Recommended panel testing at Breda Genetics for this condition:
Cystic fibrosis and its differential diagnosis (ARMC4, C21ORF59, CA12, CCDC103, CCDC114, CCDC151, CCDC39, CCDC40, CCDC65, CCNO, CFAP300, CFTR, DNAAF1, DNAAF3, DNAAF4, DNAAF5, DNAH1, DNAH11, DNAH5, DNAI1, DNAI2, DNAJB13, DNAL1, DRC1, GAS8, HYDIN, IL2RG, KTU, LRRC6, NME8, PIH1D3, RSPH1, RSPH3, RSPH4A, RSPH9, SBDS, SCNN1A, SCNN1B, SCNN1G, SPAG1, TAP1, TAP2, TTC25, ZMYND10)
Other recommended genetic testing at Breda Genetics (depending on the clinical indication):
CFTR34, CFTR139 or CFTR152: screening of the 34, 139 or 152 most common Caucasian pathogenic mutations; detection power: 85%, 95% and 97% respectively.
EXOME GENE: CFTR full gene sequencing; detection power: 98%.
MLPA GENE: CFTR del/dup testing, detection power: 2% (100% in conjunction with EXOME GENE or EXOME PANEL).
Cystic fibrosis is an inherited disease also known as mucoviscidosis. The disease is characterized by the increased density and viscosity of body mucous secretions, with main signs and symptoms on the respiratory tract, pancreatic function, intestine, hepatobiliary system, sweat glands, and male fertilty. The disorder typically express itself as progressive obstructive lung disease with bronchiectasis, pancreatic insufficiency, malnutrition, recurrent infections of the upper respiratory ways (sinusitis and bronchitis), and male infertility. Signs and symptoms of cystic fibrosis may vary in severity, going from life-threatening early onset to forms starting in adolescence or adulthood. The most common among the mild forms of the disease is Congenital (Bilateral) Absence of the Vas Deferens (CBAVD or CAVD), which manifests as isolated azoospermia in healthy adult males. The life expectancy for cystic fibrosis patients has increased dramatically over the last 30 years. The disease is autosomal recessively inherited, being caused by biallelic mutations in the CFTR gene.
Detailed clinical description
Cystic fibrosis is characterized by the obstruction of respiratory and pancreatic systems due to increased density and viscosity of mucous secretions. Signs and symptoms of cystic fibrosis vary, depending on the severity of the disease. In classic cystic fibrosis, clinical manifestations include progressive obstructive lung disease with bronchiectasis, frequent hospitalizations for pulmonary disease, pancreatic insufficiency and malnutrition, recurrent sinusitis and bronchitis. In non-classic cystic fibrosis, symptoms may not be experienced until adolescence or adulthood and are variable from mild/medium pulmonary involvement with/without pancreatic involvement (recurring pneumonia, recurring bouts of pancreatitis) to isolated male infertility due to CAVD.
The diagnosis of cystic fibrosis usually relies on typical clinical findings and genetic testing, when biallelic pathogenic mutations are found in the CFTR gene. In infants, the diagnosis can be supported by the identification of elevated level of trypsinogen at newborn screening in addition to biallelic CFTR mutations or abnormal sweat chloride.
The diagnosis of CAVD is established in a male with azoospermia and absence of the vas deferens on palpation or identification of biallelic CAVD-causing CFTR pathogenic variants.
Early diagnosis, regular follow-up, maintaining hygienic conditions, proper treatment can improve the quality of life. Classic cystic fibrosis can be treated daily with inhalation therapy, physiotherapy, pancreatic enzymes, antibiotics, other medications, and diet.
Respiratory signs and symptoms
In the classic, severe form of the disease, the abnormally thick and sticky mucus can clog the airways leading to infections and severe obstructive disease. Persistent infections cause chronic inflammation, which eventually leads to permanent lung damage. Notably, 97% of deaths in cystic fibrosis are due to pulmonary causes.
