STR Flashcards
(162 cards)
1
Q
Question
A
Answer
2
Q
What is the clinical significance of 36-39 CAG repeats in Huntington Disease?
A
Reduced penetrance - may or may not be affected
3
Q
Above what number of CAG repeats would you see fully penetrant development of Huntington Disease?
A
40 CAG repeats
4
Q
What is the significance of 27-35 CAG repeats in Huntington Disease?
A
Intermediate allele - will not cause disease but may expand to cause disease if paternally transmitted
5
Q
Gene and location associated with Frax
A
FMR1 within FRAX A fragile site at Xq27.3
6
Q
Cause of Fragile X syndrome
A
99% is CGG repeat expansion in 5’ UTR of FMR1 gene| 1% is FMR1 point mutations/deletions
7
Q
Underlying pathogenesis of Fragile X syndrome
A
> 200 CGG repeats causes hypermethylation of FMR1 promotor, turning gene off. FMRP expressed at highest levels in brain and testes > most functional in neurons where is it has role in functional and structural maturation of synapses
8
Q
Interspersions in CGG tract
A
AGG interuptions thought to confer stability| Often found in normal size alleles
9
Q
What proportion of Fragile X is mosaic?
A
15-20% of mutations are mosaic
10
Q
What types of mosaicism are there in Fragile X?
A
repeat size (full mut/pre mut)methylation (full mut meth / full mut unmeth)
11
Q
Symptoms of FXTAS
A
Late onset progressive neuromuscular disorder- cerebellar ataxia- intention tremor- parkinsonism
12
Q
Cause of FXTAS
A
CGG premutation (55-200) in FMR1 in both males and females
13
Q
Underlying pathogenesis of FXTAS
A
Transcription of premutation alleles is higher than normal resulting in increase mRNA, but translation is less efficientLess FMRP, more FMR1 mRNA; toxic - leads to cellulat injury
14
Q
Clinical features of FXPOI
A
Early menopause (<40 years)
15
Q
Cause of FXPOI
A
CGG premutation (55-200) in FMR1 in females only
16
Q
Clinical features of HD
A
Progressive neurodegenerative disorder1. Movement - chorea, dystonia2. Psychiatric - depression, mood swings3. Dementia
17
Q
Average age of onset for HD
A
35-50 years. Juvenile HD <20 years
18
Q
Cause of HD
A
> 39 CAG expansion in exon 1 of HTT gene at 4p16.3
19
Q
Intermediate alleles in HD
A
27-35 CAG| Have potential to expand into disease range in single generation
20
Q
What effects risk of expansion for intermediate alleles?
A
- Length of CAG tract - the longer the greater the risk2. Age and sex of transmitting parents - greater risk for males >36 years3. Family history4. Sequence surrounding CAG repeat, polymorphisms etc
21
Q
Molecular pathogenesis of HD
A
HTT translated into protein with extended polyglutamine tract; aggregates in neuronal nuclei to form neuronal intranuclear inclusions - interefere with transcription factorsAggregate HTT protein contains toxic N-terminal fragments
22
Q
Genetic testing for HD
A
PCR to size CAG repeat - primers flanking tract| products separated by capillary electrophoresis
23
Q
Presence of one peak in HD testing - what could this mean?
A
1) patient is hom for same sized CAG expansion2) SNP under primer binding site caused dropout of one allele3) patient has one normal allele and one allele not identifiable by the test (ie too large - juvenile)
24
Q
Presence of one peak in HD testing - reflex test?
A
Use second set of primers that flank CAG repeat AND adjacent (CCG)n repeat tract; highly polymorphic- allows you to ensure that two peaks are present (not for sizing)If juvenile HD, could do TP-PCR for large expansions
25
Exclusion testing in HD - when is it used?
When fetus has 25% chance of being affected (ie affected grandparent) but parent doesnt wish to know their HD status
26
Limitations of HD exclusion testing
1) Will only determine which grandparental haplotype has been inherited; not whether it was the disease causing allele or not so max information on risk is 0% or 50%. BUT it avoids a prenatal test for those at 0%2) risk of recombination across HTT is 2%3) testing may not be informative4) relies on grandparental sample availability
27
Expansion in FMR1
Expansion can only occur when maternally transmitted| >90 CGG have 90% risk of expansion upon transmission
28
Considerations in prenatal FraX testing (CVB)
MCC| Methylation pattern not fixed
29
Testing strategy in FraX
1) Flourescent PCR (sizing) - run with known controls, can only identify normal alleles2) TP-PCR
30
Limitations of flourescent PCR in FraX
1. preferential amplification of the smaller allele2. SNP under primer binding site could cause allele drop out3. Cannot detect full mutations, including mosaic N/FM4. Cannot detect deletion/point mutation (1%)5. Cannot distinguish N/N hom female
31
Alternative techniques in FraX
Southern blotting, methylation specific PCR
32
Methylation specific PCR - principles
Split DNA into 2 reactions; 1 normal PCR and 1 PCR following digestion with methylation specific restriction enzyme which cleaves unmethylated sequences within ROIProducts of each reaction labelled with different flourophore, peak areas compared to determine % methylation
33
How is Fragile X caused?
