Session 4 - Screening and biochemical genetics Flashcards
1
Q
Define Linkage
A
A statistical method to determine the link between a region of the genome and a given genotype in a family or group of affected patients.
It is performed on small sets of data, often large pedigrees, but can also be used for sib-pair analysis.
Used to identify genes with large effects, often Mendelian.
2
Q
Define an Association study
A
A statistical method of determining association between SNPs and disease in large populations.
Performed on large cohorts in a case-control fashion.
Can identify genes with small effects
3
Q
What are the advantages and disadvantages of Linkage analysis?
A
Advantages:
Localisation of disease region on a chromosome.
Multiple markers can be studied at once
Disadvantages:
Need multigenerational cases
Difficult to study complex traits, however data from multiple families can be combined to increase the power of the study
4
Q
What is used to measure Linkage? What is the cut off?
A
LOD scores
no linkage -2 < indeterminate < 3 Linkage
5
Q
Define linkage disequilibrium
A
The likelihood that two independent markers will be inherited together at a frequency greater than chance. This is different from Linkage.
6
Q
What can cause linkage disequilibrium?
A
Natural selection
Chance
7
Q
How is linkage disequilibrium expressed? How is it calculated?
A
D
D(ab) = P(ab) - P(a)P(b)
Alleles in complete LD have a score of 1.
8
Q
What factors affect LD?
A
Recombination Gene conversion Selection Population structure New mutations Genetic drift Gene flow Population history.
9
Q
What is the Transmission Disequilibrium Test (TDT)?
A
A method of linkage analysis that can be performed in trios or between sib-pairs. It involves calculating the likelihood of two affected individuals inheriting the same allele from parents. It is used to study complex disease traits in families. Overtransmission of an allele suggests disease susceptibility
10
Q
Name a successful Linkage study
A
Identification of NOD2 in Crohn’s disease
11
Q
Name a successful GWAS study
A
Identification of associations between genotypes and age-related macular degeneration
12
Q
Name the 4 possible explanations for finding a positive association between variant and disease.
A
- There is a true association
- variant is in LD with the true variant.
- Association by chance
- Case-controls my be selected from different populations
13
Q
Give some advantages and disadvantages of GWAS
A
Advantages:
No pre-hypothesis
Good for studying complex disease
Tend to have better genomic resolution that linkage studies.
Disadvantages:
Need large cohort of ethnically matched cases and controls
Cannot test causality
Can be expensive
Doesn’t test for CNVs
Large number of statistical tests means a very low P value is required.
14
Q
What assumptions are made in order for GWAS results to be valid?
A
Case-controls are ethnically matched
Case participants reflect disease phenotype
Genomic and clinical data are collected in the same way in cases and controls.
15
Q
How can study power be defined? What is it determined by?
A
The ability of a study design to pick up associations accurately.
The MAF of the risk allele, samples size, LD between true risk allele and another, genetic heterogeneity of the sample population, relative risk of the risk allele.
16
Q
What are the two types of linkage analysis? What information do each of them require?
A
Parametric: Gene information, MOI, penetrance, allele frequency
Non-parametric: no data.
17
Q
What type of linkage analysis can be used in consanguineous populations
A
Autozygosity mapping.
18
Q
What are the problems encountered during LOD score analysis?
A
Vulnerable to lots of sampling and experiemntal errors
Computational limitations
Locus heterogeneity
Low resolution
Parameters need to be correctly spec’d
Low penetrance and phenocopies in a family.
19
Q
What is the principle of non-parametric linkage analysis?
A
That the region of a genome in which disease-causing variants are located is more likely to be shared by affected individuals than would be expected by chance.
20
Q
What must be distinguished in non-parametric linkage analysis?
A
Whether regions are identical by descent (the patients are related and the variant has been inherited from an ancestor), or if they are identical by state (the allele has entered the family twice - not identical due to ancestral inheritance)
The frequency of the allele in the population must be taken into account.
21
Q
What can Affected Sib Pair analysis be used for?
A
To identify genes that cause multifactorial disorders in affected siblings.
22
Q
Explain the process behind ASP analysis
A
Genotype affected siblings
Look for chromosomal regions where sharing of allele is above the expected mendelian values
Identify regions of the genome that are IBD
Re-examine region to narrow down candidate area
23
Q
List some population genome studies. State their purpose
A
1000G - catalogue of human genetic variation - 26 populations ~2600 individuals
100KG - stimulate UK genomics, introduce genomics into healthcare, identify new disease, produce ethical programme based on consent.
