Dalton - Genomic Testing in the NHS Flashcards
1
Q
What pathway do samples take in the NHS from being taken to a report being made?
A
- assessment of test (input of clinical and/or diagnostic info)
- extraction of DNA/RNA/chromosome preps (or all)
- then either direct mutation analysis, genetic mutation detection or linkage analysis
- get results
- report written (so useful to patient)
2
Q
What does the clinical pathway of a sample start and end with?
A
- the patient
3
Q
When would direct mutation analysis be carried out on a sample undergoing genomic testing?
A
- if know what mutation is, eg. if in family already
4
Q
WHen would genetic mutation detection be carried out on a sample undergoing genomic testing?
A
- if query a certain condition but don’t know specific mutation
- eg. if query CF then check CFTR gene
- have to have some understanding of clinical presentation to choose right test
5
Q
What is also important to consider when deciding what tests to do on a sample?
A
- what patient has consented to
6
Q
What is the referral reason important for?
A
- to decide what to do w/ sample
- eg. may be query CF, or may be more general
7
Q
What types of sample can be taken prenatally?
A
- pre-implantation genetic diagnosis (PGD)
- free fetal DNA
- chronic villus sampling
- amniocentesis
- placental biopsy
- fetal blood sampling
8
Q
What types of samples can be taken postnatally (and through to adulthood)
A
- cord blood
- dried blood spot
- salivary brush
- bone marrow
- cell free (tumour) DNA
- skin biopsy
- venipuncture
- post mortem
9
Q
What is PGD and what can it be used for currently?
A
- take 1 cell from 8
- currently can only test specific region so have to know what looking for
10
Q
What is the future for PGD?
A
- will be able to test whole genome eventually
- but should we?
11
Q
What is free fetal DNA, and how is it sampled?
A
- DNA that circulates freely in maternal DNA
- sampled by venipuncture
12
Q
When is chronic villus sampling taken, and what can happen if its done too early?
A
- 11 weeks
- clubbing
13
Q
What does an amniocentesis sample?
A
- sampling amniotic fluid surrounding fetus
14
Q
When can placental biopsies be taken?
A
- any time after 15 weeks
15
Q
When do RBCs contain DNA?
A
- is fetuses and newborns up to 6 months
16
Q
What sample is taken from all newborns in England?
A
- dried blood spot
17
Q
What is the issue w/ amniocentesis?
A
- risk of miscarriage, but v low risk
- having termination at this time is induced labour
18
Q
How are genes labelled, ie. nts, exons, etc.?
A
DIAG
19
Q
Do cDNA and gDNA contain introns?
A
- gDNA does
- cDNA is cop of RNA so does not
20
Q
What kind of brackets are used in mutation nomenclature to indicate predicted protein change?
A
- ( )
21
Q
In mutation nomenclature how would a point change be written for cDNA, eg. C changed to T at position 145?
A
- c. 145 C>T
22
Q
In mutation nomenclature how would predicted protein be written, for eg. Arg to stop at position 49, or to Leu?
A
- p. (Arg49*)
- p. (Arg49Leu)
23
Q
What is a nonsense mutation, and how likely is this to be pathogenic?
A
- AA to stop
- more likely to be pathogenic (but not always), as disrupt prod of protein though nonsense mediated decay
24
Q
What is a missense mutation, and how likely is this to be pathogenic?
A
- changes AA
- much harder to predict, could be pathogenic or not
25
In mutation nomenclature how would a change in the intron be written for cDNA, eg. for G changed to T at position in intron before 51 in the exon?
- don't want to inc gDNA numbering as well, so use last no, from cDNA, ie -1 means go back 1 towards 5' end (can do from either end)
- eg. c. 51-1G>T
26
In mutation nomenclature how would you write an unknown predicted protein change, and why would we not know?
- p. (?)
- if splice site mutation don't know what it does, as how cell deals w/ it can vary --> exon may be removed or intron inc
27
In mutation nomenclature how would a deletion be written for cDNA, eg. a TG deletion at position 104 to 105?
- c. 104_105delTG
28
What is the effect of a 2 base deletion in mammals?
