CF Flashcards

1
Q

Question

A

Answer

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2
Q

CFTR protein function

A

CFTR protein acts as an ion channelLocated in apical membrane of epithelial cellsRegulated transport of Chloride and Sodium

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3
Q

Mutations tested for in NBS for CF

A

F508delc.621+1G>TR542XG551D

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4
Q

CFTR poly T tract

A

String of T’s in intron 8Can have 5T, 7T and 9T5T reduces efficiency of intron 8 splicing > transcripts lack exon 9

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5
Q

When should polyTG tract be analysed?

A

When polyT tract shows 5T because a longer TG tract with a short polyT (ie 5T) has reduced intron 8 splicing (and thus more transcripts lacking exon 9)

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6
Q

When should polyT tract be analysed?

A

CFTR related disorders = can be caused by severe CF mutation in trans with 5T alleleCBAVD = can be caused by above OR homozygosity of 5T12TG in the absence of other mutationsR117H = can make this mut pathogenic when in cis

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7
Q

p.Arg117His and polyT tract

A

5T in cis with R117H is pathogenic; R117H not usually pathogenic aloneR117H/5T + CF mut = classical CF R117H/7T + CF mut - CFTR-related illness such as CBAVD

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8
Q

Why is the frequency of CF stable in the population?

A

CF carriers have selective advantage to typhoid/cholera| CF present gene frequency will be maintained if CF carriers have 2% more children than normal people

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9
Q

Principle of ARMs PCR

A

Allele specific PCR. Principle is that oligonucleotide primers with 3’ mismatched residue will not function as PCR primers under specific conditionsDesign primers for mut and WT

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10
Q

Underlying pathogenesis of CF

A

When normally functioning CFTR is activated, chloride ions are secreted out of the cell. CFTR also inhibits epithelial sodium channel (ENaC) and less sodium is absorbed into the cell, leaving a greater combined ionic gradient to allow water to leave the cell by osmosis providing fluid for epithelial tissue secretions.Absent or malfunctioning CFTR = less water leaves cell by osmosis, mucus becomes dehydrated, thick and sticky

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11
Q

CF; common reasons for referral

A
?CF?CF related diseasefamily history of CFAzoospermia/CBAVDFetal echogenic bowelNewborn screening
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12
Q

Apparent homozygosity for a mut - causes

A

SNP under primer binding site for WT alleleDeletion of CFTR exon(s)UPD7 (RSS)Always perform biparental testing where possible to confirm homozygosity

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13
Q

Frequency of 5T allele in general population

A

5%

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14
Q

Therapies in CF with type 1 mutations

A

TYPE 1 = no synthesis = drugs that permit readthrough of PTC

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15
Q

Therapies in CF with type 2 mutations

A

TYPE 2 = block in processing = Lumacaftor

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16
Q

Therapies in CF with type 3 mutations

A

TYPE 3 = defect in channel activation = Ivacaftor (potentiator - increases functional CFTR at the membrane)

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17
Q

Severity of the different classes of CF mutation

A
Types 1 (no synthesis) and 2 (block in processing) are SEVERE - classical CFTypes 3-5 are milder as some residual CFTR functionality remains
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18
Q

Describe Class 1 CFTR mutations

A

Nonsense, most frameshift mutns and large del’s create premature stop codons causing defective protein synthesis and no CFTR protein expressed. Severe phenotype, W128X, R553X, G542X. Treatments - readthrough drugs - Ataluren phase 3 trials

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19
Q

Describe Class 2 CFTR mutations

A

Some missense mutns and in frame del’s disrupt CFTR protein folding and trafficking to the surface. F506del, N1303K. Treatments: correctors to promote folding - Lumacafter

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20
Q

Describe Class 3 CFTR mutations

A

Some missense mutns result in substitution of aa’s, disrupting regulation of CFTR channel, which no longer opens in response to channel agonists. G551D, G551S, G1349D. Treatment Ivacafter being trialled

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21
Q

Describe Class 4 CFTR mutations

A

Some missense mutations result in changes to CFTR protein structure that forms the pore of the channel which can restrict the movement of Cl- ions through the channel - conductance defect. Eg R117H, R334W, R347P. Ivacafter being trialled.

