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Infertility And DSD > Cystic Fibrosis > Flashcards

Cystic Fibrosis Flashcards

1
Q

CF is complex multi-system disease. What are the main clinical features?

A

It affects the mucus producing cells of exocrine glands:

  • Epithelialia of resp tract
  • Exocrine pancreas
  • Intestine
  • Hepatobiliary system
  • Male genital tract
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2
Q

What is the major cause of morbidity and mortality in CF patients?

A

Pulmonary disease (resp tract obstruction and infection)

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3
Q

What two issues can arise when CF affects the gastrointestinal tract?

A
  1. Pancreatic insufficiency with malabsorption - majority of patients
  2. Meconium ileus (blocked bowel at birth due to sticky contents)
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4
Q

What are the criteria for making a diagnosis of CF?

A

One or more phenotypic features (chronic sinopulmonary disease, gastrointestinal/nutritional abnormalities, obstructive azoospermia, salt loss syndrome) plus 1, 2 or 3:

  1. 2 disease causing mutations in CFTR
  2. 2x sweat tests with Chloride >60milliequivalents
  3. Transepithelial nasal potential difference characteristic of CF
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5
Q

What are the features of classical CF?

A
  • Obstructive lung disease
  • Bronchiecstasis
  • Exocrine pancreatic insufficiency
  • Elevated sweat chloride (>60mM)
  • Infertility in males due to CBAVD
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6
Q

What are the features of non-classical CF?

A

Chronic pulmonary disease
AND/OR Pancreatic disease
AND/OR elevated sweat chloride
AND/OR CBAVD

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7
Q

What is CBAVD?

A

Congenital bilateral absence of the vas deferens

Represents 1.2-1.7% of male infertility and 80% will have at least one mutation in CFTR

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8
Q

What are some current management strategies for the following symptoms of CF:

  1. Resp
  2. GI
  3. CBAVD
A
  1. Physio, exercise, antibiotics, bronchodilator, mucolytics, anti-inflammatories, lung or heart-lung transplant
  2. High calorie high fat diet, supplemental feeding, tube feeding, oral pancreatic enzyme replacement therapy
  3. ART (assisted reproductive technology)
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9
Q

Provide some details on the CFTR gene

A
  • Located at 7q31.2
  • Spans 190Kb
  • 27 exons
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10
Q

Where does the CFTR protein function?

A

Secretory epithelial cells

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11
Q

What does the CFTR protein have a role in ?

A

Homeostasis salt balance involving Chloride channel

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12
Q

Movement of chloride ions is regulated by 3 CFTR domains. What are these?

A

2 nucleotide binding domains and 1 regulatory domain. CFTR function requires ATP binding to the nucleotide binding domains and phosphorylation of the regulatory domain

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13
Q

What mutation accounts for ~70% of white UK mutations?

A

Phe508del - 3 nucleotide deletion causing loss of phenylalanine residue

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14
Q

Approx 30 mutations account for what percentage of total CF mutations identified?

A

Over 90%

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15
Q

What are the five different classes of CFTR mutation?

A
  • Class I: defective translation e.g. G542X causes premature stop codon
  • Class II: incomplete protein maturation e.g. Phe508del causes incorrect folding and susceptibility to proteolysis
  • Class III: protein at cell surface but cannot be activated e.g. G551D affects NTB domain
  • Class IV: decreased chloride conductance e.g. R1347P affects transmembrane segment
  • Class V: decreased synthesis of CFTR e.g. Mutations affecting splicing efficiency
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16
Q

Which of the different classes of CFTR mutation cause severe or mild phenotype?

A 
Severe = class I, II and some III
Milder = class IV, V and some III
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17
Q

What is the future direction for CF treatment?

A
  • Gene therapy

- Protein assist/repair

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18
Q

What does gene therapy involve?

A
  • Single molecules of plasmid DNA compacted into nanoparticles
  • no immune response or significant side effects
  • evidence of gene transfer transient (6-28 days)
  • efficiency of gene transfer increased if delivered with liposomes
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19
Q

What is protein repair in context of CF treatment?

A
  • Involves small novel molecule that promotes read through of premature stop codons
  • Compares favourably to gentamycin - well tolerated and non toxic
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20
Q

What is protein assist in context of CF treatment?

A
  • VX770 acts as a potentiator: shown to increase gating activity
  • Studies carried out using G551D and G1349D (Class III mutations) have shown up to 10% in FEV (forced expiratory volume)
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21
Q

mutations in another gene have been found in non-classic CF cases where no CFTR mutation is identified. What gene?

A

SCN1A

22
Q

What is the mode of inheritance for CF?

A

Autosomal recessive

23
Q

What is the UK carrier frequency for CF?

A

1/22 to 1/27

24
Q

Are de novo CFTR mutations common or rare in CF?

A

Rare

25
Q

What are the 9 most common CFTR mutations?

A
  • Delta F508
  • G542X
  • G551D
  • N1303K
  • W1282X
  • R553X
  • 621+1G>T
  • 1717-1G>A
  • R117H
26
Q

The intron 8 polyT tract adjacent to exon 9 splice acceptor site can contain how many Ts?

A

5, 7 or 9

27
Q

Which variation in the CFTR intron 8 polyT tract gives the least efficient splicing?

A

5T: highest levels of mRNA lacking exon9 - reduced production of functional CFTR

5T is present in 5% of alleles in general population and modifies the expression of R117H (linked to pancreatic sufficiency)

28
Q

What lies upstream of the CFTR intron 8 polyT tract?