- disrupts ORF as not multiple of 3
| - generally get stop w/in 100 AAs, but usually w/in 10 and often immediately
29
In mutation nomenclature how would the predicted protein change be written for a 2 base deletion, if eg. Cys changed to Tyr at position 34, and frameshift of 12 AAs?
- p. (Cys34Tyrfs*12)
| - frameshift is at 12AAs from and inc 1st AA changed --> get stop
30
Do frameshift mutations always mean all AAs afterwards are changed?
- no, sometimes can get some AAs after where maintained
31
In mutation nomenclature how would an insertion be written for cDNA, eg. of A at position 104?
- c. 104insA
32
In mutation nomenclature how would how would a duplication be written for cDNA, eg. of T at position 104?
- c. 104dupT
33
In mutation nomenclature how are larger deletions written for cDNA, eg. of bases 94-97?
- c. 94_97del4
34
In mutation nomenclature how would deletion of an exon be written for cDNA, if don't know exactly what's been lost as unsure where breakpoint is, eg. deletion of part of exon 2, between positions 51 and 141?
- c. 51-?_141+?(del exon 2)
35
In mutation nomenclature how would a 3 base deletion be written for cDNA, eg. of TTT at 133 to 135?
- c. 133_135delTTT
36
In mutation nomenclature how would a 3 base deletion be written for predicted protein change, eg. was Phe at position 45?
- p. (Phe45del)
37
What is generally the effect of 3 base deletions?
- not pathogenic and quite common, as doesn't alt ORF
38
What is the nomenclature for a known genotype?
- [genotype]
39
What is the nomenclature for 2 diff alleles in trans (give eg.)?
- p. [(Phe45Leu)] ; [(Arg49*)]
| - for AAs write name then no. but for nts write no. then name
40
What is the nomenclature for same allele in cis (give eg.)?
- p. [(Phe45Leu) ; (Arg49*)]
| - remove internal [ ] as know on same allele
41
What is the nomenclature if unsure whether on cis or trans?
- p. [(Phe45Leu) (;) (Arg47*)]
| - ; in brackets as don't know
42
What is the nomenclature for heterozygous changes in a limited screen?
- c. [94_97del4] ; [94_97 =]
| - on other allele only looked at these bases to see if WT, can't say rest is WT as we don't know
43
What is the nomenclature for heterozygous changes in a full screen?
- c. [94_97del4] ; [=]
| - no no. in front of = as looked at whole gene on other allele and found to be WT
44
What is the nomenclature for hemizygous changes?
- c. [135C>A] ; [0]
45
What is the nomenclature for somatic changes (mixed cell pop)?
- c. [51-1G>T / =]
| - / means allele diff in diff cells
46
How can you define mutation (if germline and if somatic)?
- if in germline = disease causing alteration that can be inherited
- if somatic = disease causing alteration that has occurred after meiosis
47
What is a polymorphism?
- alteration which is w/o effect or advantageous, and can be inherited
48
Does frequency of occurrence define whether something is a mutation or polymorphism, what can be looked at instead?
- no, polymorphisms can be rare and mutations can be common
- incidence = look at disease freq, ie. if have common change and rare disease then assoc not likely, so prob polymorphism
49
When are nonsense mutations less likely to be pathogenic, and what happens?
- when near end of ORF
- as need big gap between incorrect and normal stop codon for it to be targeted by NMD
- so get abnormal peptide, which may or may not be pathogenic and cause phenotypic effect
50
Why do we get splicing variants, and what is the result of this?
- can result in exon being deleted or intron inc
- if intron inc can result in stop and NMD, due to alt ORF
- if exon del can be in frame, so n stop --> get milder phenotype or none
- some genes spliced diff in diff places --> so not only have to know about what splice variant is and does, but also good knowledge of gene itself and how expressed in diff tissues
51
What is an eg. of genes spliced diff in diff place, and how is this signif when looking for variant causing symptoms?
- glycogen storage genes in muscle and liver
- so if symptoms muscular, but variant positioned in exon only transcribed and translated in liver, then prob not mutation
52
How can you tell if something is a mutation or a polymorphism - ie. what can we look at?