Expansion of unstable 5' UTR CGG repeat to >200 repeats, causing gene silencing
34
Give 3 clinical features of Fragile X in males
moderate/severe intellectual and social impairment, characteristic facial features, joint laxity, macro-orchidism
35
Give the clinical presentation of Fragile X in females
Variable phenotype - apparently normal approx 50%) through to mild/moderate mental and social impairment
36
What is the significance of repeat size of 55-200 in FMR1?
Premutation - may expand in maternal line to cause FraX in future generations. Patients may develop FXTAS or POI
37
What is the proposed disease mechanism for FXTAS/POI
NOT the same as FraX - ?toxic gain of function?
38
What is the location of FMR1?
Xq27.3
39
What is the role of FMRP protein?
RNA binding protein, present in many tissues including brain, ovaries and testes - thought to act as a shuttle within cells by transporting mRNA from nucleus to areas of cell where proteins assembled. Also helps to control when the instructions in these mRNA molecules are used to build proteins.
40
Name 2 NGS platforms
Agilent Sure Select, ThermoFisher Ion Torrent, Illumina HiSeq/MiSeq SBS seq by synthesis
41
What are the mutation ranges for SBMA?Spinal and Bulbar Muscular Atrophy
Normal - 34 CAG repeats or lessQuestionable - 35 CAG repeatsReduced penetrance - 36-37 CAG repeatsAffected fully penetrant - 38 CAG repeats or greaterSequence any 35-37 repeats
42
Name 3 CAG expansion disorders
1. Huntingtons2. Spinal and Bulbar Muscular Atrophy (SBMA)3. SCAs - 7 of them
43
Name 2 CGG expansion disorders
Fragile XFXTAS
44
Name 3 CTG expansion disorders
Myotonic Dystrophy 1Huntington Like Disease 2SCA 8
45
Name a GAA expansion disorder
Friedreich Ataxia
46
Name a CCTG expansion disorder
Myotonic Dystrophy 2
47
What additional features might you see in a juvenile HD patient with a large expansion?
Bradykinesia -slowness of movementDystonia
48
What factors can contribute to pathogenic mechanism of repeat expansion diseases
1. Sequence of repeat2. Size of repeat3. Location of repeat within gene4. Whether repeat encodes RNA or protein5. Function of repeat-containing gene6. Extent of meiosis and somatic instability
49
What causes Myotonic Dystrophy 1?
CCTG repeat in intron 1 of the CNBP gene
50
What are some clinical features of DM1?
Progressive weakness and myotoniaCataractsCardiac arrhythmiasEndocrinopathyCognitive impairment
51
What is the prevalence and penetrance of DMD and BMD?
Most common muscular dystrophy - 1 in 3500 malespenetrance is 100% in males and variable in femalesprevalence of BMD is 1 in 8000 and females rarely display phenotype
52
DMD ans X-linked cardiomyopathy?
Mutations in the dystrophin gene can also result in X-linked cardiomyopathy. This presents as cardiac disease with no skeletal muscle involvement.due to a mutation in promote and 1st exon resulting in no dystrophin present in cardiac muscle. - skeletal muscle spared as it can use other promoters - onset is 20-25yrs in males was fast progress and death in a few years- in melanges onset is 40-50 yrs and is slower
53
Describe the phenotype of DMD?
- onset < 5yrs and delayed motor development- wheelchair bound by 12 years- progressive muscle weakness and muscle replaced by fat and fibrotic tissue- gower sign- legs and pelvis affected first- scoliosis may develop- 95% of males develop cardiomyopathy- dev delay present in 30-50% and coincides with later onset of symptoms (may be referred for array for LD and DMD discovered then)- mean age at death is 25yrs due to respiratory and cardiac insufficiency- creatine kinase levels are 10-100x normal
54
what is the phenotype of BMD?
milder than DMD- 20% have dilated cardiomyopathy- may be lat learning to walk- muslce weakness onset from ~ 11yrs - lose ability to walk at 49-50 yrs- no LD- survive to middle- creatine kinase 5x normal
55
describe the DMD gene
largest human gene with 79 exons- 2.4MB in size but only 3% is coding7 tissue specific promoters including brain, cardiac an skeletal muscle
56
Describe the dystrophin protein.
dystrophin is a rod shaped cytoskeletal protein which provide structural support dystroglycan complex (DGC)the DGC forms a critical link between the cytoskeleton and the ECM and stabilizes the sarcolemma during contraction and relaxation. loss of dsytrophin disrupts the link between the ECM and cytoskeleton which increase the fragility of the cell membrane and muscle becomes mechanically damaged during contraction. This also increases the permeability to Ca2+ which activate proteases which digest contractile proteinsincreased creatine kinase levels indicate muscle damage
57
How much dystophin is expressed in DMD and BMD?
