genome amp meaning lots of DNA can be generated from small samples for genotyping multiple polymorphic markers

Answer 152

confusion of embryo and cell line number transfer of the wrong embryo mat/pat contamination allele drop-out probe/primer failure chromosomal mosaicism

Answer 153

children born from ART are at a greater risk of suffering from an imprinting disorder -> 10% increased risk of BWS this is because the epigenetic genome undergoes 2 waves of reporgramming - in primordial germ cells - during preimplantation genetic development ART is though to interfere with this however there is not a large cohort to study and this also makes analysis of trangenerational effects on imprinting difficult to study - most parents will still chose to have PGD despite the associated risks

Answer 154

- morally acceptable? many people put higher value on the fetus than embryo making PGD preferable to PND - the risk of designer babies if exaggerated and improbable providing there is adequate legislation - need to consider the severity of disease, age of onset (number of disease free years), treatments available - variable penetrance diseases?

Answer 155

not permitted in most centers client may not want to know if they have the disease but this is unethical to uphold if you find they have zero risk as they will be undergoing unnecessary and risky procedures for no reason

Answer 156

used to conceive a child that is a HLA to a sibling so they may be a stem cell donor for a critically ill sibling if no other donor is available preferential procedure may be to harvest embryonic stem cells from matched embryos

Answer 157

1-2% in CVS 0.1-0.3% in AF lower level in AF likely represents pregnancy loss in the time between CVS and AF sampling and CPM

Answer 158

fertilisation --> zygote--> early blastocyst - trophpectoderm goes on to form the placenat and extra-embryonic material - ICM form the amnion and embryo the amniotic cavity and embryo are formed from a different cell lineage from the chorion. The chorion differentiates early and is rapidly dividing do more likely to contain mitotic errors.

Answer 159

AF contains cells shed from the embryo- skin, fetal urine, cells from the fetal respiratory tract cell are derived directly from the phenotype and are more likely to be a true representative of the fetal genotype

Answer 160

Mitotic NDJ Trisomy rescue

Answer 161

level 1- 1 cell in 1 culture - pseudomosaic level 2- > 1 cell in 1 culture - may be pseudomosaic or true mosaicism level 3 - > 1 cell in > 1 culture- likely to represent true fetal mosaicism and reported type 1 and 2 may be a cultural artefact

Answer 162

basic- 20 cells from 2 cults, 1 of which may contain an abnormal metaphase moderate- 20 cells from an additional separate culture extensive - 20 cells from 2 additional separate cultures

Answer 163

single cell abn can be clinically significant and 30 additional metaphases should be screened If FISH is used 50 cells should be screened for exclusion of mosaicism

Answer 164

pseudomosaicism is considered an artefact by application of Hsu and Benn if level II mosaicism is sen on CVS it may be appropriate to mention on report and recommend additional follow-up with USS and AF for level III mosaicism the number of cells in each cell line should be included in the report with the caveat that the levle may be different in other tissues if a chr involved in UPD is involved UPD studies should be activated

Answer 165

iso(12p) - normal karyotype in peripheral blood due to the high turnover rate of cells and instability of the iso(12p) meaning it is lost from these cells - can be detected in skin, AF, CVS - only ever seen as mosaic

Answer 166

small piece of extra chromosomal material - smaller than chr 22 and unable to be clearly identified by G-banding alone - usually present in additional to the normal chromosome complement and termed supernumary marker chromosome or extra structurally abnormal chromosome

Answer 167

ring inv dup centric minute

Answer 168

size genetic content level of mosaicism euchromatic/heterochromatic result in copy number gain of the region in the marker

Answer 169

markers derived from chromosome 15 are the most common

Answer 170

+ psu idic (22)(q11.2)

Answer 171

+ der(22)t(11;22)(q23;q11)

Answer 172

prenatal or postnatal array- detect presence of duplication and investigated by follow-up karyotype to confirm as a marker karyotype investigation of an infertile male/ POF female

Answer 173

- determine genetic content - check to see if it is mosaic - test parents to inform on pathogenicity and recurrence risk - if marker involves a chromosome involved in UPD it is relevant to check for UPD for the sister chromosome for UPD

