Genetics 9 - Cystic Fibrosis and Genetic Screening

Question 1

learning outcomes

Answer 1

Question 2

why do some communities/ethnic groups have different risk of inherited genetic disorders than the general population

Answer 2

founder effect

reduction in genetic variation due to migration/isolation of a small number of endogamous individuals from a large population

Question 3

endogamy

Answer 3

marriage within a specific community

Question 4

syndrome associated with Pennsylvania Amish

Answer 4

Eilis van Creveld syndrome

Question 5

symptoms of Eilis van Creveld syndrome

Answer 5

short stature

polydactyly

abnormality of nails and dentition

cardiac defects

Question 6

allele frequency of Eilis van Creveld in Pennsylvania Amish and in General Northern European population

Answer 6

7% - Amish

0.1% - Northern European population

Question 7

gene associated with Eilish van Creveld syndrome

Answer 7

EVC gene short arm of chr 4

Question 8

requirements for founder effect

Answer 8

relatively discreet breeding sub-population (endogamy)

derived from a small group of related people (founders)

if a specific mutation was present by chance in the founders it may be disproportionately common in the expanded group derived from that population

likewise, frequent disease associated alleles that are by chance missing in the founders may be disproportionately uncommon in the expanded group derived from that population

exogamy (marrying unrelated people) will tend to dilute or diminish founder effect

Question 9

founder effect and genetic drift

Answer 9

genetic drift = change in allele frequency due to chance i.e. which copy of the allele you’re going to inherit

effect on larger populations is a lot smaller

Question 10

symptoms of Fanconi Aplastic Anaemia

Answer 10

rare genetic syndrome characterised by short stature, various congenital abnormalities, bone marrow failure and cancer predisposition

Question 11

pattern of inheritance - Fanconi Aplastic Anaemia

Answer 11

autosomal recessive

Question 12

heterozygotes - FAA

Answer 12

may have short stature, but no bone marrow aplasia or increased cancer risk

Question 13

genetic variants associated with FAA

Answer 13

lots but NB:

FANCA chr 16

FANCC chr 9q

FANCG chr 9p

Question 14

what is FA pathway responsible for

cells most affected

what is their predisposition to

proportion that develop cancer

age of onset

associated diseases

Answer 14

FA pathway - fixing DNA damage during DNA replication

quick dividing cells most affected - bone marrow, foetal

predisposition to malignancies in surviving cells

about 20% homozygotes develop cancer

median age of onset = 16

haematological malignancy (AML, MDS, ALL)

squamous cell cancers

hepatomas

Question 15

prevalence and carrier frequency of Fanconi Anaemia

Answer 15

overall prevalence - 1 in 5m

carrier freq - 1 in 200, 1 in 300

Question 16

genetic bottleneck

Answer 16

relatively small initial population - founders

Question 17

high incidence of what genetic diseases (Ashkenazi Jews)

Answer 17

Cystic Fibrosis

Tay-Sachs

Fanconi anaemia

Question 18

carrier screening

Answer 18

testing a target population to identify unaffected carriers of a disease allele

done in NA for Tay-Sachs

Question 19

homozygotes - Tay-Sachs

Answer 19

fatal

deficiency in lysosomal enzyme β-hexosaminidase A (HEX A) - substrate GM2 ganglioside accumulates

blindness, seizures, hypotonia - neuronal damage

death by 5 years

Question 20

principles of population screening

Answer 20

each screening programme needs to be appraised for viability, effectiveness and appropriateness in each target population

condition - serious, well understood, and relatively common (cost/benefit)

test - acceptable, easy and cheap, valid and reliable

intervention - effective treatment/counselling, prenatal diagnosis and ART available

screening programme - effective (clinical data), ethical, benefit outweighs harms

implementing criteria - accessibility, resources for diagnosis and treatment, communication of results, data privacy, quality assurance

Question 21

gene associated with CF

type of regulator

domains

function

Answer 21

CFTR gene

ion channel (Cl and HCO3-)

chr 7q31.2 (locus)

27 exons and 230,000 bps (large)

12 transmembrane domains, 2 nucleotide binding domains

1 regulatory domain

function = gating - ATP dependent cycling between conducting ions (open) and not conducting ions (closed)