In DMD dystrophin is virtually absentIn BMD there is between 20% to normal levels (although abnormal protein is expressed)
58
What is the mutation spectrum in DMD?
No correlation between del/dup sizer and the severity of the phenotype. Majority of pathogenic variants are exon deletion (>1 exon) clustering in exons 2-20 (20%) and 45-55 (80%). duplications are clustered in these regions with opposite frequenciesnonsense mutations are next most common mut in DMD followed by dups and indels. single exons deletions- do not routinely test asymptomatic patients therefore do not know the frequency of these in the normal population. If detected in a patient with phenotype it is considered to confirm the diagnosis but could be masking the true diagnosis
59
what is the reading frame rule in DMD?
mutations that disrupt the reading frame result in a no functionla dystrophin due to NMD and asre associated with DMDin-frame dels and dups result in the expression of a partially functional protein and are associated with BMDthe reading frame hypothesis holds true in 90% of cases therefore a diagnosis should be made on clinical assessment not the reported prediction.
60
What are the limitations to the reading frame hypothesis?
#NAME?
61
What are the genotype-phenotype correlations in DMD?
#NAME?
62
What is the inheritance of DMD?
XLR2/3 of cases are inherited from a carrier mother and 1/3 are do novo- new mutation can occur in the oocyte, embryo post conception (mosaic) or be in the mothers germline (7-10% recurrence risk)when the at risk haplotype is known the recurrence risk is 8.6%. However testing my MLPA does not revel this (need linkage) therefore the recurrence risk for a mother of an affected who has not been found to be a carrier is 4.3%sibling of an affected is also at risk of being a carrier even if mother is not. If the sibling then tests negative for the family mutation there is no recurrence risk and her risk of an affected is reduced to that of the general population. DMD has a high recombination rate- 4x greater than would be predicted by its size alone
63
Manifesting females in DMD
2-8% range from mild muscle weakness to an inability to walk. If cardiomopathy is included the incidence is 38%- therefore monitoring for cardiac involvement should be included in female carrier reports
64
How can DMD be diagnosed? CK/IHC
#NAME?
65
what is the genetic testing for DMD?
Dosage analysis (MLPA - 2 kits, can only use one if it will detect known mut in familial cases) will detect 72%- sequencing analysis for CNV negatuve cases- linkage analysis using microsatellites can be used for carrier testing and PND in cases where the pathogenic mutaion has not been detected but there is a confirmed clinical diagnosisin MLPA is a single exon del or dup is detected it should be confirmed by another method e.g. dosage pCR or sequencing across the breakpoint
66
Carrier testing in DMD?
if the mutation is not known and the poband is not available them most closley related at risk family member is tested as they have the highest risk of carrying the mutation (mother then sibling). If a mutation is not found it does not rule out that they are carriers just reduces the risk Even if the mutation is known there is still a recurrence risk in mothers if affected due to the possibility of germline mosaicism so PND is still offered
67
PND in DMD?
for all known carriers of mothers of affected. Only male pregnancies tested, NIPD for sex is becoming more common as can avoid the need for an invasive test in a female pregnancy
68
Linkage analysis in DMD
Preferably need a sample from the proband but if not available can use an unaffected male sibling for exclusion testing- 10% rate of recombination across the DMD gene- to reduce the risk of recombination markers spanning the whole gene are used not just flanking it. Still need to calculate the risk of a double recombination which could result in the mutation being present on the low risk allele- recombination risk is calculated from the average risk of a double recombination between markers and multiplying by the percentage of the coding region that is covered by the markers
69
Microarray IFs
- may be detected in a male referred for LD- explains pheno| - may be detected in females referred for LD- does not explain pheno byt result is reported for risk to family members
70
What are the differential diagnosis for DMD?
#NAME?
71
What are the treatments for DMD?
No cures, treatments aims to help the symptoms e.g. physiotherapy and steroids. Transplant in severe cases of DCM
72
What re the molecular therapies for DMD?
