Chloride Transporter Protein and Cystic Fibrosis

Complications from failure to diagnose CF during infancy?

- hypochloremia (alkalosis) - hyponatremia (dehydration, failure to thrive) - hypoproteinemia (Kwashiorkor, false negative diagnosis) - vitamin E deficiency (hemolytic anemia) - vitamin K deficiency (bleeding diathesis) - Zn deficiency (acrodermatitis) This is rare because we're picking things up earlier! They take ADEKs = fat soluble vitamins

Therapy review?

- nutritional support (calories, vitamins, enzyme replacement) - aggressive chest physiotherapy with mucolytic medications - oral, aerosolized or IV antibiotics - monitoring for complications: GI (liver, constipation), endocrine, pulmonary - frequent cultures, pulmonary function - social/emotional support

How many people are CF carriers?

1 in 20 or 25!

Incidence of CF in US?

1 in 2500-3500 live Caucasian births, most fatal recessive condition in the US Avg life expectancy early 40s Carrier frequency varies in different populations. Ashkenazi Jewish 1 in 24, Caucasian non-Jewish 1 in 25, hispanic white 1 in 58, African american 1 in 61, Asian 1 in 94

Describe the model of CFTR channel gating.

1. R domain is phosphorylated by cAMP-dependent protein kinase A (PKA) 2. Allows binding of ATP to NBD1 3. Hydrolysis of ATP by NBD1 opens channel (transient) 4. PKA phosphorylates more sites on R domain. 5. NBD2 can bind ATP, which stabilizes open channel. 6. ATP hydrolysis at NBD2 results in subsequent release of ADP and channel closing 7. R domain dephosphorylation closes the channel CFTR is cAMP activated and ATP-gated ion channel The greater the phosphorylation of the R domain the greater the probability of ATP binding to NBD domains. When both NBD domains have bound nucleotide, there's a greater probability that the channel is open and will remain open for longer. In CF: defect in channel function, loss of activity. Cholera: Channel is activated and becomes hyperactive. CFTR regulates the activity of other channels including Na+ channel and other Cl- channels.

Genetic testing methodologies?

1. targeted mutation testing - look for 1-2 specific mutations -- know what you're looking for! - done for known familial mutations - single or dual site testing - included as part of panel of common mutations 2. mutation panels - ACMG recommends a "minimal panel" for carrier screening - 23 specific mutations, including mutations frequent in several US ethnic groups - mutation detection rate differs amongst ethnic groups - many labs offer extended mutation panels to improve detection rate - available in a variety of forms, from 23-100+ mutations - number of mutations dictates residual carrier risk from negative screen (or risk to be affected) - ACOG recommends that CF carrier screening be offered to every preconceptional/pregnant woman regardless of race/ethnicity (key pt)! 3. sequence analysis - read through to look for mutations or misspellings in all important areas of the gene - exons, intron/exon border (splice site), known areas in introns, detects ~98% of mutations, includes common mutations and family-specific mutations - always better? no...can also detect unclassified CFTR variants, how do you know if it caused disease or just normal variation? unique mutation? rare polymorphism? How to interpret? - does NOT detect large deletions or duplications, only reads what is present! - same results given if 2 normal copies of CFTR is present. 1 normal CFTR and one with single exon deletion. 1 normal CFTR and one with single exon duplication. WHY 4. deletion/duplication testing - deletions/duplications account for ~2% of CFTR mutations - extra/missing exons can remove an important part of the protein, change the reading frame of the rest of the gene, affect the folding of the protein - ex: CFTRdele2,3 -- deletion of exons 2 and 3 5. sequence + del/dup testing - the most thorough genetic testing clinically available, but still could be missing other important areas of the gene. - often times has slower turnaround time, but improving with technologies. - more expensive than panel testing, insurance may balk at paying and question why doing more than 1 test. No perfect test! Dosage testing will detect del/dups of varying size, but not single BP or other mutations. Sequencing can detect single BP changes but not del/dups. Some labs include both!

Normal pathway of functional protein?

1. transcription 2. translation 3. post translation modification 4. trafficking 5. surface expression CFTR

History of CF?

