Molecular Heterogeneity in Cystic Fibrosis

Abstract

We aimed to evaluate type, frequency, and variety of pathogenic variants according to clinical and demographic features of children diagnosed with cystic fibrosis (CF). Twenty-five CF patients were evaluated retrospectively. Patients' demographics, physical examination, imaging, laboratory, and molecular pathogenic variant analysis findings were evaluated. Phe508del was the most frequently (33.3%) detected pathogenic variant, followed by point pathogenic variants E92K, 1898 + lGA/7T/7T, and 2789 + 5GA, respectively. Statistically higher rates of pathogenic variants were detected in male patients. The most frequently detected pathogenic variant was Phe508del. The identification of nine additional pathogenic variants of Phe508del revealed the heterogeneous nature of the CF.

Introduction

Cystic fibrosis (CF) is an autosomal recessive inherited disorder with multisystem disease that occurs as a result of pathogenic variant in the CFTR gene. CF occurs in approximately 1 in 4,000 newborns and more than 2,000 pathogenic variants have currently been identified. ¹ The clinical manifestations of the disease are caused by the structural or functional defects of the CFTR ion channel. ² The structural defects or the absence of CFTR protein, which is expressed in various tissues, induces complicated physiological processes leading to multiple organ failures. ^{2 3} The most common pathogenic variant encountered worldwide is phenylalanine deletion in 508th position. Homozygous or compound heterozygous “Phe508del” frequencies vary depending on the population. ⁴

The typical presenting symptoms of patients were fat malabsorption due to the pancreatic insufficiency or chronic obstructive airway disorders related to bacterial colonization. ³ The mean age of survival for CF patients has increased in recent years, a poor prognosis and increased mortality are associated with the female gender, malnutrition, low lung function, infections, and CF-related diabetes. ⁵ The diagnosis of CF is based on classic symptoms mostly. Programs, such as prenatal ultrasonography (USG) screening and newborn screening (NBS), allow for early detection of asymptomatic infants, and an opportunity for better growth and development. ⁶ As specific pathogenic variants in CFTR are identified by NBS programs and CFTR databases, the development of target-specific molecular therapies have been developed. ^{2 7} We aimed to evaluate type, frequency, and variety of pathogenic variants according to clinical and demographic features of children diagnosed with CF at our institution.

Materials and Methods

This study was performed with the Institutional Review Board protocol approval date February 18, 2019 and number 2019/13 at the Istanbul Dr. Sadi Konuk Training and Research Hospital, Department of Pediatric Gastroenterology, Hepatology and Nutrition, between January 2017 and June 2018. A total number of 25 CF patients between 0 and 18 years of age were evaluated retrospectively. Patients' demographic characteristics, age at disease onset, anthropometric measurements including height, weight, and body mass index (BMI) over a 2-year period, physical examination, imaging, laboratory findings, and molecular pathogenic variant analysis were evaluated. The patients included in the present study were 0 to 18 years old and had one or more of the followings: positive clinical symptoms, family history of CF, a sweat/chlorine test results above 60 mEq/L, or a CFTR pathogenic variant without accompanying chronic disease. CF-related pathogenic variants were examined by real-time polymerase chain reaction (PCR) technique through genomic DNA isolated from the patients' peripheral blood.

Blood cell count analysis was performed on patients' venous blood samples. Hematological parameters were analyzed using a hematology analyzer (Cell-Dyne 3700, Abbott, Abbott Park, Illinois, United States). Biochemical analysis performed from serum samples by electrochemiluminescence immunoassay on Beckman Coulter Unicel DXI 800 analyzer.

Variant Analysis

DNA, for the assessment of CF pathogenic variant test, was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) obtained from patients' peripheral blood samples (minimum 3 mL samples in tubes with EDTA as an anticoagulant). PCR was performed via some extraction, incubation, and amplification cycles according to the manufacturer's instructions. Amplification process performed by gene-specific primers targets all 27 CFTR exons. Features of the identified variants in CFTR gene were summarized in Table 1 .