A

PolyTG tract, length of which further affects efficiency of splicing

Normal = 11. 12/13 rpts in cis with 5T leads to increased skipping of exon9

29
Q

What is the incidence of cystic fibrosis?

A

Approx. 1 in 2500 live births

30
Q

What are the two common non-molecular tests for CF?

A
  1. Sweat test (gold standard): >60mEQ/l positive in 98% of CF cases
  2. Immunoreactive trypsinogen (IRT): IRT serum levels are elevated in newborns with CF
31
Q

What are the two common molecular testing methods for CF?

A
  1. Mutation specific testing (for common mutations: amplification refractory multiplex system (ARMS) or oligonucleotide ligation assay (OLA))
  2. Gene screen (for rare mutations: sequencing, MLPA)
32
Q

What is the principle behind ARMS testing in CF?

A
  • 3’ ends of primers specific for wildtype/mutant alleles
  • Extra mismatch introduced near 3’ end to increase specificity
  • PCR product is only obtained from matched primers (if 3’ end of primer not complimentary = no binding = no product)
  • Separate Wildtype and mutant in different tubes or have tails on primers
  • Require control primers to test for PCR amplification
33
Q

Provide details on the CF-EU2 kit

A
  • tests for 50 CFTR mutations (~90% of mutation in European Caucasians)
  • contains Wildtype and mutation-specific primers for all mutations (except S549R)
  • includes primers for polyT (can exclude from analysis if necessary)
34
Q

The CF-EU2 kit is a two tube reaction. How does this work?

A
  • Tube A contains all mutation-specific primers and the WT phe508del
  • Tube B contains all other Wildtype specific primers and is only required to determine zygosity of mutations identified in A
35
Q

What are included in the CF-EU2 kit in order to troubleshoot if it has worked correctly or not?

A

Hypervariable STR (short tandem rpt) markers:

  • absence = failed result
  • difference in A+B = sample mixup
36
Q

How can the presence of insertions/deletions for which primers are not included in the CF-EU2 mix be detected?

A

By the change in expected amplicon size in the Wildtype (B) mix

37
Q

One possible problem with CF-EU2 testing is the presence of a SNP under the primer-binding site preventing amplification. How can this cause an issue?

A
  • false negatives possible
  • heterozygote wrongly assigned as a homozygote
  • you would have to use another primer set to confirm homozygotes
  • a caveat is included in reports
38
Q

What is the principle behind OLA?

A
  • 2 phases: multiplex PCR amplification and multiplex OLA
  • Phase 1: PCR primer hybridised to target sequence - designed with either WT or mutant nucleotide at 3’ end and a tail of different lengths to distinguish various PCR products based on size
  • Phase 2: ligation reaction - common primer with fluorescent dye at 3’ end meets first primer right over nucleotide altered in a mutant allele. If 3’ end matches perfectly with target then it will be brought in close enough proximity to the second oligo that they can ligate together
39
Q

What cross-reactivity can be observed using the OLA testing method for CF?

A

Presence of Delta F508 mutation on one chromosome and F508C on the other may result in the failure of the normal probe to hybridise and prevent ligation. This would give a false deltaF508 homozygote

40
Q

What CF mutation is specific to the Asian population?

A

Y569D

41
Q

What does it mean if you identify two mutations in targeted CF mutation testing?

A

confirms diagnosis:

  • test parents to see if mutations are in trans
  • carrier testing offered to other family members
42
Q

What does it mean if you identify one mutation in targeted CF mutation testing?

A

Does not confirm diagnosis:

  • Test parents to see which side of family mutation is on and cascade screening
  • patient could just be CF carrier (depends on symptoms)
43
Q

What does it mean if you identify no mutations in targeted CF mutation testing?

A
  • Testing cannot fully exclude CF (~1% CF patients will test negative for common mutations)
  • extended screening can be offered
  • offer testing for population specific mutations
44
Q

If R117H is identified in CF testing what analysis should then be performed?

A

PolyT analysis

45
Q

What categories of individual are eligible for CF carrier testing?

A
  • Parents of CF-affected child
  • Parents of child with one mutation identified on neonatal screening
  • siblings of affected parent
  • cascade screening of family members
  • partner of CF carrier
  • individuals with population risk prior to fertility treatment/sperm or egg donors
46
Q

Provide some background info on prenatal diagnosis in CF

A
  • amnio or CVS can be tested if parents known CF carriers
  • negative result makes risk of foetus having CF very low (assuming paternity is correct)
  • carriers will be identified
  • maternal cell contamination checks necessary
47
Q

What is echogenic bowel?

A
  • Brightness in bowel on ultrasound of baby
  • detected on ultrasound in less than 1% of pregnancies
  • 3% of grade 2 or above echogenic bowel cases have CF
  • parental sample tested for carrier status
48
Q

What 4 mutations are included in the neonatal CF screening program?

A
  1. Phe508del
  2. G551D
  3. G542X
  4. 621+1G>T
49
Q

What are the advantages and disadvantages of neonatal screening in CF?

A
  • Advantages = lowered disease severity due to early treatment and decreased burden of care (reduced costs)
  • Disadvantages = false positives causing anxiety and identification of carriers
50
Q

A neonate is tested for CF and found to be a homozygote for phe508del. The parents are tested and mother is found to be carrier and father is negative. What are the possible explanations for this?

A
  1. Non-paternity
  2. Sample mix up
  3. Sequence variant under primer binding site on normal allele (no WT product)
  4. Deletion encompassing codon 508 on one allele (no place for WT specific primer so no product)
  5. Maternal uniparental disomy (2 copies of same chromosome inherited)

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