COSMIC - Catalogue of Somatic mutations in Cancer
HapMap - Identify linked haplotypes (international)
ExAC - Aggregation of data from multiple large scale genome studies. 60706 individuals from multiple populations. Now GnomAD
EVS - combination of multiple US studies
The Cancer Genome Atlas - somatic mutations identified in different cancers. Tumour profiling and clinical information.
CHIP - identification of mutations normally associated with haem-onc that don’t have disease
PAGE - 1000 foetus/parent trios with abscan of NT>4mm, looking to develop good exome test for prenatal testing
DGV - database of CNVs and SVs in the normal population
DDD - 12,000 trios with ID
CR-UK stratified medicine programme - aim to combine genetic testing with trial enrollment. 2 stages. First stage looked at 10 genes in multiple tumours to ID markers for disease/therapy/prognosis. Stage 2 now looking at the Lung Cancer Matrix Trial of nsc lung cancers to identify genetic faults driving their disease.
ENCODE
24
Q
What types of DTC testing are there?
A
Testing for predisposition to disease Dispositional health tests (warfarin dose, lifetime risk of dementia) Nutrigenomics Compatibility tests Paternity tests Related-ness tests Ancestry testing
25
What motivates people to chose a DTC test?
Curiosity
Taking part in research
Generate actionable knowledge
26
What concerns to consumers have?
```
Insurance
Confidentiality
Privacy
Unwanted information
Unreliable test results
Not understanding results
```
27
What impact do the results of DTC testing have?
Emotional and psychological stress or happiness if results are all low risk
Changing behaviour
Able to carry drug sensitivity card with them
28
List issues with DTC testing
Inadequate provision of pre and post test support.
People not being prepared for a bad result
False reassurance if found to be negative
Poor understanding of results by GPs
Costs mean there is no equity of access
29
What ethical considerations are their surrounding DTC testing?
1. Autonomy - The patient has the right to know
2. Beneficience - With-holding information may prevent an individual taking preventative action
3. Non-maleficence - may give false reassurance or unnecessary burden
4. Justice - inequity of access due to cost
30
Which organisation has introduced legislation to protect consumers ordering DTC genetics tests?
ESHG
31
Give the 9 points summarised in the ESHG policy on DTC testing
1. Clinical utility of a test shall be an essential criterion when deciding whether to offer the test
2. Labs should comply with accepted quality standards
3. Information about the purpose and scope of the test should be given before testing
4. Genetic counselling and pyschosocial assessment should be offered before some testing
5. Privacy and confidentiality must be secured
6. Avoid inappropriate testing of minors
7. All advertising claims should be transparent
8. Ethical principles, and international treaties/recommendations should be taken into account
9. Nationally approved guidelines should be followed
32
What regulations must companies operating in the UK adhere to?
Data protection act
| Human tissue act
33
Which UK advisory body has also drawn up guidelines to protect consumers?
UK Human Genetics Commission.
34
What are the WHO criteria for genetic screening programmes? Who were they drawn up by?
Wilson and Junger 1968.
1. Clinically and biochemically well defined disorder
2. Significant health burden on the population (disease frequency)
3. Disease is associated with significant mortality or morbidity
4. Effective treatment is available
5. Effective testing is available
6. Cost effective
7. Early treatment is associated with better outcomes.
35
What are the benefits of screening?
Improve the prognosis
Identify individuals at high risk of disease before onset
Less radical treatment needed
Reassurance for those with negative results
May inform reproductive choices in carriers
36
What are the disadvantages of screening?
Longer period of morbidity if there is no effective treatment
Overtreatment
Costly
False negative results give false reassurance
Anxiety from positive result
Unmask carriers
Testing minors
Informed consent
May be undue pressure on individuals to be screened
May be undue pressure to abort affected pregnancy
Genetic disclosure to other family members may be hard.
37
Name the 9 disorders currently screened for under the UK NSC newborn screening programme.
```
CF
MSUD
Homocysteinuria
PKU
MCADD
CHT
IVA
GA1
Sickle Cell
```
38
What screening tests may be available, pre-conception, and to what populations?
B-thal in Cypriots (1/7 carrier freq)
| AJ screening - 1/5 are carriers for a rare disorder
39
What are the ethical issues associated with screening programmes?
Autonomy
Beneficience
Non-maleficance
Justice
40
List some potential future uses of screening in genetic disease.