Table 1. Summary of the identified variants in CFTR gene (NM_000492.3) .

cDNA	Protein	GnomAD	DANN	GERP RS	Mutation taster	SIFT	Clinival variance	ACMG
Exon2 deletion	Large deletion	N/A	N/A	N/A	N/A	N/A		P
c.1521_1523delCTT	p.Phe508del	0.007172	N/A	N/A	DC	N/A	P	P
c.274G > A	p.Glu92Lys	N/A	0.9991	5.73	DC	DM	P	LP
c.2789 + 5G > A	Splice	0.00007070	0.9076	5.92	DC	N/A	P	LP
c.1898 + 1G > A	Splice	0.00003564	0.9956	4.8299	DC	N/A	P	P
c.2052A > T a	p.Lys684Asn	N/A	0.9925	-0.7089	DC	T	N/A	VUS
c.2062A > C a	p.Lys688Gln	0.000004018	0.9955	5.63	DC	T	N/A	VUS
c.2051_2052delAAinsG	p.Lys684SerfsTer38	N/A	N/A	N/A	DC	N/A	P	P
c.1397C > G	p.Ser466Ter	0.00001769	0.9959	5.4699	DC	N/A	P	P
c.3209G > A	p.Arg1070Gln	0.0006097	0.9996	5.69	DC	T	P	LP

Abbreviations: ACMG, American College of Medical Genetics Guidelines; DANN, deleterious annotation of genetic variants using neural networks; DC, disease causing; DM, damaging; GERP RS, genomic evolutionary rate profiling rejected substitution; GnomAD, genome aggregation database; LP, likely pathogenic; N/A, not available; P, pathogenic; SIFT, sorting intolerant from tolerant; T, tolerated; VUS, variant of uncertain significance.

^aIdentified only in present study.

Statistical Analysis

All the data were analyzed with SPSS (Statistical Package for the Social Sciences) software for Windows (v21.0; IBM, Armonk, New York, United States). Individual and aggregated data were summarized using descriptive statistics including mean, standard deviations, medians (minimum–maximum), frequency distributions, and percentages. Normality of data distribution was verified by the Kolmogorov–Smirnov test. Comparison of the variables with normal distribution was made with the Student's t -test. The variables which were not normally distributed, the Mann–Whitney and Kruskal–Wallis tests were conducted to compare between groups. Evaluation of categorical variables was performed by the Chi-square test. p -Values < 0.05 were considered statistically significant.

Results

A total of 17 (68%) female and 8 (32%) male patients were included in this study. The overall female-to-male ratio was 2:1. Mean age of the patients was 9.52 ± 20.06 months (range, 0–96 months). In addition, the mean age was significantly higher in male patients (16.75 ± 32.07 months) than female patients (6.12 ± 10.73 months; p  = 0.017; Table 2 ).

Table 2. Comparison of the data according to the age and gender.

	n (%)	Age (mean ± SD) in mo	p -Value
Male	8 (32.0)	16.75 ± 32.07	0.017 a
Female	17 (68.0)	6.12 ± 10.73
Total	25 (100)	9.52 ± 20.06

Abbreviation: SD, standard deviation.

^a p  < 0.05 statistically significant.

According to the evaluation of anthropometric measurements, mean height was 126.29 ± 24.20 cm, mean weight was 24.45 ± 17.43 kg, and mean BMI was 16.33 ± 3.32 kg/m ² (range, 12.90–25.31 kg/m ² ). In 16% ( n  = 4) of our sample group, a BMI of < 3th percentile was noted, and in 40% ( n  = 10) of our sample group, a BMI of < 50th percentile on the growth curves of Turkish children.

The most common symptoms reported in our patients were diarrhea and cough with a percentage of 39% ( n  = 9) each and followed by halitosis (34.7%, n  = 8), abdominal pain (26.0%, n  = 6), sour taste in the mouth (26%, n  = 6), puffiness (13%, n  = 3), dysphagia (4.3%, n  = 1), and vomiting (4.3%, n  = 1), respectively. Additionally, hepatomegaly was the most frequent physical finding (30.4%, n  = 7), followed by splenomegaly (17.3%, n  = 4), cholelithiasis (17.3%, n  = 4), and hepatosteatosis (13%, n  = 3; Table 3 ).