1938: first clear description of the disease, thought related to pancreas 1940s: antibiotics introduced to treat chronic respiratory infections associated with CF 1948: heat wave in NYC -- excessive salt wasting in sweat 1959: salty tasting skin --> diagnostic pilocarpine sweat test for CF 1980s: description of underlying pathophysiology with description of Cl- impermeability in CF sweat gland and transport across epithelia 1989: gene was cloned! CF gene discovered 2000s: newborn screening widespread 2012: first FDA approved therapy for specific CF genotype 2017: new formulations of medications/combo therapies Survival in 1938: 70% fatality w/in 1 year; 1985: 25 yr; 2008: 37 yr; this decade: ~60 years, avg 40s Target respiratory infection, inflammation, mucociliary clearance, nutritional status

Heterozygote advantage?

4-5% caucasians are heterozygous for CF. Frequency due to random mutations never exceeds 1%, why maintained? CFTR is targeted in cholera a highly lethal diarrheal disease. heterozygotes express 50% of normal CFTR gene levels, so less fluid loss and dehydration -- an adv for europeans where cholera was endemic! cholera = too much water release, this stops that

What mutation are most of the cases due to? How many known mutations?

>70% of all cases are due to mutation of a single amino acid (protein misfolded in ER, never gets to plasma membrane, even though protein is partly functional). exchange (missense) one aa for another. deletion. insertion. that's all due to deletion of 1 single aa. since single aa got deleted, look at transcription/translation. folded together. folding not done properly. not able to get to plasma membrane. if you get this mutated CFTR to surface it'll work partially. huge gene --> lots of mutations. 1900 known mutations

Relationship of clinical features to residual CFTR function?

<1% normal CFTR function: pancreatic insufficiency and below <4.5: pulmonary infection and below <5: positive sweat test and below <10: congenital absence of vas deferens 10-49: none 50-100: none more than 10% = no symptoms! first thing affected is male repro pancreatic status can be predicted from mutation class! Pancreatic insufficient is class I, II, III. PS is class IV, V.

Keys to improved survival? In chronological order

Airway clearance Anti-staphyloccal antibiotics Anti-pseudomonal antibiotics Improved nutrition enzymes DNASE (breaks up DNA in mucus, liquifies it) Inhaled tobramycin ("toby" -- preventative, kills pseudomonas) Azithromycin (=zithromax -- antiinflammatory) Quality improvements Ivacaftor (now)

What causes ASL decrease in CF?

ASL decrease is caused by decrease in Cl- secretion (mediated by apical CFTR and alternative anion channels) accompanied with the release of ENaC inhibition... If the ENaC is no longer inhibited (as it is with normal patients!), Na can flow into the cell. NaCl high in cell, water follows. It could also be caused by overexpression of ENaC.

Class VI mutation?

Accelerated turnover of surface CFTR Go to cell surface but quickly turnover Increased CFTR turnover at cell surface, protein unstable and quickly degraded. Rescued p.Phe508del. Rescue and be able to force cell to take misfolded protein and get into cell surface, but then turned over...1 hurdle not enough!

CFTR protein structure?

Activation of CFTR relies on phosphorylation through PKA and other kinases too. The carboxyl terminal of CFTR is anchored through PDZ-type-binding interaction with cytoskeleton and is kept in close approximation with other important proteins. These associated proteins influence CFTR functions, including conductance, regulation of other channels, signal transduction, and localization at apical membrane. Regulatory domain is a site of PKA and PKC phosphorylation. the common F508del occurs at NBD1, the gate. But, theres still partial function!

Pathophysiological cascade of CF lung disease?

CFTR gene defect Reduced quantity/function of CFTR protein Defective ion transport ASL depletion Defective mucociliary clearance (--> mucus obstruction, infection, inflammation) all that leads to scarring end-stage lung disease

Normal biosynthesis of CFTR?

CFTR gene undergoes transcription and is translated into protein in RER. As a glycoprotein, posttranslational modifications begin in ER lumen into an immature core-glycosylated protein. Protein is fully processed in the Golgi, where the mature fully glycosylated protein is correctly folded. Protein moves through trans-golgi and is delivered to the apical plasma membrane where it's normally expressed. At each step, defects can result in defective expression of CFTR. Even with normal conditions, only about 30% of CFTR protein is folded properly and delivered to plasma membrane as a functional protein. Remaining protein is degraded by the proteasome!

What is the CFTR protein structure?