NIPS - may replace current combined screening
Rapid screening of NICU patients
Begin screening for carriers of diseases with high UK frequency (CF, SMA)
ACMG59 screening
Screening for late-onset adult disease which may be preventable
41
How common is PKU?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/10,000, AR
PAH
Phenylalanine
MS/MS for Tyrosine:Phenylalanine ratio and absolute concentrations on day 2-5 bloodspot. Genetic testing not routine.
Consider DHPR mutations - they provide a similar phenotype, but are sensitive to BH4 supplementation (a restricted deit is not required in these patients)
42
How common is CHT?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/4000, AR. 10% due to a genetic defect
Can be primary (thyroid a-/dys-genesis) or secondary (TSH insensitivity)
Cause by mutations in 8 different genes.
Screen for present of TSH. Treat with Thyroxine to prevent serious disability
43
How common is MCADD?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/10,000
ACADM, common mutation in 88% (p.Lys304Glu) clinically diagnosed cases, but only homozygous in 50% of NBSP cases
Build-up of medium-chain fatty acids (C8) that accumulate in the lysosome.
Measure using MS/MS to get ratio of C10:C8 and quantification of C8.
Treat with diet, make sure they're fed regularly
44
How common is SCD?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/2000 UK babies, AR
HBB mutation p.Glu6Val
No accumulation, two mutant b-chains produce HbS and change shape of cells.
Tested by HPLC - will also detect multiple other Hb mutations, and can detect carriers
45
How common is CF?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/2500, AR
CFTR
Abnormal Chloride channel function
IRT test using AutoDELFIA (flurescent Ab-based assay) if high, CF4 kit - if 1 mutation found - second spot, if none found - repeat IRT
46
How common is MSUD?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/120,000, AR
BCKAD complex
Leucine, isoleucine, valine, alloisoleucine - elevated levels of all are suggestive, but not conclusive - second tier biochemical tests required.
Classical form tested using MS/MS, but rarer milder forms may not be detected.
Treat with low protein diet
47
How common is Homocysteinuria?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/100,000, AR
Defects in catabolism of homocysteine to methionine
Homocysteine accumulates
Don't test within first 24hr of life as methionine levels fluctuate. MS/MS test, quantify homocys and meth
50% of patients in UK are responsive to Vitamin B6
48
How common is Glutaric acidaemia type 1?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
```
1/100,000, AR
-
Glutaric acid, lysine and tryptophan
MS/MS to quantify C5DC in blood spot
Treat with low protein diet
```
49
How common is Isovaleric acidaemia?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/100,000, AR
-
Isovaleric acid
MS/MS for C5 in blood spot - if increased, a full acylcarnitine scan is carried out to rule out GA2
50
What three methods are responsible for inborn errors of metabolism?
Accumulation of toxic metabolite
Activation of alternative metabolic pathway > toxic
Loss of product.
51
Why is a genetic diagnosis important for IEMs?
Reproductive choice
Determine carrier status and cascade screening
An exact diagnosis is offered; genotype/phenotype correlations may exist
No invasive procedures needed
Can offer a quicker diagnosis
Promotes use of specific, targeted therapies
52
Name some IEMs
```
Pompe disease
MCADD
Wilson disease
Zelwegger syndrome
Mucopolysaccaridosis
PKU
OTC deficiency
SLO
```
53
```
What is Pompe disease caused by?
What sort of IEM is it?
What are the associated clinical features?
How is it diagnosed?
How is it treated?
```
Mutations in GAA - lead to build up of GAA
Glycogen storage disorder
Three types: classical: Present in first 2 weeks of life with muscle weakness, hypotonia, cardiomegaly, HCM, feeding difficulties, deafness and failure to thrive.
Death in first year of life.
Non-classical Pompe is less severe. Less cardiac problems and present in first year of life.
Late-onset Pompe shows progressive muscle weakness, but cardiac failure is rare.
Diagnosed by reduced GAA activity in the blood
Treat with high protein diets and enzyme replacement therapy.
Two common founder mutations.
54
Name the three major haemoglobinopathies
a-thal
b-thal
SCD
55
What is Hb normally comprised of?
2a2b chains. Though HbF has gamma-chains instead of beta chains and HbA2 has delta chains instead of beta chains.
56
What is a-thal caused by?
| Where is it common?
Loss of multiple copies of the HBA (HBA1 and HBA2) genes on chromosome 16. Deletions are most common, but SNPS and small mutations are responsible for ~10%
Common in Saudi, mediterranean
57
```
What causes the following variants of a-thal?
a-thal silent carrier
a-thal minor
HbH disease
Barts hydrops foetalis
```
loss of one copy - asymptomatic
loss of two copies - mild microcytic anaemia
loss of three copies - microcytic anaemia, hepatosplenomegaly, jaundice. Transfusion may be required
loss of all copies - hydrops, not compatible with postnatal life
58
What is visible in the cells of patients with HbH disease? How are they formed?