- look at freq vs incidence
- type of mutation
- splicing variants
- look at degree of conservation
- functional info
- structural info
- functional studies in vitro
- functional studies in vivo (not done)
- clinical info
53
How can we look at degree of conservation to see if a variant is a mutation of polymorphism?
- look at how well particular base/AA/nt conserved
- is it conserved all the way through evo history?
- does it vary when move from humans to close relatives (eg. chimps)?
- use multiple seq alignment (MSA)
54
What functional info can be looked at to see if a variant is a mutation of polymorphism?
- motifs (eg. does it bind ATP?)
| - functional domains
55
How can functional studies be carried out in vitro to see if a variant is a mutation of polymorphism?
- eg. might be enz and can look at activity or do mutagenesis
- or use Ts mutants
56
What clinical info can be looked at to see if a variant is a mutation of polymorphism?
- go back to info on patient
| - does change we've seen in gene assoc w/ symptoms in patient (if not then not helpful to patient and prob not relevant)
57
What types of software tools and packages are there for looking at variants (and often meta analyses)?
- machine learning methods
- protein seq and structure based methods (algorithms that looks at predicted/known protein structure)
- seq and evolutionary conservation based methods
- analysis and collation packages
58
How are variants classified?
- benign -1
- likely benign -2
- unknown signif -3
- likely pathogenic -4
- pathogenic -5
59
What does a variant classification of -1 mean?
- v certain is polymorphism
- high pop freq
- not linked to patients phenotype
60
What does a variant classification of -2 mean?
- some freq data
- in silico predicts neutral
- not in functional domain or likely to impact structure
- few/no pathogenic mutations in region
- but can't be certain not pathogenic
61
What does a variant classification of -3 mean?
- conflicting predictions from in silico tools
| - no freq data or lit
62
What does a variant classification of -4 mean?
- no/low pop freq
- suggestion that mutations in region identified before
- pathogenic in silico prediction
- disease specific lit
- but can't be certain pathogenic
63
What does a variant classification of -5 mean?
- segregates w/ disease
- functional study
- all evidence in agreement
- def pathogenic
64
What classification are lots of variants found, and why is this a problem?
- unknown signif (-3), esp in genes don't know lot about
| - as can't give patient info either way
65
Who's guidelines were adopted for variant interpretation?
- American College of Medical Genetics (ACGS)
66
What does linkage analysis involve following the inheritance of?
- following inheritance of particular parts of chromosome through families
67
What does linkage use as markers?
- natural polymorphisms w/in DNA
68
What samples do we still use linkage for (instead of direct mutation analysis)?
- PGD
69
When is linkage esp useful, and how?
- in patients who don't want to know things
- can track variants and how segregate to see who's inherited what chromosome from who
- look at microsatellites (/variable nt triplet repeats)
70
How many variants do people have in their DNA?
- approx 5 mil
71
What about microsatellites varies?
- no. present in indiv
72
What are looking at microsatellites used in?
- DNA fingerprinting
73
What type of microsatellites are most desirable for analysis and why?
- intragenic
- problem if not due to recomb
- greater distance from gene = greater chance of recomb, but relationship not linear (due to recomb hotspots)
74
Why are SNPs less informative than microsatellites?
- 1 of 2 bps
| - can be intronic or exonic
75
What are the methodologies of PCR based technologies?
- amp of target DNA/cDNA
(input of sequencing --> pyroseq/Sanger/NGS)
- electrophoresis
- visualisation (how is dep on size)
76
What happens to PCR if part where primers bind del?
- won't get amp
77
How can changes in DNA be identified by PCR?
- put primers where deletion suspected, and if get amp then DNA is there
78
What is the fundamental problems w/ this method of using PCR to identify changes in DNA?
- if looking for del on X chromosome and looking at male and PCR doesn't work --> is this because they're male?
- and if look at female w/ 1 normal X chromosome?
- so have to be able to distinguish between half amount of PCR product and normal amount
- but usually take PCR to completion so no longer prop to to starting material
- therefore important to consider dosage
79
What are other poss problems w/ using PCR to identify changes in DNA?
