Abstract
Cystic fibrosis is a monogenic and autosomal recessive disease. It is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene responsible for encoding the CFTR protein. Involvement of the gastrointestinal and respiratory systems is the main clinical manifestation. In this case, we report a heterozygous CFTR patient harboring class I (p.Gly542*) and class V (c.2657+5G>A) mutations. The importance of this case report lies in the clinical features because the patient, aged 3 years, presented with early exocrine pancreatic insufficiency, which can be considered atypical, as most individuals with this genotype are pancreatic sufficient or develop pancreatic insufficiency later in life. This report aims at presenting the tests requested that contributed to the patient’s diagnosis, as well as at understanding the association between these mutations and their phenotypic presentation. Interpretation of the genotype-phenotype relationship represents a challenge, as genetic analysis alone is not sufficient to clearly predict severity of the disease. This is because the significant phenotypic heterogeneity existing among patients with the same genotype may exert socioeconomic and sociocultural influences, or by the action of CFTR modifiers, such as environmental and modifying genes, which can alter the protein’s function and exert an impact on the individual’s phenotype.
Cystic fibrosis (CF) is a genetic, autosomal, recessive, inherited disease resulting from changes in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which is located on chromosome 7 and encodes chloride (Cl-) channel protein, CFTR. Epidemiological data indicate that around 70,000 people have this disease worldwide and that its incidence varies across countries or regions. The pathophysiological manifestations have multisystemic characteristics, mainly affecting the respiratory, gastrointestinal, reproductive, and endocrine tracts. However, more than 2,000 different mutations in the CFTR gene have been described; and the clinical manifestations of the disease, as well as its treatment, vary according to each type of mutation.1 Therefore, it is extremely important to describe the most diverse combinations of mutations in the CFTR gene, relating their main phenotypic manifestations and the most effective therapeutic strategies.
This case, which was approved by the local Ethics Committee (number 4,050,055) and for which the informed consent form was signed by the participants, presents a single case of a male patient with two pathological mutations in compound heterozygosity in the CFTR gene: p.Gly542* and c.2657+5G>A. The phenotype consists of early total dysfunction of the exocrine pancreas in the first year of life, without pancreatitis episodes until that moment, and the patient presented no manifestation or any respiratory impairment due to CF. The phenotype, clinical manifestations, and progression of this genetic combination were not previously published, and the combination of these two pathogenic variants is rarely present in the CFTR databases.
In this perspective, CFTR mutations affect the protein amino acids sequence, and the result is little or no functional CFTR in the apical membrane of exocrine epithelial cells. CFTR mutations are grouped into six different classes based on their known or predicted molecular mechanisms.1 Studies evaluating the genotype-phenotype relationship show that it varies considerably according to the organ. The correlation degree is higher for the pancreas and lower for lung disease, which strongly suggests an influence of the environment and of secondary genetic factors (CF modifiers).2
Pancreatic insufficiency (PI) correlates closely with the specific CFTR mutation in an individual with CF.2 In this sense, class I, II, and III disease-causing variants are associated with little or no CFTR function and are linked to a more severe phenotype. Specifically, in the Class I group, there is no effective protein synthesis, and no functional CFTR is present in the apical membrane. The type of mutations are usually nonsense, frameshift, or canonical splice,3 and examples are p.Gly542*, c.3846G>A, c.1659C>T, and 621+1G>T.1
Class IV, V, and VI variants have residual CFTR function, which is often associated with preserved exocrine pancreatic function early in life.3 In relation to Class V, reduced synthesis and trafficking results in reduced expression of functional CFTR channels in the apical membrane. Some specific Class V mutations are associated with a milder CF phenotype, such as 3849+10kbC>T, 2657+5G>A, 3120+1G>A.1 Furthermore, individuals with two severe CFTR mutations tend to have early PI, often at birth, while those with two CFTR class IV and V mutations, or with one severe and one mild mutation, tend to be pancreatic sufficient (PS) at birth.3
This case report aims at enriching the literature data regarding the phenotype associated with this specific genotype to better understand the damage it causes to the CFTR protein, as well as to trace the patient’s prognosis.