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22
Q

Describe Class 5 CFTR mutations

A

Some missense mutations result in alternative splicing that disrupts mRNA processing - extremely reduced amounts of normal CFTR protein are synthesised, less protein at cell surface. 2789+5G>A, A455E. Treatments are focussed on compounds that enhance CFTR retention/anchoring

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23
Q

Describe Class 6 CFTR mutations

A

Different types of mutations increase the turnover of CFTR protein at the cell surface, quickly removed and degraded by cell machinery. Egs include Rescued F508del, 120del23, N287Y, 4326delTC, 4279insA

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24
Q

How many classes of CFTR mutations are there?

A

6

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25
Q

Curry-Jones syndrome

A

7q32.1MosaismsRecurrent somatic variant SMO p.L412FPatchy skin lesions, polysyndactyly, cerebral malform, craniosynostosis, iris colobimas, microphthalmia

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26
Q

Potential treatment for CF

A

Gene therapy - introducing a normal copy of CFTRCFTR modulators:1. Read through -Ataluren2. Corrector therapy - corrects mts that cause misfolding and degradation eg F508del - Lumacaftor3. Potentatior therapy - improves CFTR channel function by correction mts thatvreduce gating efficiency eg G551D - Ivacafor

27
Q

NBS - CF variants

A

4 variants:p.Phe508delp.Gly542Xp.Gly551Aspc.489+1G>T

28
Q

CF intron 9 poly T & polyTG tract - significance

A

Poly T 5T - 90% cases exon 10 skippedPolyTGLonger TG associated with disease phenotypeCommon TG lenght = 11, 13 and 13Most common TG11Increased skipping of ex10 caused by TG12, TG13 and 5TTG12/T5 assoc. with CBAVDTG13/T5 assoc. with atypical CF5 T also modifies expression of p.Arg117His ( variable phenotype)9T in exclusively seen in cis with p.Phe508del

29
Q

what are the clinical symptoms of CF? (CF PANCREAS)

A

Chronic cough and wheezingFailure to thrivePancreatic insufficiency (symptoms of malabsorption like steatorrhea)Alkalosis and hypotonic dehydrationNeonatal intestinal obstruction (meconium ileus)/ Nasal polypsClubbing of fingers/ Chest radiograph with characteristic changesRectal prolapseElectrolyte elevation in sweat, salty skinAbsence or congenital atresia of vas deferensSputum with Staph or Pseudomonas (mucoid)

30
Q

what is the testing process for diagnostic CF case?

A
  1. screen for most common UK variants (Devyser, OLA, Elucigene) + MLPA (10% of CF variants are CNVs)2. If negative or only one variant identified do NGS for 27 exons
31
Q

If rare homozygous variants in non-consanguineous individuals are detected what should be considered?

A
  1. deletion2. UPD7
32
Q

Blood spots are collected on Guthrie cards at day 5 and analysed for the presence of immunoreactive trypsinogen (IRT) in duplicate – if levels of IRT are observed to be above the 99.5th centile, may be indicative of CF. What is IRT?

A

Immunoreactive trypsinogen - precursor to the enzyme trypsin. produced in pancreas and transported to small intestine where it is converted. In CF patients the mucous blocks the pancreatic ducts preventing trypsinogen from reaching the intestine and so Newborns with CF may have raised levels of trypsinogen in their blood for several months.

33
Q

what is the CF testing pathway for a newborn with high IRT on first 5 day test?what is the testing pathway for Babies with a FH, meconium ileus or echogenic bowel with a normal IRT?