- doesn't always work (need good control)
- contam (esp from PCR products)
- pol can introd mutations
- non specific binding if primers not specific enough
- repeated seqs in gDNA and pseudogenes etc.
80
What are the advs of direct mutation tests, eg. ARMS (amp refractory mutation system)?
- cheap
- quick
- don't get unwanted info
- can do lots in multiplex
81
How is ARMS carried out?
- design primer complementary to WT (control) and 1 to mutant
- so can design primer and get PCR products formed only when mutation present
- put stacker on end of 1 primer to make them diff size so can distinguish between products (as will be 2 bands)
- can test for multiple mutations at once but have to know seq info and what looking for
- have to remove pol proofreading activity as will correct extra base
82
What is the important thing to remember about ARMS?
- targeted to particular mutation
83
Why doesn't the NHS just use genomic screen for everything, as will def find what looking for?
- issues of finding out things didn't want to know
| - cost (and responsibility to deliver value for money service as publicly funded)
84
Why is automated Sanger seq used, even though so old?
- v robust --> often seen as gold standard as well worked and well understood
85
How does Sanger seq work, and how does it provide the seq?
- seq reaction w/ labelled and unlabelled dNTPS
- create ss template (invariably from PCR) by splitting PCR
- use seq primers to seq across, in presence of fluorescently labelled dNTPs (system able to detect these)
- when dNTP incorp into seq it terminates, so get group of products stopping at primer +1, primer +2 etc., w/ diff dNTP labels at that point
- carefully titrate, so get series of products that can run out through capillary gel, sep by size
- for every 1bp change know what end dNTP is as can detect fluorescence
86
What is the result of Sanger seq, ie. the trace, and how is it read?
- get trace differential, which takes 1st trace away from 2nd so can see what diff is in peak height and area
- no diff gives straight line, diff gives "bubble"
- can still get variations, as long as consistent w/in seq
87
How are heterozygotes seen in Sanger seq trace?
- 2 peaks in same location at half height
88
What does it mean if see sudden change in Sanger seq trace after certain point?
- likely caused by frameshift, ie. all bases become much lower on trace and some changed
89
What is pyrosequencing good for?
- seq lots of short fragments v quickly
90
How does pyrosequencing work, ie. what are we looking at and how do we know what dNTP incorp?
- look at incorp on dNTP, where incorp releases hydrogen which can be detected to see if seq reaction occurs
- know which dNTP incorp as presents each dNTP in seq --> ie. present dGTP, does it work? if not washes away and try next dNTP until find one that prod reaction
91
What is the role of the diff components in pyrosequencing?
- DNA pol polymerises DNA
- ATP sulfurylase quantitatively converts pyrophosphate to ATP in presence of adenosine 5' phosphosulfate
- luciferase converts luciferin to oxyluciferin w/ visible light when have dNTP being incorp
- apyrase degrades unicorp nts, ATP and substrates
92
What drives the pyrosequencing reaction?
- the fact the dNTPs being incorp
93
How do the templates and they way they are prepped differ between sanger and next gen seq?
- sanger = template prep by PCR
| - next gen = DNA seq libs clonally amp in vitro by emulsion PCR or by shearing and add of terminators
94
How does way DNA is seq differ between sanger and next gen seq?
- sanger = DNA seq by synthesis w/ fluorescently labelled chain terminators
- next gen = DNA seq by synthesis, by add of nts to complementary strand
95
How does way in which products are sep differ between sanger and next gen seq?
- sanger = products sep using gel tech (slab or capillary)
- next gen = spatially segregated, amp DNA seq simultaneously in massively parallel fashion --> no need for physical sep step
96
How does amount of DNA that can be seq per day and time taken per run differ between sanger and next gen seq?
- sanger = 1Mb/day, several hours per run
| - next gen = 25Gb/day, 2 hours per run
97
How does what can be seq differ between sanger and next gen?
- sanger = single amplicons x96 or x384
| - next gen = gene panels, whole exomes, whole chromosomes, whole genomes, multiple patients using barcoding
98
What can sanger seq not detect?
- large exonic deletions or duplications
99
What does next gen seq have the pot to replace?