Case Report
The patient was a White male, aged 3-years, with no previous family history of a similar phenotype or complications in the prenatal and perinatal period. At 7 days of life, neonatal screening indicated that the trypsinogen plasma level was increased at 76.5 ng/mL (reference <70 ng/mL), and CF was considered as a primary hypothesis. Trypsin serum was measured at day 19, obtaining a value of 57.3 ng/mL, thus discarding the previous hypothesis. However, after the parents reported that the infant had salty sweat, sweat chloride analysis was performed at the 6th and 7th months of life and showed values of 71 mEq/L and 67 mEq/L, respectively (reference <39 mEq/L). CFTR sequencing was performed by next-generation sequencing in a clinical laboratory. The CF diagnosis was confirmed, with pathological mutations p.Gly542* and c.2657+5G>A identified in trans. The monoclonal fecal elastase-1 (FE-1) test was requested because the patient had reduced weight gain at the 7th and 8th month of life, and PI was confirmed (53.48 µg/g; reference >200 µg/g). No pancreatic function secretin tests or imaging were performed. Steatorrhea was the only gastrointestinal clinical manifestation observed. Inflammatory enteric disease or other celiac diseases that cause pancreas malfunction were excluded due to normal C-reactive protein (CRP) testing (1,11 mg/L; reference ≤5 mg/L). The therapy adopted consisted of replacement of pancreatic enzymes with the use of medications that contain amylases to digest carbohydrates, proteases to digest protein, and lipases to digest fat (10.000 IU pancreatin per meal). In addition, supplementation with vitamins A, D, E, K, and ferrous sulfate was implemented. Since treatment initiation, there was an excellent response to the enzyme replacement therapy, as the patient presented normal development in relation to weight, height, and cognition.
In follow-up, the patient’s sputum culture swab was positive for Pseudomonas aeruginosa, and he was treated with inhaled tobramycin for 28 days. There were no other clinical manifestations, complications, or respiratory infections recorded resulting from P. aeruginosa, and this examination was repeated every 3 months. In summary, it is important to mention that, until this time, the patient presented no manifestation or any respiratory impairment due to CF. Thus, the only phenotype of this case is total exocrine PI.
Discussion
In Brazil, the CF diagnosis was included in the Unified Health System (Sistema Único de Saúde, SUS) in 2013 through the National Neonatal Screening Program of the Ministry of Health (Programa Nacional de Triagem Neonatal/Ministério da Saúde, PNTN/MS).4 It establishes CF screening through the dosage of immunoreactive trypsinogen (TIR), a precursor protein of the trypsin enzyme, elevated in CF neonates up to 30 days of age.5 Increase of this protein’s blood concentration occurs when there is obstruction of the pancreatic ducts, because TIR does not reach the intestine and is not converted into trypsin. Altered TIR values (above 70 ng/mL) are repeated before 30 days of age.6,7 However, TIR measurements can be imprecise, leading to false positive, false negative, or even uncertain results,8 which would justify the false negative patient test in our case report, a result collected at day 19 (57.3 ng/mL).
According to Taccetti et al9 from 1992 to 2018, they retrospectively reviewed all the results from the database of the Regional Referral CF Center in Florence (Italy). From the total CF cases, 8.7% were false negative. This study supports the claim that the false negative patients had rarer CFTR mutations when compared to the true positive cases. Furthermore, this study shows that respiratory symptoms and salt-loss syndrome (metabolic hypochloremic alkalosis) are suggestive symptoms of CF and were common events in false negative patients.9
In this sense, the patient in our case report had salty sweat, which is a common characteristic and suggestive of CF. This is because patients present a tendency towards excessive loss of sodium by the sweat glands due to dysfunction of the CFTR protein. Dosage of the electrolytes is performed through the sweat test by determining the rules for chloride and sodium ions by colorimetry and flame photometry.10,11 Respectively, Cl- ion concentrations above 60 mmol/L represent a positive test. Values below 29 mmol/L indicate improbable CF diagnosis, and values between 30 mmol/L and 59 mmol/L are intermediate, indicating a possibility of CF diagnosis, which needs confirmation through genetic testing.12
In relation to the patient’s genetic test, the p.Gly542* mutation located on Chr7:117.227.832 G>T promotes substitution of the amino acid glycine in codon 542 by a stop codon, which leads to early interruption in translation of the protein. The c.2657+5G>A mutation located on Chr7:117.242.922 G>A promotes alteration in the mRNA processing site, leading to exclusion of exon 16.1 A class I mutation, such as p.Gly542*, tends to promote a classical CF phenotype; however, compound heterozygosity with a class V mutation, such as c.2657+5G>A, which preserves a residual CFTR protein function, is frequently associated with a normal exocrine pancreatic function early in life,1 with better lung function and less bronchiectasis.13 However, the phenotypic manifestations vary, as other factors interfere with severity of the disease, such as environmental triggers and modifying genes.1
Based on the diagnosis and the case report presented, we will discuss some important points about the patient’s clinical manifestations. One of the most frequent clinical manifestations in CF is PI, as there is a high CFTR expression in the pancreas which generates the disease manifestations due to obstruction of the ducts by the thick accumulated secretion.14 When thick mucus causes obstruction, accumulation of pancreatic enzymes causes self-digestion, resulting in pancreatitis,15 cyst formation, and pancreatic tissue fibrosis.16 Recurrent acute and chronic pancreatitis can precede the CF diagnosis by several years.17
The steatorrhea presented by the patient in our case report is one of the most used parameters for the classification of exocrine pancreatic insufficiency, due to pancreatic alterations that result in lipid malabsorption associated with low body weight.17,18 Direct assessment of the pancreatic function, including secretin, secretin-cholecystokinin, or secretin-caerulein stimulation tests, is more accurate in assessing pancreatic secretion, but is invasive, time-consuming, expensive and not fully standardized. Therefore, the FE-1 test measures fecal levels of elastase-1, a proteolytic enzyme produced by pancreatic acinar cells, which binds to bile salts and passes through the intestine with minimal degradation. The result correlates well with its pancreatic output, as well as with that of other pancreatic enzymes, such as amylase, lipase, and trypsin.19
In the Clinical and Functional TRanslation of CFTR (CFTR2) database, of the 27 patients with those two mutations (p.Gly542* and c.2657+5G>A), 8 had PI. Nevertheless, the mean age of patients was 26 years, which enables a parallel with a patient younger than one year-of-age, who had a minor environmental interference on its phenotype.20 Thus, to better understand the association between genotype and phenotype, we analyzed the incidence of PI in mutations of the same classes: I and V in the CFTR2 database.