More in detail, the following clinical signs and symptoms are found at the respiratory level suggesting a diagnosis of cystic fibrosis: persistent cough with thick sputum, chronic wheeze (dyspnea), air trapping and breathlessness, exercise intolerance, repeated lung infections, inflammation of the upper ways (sinusitis and bronchitis), pneumonia, prolonged bronchiolitic type illness in infants, coexistent atopic asthma in children, nasal polyps, sputum expectoration in older children. Hospitalizations for pulmonary disease are frequent.
Indirect clinical signs of respiratory involvement in cystic fibrosis include: digital clubbing, increased anterior-posterior diameter of the chest wall (barrel chest), kyphosis, and downward displacement of the liver. Simple allergy to Aspergillus fumigatus is also common (blood and sputum eosinophilia).
Digestive signs and symptoms
Pancreatic secretions are strongly compromised in cystic fibrosis. Pancreas produces insulin and digestive enzymes. Due to the blockage of the ducts of the pancreas by thick and sticky secretions, insulin production is reduced and digestive enzymes are prevented from reaching the intestine. This causes cystic fibrosis-related diabetes mellitus (CFRDM), malnutrition with weight loss and poor growth.
In general, the following clinical signs and symptoms are found at the digestive and nutritional level: meconium ileus (which is present at birth in 15%-20% of newborns with cystic fibrosis and is consistent with a bowel obstruction at the ileum level due to thicker and stickier meconium – the meconium is the earliest stool of an infant), voracious appetite in infants, malabsorption, rectal prolapse, steatorrhoea (with passage of greasy, large, and offensive stools), distal intestinal obstructive syndrome, recurrent acute pancreatitis, chronic pancreatitis, severe constipation, distension of abdomen, vitamin E deficiency with subsequent haemolytic anaemia and neurological symptoms, vitamin A deficiency with consequent benign intracranial hypertension, vitamin K deficiency with leading to bleeding disorders; salt deficiency leading to severe hypochloraemic metabolic alkalosis and hyponatremic hypochloremic dehydration.
Notably, the pancreatic involvement is variable in cystic fibrosis patients, mainly depending from the underlying CFTR genotype (CFTR mutations can be divided in pancreatic sufficient or pancreatic insufficient). Acute or chronic recurrent pancreatitis can be a presenting manifestation of cystic fibrosis, and is much more common among those with pancreatic sufficiency than those with pancreatic insufficiency.
Cystic fibrosis is also characterized by important involvement of the liver function. Patients usually show: prolonged neonatal jaundice, hepatomegaly secondary to fatty infiltration, gallstones, and chronic hepatic disease manifested by clinical or histological evidence of focal biliary cirrhosis or multilobular cirrhosis. Cystic fibrosis-related liver disease usually shows its peak in adolescence.
Congenital (bilateral) absence of the vas deferens (CBAVD or CAVD)
Vas deferens are usually absent, atrophic, or fibrotic and lead to azoospermia and infertility in 95% of males with classic cystic fibrosis. However, male infertility may also be the only clinical sign: isolated CBAVD (commonly referred to also as CAVD) leads to severe oligospermia or azoospermia in otherwise healthy adult males. Isolated CBAVD can be seen in male patients with the combination of a classic (severe) and a mild CFTR mutation. Clinical findings commonly associated with CBAVD include low volume of ejaculated semen, which also shows a specific chemical profile (low pH, elevated citric acid concentration, elevated acid phosphatase concentration, low fructose concentration, and failure to coagulate), and evidence of abnormalities of seminal vesicles or vas deferens on rectal ultrasound examination and at palpation.
Fertility in women
Women with cystic fibrosis are mostly fertile, although a few females may have abnormal cervical mucus that may contribute to infertility. Others with severe illness and reduced body mass index may be anovulatory.
Additional clinical signs and symptoms sometimes seen include: transient non-specific arthritis, which most commonly affects the knee and ankle joints, increased incidence of gastrointestinal malignancies (especially in the post lung transplant population), complications in pregnancy (the most important predictors of pregnancy outcome are the severity of maternal pulmonary impairment and nutritional status; deterioration during pregnancy may precipitate preterm delivery).