Heinz bodies. They are precipitations of Hb beta tetramers (these tetramers form in the absence of Hb alpha).
59
What are some other alpha thal syndromes?
ATR-16 - a gene deletion syndrome of the region of chromosome 16 containing HBA1 and HBA2 genes.
60
What is beta thal caused by?
| Where is it common?
Loss of HBB expression on chromsome 11. Results in reduced Hb-beta, reduced red cell production and subsequent anaemia.
Caused by ~280 mutations.
Common in Cyprus, tropics and sub-tropics.
61
What are beta-thal major and beta-thal intermedia caused by?
beta-thal major caused by nonsense/frameshift/splise/missense variants
beta-thal intermedia cause by non-coding variants in the UTRs or promoter regions.
62
List clinical features of beta-thal major
Severe Anaemia
Severe Hepatoplenomegaly
Transfusion dependent, need iron chelation therapy to prevent iron overload.
63
List clinical features of beta-thal intermedia
```
Milder disease, transfusions can improve quality of life, but aren't required for survival.
Pallor
Jaundice
Iron overload
osteoporosis
```
64
What is responsible for the phenotypic variability in beta-thal?
Co-inheritance of alpha-thal - reduction in alpha and beta globin appears to result in a milder phenotype.
Mutations increasing the expression of gamma-globin (increase the HbF fraction)
65
What other-beta thal disorders are there?
beta-thal minor (beta-thal trait) - carriers
Dominant beta thal
Beta-thal in association with other Hb anomalies
66
List features of sickle cell disease
```
Chest pain
Stroke
Bone pain
Vulnerability to infection
Anaemia
```
67
What is the mutation responsible for sickle cell disease?
p.Glu6Val mutation in HBB
68
Apart from homozygous HbS, what else can cause sickle cell disease?
Co-inheritance with other variant HBBs: HbC, HbO, HbD, Beta-thal
69
Describe the testing procedure for haemoglobinopathies
```
MCV
MCH
FBC
RBC count
Microscopy
```
Then proceed to HPLC
Then genetic testing, if required.
70
What cut-off is used to determine whether further investigation of a Hb-opathy screen is needed?
MCH <27pg/l
71
Describe the SC and thal screening programme
All expectant mothers tested before 10wks. If carriers, try to test fathers by 13 weeks so PND can be offered.
Family Origin Questionnaire used to target screening to higher risk populations based on ethnicity.
72
What treatments are currently available for Hb-opathies?
Transfusions
BMT in very severe cases - 5-10% chance of death as a result of GVHD
Iron chelation
Induction of HbF production
73
What future, novel treatments are available for Hb-pathies?
```
Gene therapy, including CRISPR/Cas9 to edit HBB and correct SC mutatation. increase expression of gamma globin, introduce exogenous beta-globin gene.
Activin receptor ligand traps
Macrophage targeting (shown to improve haematopoesis in thalassaemia)
```
74
Name the found categories of lysosomal storage disorder, and give an example of each.
Mucopolysaccharidoses - Hurler, Morquio
Oligosaccharidoses - Fucosidoses, Mannosidosis
Sphingolipidoses - Fabry's, Gaucher;s, Neimann Pick
Other - Pompe's disease, Batten disease
75
What two types of inherited bleeding disorder are there?
Haemophilias (not enough clotting) and Thrombophilias (too much clotting)
76
```
What gene is mutated in VWD?
How is it inherited?
What are the clinical features?
What is the UK frequency?
How is it diagnosed?
```
VWF
AR
Bleeding gums, heavy periods, bleeding after minor surgery, nosebleeds, bruising.
0.6-1.2%
Platelet aggregation test, immunological quantification.
DNA sequencing to distinguish between Types 2 and 3, PND for type 3.
77
What are the three types of VWD?
Type 1 - partial loss of VWF, 70% of cases, mild qualitative
Type 2 - further broken down into A, B, M, N. N is most severe and AR. Qualitative deficiency
Type 3 - Most severe, severe quantitative deficiency, AR, 80% nonsense.
78
How can VWD be treated?
Desmopressin injections
Transexamic acid - tablets
Factor8-VWF infusion
79
```
What gene is mutated in Haem A?
How is it inherited?
What are the clinical features?