Of the 122 patients with a homozygous variant of p.Gly542*, 108 (95%) had PI, and the patients’ mean age in this category was 15 years. The homozygous 2657+5G>A mutation revealed a total of 36 patients with this variant, of which 6 (18%) had PI, and the mean age was 32 years. Of the 36 patients who carried both mutations, p.Gly542*and 3849+10kbC>T, 10 (30%) patients had PI, and the mean age in this category was 27 years. Of the 56 patients with the homozygous variant 3849+10kbC>T, 14 (30%) developed PI, and the mean age was 29 years. There were 6 patients who had both the p.Gly542* and 3120+1G>A mutations, and all (100%) had PI, with a mean age of 10 years. The same was observed with the homozygous 3120+1G>A mutation: all 47 patients (100%) had PI, and the mean age was 17 years.20
Furthermore, Masvidal et al13 analyzed the characteristics of 11 CF patients that presented the c.2657+5G>A mutation and, among them, the PI phenotype was unusual. In addition to this, another study in a tertiary-level CF center from Istanbul described that, of eight c.2657+5G>A homozygous patients, none presented with PI.21
In general, patients with two severe mutations develop PI. However, a small proportion of these individuals are PS at the time of diagnosis and experience a gradual transition to a PI status. Patients with at least one mild mutation, such as 2657+5G>A, usually remain PS (63%);17 furthermore, it should be noted that, according to the CFTR2 database, PI is an uncommon manifestation in childhood, even among the different mutations mentioned.20
To better understand the relationship between PI prevalence and mutations, Terlizzi et al22 developed and validated a novel classification system for mutation severity, known as the Pancreatic Insufficiency Prevalence (PIP) score. This classification is based on three premises: first, the well-established correlation between severity of the CFTR mutations and exocrine pancreatic function; second, the dominant phenotypic effect conferred by the milder of the two CFTR mutations; and third, the availability of a comprehensive database containing large numbers of CF patients with stringent clinical diagnosis and exocrine pancreatic status determination. The PIP score for a specific mutation is the ratio between the PI patients with the mutation (Total PI) and all PI and PS patients (Total PI+PS) harboring the same mutation in a homozygous or heterozygous state in a combination with a severe mutation, such as F508del, G551D or a Class I mutation.22
Therefore, we performed the PIP search for class I and V mutations and obtained the following results from three CF study centers: in the Verona center, PIP was 1.00 for p.Gly542* and 0.27 for c.2657+5G>A.23 In the Canadian Consortium for CF Genetic Studies, PIP was 0.99 for p. Gly542*, 0.38 for c.2657+5G>A, and 0.09 for 3849+10kbC>T.22,23 PIP in the Naples CF center was 1.00 for p.Gly542*, 0.50 for c.2657+5G>A, and 0.00 for 3849+10kbC>T.22,23 Categorically, the mutations can be classified as either mild (≤0.25) or severe (>0.25) on the basis of the PIP score.24
Based on the PIP score results, the study by Terlizzi et al22 showed that patients with CF severe mutations on both alleles, sweat chloride concentrations over 60 mmol/L, and PIP scores >0.25, presented acute recurrent pancreatitis and developed PI 6.5 years after the first pancreatitis episode. However, the patient in our case report had a PIP score >0.25 for both mutations, and sweat chloride concentrations of 71 mEq/L and 67 mEq/L. Therefore, it was expected that he would develop recurrent pancreatitis. However, the presentation is atypical, and the patient presented with PI in the first year of life.