Answer 174

arrayCGH best first option If the marker only contains heterochromatin may require FISH to determine origin chr 15 most common

Answer 175

if inherited more likely to be benign - phenotype expected to be similar to parent however may be different if mosaic as level and tissue distribution can affect penetrance

Answer 176

provide structural info main limitation is resolution can be used to determine level of mosaicism and is more sensitive than array

Answer 177

C-banding can be used to determine the amount of heterochromatic and determine if a marker is acrocentric or not. can also determine the number of centromeres to ID is mono or dicentric markers that are all het are expected to have low risk as they contain little active genetic material

Answer 178

stains ribosomal RNA in the nucelolar organiation region (NOR) - found on short arms of acrocentric chromosomes

Answer 179

can use sequential FISH - if acrocentric start with 15 - limited benefit if inhertied from normal parent so may just want to start with chromosomes involved in UPD (6,7,11,14,15,) - costly and inefficient - if abn pheno may also want to FISH for chr 12, 18 and 22 and iso chro associated with severe pheno

Answer 180

- if karyotype is correlated with TS the marker is likely to be derived from a sex chr e.g. 45,X + mar - important to determine if Y chr derived due to the risk of gonadoblastoma - if X chr derived can test for Xist- if present marker will be inactive and more likely benign XIST = X Inactive Specific Transcript

Answer 181

- can ID full extend of marker and hence gene content and clinical significance - may miss low level mosaic and het markers - can ID markers consisting or more than 1 chromosome - cannot ID the structure

Answer 182

need to distinguish between benign small marker idic(15) and larger pathogenic marker idic(15) which contains the PWS/AS region and is associated with LD, epilepsy and ASD

Answer 183

may see dynamic mosaicism and loss, gain, doubling up in different cells resulting in generalised ring syndrome and short stature

Answer 184

cells apoptosed from the outer layer of the placenta - cytotrophoblasts and syncytiotrophoblast - therefore it is at risk of CPM

Answer 185

can be observed from ~ 5 weeks but generally need to wait till 8-10 weeks before there is enough for reliable testing (high enough fetal fraction) benefit is testing in early pregnancy

Answer 186

cffDNA is ~200-300bp long shorter than mat cffDNA and this difference can be used to enrich for cffDNA for genetic testing ang

Answer 187

inner mesenchyme, diofferentiates later and more closely related to fetus outer cytotrophoblast

Answer 188

early CVS is associated with fetal limb defects taken in 1st trimester 10-13+6 weeks gestation

Answer 189

1-2% according to RCOG, risk influenced by experience of operator and due to infection, amniotic leakage or damage to the placenta

Answer 190

10-25mg of CVS is taken from the placenta via a needle and syringe under USS guidance

Answer 191

can be taken in earlier gestation giving more time for pregnancy decision making and follow-up testing Risk of CPM slightly increased miscarriage rate

Answer 192

fluid filled sac that surround the developing fetus and facilitates symmetric growth and lung development cells shed from fetal skin, fetal urine and fetal respiratory tract = more representative of true fetal genotype

Answer 193

transabdominally using syringe 10-20mls

Answer 194

0.5-1% RCOG

Answer 195

2nd and 3rd trimester from ~15 weeks, early AF has an increased miscarriage risk and low test success as not enough fetal cells. late gestation AFs are also more dirty and prone to failure

Answer 196

from ~ 18-20 weeks when veins are sufficiently developed

Answer 197

blood is taken by needle from umbilical cord or fetal blood vessels

Answer 198

most commonly for: - suspected fetal blood disorders e.g. fetal anemia - inconsistent CVS and AF result follow-up - fetocide/MTOP - immunologic tests

Answer 199

pleural effusion when excess fluid removed POC from miscarriage or TOP

Answer 200

CVS- need to carefully dissect to remove the maternal decidua AF- bloodstaining may be maternal (or fetal) also risk of tissue plug - discarding the first draw of AF can reduce the risk of MCC

Answer 201

culturing conditions for AF favors the growth of the amniocytes to reduce or eliminate mat blood cells In CVS and POC the culturing process does not reduce the risk of MCC as the maternal and fetal tissue will both be grown

Answer 202

75% confluent monolayer- can be less for a targeted test of a few cells

Answer 203

- normal female in bloodstained sample - XX/XY mixed genotype - presence of mixed genotype on PCR - mosaicism in a female - uncertainty about the ID of a tissue sample in culture - slow growth, especially when originating from a single piece of tissue or a small number of colonies.