A

screen 4 most common variants accounting for 80% of variants seen in the UK population. If a single variant detected or two variants (immediate referral to a CF specialist), screen for all variants. 2nd IRT on day 28 if first is raised. If no mutation identified but still high > CF suspected. If one mutation detected but still high > CF suspected. If one mutation detected and IRT normal on 2nd test = probable carrier.Babies with a FH, meconium ileus or echogenic bowel with a normal IRT should be investigated according to clinical circumstances as well as being screened.

34
Q

what 3 biochemical testing is available for CF?

A
  • IRT- sweat test - Individuals with CF will lose more Cl- in sweat than unaffected people. 90% of patients with have abnormal test- Transepithelial nasal potential difference measurements assesses ion conductance in the upper respiratory epithelium: separately measures transport of sodium (Na+) and chloride ions (Cl-).
35
Q

what is classical CF?

A

severe and chronic lung disease, cough, pancreatic insufficiency, pancreatitis, pulmonary infections, up to 20% of neonates meconium ileus at birth , high sweat chloride, congenital absence of the vas deferens (CBAVD). Caused by ‘severe’ mutations (no or reduced CFTR protein

36
Q

what is mild CF?

A

a milder more variable disease resulting from mild mutations - some CFTR protein.pancreatic sufficiency, onset usually after 10 years, no meconium ileus, lower sweat chloride levels and less severe respiratory disease• Patients with a class 4 or 5 mutation have a later onset of respiratory symptoms as there is some function of CFTR (although less than wild-type CFTR).

37
Q

what is the inheritance pattern in CF?

A

AR- shows horizontal inheritance with affected siblings born to generally unaffected parents- increased risk in consanguineous families- unaffected siblings of affected have a 2/3 risk of being a carrier (1/3 unaffected non-carrier)

38
Q

Describe the basics of CF

A
  • most common AR disease in UK with carrier freq of 1/25 (high carrier freq suggests het advantage like in sickle, advantage is unknown- may be associated with cholera)- affects 1 in 2500- carrier freq depends on population and so specific frequencies should be used for risk calculations where possible- there is a good genptype/phenotype correlation between the CF mutation and pancreatic sufficiency but not pulmonary disease. - phedel508 is most common UK caucasian deletion ~75%
39
Q

what is the function of the CFTR gene

A

CFTR gene encodes a cAMP activated chlorine channel located in the plasma membrane of secretory cells e.g. in the respiratory tract, reproductive tract, kidney, pancreas, vas deferens, sweat ductsCFTR channels are responsible for moving chlorine out of the cell, this creates a trans-membrane gradient. and Na+ and water flow out of the cell down the concentration gradient. Mutations that result in a loss of function or reduce ability to function result in reduced Cl- transport and hence water resulting in increased viscosity of mucus

40
Q

What is the phenotype of CF?

A

Sweat chloride concentration >60mm/L (raised compared to normal)- chronic cough and wheezing- recurrent chest infections- can be pancreatic sufficient/insufficient- CAVD/CBAVD- 95% make are infertile- failure to thrive- moconium ileus in newborns- fetal echogenic bowel- 4% of fetus with FEB have CF- salty skin

41
Q

what is the phenotype of mild CF?

A

pancreatic sufficientvariable lung diseaselower sweat chloride levels

42
Q

What is CFTR-RD?

A

due to mild CF mutations e.g, R117H/5Tresults in only mild dysfunction in one organ and may have slightly elevated sweat chlorides- disseminated bronchiecstasis- CBAVD- chronic ideopathic pancreatitis- allergic bronchopulmonary aspergillosis

43
Q

what are the diagnostics tests for CF?

A
IRT testing- used for newborn screeningSweat chlorideTrans nasal epithelial testSemen analysis- for CBAVDgenetic testing
44
Q

Describe IRT testing in NBS?