Stop codon read through using amnioglycosides to result in some functional dystrophin being produced and can convert DMD to BMD pheno. 15% of DMD have a PTC.exon skipping by mopholino ASO interferes with splicing and can skip the alterd exon to restore the reading frame and give a BMD phenotype
73
Phenotype of DM1
Can be:mild (cataracts and balding)classical (adult onset; DISTAL muscle weakness, mytonia, difficulty gripping with hands, dropping eyelids)Juvenile - same as adult but also cognitive/behavioural phenotypeCongenital - in utero polyhydramnios and poor fetal movements, neonatal feeding difficulties
74
Phenotype of DM2
cataracts, balding, variable myotoniaPROXIMAL muscle weaknessmuscle pain
75
Cause of DM1
(CTG)n expansion in 3'UTR of DMPK gene at 19q13.3 (99% cases)
76
Cause of DM2
(CCTG)n expansion in intron 1 of CMPG gene at 3q21
77
Repeat ranges for CTG expansion in DPMK
```normal = 5-34premutation = 35-49Expanded = 50+```
78
Expanded repeat ranges for CTG expansion in DMPK
51-500 = mild100-1000 = classical/adult500-2000 = juvenile1000 - 5000 = congenital
79
Allele expansion patterns in DM1
51-150 CTGs = expand in 75% cases, irrespective of transmitting parent150+ CTGs = high risk of expansion into congenital range, especially if maternally inherited
80
Repeat ranges for CCTG expansion in CMPG
```Normal = <27Premutation = 27-74 (phenotype and clinical relevance unknown)Affected = 75-11,000 (actual pathogenic threshold not determined yet!) - shows marked somatic heterogeneity due to instability```
81
Molecular pathogenesis for DM1 and DM2 (similar)
RNA MEDIATED GOF MECHANISM:mRNA trnascripts containing expansions accumulate in ribonuclear foci of cellsAlters regulation and localisation of CUG and RNA binding proteinsResults in aberrant splicing of genes expressed in tissues affected by DM
82
Testing for DM1
1) flourescent PCR specific for repeat tract (only detects normal alelles)2) triplet primed PCR (used to size repeat tract)
83
Principles of triplet primed PCR (TP-PCR)
3 primers;Forward - binds to sequence upstream of repeatReverse - 3' end is complementary to repeat, 5' end is non specific, so sticks upTail - specific to 5' end of R primer
84
Testing for DM2
Testing is for whole complex, not just CCTG (rest of complex highly polymorphic
85
Clinical features of DMD
```Gower manouveredev del/LDmuscle pseudohypertrophyIncreased CKcardiomyopathy```
86
Clinical features of BMD
motor delay| muscle weakness and cramps
87
What proportion of females are manifesting carriers
5-10%
88
Causes of manifesting carrier females
- Turner syndrome- homozygous for DMD/BMD variant- deletion on other allele; or other structural rearrangement- skewed X inactivation- X UPD
89
Cause of DMD
Dystrophin gene at Xp21.2Out of frame deletions = 60-65%Out of frame duplications = 5-10%Point mutations = 25-35%
90
Cause of BMD
Dystrophin gene at Xp21.2In frame deletions = 85%In frame duplications = 5-10%Point mutations = 10-20%
91
Hotspot exons in dystrophin gene
2-20| 45-55
92
In what proportion of cases is the frameshift hypothesis true
~90%
93
What proportion of dystrophin mutations are de novo
01-Mar
94
What is the risk of gonadal mosacism?
10%
95
When might de novo mutation occur?
1. in egg at probands conception - germline, no recurrence risk for mother2. after conception - proband mosaic3. mutation occurs in mothers egg cells pre conception; mother is germline mosaic, recurrence risl!
96
Normal function of dytrophin
Forms part of dystrophin-associated protein complex (DAPC)DAPC forms link between actin exoskeletion and extracellular component -stabilises plasma membrane in muscle during repeated rounds of contraction and relaxation --> key in maintaining muscle integrity
97
Testing strategy in DMD/BMD
MLPA, array, NGS/Sanger
98
Limitation of linkage analysis in DMD
10% chance of recombination across dystrophin gene because it is so large
99
Therapy options in DMD
1. Gene therapy - introduction of dystrophin gene2. Antisense oligonucleotides - cause exon skipping in DMD to bring back in frame3. Read through drugs - promote read through of STOP codons
100
CAG repeats caused by polyQ glutamine, give three examples, other than spinocerebral ataxias. do these mutations occur in coding or non coding regions?
1. HD - HTT gene2. SBMA - XLR - AR gene (also causes androgen insensitivity syndrome) 3. dentatorubral-pallidoluysian atrophy (DRPLA) - CAG expansion in ATN1 geneAll exonic! GOF - CAG repeat expansions result in polyglutamine aggregates
101
what are the repeat sizes for FRAX?
N up to 4546-58 intpremutation 59-200mutation >200
102
what is the phenotype of Fragile X full mutation in males? and females? why is there a variable phenotype in females?
- moderate to severe ID and social impairment, large ears, macroorchidism, hypermobile- apparently normal (about 50%) to mild to moderate mental and social impairment- skewed x inactivation
103
what is the phenotype associated with FRAX premutation in females? and in males?
- POI - cessation of menses before 40 and early menopause- occurs in 20% of casesMALES- late-onset progressive cerebellar ataxia and intention tremor- onset usually >60 years, penetrance ~50%
104
what is FRAXE? what is the mutation and phenotype?
- distal fragile site Xq28- GCC 5’ UTR expansion of FMR2- phenotype is considerably less severe than the Fragile X of FRAXA and lacks the specific syndromic features- no phenotype linked to premutation
105
what are limitation of PCR for FRAX?
#NAME?
106
what is an intermediate FRAX allele?
46-58 repeatsoften transmitted stably, but may show increasing unstable transmission with larger sizes. stability correlates with correlate with the presence of two or more interspersed AGG motifs within the CGG tract
107
what are FRAX premutations?
59-200 repeatsunmethylated- pcr can detect smaller alleles- expansion to full mutation exclusive to females and usually if >90 repeats
108
what % of FRAX full mutation patients are mosaic?
