Cystic Fibrosis Flashcards

Question

Which classes of mutations are generally associated with a severe/classic CF phenotype?

Answer 1

Class 1, 2 and some 3 are generally associated with a more severe phenotype/classic CF

Answer 2

Class 4, 5 and some 3 are generally associated with a milder phenotype

Answer 3

It is thought that the phenotype is variable because in addition to the CFTR mutations, other modifier genes and environmental influences also have an effect on the phenotype displayed.

Answer 4

The process uses single molecules of plasmid DNA with the CFTR gene in compacted into nanoparticles. These particles produce no immune response or significant side effects. There is evidence of gene transfer for several days (transient, 6-28 days). The efficiency of gene transfer is increased if delivery is conducted using liposomes. There is now ongoing work in animals to look at the viability of this in animals.

Answer 5

It has been known for some time that read through of stop codons can be facilitated by the antibiotic gentamicin. PTC124 is an analog of gentamicin and in trials it compares favourably with gentamicin (well tolerated, non-toxic). It promotes read-through of premature stop codons (such as W128X). PTC124 has been of particular interest in Israel where W1282X accounts for 60% of CF mutations in Ashkenazi Jews.

Answer 6

VX770 acts as a potentiator and has been shown to increase gating activity in studies using the mutations G551D and G1349D which are both class 3 mutations. Patients have shown up to a 10% increase in their lung function.

Answer 7

PTC124 - fixes some of the Class 1 mutations by allowing stop codon read through. VX809 - interferes with the chaperone machinery and fixes some Class 2 mutations. VX770 - aids in gating defects of Class 3 mutations. Future therapy may well be mutation specific.

Answer 8

PTC124 - fixes some of the Class 1 mutations by allowing stop codon read through.

Answer 9

VX809 - interferes with the chaperone machinery and fixes some Class 2 mutations.

Answer 10

VX770 - aids in gating defects of Class 3 mutations.

Answer 11

- CFTR mutations cause a disease spectrum - The primary pathology of CF is the loss of salt movement and homeostasis leading to viscous mucus (lung pathology/pancreatic malfunction) and developmental abnormalities (CBAVD) - CF is a serious condition with improving management - Several drugs that target specific mutations are currently being developed and are in trial stages

Answer 12

Mutations in the SCN1 gene have been found in non-classic CF cases where ni CFTR mutations have been identified (diagnostic utility of test not determined).

Answer 13

The UK incidence is about 1 in 2000 to 1 in 3000

Answer 14

The UK carrier frequency is about 1 in 22 to 1 in 27

Answer 15

It is postulated that CF must have/have had some selective advantage over normal homozygotes. There is some debate over what this advantage may be. One theory is that the CFTR membrane chloride channel is required for Salmonelli typhi to enter intestinal epithelial cells. Therefore, heterozygotes may be resistant to typhoid fever and/or to cholera. For the present CF gene frequency to be maintained even in the absence of fresh mutations heterozygotes need to have only 2.3% more surviving children than wild type homozygotes.

Answer 16

Locus homogeneity means that only 1 gene is involved and only 1 gene therefore needs to be tested (as is regarded to be the case with CF).

Answer 17

There is a lot of allelic heterogeneity in the CFTR gene. More than 1500 different mutations in the CFTR gene have been reported. These mutations are loss of function mutations including small deletions/insertions, nonsense, splice site and missense mutationsm and in addition large deletions/duplications (whole exon / multi exon) which account for about 2% of CFTR mutations.

Answer 18

There is a lot of allelic heterogeneity in the CFTR gene. More than 1500 different mutations in the CFTR gene have been reported. These mutations are loss of function mutations including small deletions/insertions, nonsense, splice site and missense mutationsm and in addition large deletions/duplications (whole exon / multi exon) which account for about 2% of CFTR mutations.

Answer 19

Given the size of the gene it is relatively time consuming and expensive to perform a gene screen of the coding region for CF mutations. In addition, some mutations are deeply intronic such as the 3849+10kbC>T mutation. Just sequencing the coding region of the gene would not detect mutations such as this.

Answer 20

Due to a founder effect and heterozygote advantage.

Answer 21

Most labs use a method based around mutation-specific testing. Because a large proportion of CFTR mutations are common mutations the majority of CF carriers can be detected using mutation-specific testing for the most common mutations.

Answer 22

R117H is a class 4 mutation meaning that the mutation alters the conductance of the chloride channel and this results in a milder form of CF.