Question 22

cystic fibrosis manifestions

Answer 22

sweat (Na+ > 60 mmol/L)

lungs - thick sticky mucus, infections

GIT (may present with obstruction in infancy - meconium ileus)

frequent greasy, bulky stools (steatorrhea)

sinuses - polyps, thick sticky mucus, infections

pancreas (malabsorptionm failure to thrive, CF)

male infertility - congenital bilateral absence of vas deferens (CBAVD) - 95%

female infertility - thickened cervical mucus

Question 23

resp mucosa - normal vs CF

Answer 23

Main differences In airway surface layer that protects airway

Reduced Cl- secretion and hyperabsorption - dehydration of layer

Question 24

CF mutations

Answer 24

hypomorphic (less function) heterogeneity - 1000s of alleles associated with disease are known

LOF mutations tend to be more heterogeneous than GOF mutations

recessive pattern of inheritance (affected people usually have unaffected parents)

CFTR has biallelic expression - limited/no clinical effect in heterozygote as CFTR protein is haplosufficient

Question 25

carrier rate for CF mutation in Ireland

Answer 25

1 in 19 (1 in 25 northern Europeans)

Question 26

most common abnormal CFTR allele

Answer 26

p.F508del = ΔF508 70% of CF alleles in northern Europeans deletion of phenylalanine at position 508 in CFTR (in nucleotide binding domain 1) altered CFTR protein is degraded IC and does not make it to the apical cell membrane (loss of function) FRAMESHIFT MUTATION deletion of 3 nucleotides in exon 10

Question 27

CF - type of mutation

Answer 27

frameshift ΔF508 deletion of 3 nucleotides in exon 10

Question 28

why is mutation in CFTR gene so common

Answer 28

heterozygote advantage - protected against dehydration due to diarrhea - cholera, typhoid fever epidemics

Question 29

screening available for CF

Answer 29

individuals/couples could be tested to determine if heterozygous (Aa) with a view to planning pregnancy testing of embryos for purposes of embryo selection prenatal testing on amniotic fluid with a view to termination of pregnancy screening of newborns to provide early detection and intervention the challenge is \> 2000 mutations * IRT - immunoreactive trypsinogen * Blood spot test * CF mutational analysis plan

Question 30

things to remember

Answer 30

Question 31

Answer 31

C?

Question 32

learning outcomes

Answer 32

Question 33

Answer 33

CF marked reduction in life expectancy duration and wuality of life critically dependent on quality of services repeated LRTI with progressive destruction of lung tissue - bronchiectasis and resp failure

Question 34

CF pulmonary infection - bacteria

Answer 34

different bacteria cause infection at different stages age at which they become permanently colonised/infected with pseudomonas aeruginosa is a critical issue bacteria comes from everywhere

Question 35

what is meconium ileus prevalence gene associated

Answer 35

obstruction of GIT of infant related to inspissated (thick, dehydrated) material occurs in 15-20% of infants with CF genotype at CF modifier 1 (CFM1) gene on chr 19 may determine risk of developing meconium ileus

Question 36

how to confirm CF if suspected

Answer 36

# define the mutations - targeted mutation panel if not a common mutation, scan exons by PCR amplification and Single Strand Conformation Polymorphism - sequence exons that look different from controls

Question 37

CF carrier - A vs a

Answer 37

A = any CFTR allele that results in a functioning chloride channel - sequence may vary a = any CFTR allele that does not code for a functioning chloride channel \> 2000 mutated CFTR alleles that have been described RECALL: LOF are a lot more heterogenous than GOF mutations

Question 38

LOF vs GOF

Answer 38

RECALL: LOF are a lot more heterogenous than GOF mutations

Question 39

5 mutation categories - CF

Answer 39

1. protein production - no functional protein produced 2. protein processing - misfolding (DF508 - doesn't go to where it is supposed to go (surface of cell) because it has not folded correctly) 3. gating - doesn't open 4. conduction - faulty channel 5. insufficient protein - splice site (Changes in sequence - not enough protein produced - often in promoter)

Question 40

CFTR pharmacogenetics - 3 categories of therapies designed to treat faulty CFTR

Answer 40

CFTR modulator therapies are designed to correct the malfunctioning protein made by a mutated CFTR * read-through compounds (non-sense) * correctors (misfolding) * potentiators (open channel/increase function) - Channel openers - constitutively open, even if protein is not working properly

Question 41

gating mutation - less common treatment

Answer 41

e.g. G551D (glycine changed to aspartic acid at position 551) 4-5% of cases of CF (depends on population) CFTR protein is in place in cell membrane but does not work because Cl- channel does not open Ivacaftor (Kalydeco) (potentiator) binds to CFTR and allows it to open does not work for DF508 but combination drug Orkambi (corrector) does

