Cholelithiasis in Cystic Fibrosis Patients in a Tertiary Care Center in Saudi Arabia

Introduction: Cholelithiasis has been reported in 12% to 24% of Cystic fibrosis (CF) patients and is usually made up of cholesterol gallstones. These abnormalities are frequently asymptomatic and can include the intra and extra-hepatic ducts, gallbladder thickening and contraction, micro-gallbladders and cholelithiasis. Abdominal sonography is routinely used in order to detect these abnormalities. Objectives: To obtain the prevalence of gall stones (Cholelithasis) in CF patients and its relation to other clinical, laboratory, radiological, and Genetic data. Methodology: A retrospective chart review as part of the CF registry data from the period 1st January 1984-1st June 2018. All confirmed CF the patients of all age groups that have US study done were included in the study. Patients with positive gallstones or sludge were evaluated and discussed. Results: A total of 391 confirmed CF patients were involved, out of them 252 patients had an abdominal ultrasound. 7 patients (3%) had gallstones on abdominal US, 8 patients (3%) were revealed to have sludge and 237 patients (94%) had normal gallbladders. Pancreatitis was found in 4 patients (2%). 191 patients (76%) had pancreatic insufficiency. 77 patients had follow up abdominal ultrasounds and 5 patients (7%) were found to have persistent gallstones, 4 patients (5%) had persistent sludge and 68 patients (88%) remained negative for gallstones. 2 patients required cholecystectomy. Conclusion: Cholelithiasis is a common complication of CF disease; its incidence is more than the general population. Thus, we recommend that every CF patient get an ultrasonography study as part of liver disease screening to rule out any Gallbladder pathology. Limitations: Our CF patients reflected approximately 80% of the CF population in KSA.


Introduction
Cystic fibrosis (CF) is a progressive disease that affects many systems including respiratory, gastrointestinal, urogenital and sweat glands [1][2][3]. It is the most common life-shortening autosomal recessive disease among Caucasian populations, with a frequency of 1 in 2000 to 3000 live births (3). CF is inherited as autosomal recessive disease due to mutation of CF transmembrane conductance regulator (CFTR) gene [1]. This causes an abnormal chloride ion transport on the apical surface of the exocrine gland ' s epithelial cells [2]. This results in abnormally thickened, viscous secretions, which affect multiple organ systems [2]. Deletion of phenylalanine in amino acid position 508 (deltaF508) on chromosome 7 is considered the most common mutation in North America and western Europe [4], in contrast to Saudi Arabia, the most common mutation was 1548delG and I1234V [5,6]. One-third of Cystic Fibrosis patients have abnormal gallbladders and cystic ducts, regardless of their age, severity of the disease and hepatic pathology [7,8]. These abnormalities can include the intra and extra-hepatic ducts, gallbladder thickening and contraction, microgallbladders and cholelithiasis [9]. Cholelithiasis occurs in 12%-24% of Cystic fibrosis patients and is usually made up of cholesterol gallstones. These abnormalities are frequently asymptomatic [10]. Abdominal sonography is routinely used to detect these abnormalities. However, Magnetic resonance (MR) cholangiography is more accurate [10]. The underlying pathophysiology of the increased cholelithiasis prevalence in Cystic Fibrosis patients is not yet discovered;