Cystic fibrosis is a common genetic disease and its incidence has long been estimated in 1:2,500 live births in the white population of Europe and United States, which leads to a healthy carrier frequency of 1:25. Therefore cystic fibrosis is one of the most common genetic disorders (although its prevalence classifies it as a rare disease). Cystic fibrosis is less common in other ethnic groups: it’s about 1 in 17,000 African Americans and 1 in 31,000 Asian Americans.
Cystic fibrosis is caused by mutations in the CFTR gene. The coded protein is a channel that allows chloride ions and water flowing into and out of cells. When the channel is quantitatively and/or qualitatively damaged due to a pathogenic CFTR gene mutation, the epithelial cells in the lungs, pancreas, and other organs produce thicker and stickier mucus, which obstructs the airways and various ducts.
CFTR is a medium-large gene, spanning 27 exons. More than 1,000 CFTR variants have been reported to date. The vast majority are missense variants or small deletions (1-84bp), but also nonsense, frame shift and splice mutations are described. The most common pathogenic variant is p.Phe508del (often referred to as DeltaF508 or ΔF508), accounting for an estimated 30%-80% of all pathogenic variants (depending on the ethnic group). Notably, not all reported CFTR pathogenic variants are proven by population or functional studies to be pathogenic. The most common 34 pathogenic mutations of the Caucasian population are listed here below (these mutations are known to be certainly pathogenic; due to their high frequency, they account for about 85% of all pathogenic alleles in this population).
The 34 most common CFTR pathogenic mutations in the Caucasian ethnic group are: G85E, 394delTT, 3849+10KbC>T, 3876delA, W1282X, 3905insT, N1303K, 621+1G>T, R117H, 711+1G>T, 1078delT, R347P, R347H, R334W, polyT polymorphism, A455E, DI507, DF508, 1717-1G>A, V520F, G542X, S549N, S549R, G551D, R553X, R560T, 1898+1G>A, 2183AA>G, 2184delA, 2789+5G>A, 3120+1G>A, R1162X, 3659delC, 2184insA.
Large exonic or multiexonic deletions/duplications not detectable by sequencing account for 2-3% of all pathogenic alleles. These mutations may be detected by specific methods (e..g. MLPA or qPCR).
CFTR pathogenic variants can be distinguished in severe (classic) mutations and mild mutations. Severe (classic) variants are typically loss-of-function mutations, whereas mild variants retain some protein function. Overall, CFTR mutations may be clustered into five main groups (classes), according to their predicted impact on the CFTR protein:
I. Reduced or absent synthesis (nonsense, frameshift, splice junction variants, large deletions)
II. Block in protein processing (some missense variants, in-frame amino acid deletions)
III. Block in regulation of CFTR chloride channel (some missense variants)
IV. Altered conductance of CFTR chloride channel (some missense variants)
V. Reduced synthesis/trafficking (some missense variants and splice site variants)
Classic (severe) alleles generally refer to Class I-III pathogenic variants; mild alleles refer to Class IV-V pathogenic variants exclusive of p.Arg117His and 5T alleles (see further below). Mutations of different classes in different combinations cause either cystic fibrosis (in its classic or milder forms) or a CFTR-related disorder (e.g. CAVD): see Table 1 below.