What is the UK frequency?
How is it diagnosed?
```
Factor 8
XLR
45% caused by inversion of intron 22; 3% caused by inversion of intron 1.. Remainder are milder phenotype and caused by point mutation.
1/5000
Lab diagnosis by clotting tests and measurement of functional Factor 8
Diagnosis by IS-PCR and LR-PCR for intron 22 inversion and intron 1 inversion. Sequencing for the rest.
80
```
What gene is mutated in Haem B?
How is it inherited?
What are the clinical features?
What is the UK frequency?
How is it diagnosed?
```
```
Factor 9
XLR
1/30,000
Diagnosis by clotting tests and quantification of functional factor 9
Standard PCR and sequencing, MLPA too.
```
81
Describe the mutation spectrum for Haem A and B.
Point mutations
Missense - severity dependent on nature of change
Splice - variable
Nonsense - severe
Deletion
Whole gene, whole exon, multigene, small deletions
Insertion
Severity dependent on position and effect
Rearrangements
Gene inversion involving intron 22, mediated by NAHR between CpG islands; introns 1-22 and moved away from their normal context
82
How can Haem A and B be treated?
Prophylactic
On-demand
Both require injections of relevant clotting factors.
83
Name other factor deficiencies leading to excess bleeding
```
Factor I deficiency
Factor II deficiency (GoF mutations cause clotting problem)
Factor V LoF
Factor X
Factor XII
```
84
Name some inherited thrombophilias
Factor V Lieden (p.R506Q)
Factor II prothrombin deficiency
Antithrombin deficiency
85
```
What gene is mutated in Factor V Leiden?
How is it inherited?
What are the clinical features?
What is the UK frequency?
How is it diagnosed?
```
F5, mutation p.R506Q
AD, heterozygotes are less severeley affected than homozygotes, and are at a lower risk of clot.
Impaired interaction between natural coagulants leads to increased formation of blood clots.
Increased risk of thromboembolism (25-40% increase over WT).
8.8% prevalence, 1/5000 are homozygous
Diagnosis by mutation analysis and APC resistance assay.
86
How can factor V leiden be protective?
heterozygote carriers lose less blood during childbirth and cardiac surgery than WT.
87
What is the common mutation associated with factor II prothrombin thrombophilia?
what is the associated phenotype?
G20210A; this is a GoF mutation in F2
Heterozygotes are at an 2-4fold increase risk of clot; homozygotes are at a 20-fold increase of clot.
Increased risk or pregnancy loss and pre-eclampsia
88
What two categories of inherited cardiac conditions are there? Give examples of each
Cardiomyopathies - DCM, HCM, LVNC, RCM, ARVC
| Arrhythmias - Brugada, LQT, SQT, CPVT
89
What are cardiomyopathies defined by?
Structural heart changes. Increase in cardiac wall thickness and composition. Patients present with shortness of breath, palpitations and SCD.
90
What are arrhythmias defined by?
Conduction defects altering electrical impulses controlling heartbeat
91
Name clinical features associated with DCM, including physiological changes.
Reduction in ventricular wall thickness, increased endocardial thickening. Reduced cardiac output, stroke, heart failure, SCD. Mainly adult onset, associated with DMD.
Most common cardiomyopathy.
92
How many genes have been associated with DCM?
| Mutations in which gene account for 1/3 or cases of DCM?
>40
TTN
LMNA and MYH7 account for ~5% of familial cases.
93
How is DCM diagnosed?
Cardiac MRI and ECG.
94
What is HCM?
A asymmetric thickening of the heart muscle; 25% of people demonstrate an outflow obstruction at rest; 70% demonstrate outflow obstruction under certain conditions (exercise).
Patients present with angina, palpitations, syncope, jerky pulse. Can lead to heart failure or SCD.
95
How is HCM diagnosed?
Enlargement of the left ventricle on CMR or ECG
| Cardiomyocyte disarray and fibrosis by histology.
96
What sort of proteins are mutated in HCM?
Sarcomeric proteins
97
Name some differential diagnoses for HCM
Fabry, Friedreich's ataxia, Noonan's
98
There are two subtypes of LongQT syndrome. Name them - what is the difference?
Type I and II - mutations in potassium channels
| Type III - mutations in sodium channels
99
Which genes are implicated in LQT?
SCN5A, KCNH2, KCNQ1, KNCE1, KCNE2
100
What is CPVT (mutations in RYR2) associated with?
Exercise, particularly swimming
101
Describe the screening strategy for Inherited Cardiac Conditions.