Answer 204

if MCC suspected for all monogenic tests- need to test mate sample against prenatal. lab use QF-PCR whihc has sensitivity to ~10% therefore can only state that significant MCC has been excluded BP for the testing lab to do the MCC and needs to be on the DNA aliquot that was tested. sensitivity of MCC assay may be lower than of the test so confirmation of result that are the same genotype as the mother may require confirmation y another method e.g. linkage following positive PCR result should not issue report until MCC has been completed

Answer 205

if MCC suspected for all monogenic tests- need to test materal sample against prenatal. lab use QF-PCR which has sensitivity to ~10% therefore can only state that significant MCC has been excluded BP for the testing lab to do the MCC and needs to be on the DNA aliquot that was tested. sensitivity of MCC assay may be lower than of the test so confirmation of result that are the same genotype as the mother may require confirmation y another method e.g. linkage following positive PCR result should not issue report until MCC has been completed

Answer 206

0.1-0.3% (lower than CVS)

Answer 207

- observation of aneuploidy in CVS but not in AF = risk of UPD by trisomy rescue (chr 6, 7, 11, 14, 15) most pregnancies with CPM continue to term with no abn but in some cases the abn in the placenta can lead to placental insufficiency and IUGR or other abn

Answer 208

 Origin of error – somatic errors are associated with less severe consequences.  Level of mosaicism – there is a correlation between a high number of aneuploid cells and poor pregnancy progress.  Specific chromosomes – the influence of CPM on fetal growth is chromosome specific. Certain chromosomes carry imprinted genes involved in growth or placental function, which may contribute to impaired pregnancy progress when CPM is detected. CPM involving sex chromosomes usually has no adverse effect on foetal development.  Type of chromosome abnormality – marker chromosomes are more often confirmed in the fetus than trisomies.

Answer 209

- test multiple villi and chop/digest to ensure mesenchyme and trophoblast are sampled - follow-up mosaic CVS with long term cult as mesenchymal core culture results are more likely to reflect a true fetal mosaicism than direct preparation - follow-up mosaic with AF and detailed USS decision regarding pregnancy should not be made on the ????

Answer 210

 Mitotic CPM – Mitotic **non-disjunction** can occur in a trophoblast cell or a non-fetal cell from the inner cell mass creating a trisomic cell line in the tissue which is destined to become the placental mesoderm. Trisomy 2, 3, 7 and 8 are derived mitotically.  Meiotic CPM –Due to **trisomy rescue of a trisomic conceptions** Trisomy 16 and 22 are derived meiotically.

Answer 211

- reduced or improved replicative rates of the normal and abnormal cell lines - abnormal cells may fail to differentiate or function properly - may be no selection against abnormal cells but there presence could compromise the pregancy e.g. placedntal insufficiency, IUGR

Answer 212

 Type I, the abnormal cell line is confined to the cytotrophoblast (40%)  Type II, it affects only the mesenchymal cells of the stromal villous core (40%)  Type III, it involves both tissues. (7%) Type I and II CPMs are usually the consequence of nondisjunction events, and type III is the result of “trisomy rescue” type III carries a risk of CPM in the rescued diploid fetus

Answer 213

trisomies 8, 9, 12, 13, 15, 18, 20, and 21

Answer 214

Karampetsou et al. (2014) report a case of CPM involving a deletion of exons 7-10 of the STS gene detected by array-CGH using DNA extracted from uncultured CVS. therefore suggested that reports on uncultured CVS should include the rider that CPM has not been ruled out and ideally confirmation should be on cultured CVS or AF