|First allele||Second allele1|
|Classic||Classic||Classic > non-classic|
|Mild||Classic or mild||Non-classic > classic|
|p.Arg117His and 5T||Classic or mild||Non-classic > classic|
|p.Arg117His and 7T||Classic or mild||Asymptomatic female or CAVD > non-classic|
|5T and 11TG||Classic or mild||Asymptomatic > CAVD|
|5T and 12TG||Classic or mild||CAVD or non-classic CF > asymptomatic carrier|
|5T and 13TG||Classic or mild||CAVD or non-classic CF > asymptomatic carrier|
|7T or 9T||Classic or mild||Asymptomatic|
|7T or 9T||7T or 9T||Asymptomatic|
|p.Arg117His and 5T||Classic||Classic > non-classic|
|p.Arg117His and 7T||Classic or mild||Non-classic > classic|
|5T and 11TG||Classic or mild||Asymptomatic female or CAVD > non-classic|
|5T and 12TG||Classic or mild||Non-classic > classic|
|5T and 13TG||Classic or mild||Non-classic > classic|
|7T or 9T||Classic or mild||CAVD or non-classic CF > asymptomatic carrier|
Table 1. Phenotypic outcome of different combinations (genotypes) of CFTR gene mutations. Individuals with mutations on one allele (chromosome) only are healthy carriers. Notes:
1 Or homozygous for the first allele.
Some variants cannot be properly defined as pathogenic, as they rather have a modifier effect. The most common modifiers are the polyT tract (sometimes referred to as polyT polymorphism) and the TG tract (or TG repeats). The polyT tract is a short string in intron 8, which is made up of 5, 7 or 9 thymine (alleles are named 5T, 7T and 9T respectively). The 7T and 9T alleles are the normal ones, whereas the 5T allele, which is thought to decrease the efficiency of the splicing process in intron 8, is a variably penetrant variant with a modifier effect. Just upstream the polyT, there is a short TG tract, which is made up of 11, 12 or 13 TG repeats (alleles are named 11TG, 12TG or 13TG respectively). A longer TG tract in combination with the 5T allele has the strongest adverse impact on the splicing of intron 8. The 5T allele cause CAVD in males who harbor this variant in compound heterozygosity with a severe pathogenic mutation. However, compound heterozygosity between a severe pathogenic mutation and 5T may also cause lung disease. Hence, caution is warranted when trying to predict the genotype-phenotype correlation of compound heterozygosity between a severe mutation and the 5T allele, since the clinical outcome can range from CAVD (with or without mild respiratory and pancreatic problems) to lung disease in adult females with cystic fibrosis-like clinical features.
The variant p.Arg117His (also known in the old nomenclature as p.R117H or simply as R117H) needs a special mention, due to its interaction with the polyT tract. When p.Arg117His and the 5T allele are in cis (i.e. on the same chromosome) in conjunction with another pathogenic mutation on the other chromosome, patients usually develop lung disease. When p.Arg117His is in cis with a 7T or 9T allele in conjunction with another pathogenic mutation on the other chromosome, patients have a highly variable phenotype that can range from no symptoms to CAVD or mild lung disease.
In cystic fibrosis, genotype-phenotype correlations are difficult in general, even for other combinations of CFTR mutations. The genotype-phenotype correlation is possible to an extent for what concerns the pancreatic function, but remains difficult in regard of the pulmonary involvement.
A useful CFTR gene mutation database is hosted by the Sickkids Hospital (Canada): http://www.genet.sickkids.on.ca/app.
Genetic testing and genetic screening
Patients with a clinical phenotype resembling classic or non-classic cystic fibrosis or CAVD are candidate for genetic testing, which is mostly performed as targeted mutations analysis (i.e. the scanning of the most common mutations). If only one or no mutations are found, full CFTR gene sequencing and large deletion/duplication testing (e.g. by MLPA) can be performed. The alternative approach is to perform immediately full gene sequencing or a multigene panel testing, which also enables the screening of the differential diagnosis of cystic fibrosis (see “Differential diagnosis” further below).
Genetic testing for cystic fibrosis is widespread also as preconceptional screening, especially in couple undergoing IVF (In Vitro Fertilization). In these couples, cystic fibrosis screening can (1) reveal the possible cause of infertility in the male partner (which may be found affected wihth CAVD) and (2) allow for the estimation of the couple reproductive risk for cystic fibrosis, which is one of most common and severe genetic disorders (the frequency of healthy carriers in the Caucasian population is 1:25 and the a priori risk of any couple to conceive a fetus affected by a CFTR-related disorder is 1:2,500).