Answer 23

There is a good correlation between genotype and pancreatic sufficiency. Individuals with pancreatic sufficiency usually have 1 or 2 mild CF alleles such as R117H or A445E. This shows that there is dominance with respect to the milder CF allele.

Answer 24

There is a poor correlation between genotype and pulmonary function and it is abnormal pulmonary function that causes the most problems for CF patients.

Answer 25

It is usually abnormal pulmonary function that causes the most problems for CF patients.

Answer 26

In intron 8 there is a tract of thymidine residues adjacent to the exon 9 splice acceptor site that can contain 5, 7 or 9 thymidine residues.

Answer 27

In intron 8 there is a tract of thymidine residues adjacent to the exon 9 splice acceptor site that can contain 5, 7 or 9 thymidine residues. The variation in the length of the thymidine tract affects the efficiency of the splice site. 5T, the shortest repeat, results in the least efficient splicing and therefore the highest levels of mRNA that lack exon 9. This in turn leads to reduced production of functional CFTR protein. The 5T allele is present in 5% of alleles in the general population.

Answer 28

The 5T allele is present in 5% of alleles in the general population.

Answer 29

The 5T allele is known to alter the expression of the mild R117H mutation. If an individual has a sever CF mutation such as deltaF508 on one chromosome and R117H in cis with 5T on the other chromosome, this can result in a patient with classical CF. The phenotype in such cases is variable and the indivicual may not have CF. The important point here is that with this genotype there is the possibility of getting classical CF. If an individual has the same phenotype but with a 7T rather than a 5T, this is normally associated with either no symptoms or CF-related disease such as infertility. R117H with 9T is rarely found but it is believed to be benign if it is found.

Answer 30

The 5T allele is known to alter the expression of the mild R117H mutation. If an individual has a sever CF mutation such as deltaF508 on one chromosome and R117H in cis with 5T on the other chromosome, this can result in a patient with classical CF. The phenotype in such cases is variable and the indivicual may not have CF. The important point here is that with this genotype there is the possibility of getting classical CF.

Answer 31

If an individual has the same phenotype but with a 7T rather than a 5T, this is normally associated with either no symptoms or CF-related disease such as infertility. (There is some debate over this phenotype, but R117H/7T with a sever CF mutation is accepted to be less likely to to result in CF-related disease. It rarely results in pancreatic suffucient CF and it is the 5T allele that we are most concerned about finding).

Answer 32

R117H with 9T is rarely found but it is believed to be benign if it is found.

Answer 33

The poly T tract can also have an effect when inherited alone and it can act as a mild mutation. the 5T in trans with a CF mutation or a 5T are often benign, but can in some cases result in CF-related disease.

Answer 34

Upstream of the poly T tract in intron 8 lies a variable poly TG tract.

Answer 35

Much like the lenth of the poly T tract, the length of the upstream poly TG tract can also affect the efficiency of splicing.

Answer 36

The most common repeat length to find in the poly TG tract is 11TG. 12 or 13 repeats in cis with 5T leads to increased exon 9 skipping and therefore you get less functional protein. Therefore, if 5T together with 12 or 13 TG repeats is inherited together with a severe CF mutation it is more likely to result in CF-related disease than 5T together with an 11TG and a severe CF mutation.

Answer 37

12 or 13 repeats in cis with 5T leads to increased exon 9 skipping and therefore you get less functional protein. Therefore, if 5T together with 12 or 13 TG repeats is inherited together with a severe CF mutation it is more likely to result in CF-related disease than 5T together with an 11TG and a severe CF mutation.

Answer 38

The sweat test is currently the gold standard for CF testing. A positive result is over 60mEQ/L and 98% of CF cases will be positive for 2 consecutive sweat tests.

Answer 39

The results are often borderline and it is not always possible to perform the testing. It relies on being able to collect enough sweat for an accurate measurement.

Answer 40

The IRT test is the Immunoreactive tripsinogen level test. It can be used in neonates. IRT serum levels are elevated in newborns with CF. The IRT test is not as relaiable as the sweat test but it is used as a starting point for newborn screening.

Answer 41

The most common approach to molecular testing for CF is via a mutation-specific test for common mutations. This can be done in multiple ways, including Amplification Refractory Multiplex System (ARMS) assay and Oligonucleotide Ligation (OLA) Assay. Most commonly for ARMS an Elucigene CF29 gel electrophoresis kit or a CFEU2 capillary electrophoresis kit is used. If only one or no mutations are found using one of these kits then a gene screen can be performed. Normally this is by sequencing or MLPA. This should detect any rare CF mutations. If no mutations are identified in a family with a clinical diagnosis of CF then linkage analysis using markers within and near the CF gene cane be used to identify the high risk haplotype. In reality though linkage analysis is rarely used.