Table 3. Distribution of symptoms and examination findings during application.

Symptoms	n	%
Diarrhea	9	39.1
Cough	9	39.1
Halitosis	8	34.7
Abdominal pain	6	26
Sour taste in the mouth	6	26
Puffiness	3	13
Dysphagia	1	4.3
Vomiting	1	4.3
Examination findings
Hepatomegaly	7	30.4
Splenomegaly	4	17.3
Colelithiasis	4	17.3
Hepatosteatosis	3	13

According to the evaluation of laboratory findings, the mean values of alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), vitamin A, and vitamin E were 27.95 ± 16.01 U/L, 15.25 ± 14.48 U/L, 396.71 ± 158.113 ng/mL, and 15.96 ± 8.92 mg/L, respectively. Additionally, vitamin D deficiency was detected in 45.4% of patients, and vitamin A deficiency in 23.8% ( Table 4 ).

Table 4. Laboratory outcomes.

	Clinical variables	n (%)
Vitamin A	< 300 ng/mL > 300 ng/mL	5 (23.8) 16 (76.2)
Vitamin E	< 3.8 mg/L > 3.8 mg/L	0 (0) 21 (100)
Vitamin D	< 30 ng/ml > 30 ng/ml	10 (45.4) 12 (54.6)
ALT a	Normal level High	22 (91.7) 2 (8.3)
GGT b	Normal level High	22 (91.7) 2 (8.3)
HBsAg	Negative Positive	21 (100) 0 (0)
Anti-HBsAg	Negative Positive	6 (28.5) 15 (71.5)
Anti-HAV IgM	Negative Positive	21 (100) 0 (0)
Anti-HAV IgG	Negative Positive	10 (47.6) 11 (52.4)

Abbreviations: ALT, alanine aminotransferase; Ig, immunoglobulin; GGT, gamma-glutamyl transferase; HAV, hepatitis A virus; HBV, hepatitis B virus.

^aALT = 56 U/L for newborn, 29 U/L for >12 months, as the reference value.

^bGGT = 0–36 U/L as normal reference value.

All cases were analyzed for pathogenic variants. No pathogenic variants were detected in seven cases (28%). Pathogenic variants were homozygous in 40% ( n  = 10), heterozygous in 8% ( n  = 2), and compound heterozygous in 24% ( n  = 6) of the patients. Phe508del was the most frequently detected pathogenic variant with a percentage of 33.3% ( n  = 6). Of the Phe508del pathogenic variants, 66.6% ( n  = 4) were homozygous. The Phe508del pathogenic variant was followed by E92K (16.7%), 1898 + lGA/7T/7T (11.1%) and 2789 + 5GA (11.1%) point mutations, respectively. The 2051–2052del AA ins G and 7T/7T, exon 2 del., 2051–2052del AA ins G and S466X, and R1070Q pathogenic variants were also identified (each 5.6%, n  = 1) in our study ( Table 5 ). The c.2052A > T pathogenic variant was identified for the first time in the present study. In addition, pathogenic variants were significantly higher in male patients than females ( p  = 0.027). Such that all male patients had pathogenic variants; on the other hand, pathogenic variant frequency was detected in only 68.8% of females. Moreover, when laboratory outcomes evaluated according to the pathogenic variant presence, there were no statistically significant differences found in ALT, GGT, vitamin A, and vitamin D levels ( Table 6 ).

Table 5. CFTR pathogenic variants identified in patients .

Pathogenic variants	n	%
Phe508del	6	33.3
E92K	3	16.7
1898 + lGA and 7T/7T	2	11.2
2789 + 5GA	2	11.1
K684N and K688Q	1	5.6
2051–2052 a del AA ins G and 7T/7T	1	5.6
exon 2 del	1	5.6
2051–2052del AA ins G	1	5.6
S466X, R1070Q	1	5.6

^aIdentified only in present study.