When performed as preconceptional screening, CFTR analysis mostly comes as targeted mutation scanning. A common panel include the 34 most frequent Caucasian mutations: G85E, 394delTT, 3849+10KbC>T, 3876delA, W1282X, 3905insT, N1303K, 621+1G>T, R117H, 711+1G>T, 1078delT, R347P, R347H, R334W, polyT polymorphism, A455E, DI507, DF508, 1717-1G>A, V520F, G542X, S549N, S549R, G551D, R553X, R560T, 1898+1G>A, 2183AA>G, 2184delA, 2789+5G>A, 3120+1G>A, R1162X, 3659delC, 2184insA. Such a panel covers about 85% of all pathogenic alleles in that population. A patient who is negative to such a panel has a residual risk of being a carrier of about 1:165. So, if the other partner didn’t take the test (a priori risk: 1:25), the couple reproductive risk becomes 1:16,500 (instead of 1:2,500, which is the a priori risk of the general population). If both partners are negative to the screening, the final reproductive risk for cystic fibrosis can be estimated in about 1:108,900. In Table 3, the residual risk of being a carrier and the residual couple reproductive risk are calculated for screening tests with different detection power.
|Test detection rate1||Carrier residual risk2||Residual reproductive risk3||Available at Breda Genetics|
|One negative and one carrier4||One negative and one no test5||Both negative6|
Table 3. The residual risk of being a carrier and the residual couple reproductive risk for cystic fibrosis and CFTR-related disorders are given for screening tests with different detection power. Notes:
1 Test detection rate (i.e. the percentage of all pathogenic alleles of the Caucasian population detectable by the test).
2 Residual risk of being a healthy carrier of a CFTR pathogenic mutation if the test is negative.
3 Residual reproductive risk of having a child affected with cystic fibrosis or any CFTR-related disorder.
4 When parent is negative to the test and the other parent is a proven healthy carrier of one CFTR pathogenic mutation.
5 When one parent is negative to the test and the other one didn’t take the test, having therefore a priori risk of being a healthy carrier of 1:25.
6 When both parents are negative to the test.
The genetic differential diagnosis of cystic fibrosis includes the following clinical entities, which may show some partial clinical overlap:
- Primary Ciliary Dyskinesia: in primary ciliary dyskinesia, cilia in the respiratory tract stop functioning and prevents mucous clearance from nose, paranasal sinus and ears, leading to chronic oto-sino-pulmonary disease. Respiratory distress in infancy, cough, sputum production, choking, recurrent pneumonias, chronic bronchiectasis, Pseudomonas aeruginosa or other opportunistic bacterial infections are seen. Situs inversus is present in 50% of patients. Primary ciliary dyskinesia is a highly heterogenous disorder: pathogenic mutations have been so far identified in the following genes: ARMC4, C21ORF59, CCDC103, CCDC114, CCDC151, CCDC39, CCDC40, CCDC65, CCNO, CFAP300, DNAAF1, DNAAF3, DNAAF4, DNAAF5, DNAH1, DNAH11, DNAH5, DNAI1, DNAI2, DNAJB13, DNAL1, DRC1, GAS8, HYDIN, KTU, LRRC6, NME8, PIH1D3, RSPH1, RSPH3, RSPH4A, RSPH9, SPAG1, TTC25, ZMYND10.
- Shwachman-Diamond syndrome (SDS): the disease is characterized by exocrine pancreatic dysfunction with malabsorption, malnutrition, and growth failure. SDS is caused by biallelic pathogenic variants in the SBDS gene and it is inherited in an autosomal recessive
- Cystic fibrosis-like syndrome: the condition is also known as “bronchiectasis with or without elevated sweat chloride”. The clinical phenotype is consistent with a non-classic cystic fibrosis-like phenotype in individuals. The disease can be caused by mutations in either the SCNN1A, SCNN1B, or SCNN1G gene. Mild lung disease, elevated sweat chloride concentration and reduced fertility in males can be found. Because of elevated sweat chloride, patients may be mistakenly diagnosed with cystic fibrosis.