Large panels, depending on clinical features: Aortopathy, arrhythmia, cardiomyopathy.
Clinicians may refer for smaller panels if large panels too costly.
Panels are good because there are significant phenotypic overlaps between all diseases.
102
What is the aim of testing for Inherited Cardiac Conditions?
Risk reduction - avoid sports/swimming
Cascade testing in family
Treatment - Beta blockers, pacemaker, heart transplant.
103
What general features do patients with cardiac arrhythmias present with?
Palpitations, dizziness, blackout, SCD.
104
How does WHO define infertility? How many couples does infertility affect?
The failure to conceive after 12months or regular intercourse. 15%
105
List causes of male infertility (non-genetic)
Oligozoospermia, Asthenozoospermia, Teratozoospermia, Obstructive azoospermia, chemical exposure, anabolic steroids, low testosterone, trauma, lifestyle, Mumps
106
List causes of female infertility (non-genetic)
PCOS, environment/lifestyle, age, chemical exposure, antiphospholipid syndrome
107
List genetic causes of male infertility
Sex chromosome aneuploidy - 47,XXY, 45,X/46,XY mos
Sex chromosome translocation - t(X;Y)
Robertsonian translocation - t(13;14) - 9x more frequent in infertile males
Marker chromosomes - can interfere with sex vesicle formation
AZFabc deletions
Single gene disorders
Ring chromosomes
Inversions - inversion loops may lead to meotic arrest
Insertions
iso(Y)(p)
108
What is the most frequent genetic cause of male infertility? is pregnancy possible?
Klinefelters (1/500)
| 15% of patients are mosaic and are more likely to have residual spermatogenesis.
109
How do patients with 45,X/46,XY mosaicism present?
90% normal male external genitalia; 10% ambiguous or female, depends on level or mosaicism.
110
How do 46,XX males arise?
SRY+ testicular DSD - 75% of cases
presence of Y material on an X - often submicroscopic
occult XX/XXY moscaicism
Present with normal puberty, but oligozoospermia and infertility. 10% have ambiguous genitalia
SRY-
hyperactivation of a gene normally switched on in response to SRY expression
Tend to present with hyopspadias and ambiguous genitalia at birth
111
Why are male translocation carriers more susceptible to infertility that female carriers?
Translocations result in the failure to complete synapsis in pachytene - this activates the meiotic pachytene checkpoint resulting in cell death - spermatogenic cells are more vulnerable to this check point, hence low sperm count.
Disruption of the sex vesicle may also lead to spermatogenic arrest. - Robertsonian translocations are particularly good at interfering with the formation of the sex vesicle.
112
What is detected in 13% of men with no-obstructive azoospermia?
AZF deletions
113
Which genes are contained within AZFa cluster?
UPS9Y and DDX3Y
114
What are interstitial deletions between b/b+c mediated by?
Palindromic repeats
115
Which genes are located in the AZFc region?
DAZ gene cluster
116
Why might AZFc deletions allow some residual spermatogenesis?
There are additional autosomal copies of the DAZ genes.
117
State the genotype/phenotype correlations between the different AZF microdeletions
AZFa - SCOS
AZFb - azoospermia possibly some residual spermatogenesis, but unlikely to be a good candidate for PESA
AZFc - some residual spermatogenesis seen, may be suitable for ICSI, but counselling needed as all sons will inherit the Y deletion.
gr/gr deletions are not recommended to be reported (BPG)
118
List genetic causes of female infertility
45,X
TS variants - including mosaicism for X chromosome aneuploidies. range from TS to minor menstruation abnormalities.
X chromosome structural abn
X-autosome carriers - 50% infertile
(X:X)(p11.23;q21.3) translocation carriers - abn puberty and infertiltiy certain
XY complete gonadal dysgenesis (46,XY females with mutation in SRY)
AIS - mutation in AR gene.
FraX - premutation carriers
119
How can X-chromosome structural abnormalities lead to infertility?
They may disrupt genes involved in ovarian function: POF1 and POF2
POF1 - Xq22-27; POF2 - Xq13.3-21.1
Amenorrhea to premature menopause.
120
Describe the testing strategy for females referred with POF
Karyotype to identify any X chr abn - look at 30 cells to screen for mosaic TS, refer to Hook's table to determine AR loss.
FraX premutation test (55-200rpts)
Mutations in other genes: BMP16, FSHR, FOXL2, INHA. Also not routinely available.