20% of full mutation patients also show some mosaicism for a premutation some are full mutation methylation mosaics
109
describe Amplidex for FRAX testing?
TP-PCR methodallows determination of CGG repeat length up to 200 CGGs and non-quantitative detection of alleles greater than 200 CGGs (i.e. full mutations)- can detect interrupting AGG sequences and detect size mosaics- A methylation sensitive kit is also available for the detection of methylation status in the FMR1 gene- able to detect full mutations and resolve female zygosity (one peak on PCR - homozygous or missed full mutation?)
110
what testing methods are available for FRAX? what are advantages and disadvantages
1. direct PCR - quick and cheap. only detects up to 120 repeats, preferential amplification, doesn't detect premutation/full mutation mosaics. males with no peaks and females with one peak should have further testing. 2. southern blot - detects all sizes and methylation but laborious, large amounts of DNA required, doesnt have the resolution to detect size3. Amplidex - detects normal, premutation and full mutations +some detect methylation, AGG repeats but expensive as a first-line test4. linkage - STR markers. may be useful in prenatal eg. if southern blot fails. 5. NGS - SNVs, deletions, duplications
111
What is Freidreichs ataxia?
Most common inherited ataxia- incidence of 1/50,000 with carrrier freq ranging from 1/50-100- AR- Due to a LOF GAA repeat expansion in intron 1 of the FRXN gene
112
what are the symptoms of Freidreichs ataxia
characterised by progressive ataxia and hypertrophic cardiomyopathy- average age of death is 37 from cardiac dysfunction- pateints become wheelchair bound within 10 yrs of onset of symptoms from muscle weakness
113
What is the genetic cause of Freidreichs ataxia?
98% have an expansion mutation on both alleles, remainder have 1 expasnion allele and 1 point mutation. No reports of patients with 2 point mutations- assumed to be embryonic lethal as FA results from a deficiency of FXRN protain not a complete absence
114
What is the role of the FRXN gene?
Nuclear encoded mitochondrial proteinbind Fe and is required for the formation of FE-S clusters and therefore for synthesis of respiratpry chain complexes I, II, III and aconitase shares symptoms with mitchondrial diseases MELAS and MERRF
115
What is the molecular pathogenesis of the GAA expansion in FRXN?
RNA-DNA hybrids are formed called R-loops during transcription when the nascent RNA bind to the DNA template behind the RNA pol- these block the transcriptional machinery from proceeding along the DNA = reduced FRXN protein due to block of elongationtranscriptional initiation is also affected the mechanism is unclear
116
What are the different repeat sizes in Freidreich ataxia?
Large overlap between repeat sizes and demarcation between normal and FM is not determinednormal = 5-53PM = 34-64- not associated with FA but may expand on transmissionBorderline = 44-64- shortest allele reported with FA is 44FM 66-~17,000
117
GAA expansion instability in FA- transmission
#NAME?
118
what are the genotype-phenotype correlations in FA?
Complete penetrance but age of onset varies from 5-50 due tot the large range of expansion sizes. In general the size of the smaller expansion accounts for 50% in the variation in age of onset. - late onset > 25 yrs 1 allele < 500rpts- v. late onset > 40 yrs 1 allele <300rpts
119
what are the testing strategies for FA?
- Sizing PCR across GAA repeat can exclude a diagnosis in the presence of 2 normal alleles- to detect and expansion allele TP-PCR or southern blot is performed- sequencing or MLPA may be required is only 1 expansion is detected but there is strong clinical suspiscion of a second mutation. Need to be wary of high carrier freq and the possibility of an alternative diagnosis. Protein based assay can also be used to determine the conc of FRXN protein in the blood as a deficiency of the protein is causative
120
what re the possible therapies for FA?
Carriers are unaffected so aim is to increase protein levels to that of a carrier1. increase transcriptions of FRXN1 using a histone deacetylase inhibitor to increase initiation. But non-specific2. Use synthetic nucleaic acids to bind to the GAA repeat and inhibit R-loop formation3. INFgamma upregulates FRXN1 levels in all cell types and clinical trials in children are promising 4. Fe transport molecules and antioxidants
121
What are the theories of pathogenesis for polQ disorders?
1, aggregation of toxic products- but in HD the presence of NI does not correlate with the location of neuronal death (pathognomic but not causative2. toxic fragments- cleavage of mutant Htt by caspases results in the formation of toxic n-terminal fragments3. transcriptional dysregulation - polyQ expanded proteins accumulate in the nucleus and interfere with transcription factors and regulators4. mutant proteins interfere with cytoskeletal and axonal transport resulting in accumulated cargo5. mutant proteins affect non neuronal cells types e.g. microglia are the immune cells of the CNS and are activated in HD
122
What therapies are available for HD?
IONIS-HTTRX (roche) is designed to inhibit expression of the expanded mRNA to reduce the concentration of mutant Htt
123
What is the hypothesis for repeat expansion in TNR diseases?