- microarrays
- chromosome analysis
- dosage
- methylation analysis
- southern blotting
100
How does ion torrent work?
- pol integrates a nt
- hydrogen and pyrophosphate released
- then look at change in pH --> converted using microfluidics into next gen sequencer which prod sequential frol of dNTPs onto template and constantly measure H+ release
101
What happens in ion torrent if nt compliments/doesn't compliment template?
- doesn't compliment = no release of H+
- does compliment = H+ released
- if nt compliments several bases in a row = multiple H+ released
102
What do most NGS techs involve doing (inc Illumina), due to wanting to seq large amount of DNA?
- shear gDNA into 200-300bp fragments
- ligate vectors onto ends
- apply tagged fragments to flowcell --> act as linkers, so each locks into 1 place
- anchor points placed approp far enough apart, so detection systems can look at each individually
103
After adding anchor points (in Illumina), what needs to be done so activity can be detected?
- duplicate 1 fragment in controlled way --> by looping it over to its primer compliment, then amp back and forth in loop, then cleave 1 end, end up w/ block of fragments all w/ same seq
- if split again , by denaturing, can seq along 1 side all at same point and detect changes in nts that are being incorp at each indiv point
104
What do we see in Illumina seq if apply multiple cells the same?
- then this seq amp
105
What do we see in Illumina if apply cancer cells?
- diff seqs
106
What do we see in Illumina if looking at heterozygote?
- 50% diff seq
107
How can we also use Illumina to enrich for the things we want to look at?
- select out bits of genome by looking for seq of interest
| - get something complimentary and something that can pull it out by hybridising to it (eg. magnetic bead)
108
What property of DNA drives PCR?
- complementarity
109
What is the template in PacBio (SMRT), and what is the consequence of this?
- single mol of DNA
| - means don't need amp
110
How does PacBio (SMRT) work?
- SMRT = single mol real time
- uses zero-mode waveguide (ZMW), illumination chamber
- single DNA pol in bottom of ZMW and single molecule of DNA as template
- ZMW can detect single labelled base being incorp
- CHIP containing multiple ZMW which consist of nanophotonic confinement structures approx 70nm in diameter and 100nm deep, w/ obs vol of 20x10^-21 l
111
How do the read lengths from PacBio comp to sanger seq, and what assays is there pot for?
- comparable or greater than sanger
| - methylation assays
112
Why is PacBio good for heterozygotes?
- can see them as 50/50
113
What can PacBio be misleading for, so what do you need to be able to distinguish between?
- false +ves, esp in cancers
| - need to be able to distinguish between mistake made by sequencer and a real mutation
114
What does coverage mean in terms of NGS?
- how many times has particular base been seq
115
How much coverage is needed in diagnostic work, and why?
- at least 30x
| - need to be certain, want 100% coverage
116
Is WGS carried out in NHS?
- no, seq whole genome but only look at small part of DNA gen
117
What organs can glycogen storage disorders involve?
- liver, heart, muscle or generalised
118
For glycogen storage diseases how many of gene are seq and analysed, and what is the adv of this?
- all genes seq
- but only those relevant to presentation of diseases are analysed
- adv = saves money and time,
- if result -ve then see if any dev in phenotype and test other groups of genes that could be assoc
119
What does a half height peak mean in terms of dosage?
- heterozygote --> deletion on 1 chromosome
120
What peak would be seen in terms of dosage if homozygous for deletion?
- no peak as no PCR product
121
When looking at dosage what provides an internal control that PCR is working?
- pattern seen
| - big products at beginning/small at end
122
If whole of seq the same, but 1 peak higher, what does this mean, and what can be predicted from this?
- duplication
| - harder to predict whether relevant, unless well characterised
123
If same finding about peak found in other exons, what does this mean?
- more likely to be real finding
| - but doesn't tell you its pathogenic
124
What is MLPA?
- multiplex ligation-dep probe amp
125
What do the probes used is MLPA consist of?
consist of 3 parts:
- probe
- stuffer --> to make all probes diff sixe to create diff size fragments
- universal primer --> can PCR across every probe annealed
126
How does MLPA work?