CFTR is made up of 5 domains: 2 membrane-spanning domains (MSD1 and MSD 2) that form the chloride ion channel, 2 nucleotide-binding domains (NBD1 and NBD2) that bind and hydrolyze ATP to control ion channel opening/closing, and a regulatory (R) domain responsible for CFTR activation. R domain is unique to CFTR, not in other ABC transporters. Ion channel only opens when R domain has been phosphorylated by PKA and ATP is bound at the NBDs. The carboxyl terminal is anchored to cytoskeleton by PDZ-interacting domain.

CFTR in airway epithelium/pancreas? Normal vs CF?

Cl entry across the basolateral membrane is driven mostly by Na+ cotransport. The increased intracellular Cl concentration allows apical Cl efflux by CFTR! (normal) -- secrete Cl to lumen. Normal lung: CFTR lets Cl- go out of cell into lumen. CFTR suppresses the Na+ channels so Na+ stays out too! High NaCl concentration outside of the cell allows subsequent water flow by osmosis that contributes to normal airway surface liquid (ASL). Mucus traps inhaled bacteria/foreign particles. ASL provides microenvironment for beating cilia to clear mucus with assistance of coughing. Cilia are fine hair like structures which cover lining of airways like a carpet, beat continuous and remove bacteria/particles driving from lungs to mouth. In CF, no Cl secretion, Na+ comes into cells, high intracellular NaCl conc resulted in increased fluid absorption, resulting in reduced ASL. This reduces the normal ciliary clearance of bacteria/foreign particles! Lung disease in CF dominated by infection/inflammation. Even normal lungs prone to infection. Researchers think CFTR mutations impair several lung defense mechanisms.

What is the normal CFTR function?

Conducts chloride across the plasma membrane. Regulated by PKA (phosphorylation increases chloride ion transport) Phosphorylation by PKA is regulated by cAMP CFTR regulates the activity of other ion channels including Na+ and Cl-. CFTR functions as a cAMP activated ATP-gated anion channel, increasing the conductance for certain anions to flow down their electrochemical gradient. ATP-driven conformational changes in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. This is in contrast to other ABC proteins, in which ATP-driven conformational changes fuel uphill substrate transport across cellular membranes. Essentially, CFTR is an ion channel that evolved as a "broken" ABC transporter that leaks when in open conformation. CFTR found in epithelial cells of many organs like lung, liver, pancreas, digestive tract, reproductive tract, and skin. Normally, it moves Cl- and thiocyanate ion (with negative charge) out of epithelial cell to the covering mucus. Positively charged Na+ ions follow these anions out to maintain electrical balance. This increases total electrolyte concentration in the mucus, resulting in the movement of water out of cell by osmosis.

Class I mutation?

Lack of CFTR synthesis, premature stop codon Reduced or absent CFTR transcription. Nonsense, frameshift, splice-site mutations. Ex: p.Gly542* (or any mutation ending in X), c.3659delC, c.621+1G>T Anything that changes reading frame, non multiple of 3 deletion/insertion, anything affecting splice site junction...

How does CF affect lungs? Symptoms?

Sticky mucus builds up in airways, reducing flow of air into alveoli. Lungs gradually fill up with mucus, making them less effective for gas exchange. Symptoms = severe coughing (to remove excess mucus), breathlessness (shortage of oxygen increases tiredness/lack of energy), infections (bacteria trapped in mucus)

What are the correlations like between genotype and phenotype for pancreas, sweat glands, lung?

Good correlation with pancreatic status (insufficient v sufficient) - Pancreatic insufficiency is well-predicted by genotype. severe (classic) mutations more likely to be PI. Milder mutations more likely to be PS. Moderate correlation with sweat chloride concentration when patients are grouped according to pancreatic status Weak correlation with lung function! Lung very difficult to correlate! Chronic inflammation complicates it - poor genotype-phenotype correlation, high intrafamilial variability fertility - male infertility due to CBAVD nearly always occurs - CBAVD may be present even in the absence of pancreatic/lung findings = congenital bilateral absence of vas deferens

Defect and therapy for Class I, II, III?