Answer 42

mutations. This can be done in multiple ways, including Amplification Refractory Multiplex System (ARMS) assay and Oligonucleotide Ligation (OLA) Assay. Most commonly for ARMS an Elucigene CF29 gel electrophoresis kit or a CFEU2 capillary electrophoresis kit is used.

Answer 43

If only one or no mutations are found using one of these kits then a gene screen can be performed. Normally this is by sequencing or MLPA. This should detect any rare CF mutations.

Answer 44

If no mutations are identified in a family with a clinical diagnosis of CF then linkage analysis using markers within and near the CF gene cane be used to identify the high risk haplotype. In reality though linkage analysis is rarely used.

Answer 45

The principle of ARMS testing is that a primer will only bind to a sequence if it is exactly complementary at its' 3' end. Therefore, if the 3' end of the forward primer is specific for a mutant allele then we will only get primer binding and PCR product formation if the mutation is present (there is a common reverse primer). If the specific mutation is not present the we will get no binding of primer as the 3' end of the primer is not complimentary and therefore no PCR product. The PCR product can either be visualised on a gel or by using capiliary electrophoresis. A large number of primers can be multiplexed together so that many mutation positions can be interogated at one time. The length of the PCR products is designed to be different so that the products can be separated by size. In addition, primers can be included that are complimentary to the wild type sequence. This allows heterozygotes to be distinguished from homozygotes. In some ARMS primers extra mismatches are incorporated into the primer binding sequence in order to increase the specificity of binding.

Answer 46

ARMS29 tests for 29 of the most common CF mutations in the European population. This will detect about 90% of mutations in European Caucasian populations. The test is performed in 4 seprarate reactions containing different combinations of mutation-specific primers. The kit contains wildtype and mutation-specific primers for deltaF508 mutation but cannot determine heterozygosity of other mutations. There are upper and lower control bands which should amplify in all tubes for all patients to ensure that the PCR is working. Occassionally spurious bands arise and can be seen in the tubes and can be seen in every patient. Altering the concentrations of DNA can eliminate these bands. Allele-specific PCR tests such as ARMS are sensative to salt concentrations and contaminants and this can be a problem with ARMS testing. A gel-based poly T ARMS kit is also available and the principle is the same. The limitation of the ARMS test with regards to how many mutations can be tested is what resolution can be achieved on the gel making it possible to differentiate the different mutations.

Answer 47

- Allele-specific PCR tests such as ARMS are sensative to salt concentrations and contaminants and this can be a problem with ARMS testing. - The limitation of the ARMS test with regards to how many mutations can be tested is what resolution can be achieved on the gel making it possible to differentiate the different mutations.

Answer 48

The CF-EU2 kit works on the same principle as ARMS29 but more mutations can be detected as the products are separated by capillary electrophoresis which has far greater resolution. Also, multiple dyes can be used. The CF-EU2 kit tests for 50 CFTR mutations. This also accounts for about 90% of mutations in European Caucasian populations (the extra mutations in the CF-EU2 kit are very rare so does not increase the percentage detected very much over the ARMS29 kit). The kit contains wild type and mutation-specific primers for all 50 mutations (except the S549R mutation for which there is no WT primer). The CF-EU2 kit works using a 2 tube reaction with all the mutation-specific primers in tube A in addition to the WT delftaF508 primer. All other WT-specific primers are in tube B but are only required to determine zygosity of a mutation identified in tube A. The CF-EU2 kit also includes primer for poly T in tube A but these primers are labelled with a different coloured dye and so we can exclude them from analysis if required. Hypervariable STR markers are included and act as controls. If these markers are absent then the test has failed. If the STR marker peaks are different in the A and B tubes then this can indicate a sample mix up. You can get cross-reactivity in the CF-EU2 kit due to the large number of primers in the kit. The presence of on CF mutation can affect other primers and prevent them binding. For example, the presence of the R117H mutation will result in the reduction of the height of the R117C mutation peak in the CF-EU2v2 B mix. Furthermore, in a sample homozygous for R117H the R117C peak will be absent. The presence of insertions or deletions for which primers are not included in the mix can be detected by the change in expected amplicon size in the WT (B) mix. If heterozygous then extra peaks will be present and if homozygous then the peak will be shifted. It is therefore important to look at the B tube results closely. There are a number of possible problems with the CF-EU2 kit. Firstly, there is the posibility of the identification of extra peaks in tube B when no mutations are detected in tube A. All we can tell from the analysis is that there is a shift in size. The region amplified can be exonic or intronic and there is no way of knowing if the peaks present a pathogenic rare mutation or a neutral polymorphism. Interpreting such results would require further investigations such as sequencing. The possibility of identifying mutations of unknown significance illustrates a testing dilemma, should we run the B tube for all referral reasons? and what is the benefit of identifying these extra peaks? Another possible problem with the CF-EU2 kit is the possible presence of SNPs under the primer-binding site which could prevent amplification. This could lead to false negative results, or a heterozygote wrongly being assigned as a homozygote. As these kits are commercial the primer sequences are not available to SNP check. One way of dealing with this issue is to use another set of primers to confirm any homozygotes. In addition a caveat should be included on the report. It is important to consider whether the result fits with the referral reason as this can give an indication of whether the result is likely to be correct.