- denaturation
- hybridisation
- ligation = only when 2 probes align and is quantitative
- amp = PCR from universal primers when ligation completed
- can convert into bar chart to visualise eg. exons deleted
* DIAG*
127
What is a flaw of MLPA, ie. when might it appear to be an exonic deletion but isn't?
- probes anneal to DNA, so if polymorphism under probe then prevents/decreases capacity to anneal
- so may look like exonic deletion, but actually polymorphism
128
What do NF2 and BRCA mutations have in common?
- autosomal dominant
| - give predisposition to cancer
129
What type of mutation is NF2 (Neurofibromatosis type 2) adn what protein does it affect?
- single gene
| - Schwannomin protein
130
What is the penetrance of the NF2 mutation, and what does this mean?
- fully penetrant
| - if have mutation will get disease, but don't know when
131
What is the expression of NF2 mutations like, and what does this mean for patients?
- clearly defined range of expression
- almost all affected dev bilateral schwannomas (acoustic neuromas) by age 30 (useful for diagnosis if this is presenting complaint)
132
Why can not having a clearly defined range of expression be a problem?
- don't know where to look
| - lots of people have sub-clinical disorders --> don't cause problems, so not pathological
133
What important rules are there to remember when drawing a pedigree?
- men on left, women on right
- children in birth order
- no. gens w/ roman numerals
- no. indivs w/ 1, 2, 3 etc.
- label affected and if diff affectedness then label approp
- diamond if unknown sex
- broken lines if fetus in utero
134
What is a sign of a classic autosomal dominant pedigree?
- present in at least 2 gens
135
Can a disease present in only 2 gens also be recessive?
- yes, if common enough
| - esp in consanguineous pops
136
If 2 siblings have the same mutations, is it likely to be de novo, and why?
- no, de nove likely to have occurred in parent or higher up pedigree
- unlikely for same mutation to occur twice
137
Do you see de novo mutations in autosomal recessive conditions?
- no, they are v v rare
138
What % of mutations in NF2 are de novo/not?
- 50% inherited
- 50% de novo
- 25% mosaic when only 1 person affected
139
What is importance of being able to test for NF2 breast cancer mutations in children?
- may be able to monitor and make lifestyle changes
140
How many genes are involved in BRCA mutation?
- at least 2
141
What is there a high incidence of in the pop for BRCA?
- phenocopy
| - as 1 in 12 don't have mutation but still get breast cancer
142
What is the penetrance of BRCA mutations, and in diff presentations of the mutations?
- not fully penetrant, esp in males
- 40-80% breast
- 11-40% ovaries
- 1-10% male breast
- up to 39% prostate
- 1-7% pancreatic
143
Is the risk of breast cancer still high in the absence of an identified mutation?
- yes
144
How does risk to males compare to risk to females?
- less risk to males, but STILL AT RISK
145
Apart from genetics, what needs to be considered when looking at pedigrees for breast cancer, and what does this inc?
- consider family circumstances, as tend to live together
- inc. similar diet, similar weight/weight problems (obesity related to higher risk of breast cancer), breastfeeding (decreases risk) habits tend to be same, age have 1st child, no. children, env and env toxins
146
What is the consequence of low penetrance risk factors?
- get accum of these, as well as accum of high risk autosomal dominant factors
147
If there is a known mutation, eg. BRCA 1, in a fam and identified in all affected fam members, then relative tests -ve, how much is her risk decreased?
- decreased, but not to general pop risk --> as fam history and cannot explain all of this through a single mutation
148
In the UK would CF be identified at birth, and what would be done?
- almost certainly identified through newborn screening at 5-8 days
- given prophylactic antibiotics to protect from large no. resp infections
149
What is the genotype:phenotype correlation for CF, ie. what is the phenotype in indivs homozygous and heterozygous for p. (Phe508del)?
- homozygous for p. (Phe508del) gen pancreatic insufficient but lung disease varies widely
- compound heterozygotes for p. [(Phe508del)] ; [(Arg117His)] range from asymptomatic females to pancreatic sufficient CF
150
What does it mean if a CF patient is pancreatic insufficient?