TNR are dynamic mutationsshow anticipationexpansion or contraction thought to occur due to strand slippage during replication in actively dividing cells e.g. germ cells. In non actively dividing cells e.g. neurons expansion occurs by transcription mediated repair pathways
124
```what TNR diseases are due to expansion in the:5'UTR1st exon1st intron3'UTR```
5'UTR- fragile X CCG - LOF- SCA 8, 10, 121st exon- HD CAG- GOF- SBMA CAG - SCA 1,2,3,6,7,17 CAG- DRPLA1st Intron- Freidreich ataxia GAA- LOF3'UTR- DM1 CUG- GOF
125
what are the features of PolyQ diseases?
- adult onset (juvenille cases rare and if seen are paternally transmitted due to the high level of meiosis in spermatogenesis- progressive- show anticipation with a threshold level of repeat that must be met for the disease phenotype to present. - gene is ubiquitously expressed- mutant proteins accumulates in ubiquitinated neuronal intranuclear inclusions
126
what are the 9 PolyQ disrders?
SCA- Ataxia, abnormal limb movements, slurred speech and lack of coordinationDRPLA- CVAG expansion in ATXN1 and result in ataxia, choreoathetosis and dementia. Myoclonus epilepsy also seen in cases under 20yrs and associated with increased expansion size SBMA- CAG expansion in AR gene. Affects males but females spared due to low levels of circulating androgens and lower level of AR recpetor stimulation. Males may also present with gynaecomastia, reduced fertility and testicular atrophy as a result of mild androgen insensitivity
127
Describe HD
HD is a AD disease caused by a CAG repeat expansion in the first exon of the Htt gene. Incidence of 5-10 per 100,000. Mean age of onset i3 30-60 with a peak at 40-45yrsaccount for 90% of chorea with a genetic causecharacteristic finding is the present of ubiquitinated neuronal intranuclear inclusion in the brains of HD pateints (insoluble aggregates of mutant Htt and ubiqutiin)
128
what are the clinical features of HD?
Movement- chorea, dystonia, ataxiaPsychiatric- depression, irritability, psychosis- may proceed movement changesneurologic- dementia
129
what are the features if juvenile HD
- rare and associated with paternal transmission- onset of symptoms <20- first signs are usually behavioural disturbances at school and the development of twitchy movement, chorea is rarely seen in the 1st decade.
130
what is the location and function of the HTT gene?
HTT is found at 4p16.3HTT protein is ubiquitiously expressed and involved in normal development
131
What are the pathological finding in HD?
The CAG repeat is transcribed and this results in a conformational change in the Htt protein- it becomes sticky and forms aggegates NII'sClinical features are associated with the degeneration of the CNS with loss of striatal neurons whilst large interneurons are spared. It is unclear why cell death is confined to particular neuronal cell types.
132
what are the repeat size ranges in HD
```Normal - up to 26 repeatsIntermediate - 27-35reduced penetrance 36-39Full penetrance - 40+Juvenile - 60+```
133
What are the considerations for intermediate alleles?
Found in 6% of the general population- relatively common and so the possibility fo detecting one should be covered in pre-test counselling. May be found in a diagnostic case with 1 allele in the affected and 1 allele in the IA range, therefore identifying a risk of HD in the non-HD side of the family.Not associated with phenotype but risk of expansion to HD in single transmission so PND can be offererdrisk of expansion depends on:sex- more likely in male transmission- family history, more likely if previous expansion of an IA in the family- genetic variation - Glu2645del poly more common in HD alleles than general population- If the 30 CAA has changed to CAG expansion is more likely
134
what are the considerations of a reduced penetrance result?
36-39Low penetrance and may never develop HD in their lifetime. - 40% will be HD free at 65yrs and 30% at 70yrs- unstable and risk of HD in offspring so PND available- if detected in a diagnostic case reported as being consistent with a diagnosis of HD- if detected in a PST reported to have in increased risk of developing HD- can offer cascade testing to family members
135
what are the differential diagnoses for HD?
1% of HD cases are not confirmed molecularily- phenocopies```differential diagnoses includes:HD-like 1HD-like 2DRPLASCA17Freidreich ataxiaWilson diseaseNeurocanthosisbenign familial chorea (```
136
what are the consideration of a bi-allelic HD result?
Homozygous HD has same onset (depending on repeat length plus other modifiers) and severity but the progression may be faster- reveals risk of HD on non-HD side of the family. both parents are either carriers of affectedsiblings have a 75% of risk of carrying one or both alleles
137
what is the diagnostic testing method in HD
1. sizing PCR across the CAG repeat- can size alleles up to ~80rpts so usually sufficient for exclusion or confirmation of a diagnosis- uses 2 primers, 1 amplifies the CAG repeat to accurate sizing. a second amplifies the CAG repeat plus a polymorphic GC rich repeat. This is beneficial as patients unlikely to be homozygous for CAG and GC repeat so can confirm if a patient is homoxygous for a normal allele- rare poly can cause primer looping and 2 rpoduct peaks one repeat apart for a single alleleTP-PCR is therefore used in cases when there is:- only 1 allele detected using both primer sets- 2 peaks 1 rpt apart as may be from the same allele- generally only required for juvenile cases as 55+ repeats expected to have onset by 35 and would be detected by sizing PCRneed to use controls are the borders of the size ranges with known repeat sizes as CAG repeats run anonymously in electrophoresis which can make the size ladder unreliable
138
what are the different referral categories and accepted referrers in HD?