Defect: Truncated CFTR is produced and degraded. Therapy: Compounds that allow read-through of premature stop codons result in full-length CFTR that can locate to cell surface. Defect: CFTR is not folded properly and is degraded. Therapy: Correctors help properly fold and locate CFTR to cell surface. Defect: CFTR reaches cell surface but does not function. Therapy: potentiators improve CFTR function to transport Cl-. Correctors vs potentiators

Pathophysiologic cascade in CF? (And what treatments can help at which steps?)

Defective CFTR gene (gene transfer) Defective/deficient CFTR (activate CFTR, curcumin, phenylbutyrate) Decreased Cl secretion, increased Na absorption (restore ion traffic, INS) Dessicated mucus Bronchial obstruction (early treatment to avoid infections, pseudomonas vaccine, rehydration) Inflammation/infection (control/delay chronic infection) Bronchiectasis (secretion removal, anti-infectives, avoid selection of resistance) Death (lung transplant) vicious cycle, focused on chest therapy. therapy vest where shaked high frequency, huffing coughs. shake chest then cough. high dose ibuprofen (GI side effects) Ciprofloxin is oral drug for pseudomonas Transplant patients live 6-7 years Other complications: pneumothorax, sinusitis

Class IV mutation?

Defective Cl- conductance (restricted Cl- movement through channel) Altered conductance of CFTR Cl- channel Missense mutations p.Arg117His

Class III mutation?

Defective channel regulation/gating (gating defect) Go to cell surface but cannot properly function Block in regulation of CFTR Cl- channel. Missense mutations. p.G551D = second most common (4%) -- 70% have F508del class II protein has gotten to the location, no other hurdles except open that gate! have been pretty successful in doing so.

CF Treatments?

Gene therapies to replace defective CFTR protein have not been successful to date due to difficulties in transfection of epithelia across mucus layer. Focus on pharmacotherapy that will rescue CFTR function, variety of trials underway, focused on F508del mutation and trafficking to membrane and functional enhancement. Restoration of only 10-35% of function is needed to impact pulmonary disease. only way to cure CF is gene therapy major goal in treating is to clear abnormal and excess secretions and control infections in the lungs, prevent intestinal obstruction adv stages: lung transplant treating symptoms doesn't cure but can greatly improve quality of life! increased lifespan GI treatment modified diet need vitamin supplements and pancreatic enzymes. essential to maintain adequate nutrition, high-caloric contact low in fat and high in protein.

Class II mutation?

Defective protein processing (misfolded CFTR) >85%! Immature forms of protein synthesized. Protein synthesized remains as an immature form (partial glycosylation, misfolded, not released from ER), most is degraded by ubiquitin-proteasomal pathway Never delivered to plasma membrane (F508del/phe508del/ΔF508) Among 1900 mutations found, most of the mutations involve a single amino acid change...However, fewer than 20 mutations occur at a frequency greater than 0.1% and only 5 mutations at a frequency >1%! The most common of these mutations is F508del which accounts for approximately 70% of CFTR genes in patients with CF worldwide. When a CFTR protein with F508del mutation reaches the ER, the quality-control mechanism of this cellular component recognizes that this protein is folded incorrectly and marks it for degradation, so it never reaches the cell membrane. People homozygous for F508del mutation have the most severe symptoms due to critical loss of Cl ion transport. F508del accounts for 66-70% of CF cases worldwise and 90% of CF cases in USA. Mutation over 52,000 years ago in N. Europe! Block in protein processing. Untranslated protein, protein misfolding, incomplete/incorrect posttranslational modification, error in trafficking Missense mutations, aa deletions ex: p.Asn1303Lys, p.Phe508del

Why does sweat taste salty in CF patients?

Due to failure of chloride ion absorption from the duct of sweat glands.

Patterns in classes of mutations?

I, II, III: little or no functional CFTR at cell surface; more severe mutations IV, V, VI: have functional CFTR at cell surface, affected function, might not be diagnosed til later! Associated with some residual protein function and milder disease!

IRT/DNA approach to CF newborn screening?

Immunoreactive trypsinogren (pancreatic enzyme precursor, elevated in CF infants) Mutation analysis (F508del or a panel of mutations) Sweat chloride testing (can do by a few days old) Trypsinogen is a precursor to trypsin, a digestive pancreatic enzyme Pancreas gets autodigested, we don't have trypsin to work Elevated precursor, measure IRT in blood, if it falls within the curve they do mutation analysis

False positives and false negatives?

