The Spectrum and Frequency of Cystic Fibrosis Mutations in Albanian Patients

Cystic fibrosis (CF) is a genetic multisystem disease resulting from the inheritance of a mutant allele of the gene for cystic fibrosis transmembrane conductance regulator (CFTR) from each parent. Cystic fibrosis (CF) is the most common life shortening condition in Caucasians [1]. About 162,428 people are estimated to be living with CF worldwide, of which 37,002 are estimated to be diagnosed in North America and more than 47,650 in Europe, while an estimated 57,076 CF patients are undiagnosed. [2]. Whilst prevalence is broadly similar in populations which had their origin from northern Europe, there are considerable variations through Europe from as high as 1 in 1,400 live births in Ireland, 1 in 4,200 in Italy and 1 in 25,000 in Finland [3]. Prevalence rates are much lower among non-Caucasian populations [4,5].

A major step forward was achieved by grouping CFTR mutations with a similar effect on CFTR protein synthesis or function in the same mutation class [3]. In view of drug development and drug distribution, it is therefore also useful to know the relative prevalence of these mutation classes [3].

In Albania since 1992, cystic fibrosis patients are followed up by the Department of Pediatrics, in UHCMT. The CF diagnosis is based predominantly on clinical criteria and two positive sweat test results for patients suspected of the disease. The diagnosis is confirmed by the identification of two CF causal mutations. Genetic assessment has been in practice since 2004, when the search of the genetic mutations in patients with cystic fibrosis began to be recorded.

Unfortunately, there was no national registry for Cystic Fibrosis in Albania until 2017. However, every pediatric patient diagnosed in the Department of Pediatrics in the UHCMT has been documented throughout the course of the disease. Practically, this is the only center which diagnoses and follows cystic fibrosis patients in our country. Since 2017 Albania is part of the European Cystic Fibrosis Society Patient Registry (ECFSPR) which collects anonymized demographic and clinical data from consenting people in Europe with CF [6].

The aim of this paper is to present a summary of the distribution of the CFTR mutations in 133 CF patients diagnosed in the Pediatrics Department in the UHCMT. Furthermore, we aim to compare our data with other countries in Balkan Peninsula and Europe.

This retrospective study takes into consideration 133 pediatric patients diagnosed with Cystic Fibrosis followed in the Department of Pediatrics in the UHCMT, during the year (2019) of follow up and who have been included in the European Patient Registry of Cystic Fibrosis.

The examination of the CF mutations is conducted over a 25-year period from 1994–2019 for 116 patients at the Center of Molecular Diagnosis and Genetic Research in the University Hospital of Obstetrics and Gynecology “Queen Geraldine”.

Whole blood was collected into EDTA anti-coagulant tubes. The genomic DNA was isolated from peripheral blood using Qiagen extraction kit (DNA Blood Isolation Kit, Qiagen, Valencia, California, USA). CFTR gene mutations were analyzed by PCR/OLA protocol (Abbott Applied Biosystems), testing for 33 mutations (Cystic Fibrosis V3 Genotyping Assay).

Undetermined samples of 15 cases were sent to the University Hospital Motol, in Prague, Czech Republic as part of the project: GENOTYPING IN UNDERTESTED / UNTESTED CYSTIC FIBROSIS POPULATIONS IN MIDDLE EAST, TRANSCAUCASIA, TURKEY & EASTERN NORTH AFRICA (2015–2019; Vertex grant scheme - CG-2015-104643).

Firstly, the most common mutations of the CFTR gene were examined using the commercial Elucigene CF-EU2 kit, where 50 mutations are simultaneously tested (CFTRdele2,3 / 21kb /, I507del, 2789 + 5G> A, E60X, F508del, Q890X, P67L, 1677delTA, 3120 + 1G > A, G85E, V520F, 3272-26A> G, 394delTT, 1717-1G, A, R1066C, 444delA, G542X, Y1092X (C> A), R117C, S549N, M1101K, R117H, S549R (T> G), D1152H, Y122X, G551D, R1158X, 621 + 1G, T, R553X, R1162X, 711 + 1G, T, R560T, 3659delC, L206W, 1811 + 1.6kbA, G, 3849 + 10kbC, T, 1078delT, 1898 + 1G> A S1251N, R334W, 2143delT, 3905insT, R347P, 2184delA, W1282X, R347H, 2347delG, N1303K, A455E, W846X, including IVS8-T variants (5/7/9).