- no pancreatic enzs and have to take supplements several times a day
151
What does it mean if a CF patient is pancreatic sufficient?
- some pancreatic activity, but lung disease still varies and can have CF
152
How is CF phenotype sex-dep?
- if male, vas deferens in dev acutely sensitive to decreased CFTR levels, so vast majority of boys have congenital bilateral absence of vas deferens, so are infertile
153
How does the importance of infertility vary?
- in some societies and to some people can be v important or not as important
154
What can provide further info of CF phenotype (but are not conclusive)?
- variants w/in CFTR gene
155
What is the problem w/ many new drugs that target specific CF mutations?
- v expensive --> £100,000 - £200,000/yr
| - as a society can we afford them, esp for other poorer countries
156
What do a third of isolated DMD cases arise from, and what does this mean we would not know?
- from mother w/o mutation IN BLOOD (but could be mosaic)
| - so wouldn't know if she was a carrier from this info alone
157
What % of mutations do deletions constitute in DMD?
- approx 70%
158
If a mother is carrier of DMD, what is the risk to her daughter of being a carrier?
- 50%
159
What is the order of testing for ascertaining a DMD mutation?
- look for deletions
| - then look for nonsense mutation
160
If DMD is only on the other side of the family tree to the proband, how does this affect their risk?
- much lower
| - as potential for carrier on other side to be germline mosaic, so not inherited from a common relative
161
Are de novo mutations poss in X linked conditions?
- yes
162
What options is there in terms of testing for DMD (/other X linked conditions)?
- dosage analysis
- mutation analysis
- dep what looking for
163
What is an obligate carrier?
- indiv who may be clinically unaffected, but must carry mutation based on analysis of fam history
164
What % of DMD deletions/duplications are point mutations?
- 72%
165
What type of inheritance does ALD (adrenoleukodystrophy) have?
- X-linked dominant
166
What is ALD a disorder of?
- fat metabolism
167
What are the diff types of ALD, how does their onset and dev vary?
- onset in early childhood, w/ progressive neurological decline (in 35%)
- AMN (adrenomyeloneuropathy) = late onset (3rd/4th decade), milder but still progressive (in 40-45%, but w/ 10-20% more severe w/ decreased life expectancy
- adrenal insufficiency (Addison's disease) = presents early w/ AMN occuring later (in 10%)
168
What is the classic pattern of X-linked dominant conditions when looking at pedigree?
- more mildly affected females (and later onset) due to X-inactivation
169
Is X-inactivation always a random process?
- not in 10% pop, where get preferential inactivation of 1 of X chrom
170
Why does X-inactivation mean you see more mildly affected females in X-linked dominant conditions?
- if X-inactivated is 1 w/ symptoms then won't have many symptoms (and opp true)
- so get range of symptoms
171
What is the risk to a son of getting ALD if his father is a carrier, and mother is unaffected?
- none
| - can't get male to male transmission (unless pass on x and y)
172
What are the main questions people may want answered when getting tested for a condition?
- risk of getting it
- when
- how severe
- risk of passing on
- how can be treated
173
Why might males pass on ALD w/o knowing?
- may not present until adulthood, so may have already had children
174
What organisms can mosaicism exist in?
- any
175
What types of mosaicism is there?
- gonadal
| - somatic
176
What is mosaicism and how does this affect symptoms?
- some tissues have mutations, some don't, so dep what tissues have it whether have symptoms
177
How does gonadal mosaicism affect recurrence?
- still at risk of it
| - but dep on pattern of mosaicism in gonadal tissue
178
What type of disorders does gonadal mosaicism lead to?
- autosomal dominant
179
What prop of mutations in DMD/BMD are de novo?
- third
180
What type of mosaicism occurs in brittle bone parents, and on which side are mutations more likely?
- gonadal or somatic
| - paternal
181
For genomic testing why must posterior risk be weighed against the priori risk?
- if risk of intervention far greater than risk of having affected child, then may decide against
- have to think of downstream costs --> eg. if test +ve for long QT syndrome may need intervention (pacemaker)
182
What is imprinting?
- determination of expression of gene by parental origin
183
What are 2 eg.s of conditions involving imprinting, and what chromosome do they affect?