- Isolated hyperchlorhidrosis (caused by pathogenic variants in CA12): in this condition excessive salt wasting in sweat can result in severe infantile hyponatremic dehydration and hyperkalemia with gastroenteritis and failure to thrive in infancy. Since testing of sweat shows increased chloride levels, patients may be initially suspected of having cystic fibrosis. However, pulmonary involvement is absent and the condition appears to be extremely rare.
- Young syndrome: this syndrome is characterized by progressive obstruction of the epididymis by inspissated secretions and chronic sinopulmonary infections. Patients have persistent azoospermia, but normal spermatogenesis. No gene has been thus far identified.
- Mayer-Rokitansky-Kuster-Hauser syndrome (hereditary urogenital adysplasia): females may have a range of uterine anomalies, from upper vaginal atresia to total mullerian agenesis with urinary tract abnormalities; males may have Wolffian duct anomalies including unilateral or bilateral absence of the vas deferens. Inheritance is suggested to be autosomal dominant, but no gene is known to date.
- Severe combined immunodeficiency: in X-linked severe combined immunodeficiency (X-SCID, caused by hemizygous pathogenic variant in IL2RG) males are diagnosed between three and six months of life with failure to thrive, oral/diaper candidiasis, absent tonsils and lymph nodes, recurrent and persistent infections (also by opportunistic organisms such as Pneumocystis carinii), rashes, diarrhea, cough and congestion, fevers, pneumonia, sepsis, and other severe bacterial infections. The disease is almost invariably fatal if no treatment is done (bone marrow transplantation and gene therapy).
- Bare lymphocyte syndrome type I: the involvement of the respiratory tract in TAP1 and TAP2 deficiency (caused by TAP1 and TAP2 gene mutations) is indistinguishable from two other autosomal recessive disorders, namely cystic fibrosis and primary ciliary dyskinesia. Sweat chloride concentration is normal in TAP deficiency, excluding cystic fibrosis, while electron microscopy of the ciliary apparatus may be needed to exclude primary ciliary dysfunction.
Immunodeficiency disorders other than X-SCID or bare lymphocyte syndrome type I, asthma, congenital airways anomalies, pediatric apsergillosis and primary biliary atresia are also part of the general differential diagnosis of cystic fibrosis.
Genetic testing strategy
Patients with a clinical phenotype resembling classic or non-classic cystic fibrosis and positive biochemical tests are candidate for targeted mutation analysis (i.e. the scanning of the most common mutations) or full gene sequencing. Multigene panel testing, possibly based on whole exome or whole genome sequencing, is recommended to include the differential diagnosis immediately.
Recommended genetic testing at Breda Genetics for this condition (depending on clinical indication):
CFTR34: screening of the 34 most common Caucasian pathogenic mutations. Detection power: 85%.
CFTR139: screening of the 139 most common Caucasian pathogenic mutations. Detection power: 95%.
CFTR152: screening of the 152 most common Caucasian pathogenic mutations. Detection power: 97%.
EXOME GENE: Cystic fibrosis and CFTR-related disorders (CFTR). Detection power: 98%.
Detection power: 2% (100% in conjunction with EXOME GENE or EXOME PANEL):
EXOME PANEL: Cystic fibrosis and its differential diagnosis (ARMC4, C21ORF59, CA12, CCDC103, CCDC114, CCDC151, CCDC39, CCDC40, CCDC65, CCNO, CFAP300, CFTR, DNAAF1, DNAAF3, DNAAF4, DNAAF5, DNAH1, DNAH11, DNAH5, DNAI1, DNAI2, DNAJB13, DNAL1, DRC1, GAS8, HYDIN, IL2RG, KTU, LRRC6, NME8, PIH1D3, RSPH1, RSPH3, RSPH4A, RSPH9, SBDS, SCNN1A, SCNN1B, SCNN1G, SPAG1, TAP1, TAP2, TTC25, ZMYND10)
Detection power: 98% plus exclusion of the differential diagnosis
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