Answer 49

See lecture.

Answer 50

The CF-EU2 kit works using a 2 tube reaction with all the mutation-specific primers in tube A in addition to the WT delftaF508 primer. All other WT-specific primers are in tube B but are only required to determine zygosity of a mutation identified in tube A. The CF-EU2 kit also includes primer for poly T in tube A but these primers are labelled with a different coloured dye and so we can exclude them from analysis if required.

Answer 51

The CF-EU2 kit also includes primer for poly T in tube A but these primers are labelled with a different coloured dye and so we can exclude them from analysis if required.

Answer 52

Hypervariable STR markers are included and act as controls. If these markers are absent then the test has failed. If the STR marker peaks are different in the A and B tubes then this can indicate a sample mix up.

Answer 53

You can get cross-reactivity in the CF-EU2 kit due to the large number of primers in the kit. The presence of on CF mutation can affect other primers and prevent them binding. For example, the presence of the R117H mutation will result in the reduction of the height of the R117C mutation peak in the CF-EU2v2 B mix. Furthermore, in a sample homozygous for R117H the R117C peak will be absent. The presence of insertions or deletions for which primers are not included in the mix can be detected by the change in expected amplicon size in the WT (B) mix. If heterozygous then extra peaks will be present and if homozygous then the peak will be shifted. It is therefore important to look at the B tube results closely.

Answer 54

There are a number of possible problems with the CF-EU2 kit. Firstly, there is the posibility of the identification of extra peaks in tube B when no mutations are detected in tube A. All we can tell from the analysis is that there is a shift in size. The region amplified can be exonic or intronic and there is no way of knowing if the peaks present a pathogenic rare mutation or a neutral polymorphism. Interpreting such results would require further investigations such as sequencing. The possibility of identifying mutations of unknown significance illustrates a testing dilemma, should we run the B tube for all referral reasons? and what is the benefit of identifying these extra peaks? Another possible problem with the CF-EU2 kit is the possible presence of SNPs under the primer-binding site which could prevent amplification. This could lead to false negative results, or a heterozygote wrongly being assigned as a homozygote. As these kits are commercial the primer sequences are not available to SNP check. One way of dealing with this issue is to use another set of primers to confirm any homozygotes. In addition a caveat should be included on the report. It is important to consider whether the result fits with the referral reason as this can give an indication of whether the result is likely to be correct.

Answer 55

The Oligonucleotide Ligation Assay (OLA) is very similar to MLPA but in OLA the PCR preceeds the hybridisation and ligation reactions (rather than happening after the hybridisation and ligation reaction). 1) . Genomic DNA is amplified using 15 pairs of primers (number depends on kit used). Primers flank regions of the CFTR gene where common CFTR mutations may be detected. 2) . Each amplicon is probed by 3 oligonucleotide probes. One of the probes, called a common probe, hybridises to the amplicon at common sequences found in both the mutant and normal alleles and is labelled with fluorescent dye. The mutant and normal probe then compete for binding to the PCR amplicon. The probe that is the perfect complement to the PCR amplicon will hybridise. 3) . The bound probes will then be ligated to the common probe in a ligation reaction. DNA ligase facilitates ligation of probes that are perfect complements and an OLA product is formed. 4) . Normal and mutant probes have varying numbers of non-nucleotide mobility modifying tails. The unique combination of electrophoretic mobility and fluorescene permits identification of the CFTR genotype. This means that products can be separated by capillary electrophoresis. OLA can determine the zygosity of all the mutations, unlike ARMS 29. There are also problems with the OLA technique. Due to the use of primers SNPs under the primer binding site could alsoi cause false negatives in OLA as in ARMS. Also, you can get cross-reactivity. For example, the presence of the F508 mutation on one chromosome and F508C on the other may result in the failure of normal probe to hybridise and prevent ligation. This would result in the false identification of a deltaF508 homozygote. This also occurs for I507V and I507del.