A

IRT- immunoreactive trypsinogenin newborn it is raised compared to normal neonates and is tested as part of the NBS blood spot screening using a guthrie card.Trypsinogen is released by the pancreas and is increased in CF regardless of pancreatic sufficiency- If the level is raised of the 95th percentile a second IRT measurement is taken. If still raised generic testing for the 4 most common CF mutations is carried out- F508del, Gly542X, Gly551Asp, c.489+1G>T2 mutations = confirms CF1 mutation - test for other common mutations- if not second mutation do another IRT. If raised = Cf suspected, if not raised = carrier suspectedno mutation = do another IRT test- if above threshold CF susupected and if not it is not suspectedtesting offered as no cure but early treatment benefits outcome. Set-up of NBS aims to maximize sensitivity for CF whilst reducing the detection of carriers.

45
Q

What is the diagnostic measure for sweat chloride testing?

A

gold standard test for diagnosing CF. 2 reading >60mm/L is diagnostic of CF in 98% of patients

46
Q

What is the genetic testing in CF?

A

In UK ARMs PCR using the commercial CFEU2v1 kit is generally used diagnostically. Detects the 50 most common mutation in UK caucasian population - will diagnose 90% of cases (differs for other ethnicities)

47
Q

what are the considerations of interpreting ARMS PCR results?

A

shift in the B tube may indicate the presence of a insertion or deletion so the affected exon can then be sequenced to identify the mutationSNP under the primer binding site can result in a false result and appearance of homozygosity of the other allele (may miss a mutation)- this is why a positive control should be used to confirm that the familial mutation can be detected.

48
Q

Describe ARMs PCR?

A

expploits the observation that a DNA primer can only be extended from with a perfectly matched 3’ end- primers for the mutant and WT alleles are contained in 2 mixes and the presence of an allele peak indicates the presence of that allele (alleles allele zygosity to be determined)- products differentiated by different sized stuffer fragments- weak mismatches may have a second mismatch in the adjacent base- requires a taq pol which lack 3-5’ exonuclease acitvity-

49
Q

what are the limitations of ARMs PCR?

A
  • misses 10% of mutations- routine testing for all possibel mutations is not practical or cost affective but some cases may go on to have sequencing if there is a diagnosis of CF but no second mutation detected- Y659D is not tested for by kit but is common (9.6%0 in indian sub continent so may need to be tested for separately in this population- cant detect deletions as the other allele will be amplified and will appear homozygous- wont detect copy number changes- there is an MLPA kit if this is suspected
50
Q

When is linkage analysis used for CF testing?

A

Linkage may be used in families with a confirmed clinical diagnosis where both mutants have not been identified. Is used for PND and carrier testing. Need DNA from the affected individual and parents to determine phase- small risk of a double recombination and does not test for de novo mutations so can only reduce the risk of being affected and not completely exclude it

51
Q

What may cause a failure to confirm carrier status in the parents of an affected?

A

Important to test parent for cascade testing of family members and PND but it also allows confirmation if phase to be sure the detected variants are in trans

May not be confirmed if:- non paternity- one parent is a deletion carrier- UPD7 later 2 only possible for a proband with a homzygous result
52
Q

what is the importance of the polyT tract and TG tract?

A

The length of the polyT tract at the splice acceptor site of intron 8 has an affect on the splicing on exon and the shorter the tract the more exon 9 is skipped and missing form the mature transcript. the penetrance of the 5T allele is affected by the number of TG repeats (11-13) in this case the longer the TG tract the more exon 9 is skipped. 9T/F508 is in strong linkage disequilibrium so when detected together can assume they are on the allele.