- Prader-Willi
- Angelman
- chrom 15
184
What are the symptoms of Prader-Willi?
- polyphagia (and therefore obesity)
- small hands/feet
- genital abnormalities
- dev delay
185
What are the symptoms of Angelman?
- sig dev delay
- sig intellectual disability
- unlikely to be able to survive indep
186
What is the issue of perception, w/ eg. trisomy 21?
- many people w/ it can function well
| - but also can have poor prognosis (eg. congenital leukemia)
187
Where is the PWS/AS locus on the chromosome?
- close to centromere and far from telomere
188
What locus is involved in PW and Angelman?
- PWS/AS
189
What is the pattern of methylation/repression in the PWS/AS locus?
*DIAG*
190
What diff mutations can occur at the PWS/AS locus?
- deletions (tendency towards this)
- uniparental disomy
- mutations in AS gene
- imprinting defect
191
Why do you need to know what specifically has happened at the PWS/AS locus?
- each mutation assoc w/ diff recurrence risk
192
How can deletions be detected?
- chromosome analysis
- FISH
- S blotting/bisulphite mod PCR
- absence of PCR product
193
Why do you need v good controls when looking for absence of PCR product?
- so know absence isn't just because PCR didn't work
194
How does bisulphite mod PCR work?
- wherever there is meth CpG, methyl group converted to U --> becomes T when translated back to DNA and rep
- so if look at treated DNA and use carefully designed primers to look for changes in methylation
195
In meth CpG, where is the methyl group?
- on C --> always on CG in 5'-3' order
196
What diff types of uniparental disomy are there?
- heterodisomy or isodisomy
| - maternal or parental
197
How can uniparental disomy be detected?
- microsatellite analysis
| - S blotting
198
What is the recurrence risk of uniparental disomy?
- v low, as generally unique event
199
When does uniparental disomy occur?
- after the 1st meiotic division
200
What are the diff outcomes of uniparental disomy?
*DIAG*
201
Why does uniparental disomy occur more freq than we anticipate?
- usually no phenotypic effect
202
What do we know about gene and mechanism for PW?
- unknown, just know that SNP region important
203
What gene is important in inheritance of Angelman, and how?
- UBE3A
204
How is Angelman inherited?
- to be affected must inherit active UBE3A w/ mutation, so must inherit through maternal line (as paternal would meth and silence)
- so if not de novo, must be through maternal line
- father can pass to sons and daughters w/o having effect
- BUT only sons can pass it to their children w/o them being affected
205
Why is how Angelman inherited so important to a pedigree w/ 1 affected indiv?
- as have iso case of v severe disorder w/ big impact on affected and family
- can track through many gens, and connect diff parts of pedigree, so may be carriers who would not know they were affected
206
What are we unsure about the role of imprinted genes in, and why would we not necessarily find out?
- dev disorders/intellectual disabilities
| - don't detect meth stated in routine seq
207
How are imprinting defects detected?
- combo of abnormal S blotting and normal pattern of inheritance on microsatellites
208
What is the recurrence risk of imprinting defects?
- can be 50% if caused by mutation (usually microdeletion) in imprinting centre
209
How much of genome do imprinting defects involve?
- only small sections (as far we know)
210
What is a key factor in imprinting defects?
- methylation
211
What type of expression do regions that are imprinted often have in other tissues?
- biallelic expression
212
How can imprinting alt in lifetime?
- may dev or disappear during life
| - symptoms may dev later
213
How does invasiveness of procedure affect its replaceability?
- more invasive = more irreplaceable
214
What does interpretation of genomic testing results have to inc knowledge of?
- variant
- gene, inc inheritance pattern
- splicing
- imprinting
- penetrance
- expressivity
- clinical info is vital
215
What ethical issues are assoc w/ parents wanting to know the carrier status of their unborn child?
- in UK unborn fetus doesn't have rights over mother, but will become person in own rights
- right not to know
- reporting carrier status before actionable and relevant
- pot for info to be misreported by parents
216
Why is gender very important in some societies?
- don't have pensions etc. so rely on children to look after when older
- female often moves to husbands family when married