Regarding lung function, it is known that patients with only one severe mutation have better lung function and less bronchiectasis.1,13 However, except for one episode of Pseudomonas aeruginosa colonization, the aforementioned patient did not present with noticeable lung disorders in clinical signs or imaging examinations. Despite absence of pulmonary symptoms, in the long term, these symptoms can get worse. In older adults, this class V mutation causes severe chronic lung disease.13
In relation to the therapeutic options, the administration of a new drug, ivacaftor, has become more widespread since its approval in 2012 by the US Food and Drug Administration (FDA).25 In a retrospective observational study involving patients enrolled in an ivacaftor compassionate use program in Italy, which included patients with at least one CFTR allele 2789+5G>A, 3849+10kbC>T, none of the patients had PI. The result indicated that ivacaftor improves lung disease in patients with advanced CF harboring CFTR mutations that confer residual function.26
On the other hand, case and case series reports have emerged describing PI patients with acute or recurrent pancreatitis; therefore, ivacaftor can improve the frequency of recurrent pancreatitis episodes in CF patients with residual function mutations.23 Furthermore, some studies provide consistent evidence that when the CFTR modulators act, the risk of pancreatitis can be reduced with improvement in the number of pancreatitis episodes.27–29 However, CFTR modulators are expensive and not yet available in Brazil.
Our research group is investigating the effects of natural compounds, such as caffeic acid phenethyl ester (CAPE), due to their antioxidant and anti-inflammatory properties. This is because it is known that the clinical manifestations of CF are triggered by oxidative stress and exaggerated inflammation, which play a fundamental role in the pathophysiology of CF. CAPE acts in the arachidonic acid cascade and inhibits nitric oxide (NO) episodes by the inducible NO synthase (iNOS). In addition, CAPE also inhibits enzymes responsible for the production of free radicals, such as myeloperoxidase and iNOS, as well increasing the activity of antioxidant enzymes capable of reducing the harmful effects of free radicals on the cells, such as heme oxygenase 1, catalase, glutathione peroxidase, and superoxide dismutase.30
In this research line, another study by our group revealed that modulation of the purinergic system is a potential therapy for CF. This is because the disease affects the osmotic balance of mucus and mucociliary clearance, which favors infections and inflammation. Thus, we propose several ways to modulate the purinergic system, through inhibition of the P2X7 receptors, activation of the P2Y2, A2A and A2B receptors, and blockade adenosine deaminase; it would result in an increase in adenosine with anti-inflammatory action and improve clearance and control of airway surface liquid hydration.31 Therefore, new therapies are needed to correct the main mechanisms that contribute to CF progression.
Conclusions
CF is characterized as a disease with multisystemic consequences and a broad phenotypic spectrum, as several mutations and factors are involved in the onset of signs and symptoms and in evolution of the disease. In this sense, this clinical case presents, in an unprecedented way, the compound heterozygosity between the p.Gly542* and c.2657+5G>A mutations and its clinical findings in a Caucasian patient diagnosed early, at 6-months-of-age, and it contributes information regarding evolution of the disease in patients with this genotype which is lacking in the current literature.
There are limited data in the literature regarding the phenotype related to a combination of class I and class V mutations, which by itself, would justify the importance of this report. However, we also emphasize that a class V mutation usually progresses to PS, and that a class I mutation usually progresses to PI, pointing to the hypothesis that a combination with these two mutations is enough to worsen a patient’s prognosis.
The phenotype of the aforementioned patient consists of exocrine pancreas total dysfunction in the first year of life, without pancreatitis episodes. The child did not present any manifestation or respiratory impairment due to CF. Regarding PI, it was expected that the patient would develop this condition in adulthood.
Although this case report allows establishing the genotype-phenotype association of the fibrocystic child, the limited number of patients with this same condition hinders this correlation. Therefore, interpretation of this relationship represents a challenge, as genetic analysis alone is not sufficient to clearly predict severity of the disease. This is because the significant phenotypic heterogeneity existing among patients with the same mutations may exert socioeconomic and sociocultural influences, or by the action of the CFTR modifiers, such as environmental and modifying genes, which can alter the protein’s function and exert an impact on the individual’s phenotype. Further studies on the heterozygosity in question are suggested to expand knowledge about the pathophysiology of CF, as well as to develop alternatives for treatment.
Acknowledgements
We would like to express our appreciation to the patient’s family for their cooperation.
Footnotes
Disclosures: The authors have reported no conflicts of interest or financial support for this work.
- Received September 3, 2020.
- Accepted January 25, 2022.
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