Question 42

triple therapy used to treat CF

Answer 42

available for patients 12+ with at least 1 DF508 and 1 minimal function mutation increase quantity and function of F508del-CFTR protein at cell surface may be extended to patients under 12 in the future

Question 43

elexacaftor, tezacaftor

Answer 43

correctors

Question 44

ivacaftor

Answer 44

potentiator

Question 45

implications for family of a person with CF

Answer 45

carrier analysis may be requested - cascade screening carrier testing available to adults \> 16 where there is a family history of CF or where a family member has been found to carry a CF mutation (If they are carrier - IVF, embryo selection, gene therapy (CRISPR-Cas))

Question 46

chance of getting CF if both parents are carriers

Answer 46

25%

Question 47

purpose of genetic screening for CF

Answer 47

carrier screening - to plan pregnancy testing of embryos - embryo selection prenatal testing on amniotic fluid/CVS - termination of pregnancy newborn screening - early detection and intervention → high energy diet, physio, medicines for lung function and gut absorption

Question 48

treatment for newborns with CF

Answer 48

high energy diet physio medicines for lung function and gut absorption

Question 49

Heel prick test

Answer 49

Question 50

newborn screening for CF

Answer 50

IRT screening has low specificity relatively high false +ve rate combined with mutational analysis vastly improves specificity 2% of carriers of CF born each year will also be detected

Question 51

why is there no CF population carrier screening

Answer 51

Question 52

how to screen adults for carriage of CF gene

Answer 52

testing just for DF508 is straightforward amplify relevant sequence from genomic DNA assess for wt or DF508 sequence (restriction enzyme/oligonucleotide probe) assess for DF508 during amplification using real time approach other common mutations - 38 mutation panel detects 93.5% of the CF mutations found in Irish population

Question 53

NGS and CF

Answer 53

affordable genetic testing based on the identification of variants in extended genomic regions - 99% detection rate also being used to design custom CFTR mutation panels for different geographic regions - 95% detection rate

Question 54

screening for carriage depends on

Answer 54

ethnic background

Question 55

why is DF508 not the predominant mutation across the globe

Answer 55

founder effect

Question 56

Haemochromatosis

Answer 56

accumulation of iron - liver, skin and other tissues manifestations develop in adult life - age of onset is variable hepatic and cardiac failure skin pigmentation joint disease common

Question 57

diagnosis of haemochromatosis

Answer 57

elevated transferrin saturation elevated serum ferritin levels

Question 58

haemochromatosis treatment

Answer 58

phlebotomy

Question 59

with whom is haemochromatosis more common and why

Answer 59

More common in men than women due to menstruation

Question 60

gene - haemochromatosis pattern of inheritance

Answer 60

variants alleles of the HFE gene 6p21.3 autosomal recessive HFE regulates iron absorption from the diet and iron storage deficiency = iron overload

Question 61

2 key mutations - haemochromatosis

Answer 61

1. G to A transition at nucleotide 845 (c.845G\>A) - cysteine to tyrosine (p.C282Y) 2. C to G at nucleotide 187 (c.187C\>G) - histidine to aspartic acid 63 (p.H63D)

Question 62

most common mutations

Answer 62

C282Y homozygous in 80-85% haemochromatosis cases (3x increase Fe absorption) also C282Y and H63D compound heterozygotes homozygous H63D does not result in clinically manifested haemochromatosis

Question 63

penetrance of haemochromatosis

Answer 63

as low as 1%

Question 64

is haemochromatosis a hereditsry disease

Answer 64

outcome critically dependent on lifestyle factors - exposome

Question 65

should there be a screening program for haemochromatosis

Answer 65

a useful diagnostic test does NOT ⇒ a good population screening test likelihood of discovering undiagnosed patient is \<1 in 1000 no evidence of clinical benefit for treatment of asymptomatic carriers

Question 66

contraindications for screening

Answer 66

low positive predictive value e.g. haemochromatosis H63D low population attributable risk (PAR) - proportion of total disease risk in the population attributable to the factor being screened for e.g. G6PDD mutations low absolute risk e.g. FV Leiden thromboembolism relative risk for oral contraceptive users high, but absolute risk low as most are young people no actionable knowledge - no way to improve prognosis

Question 67

principles of screening

Answer 67

Question 68

things to remember

Answer 68