121
Name syndromes associated with infertility and other features
Noonan syndrome (PTPN11, NRAS, KRAS, SOS1, MAP2K2, MAP2K1, RAF1, BRAF)
Kallman's syndrome
Congenital Adrenal Hyperplasia
PWS
AIS
122
Describe an overall genetic testing strategy for infertility.
Recurrent miscarriage investigations - karyotype 3rd misscarriage, investigate for thrombophilia and antiphospholipid syndrome.
```
No pregnanies:
Karyotype both parents
AZF mictodeletions
CF
FraX
```
123
Where is SRY found?
Yp11.2
124
How much of the Y chromosome is male specific?
95%
125
What normal variants involving the Y chromosome are seen?
```
Yqh variation
PEricentric inversion (seen in 1/200)
Satellited Yq
```
126
What pathogenic Y chromosome variations are seen?
```
Yp-
Yq-
I(Y)p
idic(Y)p
i(Y)q
idic(Y)q
Yq:Autosome translocations (1/2000), fertility variable, may cause azoospermia.
Yp:Autosome translocations - rare
X:Y translocations
ring(Y) - rare, present with 45,X cell line
```
127
What is the most common Y:autosome translocation?
t(Y:15), then t(Y:22). May be polymorphic.
128
What is the classical X:Y translocation?
How does it arise?
Which genes may be lost as a result of the translocation?
t(X:Y)(p22.3;q11)
Through NAHR during spermatogenesis between homologous sequences on the X and Y in PAR1: PRKX and PRKY in 30% of cases
Important genetic defect is loss of distal Xp - ARSE, SHOX, STS, KAL, MRX, OA1
129
How does the typical female carrier of the t(X;Y)(p22.3;q11) manifest?
fertile and of normal intelligence
| IF SHOX lost then Leri-Weill dyschondrosteosis phenotype
130
How does the typical male carrier of the t(X;Y)(22.3;q11) manifest?
Phenotypically male, if MRX lost may have intellectual disability
Infertile
131
What are the phenotypes typically associated with i(Yp) and i(Yq)?
i(Yp) - SRY+, infertility, ambiguous genitalia, TS abnormalities, hypogonadism, short stature
i(Yq) - SRY-, phenotypically female, streak gonads, TS features, short stature.
132
Where is XIST located?
Xq13
133
Describe the process of X-inactivation
XIST is transcribed from the XIC of the inactive X. the XIST RNA coast the inactive X, CpG islands are methylated and histones H3 and H4 are deacetylated.
134
List known structural abnormalities of the X-chromosome
```
X-autosome translocations
X/Y translocatiojns
XX/Translocations
Deletions
Duplications
Ring (X)
Isochromosomes
```
135
Which X is preferentially inactivated when a t(A:X) is present?
The normal X
136
How can females manifest XLR disease?
Turner syndrome
Genuine homozygote/Compound Het
Skewed X-inactivation - Balanced translocations involving the X cause inactivation of the normal X
137
Which two regions are associated with infertility and gonadal dysgenesis?
Xq13-22 (POF1) and Xq22-27 (POF2)
138
How do males manifest with balanced t(A:X)?
Spermatogenic arrest as the sex vesicle is disrupted. during pachytene, preventing passage through the pachytene checkpoint.
139
What factors need to be considered when performing prenatal diagnosis on a female carrier of a t(X:A) (male carriers are invariably infertile)?
High risk of abn foetus - up to 40%.
| Phenotype of the child may not be the same as the phenotype of the mother.
140
Which diseases can be associated with deletions of Xp22.3?
STS
KAL
SHOX
141
Which disease may be associated by microdeletion of Xp21?
DMD
142
What can duplications of Xp22 cause? Which gene is in this region?
PMD - an X-linked demyelinating disorder of the CNS. PLP1 in in this region
143
What disease is associated with Xp28 duplication?
Rett syndrome - MECP2
However the Xq28 duplication can be seen in males - absent speech, seizures, recurrent infection, ID, hypotonia. Phenotype is variable in females, and tends to be less severe.
144
How does the MECP2 duplication arise?
FoSTeS
145
What determines if a Ring(X) is likely to be pathogenic?
If XIST is retained. If XIST is lost, phenotype may be severe.
146
Aside from 100KGP, name some WGS projects
```
UK10K
ENCODE
BiLEVE - Biobank lung disease study
WGS500
PGP-UK
Scottish Genome Project
Saudi Genome Project
All of Us (US project)
EU Personalised Medicine programme
DDD
```
147
What additional thing needs to be considered if a 45,X/46,XY cell line is found?