Diagnostic- patients with features of HD- accepted from neurologist or other relevant speciality. Referral from clinical genetics if patient is under 16PST- must come from clinical genetics and requires at least 2 counselling sessions a couple of months apart. Preferably need a confirmed diagnosis in a family memeber, if not a negative result cannot rule out them being at risk of a different disorderPND- can be direct if affected parent has been tested or indirect for fetuses at 25% risk if the at risk parent does not want to be tested.
139
What are the considerations for prenatal direct testing?
Use family member as a positive control. If mother is affected need to be wary of MCC- PCR test is more sensitive than the MCC test so may be affected by levels not detected by MCC exclusion. Should be wary of results with the same expansion size as the mother or very low peak and may wish to confirm by linkage which is less sensitive to MCC
140
What are the consideration for indirect prenatal testing?
need a sample from parents of parent at 50% risk to determine the high risk haplotype- preferably affetected parent- if fetus inherits high risk haplotype 50% risk of being affected and parents counselled that if they are undertaking the test they should terminate in this circumstance if not it is a PST on a minor without permission which is unethical- low risk haplotype reduced risk of HD- small risk that other parent carries an IA (65) or recued penetrance allele and is unaware- 2% risk of recombination so need to use marker across the gene to reduce the risk of a double recombination- ideally work up should be done before getting pregnant
141
Is PGD available for HD
yes embryos biopsied at the 8 cell stage and use linked markers in karyomapping to see if the high risk haplotype has been inherited
142
What is the evidence that HD is GOF?
Dominant inheritancenot found in patient with LOF mutations or delsCAG repeat is trascribedHD hets are clinically the same as HD homs
143
what are the pathogenic therories for HD?
1. accumulation of toxic aggregates- NIIs are characteristic but are not causative as they do not cooincide with the timing or location of pathogenesis- part of the process but may not be toxic per se2. toxic fragments- abnormal HTT is a substrate for proteolytic cleavage by caspases and calpains. amnio teminal fragment expessed in mice is sufficient to induce a HD like phenotype3. transcription dysregulation- ployQ expanded fragments accumulate in the nucleaus and interfere with normal transcription4. defect is cytoskeletal and axonal transport- disruption of transport results in aggregation of accumulated cargo and neuronal death ad dysfunction5. effects on non-neuronla cell types - astocytes and microglia. Strong evidenc that microglia (neuronal immune cells) are activated in HD and level of activation correlates with level of neuronal pathology
144
What is DM1
Most common form of adult onset dystrophy1 in 8,000 characterized by myotonia and progressive muscle weaknessmean onset at 20-50yrs with death at 60 from cardiac failureAD and shows anticipation
145
What are the different types of DM1?
Mild- late onset with and may only show mild myotonia, cataracts and normal life spanClassic- myotonia, progressive muscle weakness, gait disturbances- cardiac conduction abnormalities- baldness and testicular artrophy- may also include catarcats, IDDM, digestive tract problems and hypersomina- onset in 2nd or 3rd decadeJuvenilesimilar to classic DM but earlier onset and can include cognitive and behavioral difficultiesCongenital- presents at birth with extreme hypotonia- prenatal polyhydramnios and reduced fetal movement- often have respiratory insufficiency resulting in early death- ID and motor developmental delay
146
Describe the DMPK gene
19q13.398& of cases are due to CTG expansion in the 3'UTR, does not affect coding sequence and is transcribed as a CUG repeat in mRNA
147
what is the pathogenesis model for DM1
- not due to HI as point mutations not detected in DM1 patients and KO mice do not show DM1 phenotype- CTG repeats may lead to changes in the chromatin structure and silence neighboring genes but this does not explain the full phenotype- RNA GOFexpanded CUG repeats accumulate in nuclear foci and have been visualised in affected tissues - increased repeat length correlates with the accumulation of mutant transcriptsexpression of expanded 3'UTR is sufficient to inhibit myogenesis- DM2 produces a similar phenotype but due to an expansion in a different geneCUG repeat regions interacts with RNA binding proteins which are involved in splice regulation. MBNL1 and CUGBP1- these usually act antagonistically and interaction with CUG rpt results in functional down-regulation of MBNL1 and up-regulation of CUGBP1. This results in aberrant splicing of targets and embryonal splicing patterns are seen patients instead of adult splicing patterns.
148
Why is the CUG tract instable?
expansions form as a result of the generation of secondary structures during processes which separate the DNA strands e.g. replication. once a CUG has at least 11 repeast it has the propensity to fold into a hairpin like secondary structure with pairing between C-G and U-U mismatches.