Curr Pediatr Res 2020; 24 (3): 199-203
ISSN: 0971-9032 www.currentpediatrics.com nonetheless, multiple theories have been developed. One of these theories suggests that the disturbed bile acid metabolism in CF patients causes increased bile acid fecal losses, leading to cholesterol super saturation in the gallbladder and subsequent stone formation [11]. Nucleating factors, such as mucin glycoproteins can aid in the process of stone formation [12]. Wasmuth et al. [13], proposed that the coinheritance of the common UGT1A1 promoter mutation associated with Gilbert syndrome is an additional lithogenic risk factor for gallstone formation in CF. The study included 52 patients with CF. Twenty-six patients were heterozygous for the A(TA)7TAA allele, while five patients were homozygous for the A(TA)7TAA allele. Compared with stone free patients with CF, significantly more CF patients with gallstones either carried the heterozygous A(TA)7TAA allele or the heterozygous and/or homozygous A(TA)7TAA allele. Only one patient with gallstones did not carry the mutated UGT1A1 allele. The study concluded that CF patients with homozygous Gilbert mutation appear to have an increased risk for gallstones, followed by CF patients with the heterozygous mutation and homozygous wild type alleles. Hence, genetic factors which increase the biliary secretion of bilirubin monoglucuronide raise the risk of forming gallstones [14]. Philippe et al. [8] reported the incidence of gallbladder diseases in 84 cystic fibrosis patients who underwent cholecystographic evaluation, which evaluated the opacification of the gallbladder, gallbladder size and the presence of calculi. Thirty-nine patients (46.4%) had abnormal cholecystograms, mostly due to non-visualizations of the gallbladder. Ten of the 84 patients (11.9%) had gallstones; most of which were found in patients aged 10-20 years. It was suggested that this incidence could be attributed to some factors promoting stone development in CF patients, such as biliary stasis and as a result of cholestatic drugs like sulfonamides, anabolic agents and oral contraceptives [11,14,15]. It has been proposed that the proper use of pancreatic enzymes in these patients can help in fat absorption, hence, decrease the lithogenic bile [16]. In the case of symptomatic gallstones in CF patients with suitable pulmonary function, cholecystectomy has been favored [17]. One case report has described the dissolution of gallstones in two CF patients post the administration of ursodeoxycholic acid. The authors explained that the use of ursodeoxycholic acid in selected cases, where cholesterol gallstones are established and the gallbladder shows opacification on oral cholecystography could result in dissolution or reduction in the size of the stones [18]. Our objective is to obtain the incidence and the prevalence of gallstones (Cholelithiasis) in CF patients and its relation to genetic data from the period 1st January 1984-1st June 2018.

Research Methodology
A retrospective chart review of 391 confirmed CF patients from the period 1st January 1984-1st June 2018.

Definition
The diagnosis of CF was established in all patients according to one of the following: • Typical pulmonary manifestations and/or typical gastrointestinal manifestation and/or a history of CF in the immediate family in addition to sweat chloride concentration >60mmol/L. • Pathologic mutation in both chromosomes.

Inclusion criteria
All confirmed CF patients of all the age group that have positive gallstones in their radiological investigations namely ultrasound of abdomen (US) during their follow up period in CF clinic from the period 1st January 1984-1st June 2018. The US was considered to be abnormal if it confirmed the presence of either gallstones or biliary sludge.

CFTR identification
CFTR Gene Screen Methodology: DNA Isolation, PCR amplification of genomic DNA, mutational analysis, and sequencing methods have been described before in a previous study from the same center [6]. Genomic DNA from patient's lymphocytes was used to amplify the 27 exons and flanking sequences of the CFTR (NM_000492.3; NP_000483.3). The PCR products were analyzed by sequencing in both the forward and reverse directions. Polymerase Chain Reaction (PCR) [19] PCR evaluation, 19 purification of PCR products for direct, and Direct Sequencing method were applied according to the recommended methods [19].

Mutation detection
Mutation detection was scored using publicly available mutation database for Cystic Fibrosis such as "Cystic Fibrosis Mutation Database" (http://www.genet.sickkids.on.ca/ CFTR / Home.html) and " The Human Gene Mutation Database -Professional Edition " (http://www.hgmd.cf.ac.uk/ac/ index.php). Both mutation databases provided extensive repertoire of up-to-date sequence variants, deletions and insertions for the CFTR gene.

Ethical considerations
This is a retrospective study. All data were stored in pediatrics research unit, accessed only by the principle investigator and the assigned Clinical Research Coordinator. The entire patient ' s information was kept strictly confidential. Each patient was given a study number, and all patient data were entered into the designated data sheet (EXCEL) without any patient's identification. The Declaration of Helsinki and GCP guidelines were followed.

Statistical statement
The data collected from this study were electronically entered into a database. Multiple variables were collected and analyzed such as: Abdominal Ultrasound findings, presence of pancreatitis, presence of pancreatic insufficiency based on chart review and the administration of ursodeoxycholic acid.