10-30% risk of gonadoblastoma in streak ovaries - remove gonads.
148
Outline the process of Sex determination
At 4 weeks XX and XY foetuses have the urogenital ridge formed and the bipotential gonad. This is driven by expression of WT1 and SF1 (NR0B1)
At 6-7 weeks, the Mullerian ducts and Wolffian ducts are present and can form either the ovaries or testes, respectively. This is dependent on expression of female or male genes.
To form the testes, SRY is expressed, drives SOX9 expression. This expression is maintained by the expression of FGF9 and PDG2 (these act in a positive feedback loop to further increase SOX9 expression). This promotes growth of the Wolffian ducts. The wolffian ducts form the epididymis and vas deferens.
To form the ovaries, the RSPO1/WNT4/B-catenin pathway is activated to promote stabilisation of the Mullerian ducts. The Mullerian ducts form the uterus and Fallopian tubes.
Leydig and Sertoli cells differentiate. Sertoli cells produce AMH to cause degeneration of the mullerian ducts. The leydig cells produce testosterone and stimulates the generation of the internal male genitalia. Testosterone is converted to DHT and stimulates the growth of the external male genitalia.
In the ovary, Theca and follicular cells develop to form the ovary. In the absence of AMH the Mullerian ducts develop into the uterus. The follicles secrete oestrogen and promote the development of the female external genitalia.
149
What is the difference between Sex determination and Sex differentiation?
Sex determination is controlled by gene expression.
Sex differentiation is controlled by hormonal systems.
150
List the genes involved in female and male sex determination.
Overlap:
WT1, SF1
Female:
DAX1, RSPO1, B-catenin, WNT4, FOXL2
Male:
SRY, SOX9, SOX3, TES, FGF9, PDG2, DHH, ATRX, GATA4. TESCO enhancer sequence (upstream of SOX9).
151
Name causes of 46,XY DSD
These are developmental disorders causing undervirilisation of an XY foetus
Disorders of testicular development:
Complete gonadal dysgenesis - mutations in SRY, DHH, SF1 and duplications of DAX1, WNT4 and MAP3K1. Phenotypically female, streak gonads with uterus and fallopian tubes. Lack of male developmental stimulants.
Partial GD - less severe mutations in SRY, DHH and SF1. Ambiguous genitalia, mullerian structures may be absent or partially present.
Testicular regression - no known cause
46,XY ovotesticular DSD - very rare.
Disorders of Androgen synthesis
P450scc, 17a-hydroxylase deficiency, cholesterol deficiency (SLO, DHCR7), StAR deficiency.
5a-reductase deficiency - can't convert T > DHT
Defects in AMH synthesis or receptor
Disorders of Androgen action
AIS - complete, partial or minor - caused by mutations in the AR.
Other:
WT1- related disorders (Denys-Drash, WAGR, Frasier)
ATRX
Campomelic dysplasia.
152
Name causes of 46,XX DSD
These are developmental disorders causing overrvirilisation of an XX foetus
Disorders of ovarian development:
Testicular DSD - 46,XX males. Usually cryptic SRY+, may have activation of SOX9 pathway with SRY stimulation - SOX9 duplication, WNT4 deletion, RSPO1 mutation etc. Phenotypically normal males with azoospermia, short stature and infertility.
FOXL2 mutations lead to BPES
Ovotesticular DSD (46,XX) - true hermaphrodism - ovarian and testicular tissue, mixture of wolffian adn mulerian ducts, ambigious genitalia, 1/3 are SRY-
Gonadal dysgenesis - female phenotype but pubertal failure, no secondary sexual characteristics, streak gonads with hypogonadotrophism. Mutations in FSHR and BMP15
Disorders of androgen synthesis - Foetal androgen excess:
CAH - CYP21A2 (pseudogene CYP21A1P), CYP11B1 and HSD3B2 - AR disorder caused by mutations in the cortisol synthesis pathway leading to increased androgen synthesis. Increased androgen synthesis in a foetus results in external virilisation. Three forms:
Classic
Salt-wasting
Atypical
```
Non-CAH:
Aromatase deficiency can cause virilisation of a foestus and the mother during pregnancy - the placenta cannot convert steroid hormones to oestrogens
POR defect
Iatrogenic virilisation (drug-induced)
Luteoma of pregnancy
```
Gene dosage in DSD
46,XY gonadal dysgenesis - deletion of SRY, SOX9, WT1 or SF1; duplication of WNT4, DAX1
46,XX testicular DSD - duplication of SOX9 or SOX3.