In general, 90% of all abnormal newborn screens regardless of condition are false positives. Cannot know which are true or false positives. ALL abnormal newborn screens warrant follow-up. False negative rate for CF NBS is as high as 3%. many patients with meconium ileus will have normal IRT, most false negatives are PS, milder cases...

Best test to order?

Keep it simple, use the simplest methodology to answer question. Avoid pitfalls. Panels more likely to be covered by insurance, more detailed testing may detect variants of unknown significance. Consider family's level of concern. Residual carrier risk dependent upon number of mutations tested. Negative panel reduce risk to 1 in 240. Negative sequencing reduce risk to 1 in 2500.

Molecular genetics? Where is CFTR gene located, how big? Protein function?

Locus: 7q31.2 Structure: 250,000 bp with 27 exons Function: Cl- channel protein found in membranes of cells that line passageways of lungs, liver, pancreas, intestines, reproductive tract, and skin. CFTR is also involved in regulation of other pathways.

three main effects on body?

Lung disease due to increased mucus Pancreatic insufficiency Infertility in males

2nd most common type of mutation of CF? Therapy?

Missense G551D 4% Block in gating/regulation (Class III) Ivacaftor was made to cure!! Potentiator? Ivacaftor is an oral agent which potentiates CFTR function. FDA approved 2012 for twice daily therapy in patients with G551D mutation (class III) Take 2x per day with 16g of fat! Improved sweat chloride/pulm function. Kalydeco (ivacaftor) recently FDA approved to patients >2 years with at least 1 copy of G551D. potentiator!!!!! first drug to address underlying defect of CF. G551D is a gating mutation, CFTR is made and gets to surface but channel stays closed. kaydeco unlocks the gate to allow tranport! Outcomes: lowers sweat chloride, improves lung function, helps patients gain weight. game changer. only need 1 copy of mutation to qualify.

Talk about prevalence?

Most common life shortening inherited disease among Caucasians (~30,000 US patients) Worldwide, but inheritance is variable -- mostly Caucasians Predicted survival age is 39.3, today 45.2 Most common cause of chronic lung disease in children

After twins diagnosis...

Nasojejunal feeds are begun Enzyme replacement (pancreatic enzymes) vitamins added (ADEK) IV antibiotics begun (cephalosporins and aminoglycosides) mucolytics started (doronase alfa, hypertonic saline) aggressive chest physiotherapy (vest/hand PDs/huff cough) all symptoms resolve, weight gain is steady

KEY POINTS

Negative DNA screen/mutation panel does not eliminate the risk to be affected with CF or a CF carrier, some mutations will be missed. intronic mutations and mutations in regulatory elements. Sweat Cl- testing is the gold standard diagnostic test! Should be performed first to establish diagnosis, THEN proceed to genetic testing!

Compound heterozygotes? Two mutations are required for the disease. What happens when the individual mutation predictions conflict?

Nonclassical (mild) mutation typically "trumps" classical severe mutation. Autosomal recessive conditions due to LOF. Nonclassical mutations retain some residual function, only 5-10% gene function needed. Some residual function in milder allele picks up the slack, that one will make up the difference and give some residual function.

New potential drug for F580del?

Orkambi lumacaftor/ivacaftor combines lumicaftor (a corrector) designed to move defective CFTR to proper place with "potentiator" ivacaftor to improve function of CFTR as Cl- channel FDA approved this Orkambi use in CF patients who have 2 F580del CFTR mutation and are 12+. phase 3 trial in ages 6-11 approved to treat patients >6 with 2 copies of F508del. 38% of CF population. combination therapy because corrector and potentiator! not as striking clinical outcomes, see some lung function improvement and some reduction in rate of pulmonary exacerbations.

How is pancreas impacted in CF?