Table 6. Evaluation of laboratory outcomes according to the pathogenic variant presence.

Pathogenic variant	ALT		p -Value	GGT		p -Value	Vitamin A		p -Value	Vitamin D		p -Value
	Normal n (%)	High n (%)		Normal n (%)	High n (%)		Normal n (%)	Low n (%)		Normal n (%)	Low n (%)
Absent	6 (100.0)	0 (0.0)	0.554	6 (100.0)	0 (0.0)	0.554	3 (75.0)	1 (25.0)	0.696	3 (60.0)	2 (40.0)	0.781
Present	16 (88.9)	2 (11.1)		16 (88.9)	2 (11.1)		13 (76.5)	4 (23.5)		9 (52.9)	8 (47.1)

Abbreviations: ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase.

Note: All patients' vitamin E results were within normal levels.

Discussion

The prevalence of CF, which is present in approximately 70,000 people worldwide, has given the disease the title of the most lethal genetic disorder in caucasians. ⁸ According to a multicenter study conducted by MacKenzie et al in cooperation with 110 CF institutions between 2000 and 2010, CF incidence increased from 21,000 to 26,000 and the mean age of the patients increased from 14.3 to 16.7 years. The same study documented the mean age of 0.5 years (range, 0.1–3.2 years), and the female percentage of 47.7% at the time of CF diagnosis. ⁹ In a study, consisting of 35 European countries including Turkey, Mehta et al evaluated 29,095 patients diagnosed with CF within a 5-year period based on demographic data. Researchers reported gradually increased male prevalence from the age group 0- to 5-year olds and in 15- to 20-year olds. ¹⁰ Similarly, in our study, mean age of patients was 9.52 ± 20.06 months at the time of diagnosis. Additionally, the female/male ratio was 2:1 and male patients were found to be significantly older than females at the time of diagnosis. Considering the variation in demographic features of CF patients, depending on geographical and ethnic status, no inconsistency was found between our findings and the published data.

CF, with its underlying complex phenotypical characteristics, demonstrates a wide spectrum of clinical manifestations with varying severity depending on the system involved and pathology. CF is typically characterized by increased susceptibility to inflammation and infection as a result of the accumulation of dehydrated and hyperviscous mucus in airways. Moreover, gastrointestinal system (GIS) pathologies manifest as malabsorption and pancreatic insufficiency. While cough and wheezing have been reported as the most common symptoms for respiratory tracts, diarrhea, and abdominal pain are regarded as the most frequent GIS-related symptoms. ¹¹ Gangell et al examined the symptoms of 215 children, diagnosed with CF between 3 weeks and 7 years of age at the time of admission, who have reported gastrointestinal complaints as the most common symptoms with a percentage of 35%, followed by GIS and respiratory symptoms combined (13%), and respiratory symptoms (9%), respectively. ¹² Consistent with previously reported data, the most common symptoms reported in our patients were diarrhea and cough with a percentage of 39% ( n  = 9) each and followed by halitosis (34.7%) and abdominal pain (26.0%), respectively.

CF is a systemic disease involving multiorgans which mostly affects lungs, liver, pancreas, and sweat glands. Liver involvement ranges between 27 and 35% in CF patients aged 18 years and younger. ¹³ Similarly, epidemiological studies documented isolated hepatomegaly frequency ranged between 7 and 30% among the cases. ¹⁴ In addition, in a study conducted by Monti et al, consisted of 75 CF patients, researchers have reported splenomegaly in 22.2% of the patients. ¹⁵ Supportively, the most common physical finding in our patients was hepatomegaly, followed by splenomegaly, cholelithiasis, and hepatosteatosis.