53
Q

What are the clinical pehnotypes associated with the different polyT alleles

A

5Tis not a classical Cf mutation but can be associated with CFTR-RD when found with a classic mutation e.g. F5085T is known to modify the expression of R117H and is commonin CBAVD. R1117H is not considered a CF mutation when in cis with 9T- 5T/R117H & typical severe mutation- CF with variable severity (acts as a compound CF causing mutation)- 7T/R117H & typical severe mutation - CFTR-RD e.g. CBAVD- 9T & atypical severe mutation = not considered disease causing (benign)5T/5T are very rare and variable phenotype- may be asociated with CFTR-RD5T in cis withg TG 12 or 13 and in trans with a classic mutation can result in CFTR-RD

54
Q

When is 5T allele tested for?

A

The 5T allele is not routinely tested for (polyT and TG tract is included in the ARMs PCR kit but not rouitnely interpreted or reported). Reflexively tested for:- in all males referred for infertility- in patients with disseminated bronchiecstasis and only 1 pathogenic mutaion- when R117H is detectedbecause of the importance of the phase of the polyT allele parents may be required to determine phase (except for when F508 is detected as always with 9T)NOT TESTED FOR PRENATALLY unless 5T in cis with R117H as will not result in severe CF

55
Q

Describe CBAVD and male infertility in CF

A

Normal spermatogenesis but absence of the vas deferens results in obstructive azoospermia - 1-2% of infertile males and 95% of CF males- karyotype and y del testing should be offered to males with infertility and a negative CF result- If CF mutation is detected, partner can be screened to determine risk of affected offspring- if infertile male only has a 5T allele cascade testing to family members is not offered as it is unlikely to result in classical CF and counselling the reproductive risk to them is unclear- cascade testing can be offered for R117H/5T- for other mild mutations, the choice on whether to offer cascade testing is on a case-by-case basis ans requires referral to clinical genetics for accurate risk counselling.

56
Q

when is CF testing offered?

A

To confirm a suspected or clinically confirmed diagnosis- suspiscion of CFTR-RD or CBAVD- PND- Carrier testing including partner of carrier without family history

57
Q

What are the genetic treatments for CF?

A

CFTR modulators- readtrhrough therpay to correct NMD of truncated CFTR- amnioglycosides e.g. genatmicin can be used for readthrough of PTCs fo that the full length protein is produced- issues with ototoxicity with long term usecorrector therapy- Lumacaftor: facilitates proper maturation if the CFTR protein and transport to the membrane. Corrects mutations that result in misfolding and degredation e.g. F508potnetiator therapy- Ivacafer corrects mutations that cause gating inefficiency e.g. G551DDNA editing e.g. CRISPR-CAS9 to remove mutated CFTR followed by HR with WT

58
Q

What are the 5 classes of CF mutations?

A
  1. no synthesis- severe pheno due to nonsense, framshift, splice site mutations2. Block in processing3. block in regulation4. altered conductance5. Regulation of other ion channels
59
Q

Give an example of a class 1 no synthesis CF mutation and targeted drug

A

No synthesis mutation reduces the amount of CFe.g. Gly542Xtreatment- gentamicin amnioglycosidase readthrough of PTC e.g. Gentecin

60
Q

Give an example of a class 2 block in processing CF mutation and targeted drug

A

result in protein misfolding and defective maturation- retention in ER and degredatione.g. F508deltreated by correctors e.g. Ivacaftor (phase 3 trials = improved lung function)

61
Q

Give an example of a class 3 block in regulation CF mutation and targeted drug

A

reduce capacity to secrete Cl= due to defects in channel activation e.g G551D(Phase 3 trials- Ivacaftor = improved lung function, reduced salt in sweat and weight gain)

62
Q

Give an example of a class 4 altered conductance CF mutation and targeted drug

A

reduce the capacity for chlorine conductance across the membrane e.g. R117H, Asp1152HIsFlavanoids can act directly on the channel to increase the chance of it being oenIvacaftor also in trials for R117H

63
Q

Give an example of a class 5 regulation of other ion channels CF mutation and targeted drug

A

mutations that affect regulation of other ions channels e.g. ENAC Na+ channeltreated by compound that enhance CFTR retention/acnhoring.