Pancreatic juice to small intestine is important for fat, protein, vitamin absorption! Similar to airway but with HCO3- anion! CFTR pumps out Cl-, Na+ and H20 follow out to increase volume of pancreatic juice, then HCO3- comes out to modulate the pH! When pancreatic volume is too much, Na+ channel at basal membrane pumps out Na+ to create a gradient so Na+ can get into cell by Na+ channel, Cl- and H20 follow In CF... pancreatic duct obstruction! Volume of pancreatic juice is decreased because Cl- has to stay inside, Na inside, water inside, surface liquid volume decreased and becomes viscous, pancreatic duct obstruction. sticky mucus, scarring, fibrosis Overall: - Pancreatic acinar cells secrete digestive enzymes, diluted by the bicarbonate rich fluid made by duct cells. - In CF, exocrine pancreatic secretions have lower bicarbonate, lower volume, that blocks the ducts and obstructs flow of digestive enzymes. - This, in turn, causes degeneration of acinar cells and pancreatic fibrosis. - CF patients classified as having pancreatic sufficient (mild) or insufficient (severe) disease - Pancreatic insufficiency results in maldigestion of fats and proteins with increased fecal loss and steatorrhea (pale loose foul smelling stools associated with poor weight gain) - LEads to chronic pancreatitis which can progress to pancreatic atrophy leading to endocrine pancreatic failure...diabetes!!!

Diagnostic testing for positive NBS (new born screening) and older patients with suspicious symptoms? Starts with what? Then what?

Pilocarpine iontophoresis (sweat test) - considered the gold standard for CF diagnosis! <30 in newborns is normal, 30-59 in newborns is gray zone, 60+ is positive diagnosis. - Sweat chloride is always the first test before genetic testing when there's clinical suspicion. - 1. the electrode drives the medicine into skin. 2. sweat is collected on filter paper or gauze. 3. sweat is tested for Cl- concentration. 4. high Cl- concentration likely due to CFTR mutation. Then, nasal potential difference (NPD): hard to perform, only done at a handful of CF centers. Measures salt transport in and out of nasal cells with different salt solutions. Cellular response can indicate malfunctioning Cl- channels! Genetic testing!

Main pathogens?

Pseudomonas aeruginosa S. aureus H. influenza S. maltophilia B. cepacia (gram -) patients don't go to clinics with others. no such thing as CF camps anymore.

Class V mutation?

Reduced amount of CFTR protein (alternative splicing) Cutting down quantity Reduced synthesis, often due to alternative splicing some missense, some splice mutations sometimes you have the right one! sometimes not

Guidelines for selection of lung transplant candidates?

Referral: exacerbation FEV1 < 30%, rapid decline FEV1, increased frequency of exacerbations requiring antibiotics, refractory/recurrent pneumothorax, recurrent hemoptysis Transplant: oxygen-dependent resp failure, hypercapnia, pulmonary hypertension ECMO = extra corporeal membrane oxygenation to prolong life support -- bridge to transplant to keep her alive

How does CF affect the digestive system?

Thick sticky mucus blocks the pancreatic duct preventing enzymes from reaching the duodenum. Food is not digested and absorbed. CF patients fail to put on body mass and suffer malnutrition. Trapped digestive enzymes damage the pancreatic cells affecting cells producing insulin resulting in diabetes!!!!

Symptoms of CF?

Salty sweat Progressive damage to the respiratory system and chronic digestive problems Production of abnormally thick mucus leading to obstruction of airways and increased bacterial infections Block ducts of pancreas preventing digestive enzymes from reaching the intestines Pulmonary disease accounts for 90% mortality

Explain the salt reabsorption from the sweat duct -- normal and in CF patients.

Secretory coil and reabsorptive duct in sweat gland. Normally, NaCl is reabsorbed in the reabsorptive duct, but due to low permeability for water, water is excreted as sweat In CF, Cl- cannot be reabsorbed because CFTR is the only apical pathway for chloride! Na+ tries to go in but because this leaves preponderance of - charge on duct side, it goes back out. Excrete NaCl in sweat!

Pilocarpine iontophonesis?

The sweat test!

Cis and trans for 5T?

Typical mutation analysis will not give you info about cis vs. trans If you know the patient has p.Arg117His and 5T/7T, is it... - p.Arg117His-5T and 7T -- CF carrier - p.Arg117His-7T and 5T -- risk for CBAVD (=congenital bilateral absence of vas deferens)/nonclassic CF How to determine? Phase must be determined by parental testing Testing a single parent may supply sufficient information F508del is always with 9T A single 5T variant with an R117H mutation on the same chromosome (in cis) acts as a classic CF mutation. Thus, an individual with this genotype is a CF carrier. A 5T variant occurring in trans (on the opposite chromosome) with an R117H mutation may result in CBAVD. A 7T or 9T variant in cis with an R117H mutation acts as a mild CF mutation. Thus, an individual with this genotype is a CF carrier. When coupled with a classic CF mutation, male patients may have CBAVD. Identification of an R117H mutation is followed by testing for the 5T/7T/9T variant in intron 8. If a 5T variant is identified, testing of family members is required to determine if the variant is in cis or trans. Genetic counseling is recommended.