Answer 56

There are also problems with the OLA technique. Due to the use of primers SNPs under the primer binding site could alsoi cause false negatives in OLA as in ARMS. Also, you can get cross-reactivity. For example, the presence of the F508 mutation on one chromosome and F508C on the other may result in the failure of normal probe to hybridise and prevent ligation. This would result in the false identification of a deltaF508 homozygote. This also occurs for I507V and I507del.

Answer 57

As well as testing for the European-specific mutations, population specific tests are also performed in some labs. One of the most common population-specific tests is for Y569D which is a CF mutation specific to Asian populations. The Y569D mutation accounts for about 9.6% of CF mutations found in the UK Indian Sub Continent (ISC) population. Testing for this mutation may use a restriciton digest, sequencing, or pyrosequencing. Some labs offer this testing up front to Asian referrals, whilst others offer it as a reflex test. The problem with this is that we often don't get ethinicity details with referrals and using a surname as an indicator of ethnicity is not a very accurate method.

Answer 58

One of the most common population-specific tests is for Y569D which is a CF mutation specific to Asian populations. The Y569D mutation accounts for about 9.6% of CF mutations found in the UK Indian Sub Continent (ISC) population.

Answer 59

Testing for this mutation may use a restriciton digest, sequencing, or pyrosequencing.

Answer 60

- Meconium ileus - Failure to thrive - Respiratory infections

Answer 61

- Respiratory problems, allergic bronchopilmonary aspergillosis (ABPA) - Bronchiectasis - Idiopathic chronic pancreatitis - Obstructive azoospermia (CBAVD)

Answer 62

Meconium ileus is when the content of the baby's bowel (meconium) is extremely sticky and causes the bowel to be blocked at birth. Approximately 90% of babies with meconium ileus have CF. Approximately 20% of CF cases present with meconium ileus.

Answer 63

In diagnostic cases targeted mutation testing is performed initially. If 2 mutations are identified then this confirms a diagnosis of CF. The next steps are to test the parents to confirm that the mutations are in trans and to offer carrier testing to other family members. If 1 mutation is identified this does not confirm a diagnosis but this does make a diagnosis of CF more likely. The next steps are to test the parents to see which side of the family the mutation is on and then offer cascade screening to family members. It could be that the patient is just a CF carrier. The likelyhood of this depends on the symptoms. Extended screening can be offered in these cases. If no mutations are identified, testing cannot fully exclude CF. About 1% of CF patients will test negative for the common mutations. If the clinicians really expect a diagnosis of CF then extended screening can be offered. In addition, testing can be offered or performed for population-specific mutations. If R117H is identified then poly T analysis should be performed. To work out phase it is important to note that the 9T allele is almost always in cis with deltaF508.

Answer 64

The TAT for the initial screen for diagnostics and carrier referrals is 10 days.

Answer 65

For carrier testing patients must be greater than 16 year of age.

Answer 66

- Parents of a CF affected child where each parent must be an obligate CFTR mutation carrier (assuming paternity). - Parents of a child with 1 mutation identified on neonatal screening. In such cases 1 parent will carry a mutation. If child has CF symptoms or develops CF symptoms then the other parent is an obligate carrier of a rare mutation, otherwise risk is low. Depends on symptoms, clinical info important. - Siblings of affected patient. Have a 2/3 risk of being carrier. - Cascade screening for family members. If mutation information is not known we cannot reduce the risk much in the event of a negative result. - Partner of CF carrier. Partner is at population risk if there is no family history of CF. We can then determine the couple's risk of having a CF-affected child. - Testing individuals with population risk either prior to fertility treatment or sperm/egg donation.

Answer 67

An amnio or CVS can be tested if the parents are known CF carriers. If the mutations in the family are not known then linkage analysis can be used. A negative result makes the risk of the fetus having CF very low assuming paternity. Carriers will also be identified. Maternal cell contamination (MCC) checks are necessary (also required if cord blood is tested) in order to exclude the possibility of the mother's genotype masking the fetal genotype.