Insufficiency of the exocrine pancreatic enzymes in CF patients causes intestinal malabsorption of fat soluble vitamins. Moreover, the multifactorial etiology of vitamin D insufficiency prevails in CF patients, such as impaired hepatic hydroxylation and deficiency of the vitamin D binding protein, which are prominent steps in vitamin D metabolism. Furthermore, it is noted that CF patients often avoid sunlight, since the drugs they are prescribed, especially antibiotics, can lead to photosensitivity. ¹⁶ Vitamin D deficiency in CF patients has been reported in multiple publications. ¹⁷ In a study by Rovner et al, which compared a CF-diagnosed sample group consisting of 101 children, adolescents, and adults to a healthy control group of 177 individuals, frequencies of vitamin D deficiency (<11 ng/mL) and vitamin D insufficiency were 70 and 90%, respectively. These frequencies were noted as 2 and 74% for the control group, and reported to be statistically lower than CF group. ¹⁸ The vitamin A deficiency among children diagnosed with CF is reported between 29 and 51% at the time of diagnosis, and it might only be reduced to a level of 11 to 33% after treatment. Sapiejka et al reported a frequency of 16.3% vitamin A deficiency, and stated the ALT and GGT values as 24.0 ve 12.0 U/L, respectively, in 196 CF patients. ¹⁹ Similarly, in our study, 45.4% of patients with CF had vitamin D deficiency and 23.8% vitamin A deficiency. The mean values of ALT and GGT were reported as 27.95 ± 16.01 U/L, and 15.25 ± 14.48 U/L, respectively.

The most commonly (70%) documented pathogenic variant of CF worldwide is Phe508del, and approximately half of the Phe508del pathogenic variants are homozygous. This pathogenic variant causes an almost total loss of the CFTR channel funcion. ²⁰ A more severe pancreatic insufficiency, lung involvement, and higher risk of mortality have been frequently reported in Phe508del positive patients. Santos et al reported a total percentage of 75% Phe508del pathogenic variants in 36 CF patients; of which 27.7% were homozygous and 47.3% were heterozygous. ²¹ Similarly, homozygous and heterozygous Phe508del pathogenic variant rates were reported as 53 and 39%, respectively, in a study by Gangell et al analyzing 215 children diagnosed with CF. ¹² MacKenzie et al reported 46.4% homozygous and 39.3% heterozygous Phe508del pathogenic variants among their patients reached several 26,000 in 2010 from the multicenter research that was conducted between 2000 and 2016 in collaboration with 110 CF institutions. Moreover, the decrease in homozygous pathogenic variants between 2000 and 2010 was also highlighted in same study. ⁹ Nevertheless, thousands of non-Phe508del pathogenic variants have been documented in CF pathogenesis. Although relatively common pathogenic variants are observed in certain populations, the majority of pathogenic variants are rare; either specific or limited to a small number of individuals. Radivojevic et al reported 18 different pathogenic variants in 175 CF-patients and researchers pointed out that CF disease is highly heterogeneous at molecular level. ²² Variation of diversity, frequency, and heterogeneity of pathogenic variants manifested on a social basis is highly unpredictable such that the E92K pathogenic variant, which is almost never identified in some studies, was reported as the primary CF pathogenic variant with 66% in some societies. ²³ Consistently, Phe508del pathogenic variant (66.6% homozygous) was identified as the most common pathogenic variant among nine different pathogenic variants in our study, followed by E92K (16.7%), 1898 + lGA/7T/7T (11.1%), and 2789 + 5GA (11.1%) point mutations, respectively. On the other hand, although published data indicated that CF pathogenic variants are inherited more frequently among male population, particularly in Europe and North America, the gender prevalence is still controversial. ^{10 24} In present study, a significantly higher pathogenic variant rate was detected in male patients. Furthermore, all male patients had pathogenic variants and pathogenic variant frequency was 68.8% in female patients.

Conclusion

In conclusion, although the Phe508del pathogenic variant was the most common pathogenic variant identified in our study, identification of nine different pathogenic variants, which were predominantly non-Phe508del, highlighted the heterogeneous genetic background of the disease. Further research with larger study groups may provide a new approach to the management of disease in CF patients.