If just one mutation was detected or both causal mutations are not detected, but the patient shows clear clinical and laboratory signs, next generation sequencing of the whole coding region of CFTR (NGS CFTR Devyser) was provided. This ensures maximum capture of causal mutations within the diagnostic process we provide. All pathogenic variants were confirmed by Sanger sequencing or MLPA protocol https://www.mlpa.com/.

Also, the databases CFTR1 http://www.genet.sick-kids.on.ca/, CFTR2 https://cftr2.org and CLIN Var https://www.ncbi.nlm.nih.gov/clinvar, were used for clinical interpretation.

Based on the number of pediatric patients diagnosed with CF every year from 1992 to 2017, and the birth rate during the same period [7], the incidence of CF in Albania is 1:4041. It is important to note that this incidence is not entirely correct since in the UHCMT, there are also CF patients from Kosovo, or Albanian children born in other countries.

We have presented in Table 2 an overview of the demographic as well as the clinical and laboratory characteristics of Albanian patients diagnosed at UHCMT who were included in the study, although data on some births are missing.

This study aims to shed some light on the current situation about CF mutation pattern, presenting data from patients with CF followed during 2019 in the Department of Pediatrics in the UHCMT. Practically, UHMCT is the only center which diagnoses and follows cystic fibrosis patients in Albania. It is important to note that in Albania no CF national neonatal screening has been applied until now. It is assumed that the incidence would be higher if neonatal screening program would take place.

The Albanian population is composed of an ethnic group originating in the Balkan peninsula. There are approximately five million Albanians in this region, with roughly half living in Albania and the other half in Kosovo, Northern Macedonia, Montenegro and smaller populations in Croatia and Serbia. Significant numbers of the Albanian population are also found in Greece and smaller insignificant communities in Bulgaria and Romania as well. Albanians also make up a significant diaspora, spread all over the world, especially in North America, Europe and Oceania.

According to the latest census of the National Institute of Statistics Albania (INSTAT), the current population of Albania is 2,876,591 inhabitants [7].

The most common CFTR mutation is F508del, either in homozygous or heterozygous form. In Europe, this mutation is met in 82% of patients with CF (41% homozygous and 41% heterozygous) [9]. Among European countries, there are different variations of this CFTR mutation prevalence, from Denmark 83.2% [10], to a minimal prevalence in Turkey of only 24.5% [11]. This confirms the northwest to southeast gradient in the F508del distribution in Europe [11]. The Albanian population expresses a high prevalence of F508del mutation, in comparison to other populations in the region. Thus, in neighboring Italy is 43.9% [12], in North Macedonia is 75.9% [13], in Serbia and Montenegro it is 72.28 % [14].

In Albania, there is a high prevalence of this mutation which is characteristic of Central, Northern and Northeastern Europe. This fact is well known and previously explained due to early migrations of the Caucasian population towards Northwestern Europe and Southeastern Europe, and thereafter the migration through the Mediterranean routes from Middle East and Africa towards Europe [11]. The second migration seems to have left the Albanian population unaffected.

The second most prevalent mutation found in the Albanian population is 621+1G>T (2,56%). This mutation is most prevalent in Southern Europe, found more frequently in our neighboring country Greece (6.4%) [10], while in North Macedonia this mutation is under 2% [15].

The third most prevalent mutations found in the Albanian population are G85E (2.13%) and E822X (2.13%). The G85E mutation, is most frequent in Israel (2.6%) [10].

E822X, R1066C, G1349D, S466X, 1811+1G->C, E831X, CFTRdele2-3(21kb) mutations were newly found in the patients of our study. The latter, the CFTRdele2-3(21kb) mutation, was found in only one patient in this study, is found mainly in Slavic and Eastern and Central European populations [16]. The most frequent result of this mutation is in Belarus (11.2%) [10]

There are 14 CFTR mutations and 18 confirmed genotypes. To define the optimal treatment for an individual patient, genotyping of CFTR is clearly the first step [17].

This information suggests that most of these patients can benefit from new modulatory therapies targeting CFTR mutations, which translates to very hopeful prospects for these patients [18, 19]. The Albanian population would benefit from Cystic Fibrosis neonatal screening, since outcomes can be improved by early diagnosis.

Despite a lack of discussion on the topic of a neonatal screening program, it is crucial to improve diagnosis and allowing optimal treatment of patients with CF before overt disease progression sets in.