Answer 68

Echogenic bowel can be detected on ultrasound. This is classified as when the brightness in bowel is equal or greater than the surrounding bone. It is detected on ultrasound in 0.2-1.2% of pregnancies, although there is variation depending on studies used and the particular ultrasonographers performing the scan. About 3% of grade 2 or above echogenic bowel cases have CF. When echogenic bowel is detected in a pregnancy then parental samples can be tested for carrier status. If this is negative prenatal testing is not recommended as the risk to the fetus is low. If one parent is found to be a carrier and the fetus has inherited the mutation then there is a 1 in 6 risk that the fetus has CF. The best practice guidelines don't recommend testing of the fetus but practices differ between labs. If both parents are carriers then the risk to the fetus is more than 1 in 4 and prenatal diagnosis can be offered.

Answer 69

About 3% of grade 2 or above echogenic bowel cases have CF.

Answer 70

It is detected on ultrasound in 0.2-1.2% of pregnancies, although there is variation depending on studies used and the particular ultrasonographers performing the scan.

Answer 71

Echogenic bowel can be detected on ultrasound. This is classified as when the brightness in bowel is equal or greater than the surrounding bone.

Answer 72

When echogenic bowel is detected in a pregnancy then parental samples can be tested for carrier status. If this is negative prenatal testing is not recommended as the risk to the fetus is low. If one parent is found to be a carrier and the fetus has inherited the mutation then there is a 1 in 6 risk that the fetus has CF. The best practice guidelines don't recommend testing of the fetus but practices differ between labs. If both parents are carriers then the risk to the fetus is more than 1 in 4 and prenatal diagnosis can be offered.

Answer 73

A large number of the referrals recieved for CF testing are tests in infertile males. CBAVD occurs in 1-2% of men with azoospermia and infertile males with CBAVD have increased risk of carrying CFTR mutations. According to Chillon et al. CFTR mutations are identified in 50-70% of men with CBAVD. However, many of the mutations are not detected by the commercial testink kits. CF testing in infertility referrals is carried out to support a diagnosis of infertility arising from CFTR mutations and also to alert the clinician to the risk of CF in offspring. Poly T testing should be performed in cases of obstructive azoospermia or if R117H is identified. The problem is that azoospermia or oligospermia is often the only thing written on the referrals and they do not usually state whether CBAVD or obstructive azoospermia is present. In addition, patients may have rare CF mutations. Therefore, if no mutation is identified we still cannot exclude that the infertility is due to CFTR mutations.

Answer 74

CF testing in infertility referrals is carried out to support a diagnosis of infertility arising from CFTR mutations and also to alert the clinician to the risk of CF in offspring.

Answer 75

Poly T testing should be performed in cases of obstructive azoospermia or if R117H is identified.

Answer 76

Poly T testing should be performed in cases of obstructive azoospermia or if R117H is identified. The problem is that azoospermia or oligospermia is often the only thing written on the referrals and they do not usually state whether CBAVD or obstructive azoospermia is present. In addition, patients may have rare CF mutations. Therefore, if no mutation is identified we still cannot exclude that the infertility is due to CFTR mutations.

Answer 77

CF testing is one of 5 diseases included in the UK neonatal screening programme.

Answer 78

Blood spots are taken on Guthrie cards on day 5 and analysed for IRT levels followed by mutation analysis for the 4 most common CF mutations in individuals with levels above the 99.5 percentile. The testing process is designed to detect the highest nuber of CF cases and fewest number of carriers possible.

Answer 79

The benefits of neonatal screening include: - Lowered disease severity due to early treatment - Decreased burden of care due to reduced healthcare cost

Answer 80

The disadvantages of neonatal screening include: - The identification of false positives that can lead to anxiety - The unwanted identification of carriers

Answer 81

1) . Non-paternity 2) . Sample mix up 3) . Sequence variant under primer binding site on the normal allele meaning no WT amplicon is produced even though there is a normal allele present 4) . Deletion emcompassing codon 508 on one allele meaning that there is nowhere for the primer to bind to the Wt alleles we only get amplicons produced for the mutant product and it looks homozygous 5) . Maternal uniparental disomy where 2 compies of the same chromosome are inherited

Answer 82

1) . Maternal cell contamination - unlikely, hard to explain the peak pattern, all other peaks look as they should and there is no evidence of mcc using QF-PCR markers. 2) . Variant under primer binding site for peak 12 (V520F) - most likely explanation. Variant would be on normal allele. Could variant be pathogenic? 3) . Parents tested - father had same peak pattern and unaffected, therefore fetus should have same phenotype. If the patient has the variant in cis with V520F then there may be no amplification of the mutant allele and it would produce a false negaive result.