Newborn screening for CF

Varies state-to-state Ohio started CF NBS in 2006 Starts with IRT from a heelstick -- immunoreactive trypsinogen - pancreatic enzyme precursor elevated in babies with CF - pancreatic ducts partially blocked, enzyme drainage not as efficient --> higher levels of IRT in blood Ohio follows IRT-DNA method of screening Top 4%ile of IRT measurements for the week are reflexed to DNA testing for 39 common mutations (including 23 mutation panel) at least 1 mutation is flagged as abnormal those with very elevated IRT have recommended follow up even with no found mutations -- repeat NBS at 4 weeks, refer to CF center if IRT still elevated Basic timeline: at about 2 weeks of age, abnormal NBS results given to PCP and CF center, inform family, CF center schedules sweat test/diagnostic evaluation. at about 5 weeks. full term infant for enough sweat. 1 trip. IRT cutoff usually like 55-60 over cutoff you're responsible If IRT Top 4th percentile, analyze the 39 common mutations, if no mutations low risk, 1 mutation moderate risk, 2 mutations high risk

CFTR gene stands for? What family does it belong to and what does it do?

cystic fibrosis transmembrane conductance regulator member of ATP-binding cassette (ABC) transporter superfamily (active transporter), BUT acts as a Cl- channel

What is cholera?

infectious disease caused by vibrio cholerae bacterium causes massive secretory diarrhea that can be rapidly fatal bacterium produces a protein toxin cholera toxin responsible for causing disease symptoms to induce disease the cholera toxin must cross the intestinal epithelial cell membrane and gain access to cytoplasm. it ultimately increases cAMP, PKA, overfunction CFTR, Cl and water follow suit!

Clinical clues to diagnosis?

meconium ileus (neonate) -- sticky/thick poop because dehydrated --> intestinal obstruction. If a baby has meconium ileus they have CF until proven otherwise. failure to thrive (any age) chronic respiratory disease with sinus disease rectal prolapse (inc abdominal pressure, muscle gets loose and they get rectal prolapse, push in with warm washcloth) polyps (sinus disease) newborn screening (prenatal too)

Complex allele - p.Arg117His + polyT tract

p.Arg117His is known CFTR mutation each CFTR gene has polyT tract: 5T, 7T, 9T Different combinations of p.Arg117His and polyT can lead to different outcomes Arg117His-5T LIKELY to act as CF causing mutation Arg117His-7T UNLIKELY to act as CF-causing mutation, maybe Arg117His-9T HIGHLY UNLIKELY to act as disease-causing mutation

Complex allele - 5T and TG tract?

sometimes cis-acting variants can cause more significant symptoms that each variant alone leads to more challenging counseling scenarios each CFTR gene has a polyT tract: 5T, 7T, 9T polyT tract is one of the most common cis acting variants every single one of us has the tract 5T is the one we worry about, it affects splicing of the exon. When you have 7T or 9T it is normal, does not affect splicing of exon 9! With 5T, we see that you get reduced amount of full length protein, but sometimes you can make the full length. If you have 5T 5T, you have normal CFTR channel but reduced amount at the cell membrane since it can affect the splicing! When you have 7T or 9T, the TG number is irrelevant. However, when you have 5T and it is paired with a higher number of TGs like 12 or 13, this acts like another CF mutation. 5T with 11 likely mild and asymptomatic. 5T with 12 or 13 moderate and when paired with CF can lead to CF. The lower number of T's and higher number of TG's the more detrimental! 5T is newly-thought to cause disease under certain circumstances. Each CFTR gene has a TG tract: 11TG, 12 TG, 13TG different combinations of 5T and TG can lead to different outcomes: 5T/IITG = unlikely to act as CF-causing mutation, but still risk for male infertility* 5T/12TG or 13TG = may act as CF-causing mutation, but milder symptoms. Risk for male infertility* *Note, this is when there's a second CFTR mutation on the other allele