Results
A total of 391 CF patients were included in our study. However, some patients were excluded due to the lack of abdominal sonography, out of them 252 patients had an abdominal ultrasound. Out of the 391 patients, there were 196 females (50%) and 195 males (50%) patients.
The Eastern region had the highest percentage of CF patients (38%), followed by the central region, which involved (25%) of the patients. The Southern region had the least number of patients (11%) (Figure 1).

Figure 1. Geographic distribution of CF population for total 391 patients.
The initial abdominal US of these patients revealed that 7 patients (3%) had gallstones (2 females and 5 males), 8 patients (3%) had findings consisting with biliary sludge (2 females and 6 males) and 237 (94%) patients had normal gallbladders on US. Review of Ultrasound studies showed that: 86 (34%) had normal US liver, 28 (11%) mild hepatomegaly, 40 (16%) echogenic liver, 33 (13%) fatty infiltration, 1 (0.5%) peri-portal fibrosis, and 5 (2%) developed liver cirrhosis Pancreatitis was found in 4 patients (2%). A total of 191 patients (76%) had evidence of pancreatic insufficiency. Furthermore, nineteen patients were on Ursodeoxycholic acid because of concurrent hepatic enzyme abnormalities. Moreover, 77 patients had follow-up abdominal ultrasounds and 5 patients (7%) either developed or remained positive for gallstones, 4 patients (5%) had sludge and 68 patients (88%) remained negative for gallstones. Only two patients with cholelithiasis underwent cholecystectomy. Screening for CFTR mutations for both gallstones and sludge groups showed that only one patient from each group showed similar CFTR mutation (c.2988+1G>A), otherwise the remaining patients from both groups showed different mutations as shown in Tables 1 and 2. Furthermore, there were 4 patients with splice donor-site mutational effect (2 in intron 18 and 2 in intron 5) ( Table 2). Its significance has not been reported before.

Discussion
In this retrospective study we tried to determine the involvement of gallbladder abnormalities in CF patients via the use of abdominal sonography. Furthermore, we tried to report some findings related to CF in our patients such as pancreatitis and pancreatic insufficiency. Nazer et al. showed that Cystic fibrosis has an estimated incidence of 1:4243 live births in Saudi Arabia [20]. Agrone showed that Cholelithiasis occurs in 12%-24% of Cystic fibrosis patients [10]. In comparison to our study, which  [20]. Many of our patients were asymptomatic at the time of diagnosis. Two out of our 7 patients with gallstones were females, and five were males, which is contrary to the common 4:1 female to male ratio which was shown by Nazer H and his group [20]. Likewise, 2 out of the 8 patients who had sludge were females and 6 patients were males. Zimmerman et al. has recommended that adolescent females with cystic fibrosis should avoid oral contraceptive pills as it can increase their chances of having cholelithiasis through cholesterol supersaturating in the bile [14]. The pancreas is involved in 85-90% of CF patients, commonly in the form of exocrine insufficiency while 30-50% only is in the form of endocrine insufficiency [8,21]. Our study showed similar prevalence of pancreatic insufficiency 191out of 391. Pancreatitis is usually an infrequent finding in CF patients with an estimated occurrence rate of 1.2% [22]. Compared to 3% in our study. one case report which demonstrated the complete dissolution of gallstones after the administration of ursodeoxycholic acid in two CF patients [17]. Whereas 19 patients of our cystic fibrosis papulation were on ursodeoxycholic acid for cholestasis, which may have contributed to the low prevalence of cholelithiasis in our study [23][24][25][26].
We have reported for the first time the CFTR results of our CF gall stones/ sludge group. We have shown that there were 4 patients with splice donor site CFTR mutations (2 in gall stones group and 2 in sludge group) ( Table 2). Its effect is unknown if it has any relation to cause liver cirrhosis [26][27][28][29][30].

Conclusion
We recommend that abdominal sonography should be done in every CF patient in order to rule out gallbladder and pancreatic diseases. As most of the cholelithiasis patients are asymptomatic, medical treatment is probably sufficient to improve the symptoms. However, surgical intervention should be considered in some cases.