149
What are the therapeutic approaches to DM1?
#NAME?
150
what are the repeat length ranges in DM1?
Normal - 5-35Intermediate 36-50 No DM1 but may be unstabe;Full mutation 50-150 associated with mild, classi or asymptomatic DMFull mutation >150 associated with classic, juvenile and congenital DM
151
what affect is the sex of transmission in DM1 repat instability?
Instability is skewed toward expansionintermediate alleles show highest instability when paternally inherited. in the disease range all expansion are likely to be unstable regardless of parental genderCongential DM1 is almost exclusively inherited maternally in a mechanism which is not well understoodsomatic instability results in somatic mosaicism especially in cardiac and skeletal muscle5% of DM1 patients have non CTG interruptions. - these are generally more stable on transmission and gross changes in expansion length occur in the uninterrupted part of the rpt proximal to the interruption.
152
What are the clinical features of DM2
Clinical features:- muscle pain and stiffness- progressive muscle weakness and myotonia- male hypogonadism and azoospermia- may also have ID, hypersomnia, tremor, hearing loss and male frontal balding
153
what gene caused DM2
CCTG repeat in intron 1 of the CNBP genenormal alleles have 26 rptsdisease alleles have 75->11,000 (mean 5,000) repeatsdoes not show anticipationlower prevalence and more common in the Finnish and German populations
154
What is the genetic testing in DM1?
1. Sizing PCR using primers which flank the CUG rpt- uses 2 primer pairs to give a forward and reverse amplification- 2 primers reduce the chance of false -ves whihc can occur if there a non-CTG interruptions in the 3' of the expansion (5%)2. TP-PCR - used if a sinlge normal allele is detected by sizing PCR whihc could indicate:- homoxygous for a normal allele- undetected large expansion allele- undetected second allele sue to SNP under primer binding site(long range PCR and southern blot can also be used but lonog range PCR may fail to amplify very large expansions and southern blot is time consuming and requires a lot of high quality DNA)
155
Describe prenatal testing in DM1?
offered to carriers of IA and FM alleles- CTG PCR and TP-PCR- dilute CVS samples to prevent inhibition from heparin media- MCC- TP-PCR is more sensitive to MCC that the MCC assay, therefore may need to confirm a +ve result by linkage or southgern blot whihc are less sensitive to MCC Confirmation not required if:- father is affected/carrier- allele is a different size to the maternal expansion- fetus has not inherited the mothers normal allele- negative result and 2 normal alleles in fetuslinkage - use linked markers. Aim to use multiple markers to reduce the chance of a double recombinations
156
What is the genetic testing for DM2?
PCR can be used to detect smaller alleles. Accurate sizing is not required due to the large size difference between the normal and affected ranges- quadruplet primed PCR to detect large expansions- expansions may appear as a diffuse smear by southern blot sue to somatic mosaicism- this limits the sensitivity of detection and requires good quality DNA
157
what is FSHD?
Facioscapulohueral muscular dystrophy- assymetric and progressive proximal muscle weakness presenting in the face and progresssing to the scapula, arm, hip and legs. Often show scapular winging
158
What is the genetic causative change in FSHD
- due to expression of the DUX4 transcript on chr 4q35| - due to 2 different mechanisms in FSHD1 and 2
159
Describe the molecular pathogenesis of FSHD?
there is a tandem array of D4Z4 repeats at 4q35- each contains a copy of the gene DUX4- DUX4 is normally epigenetically silenced in the majority of adult cells and tissues. - DUX4 repeat size is normally 11-100, in the disease range it is reduced to 1-10a reduction in the DSUX4 repeat length is associated with a loss of chromatin compaction and CpG methylation resulting in DUX4 expression from the final D4Z4 unitbroad inverse correlation between the repeat length and disease severity and patients with intermediate length repeats have a milder form without facial involvementRequires a permissive allele to develop disease. There is a 4qA or 4qB poly distal to the D4Z4 repeat. FSHD1 is only seen when there is repeat length contraction occurs on the permissive 4qA allele.AD inhertitcance
160
What is the genetic testing for FSHD1?
southern blot to detect repeat length
161
What is the moelcular pathogenesis of FSHD2?
Digenic inheritance1-5%of FSHD (majority is type 1)due to mutations in SMCHD1 + 1 permissive allele- SMCHD1 is an epigenetic modifier of D4Z4 and LOF mutations result in chromatin relaxtion and hypomethylation allowing expression of DUX4digenic inheritance means risk to offspring is 25-50% depending ont he number of pathogenic haplotypes in the family and can make genetic counselling difficultFSHD1 risk to offspring is 50%SMCHD1 can also be a disease modifier in FSHD1- results in ealrier onset and more severe disease and there is DUX4 expression0 by 2 mechanisms.
162
What is the genetic testing for FSHD2?
Methylation specific pyrosequencing to detect the methylation status of D4Z4 and SMCHD1 screening to identify pathogenic mutations
