The L138ins mutation, found in Russian cystic fibrosis (CF) patients, is a duplication of three nucleotides (CTA) in exon 4 of the CFTR gene and is categorised as a small in-frame insertion/deletion. As a result, the CFTR protein molecule elongates by one amino acid residue, leucine, at position 138 (codon 138 (CTA)). In accordance with the new nomenclature, it should be called c.411_412insCTA (p.Leu138dup). The c.411_412insCTA (p.Leu138dup, L138ins) mutation is found in CF patients of Slavic origin (Russians, Ukrainians) and has been linked to a single haplotype of the intragenic DNA markers IVS1CA-IVS6aGATT-IVS8CA-IVS17bCA - 22-7-16-13.
Cystic fibrosis (CF; OMIM # 219700) is an autosomal recessive disease caused by a mutation in the CFTR gene (OMIM * 602421), characterised by a variable clinical picture ranging from a relatively mild disease course with monosymptomatic manifestations to severe multiorgan lesions []. The prevalence of CF in European countries is about 1 in 2500–4500 newborns and in the Russian Federation, it is 1 in 10,000 newborns . The spectrum and frequencies of CFTR mutations vary widely among different populations and ethnic groups.
The spectrum of CFTR mutations specific to Russian CF patients has been recently identified. Moreover, the creation of the Russian Cystic Fibrosis Patient Registry (RCF-PR) and the provisions of the National Consensus documents on CF care have made it possible to combine data from clinical trials and researches conducted in different centres to better clarify the frequency of mutations both in the Russian Federation itself and in specific regions within the Federation [2,3]. According to the RCF-PR 2016,the ten most common mutations (frequency) are: F508del (52.06%), CFTRdele2.3 (5.71%), E92K (2.67%), 2143delT (2.06%), 3849+10kbC>T (2.04%), 2184insA (1.87%), W1282X (1.82%), N1303K (1.47%), 1677delTA (1.44%), and G542X (1.35%). Mutation c.411_412insCTA (p.Leu138dup, L138ins) can be considered common in Russian patients since its frequency exceeds 1% (1.15%) of the total number of identified mutant alleles (ranging from 0.29–2.89% across different regions) [2,3]. The frequency of this mutation in Europe has not been determined due to the rarity of this particular pathological allele in European populations . Indeed, the L138ins mutation is not included in the panel of routinely analysed CFTR mutations in European countries. The purpose of this study is to describe the genotypic features of the L138ins mutation in Russian CF patients.
Material and Methods
DNA was isolated from whole blood samples of CF patients using a DNA extraction kit (Promega, USA). Molecular genetic testing for the c.411_412insCTA (p.Leu138dup, L138ins) mutation was performed using amplified fragment length polymorphism (AFLP) analysis as part of the testing for frequent CFTR mutations in 1700 CF patients and was conducted in the Laboratory of Genetic Epidemiology at the Research Centre for Medical Genetics, Moscow . Sanger sequencing to confirm the c.411_412insCTA (p.Leu138dup, L138ins) mutation presence was performed in 37 CF patients. Analysis of DNA markers IVS1CA, IVS6aGATT, IVS8CA, and IVS17bCA was performed in 24 CF patients carrying the c.411_412insCTA (p.Leu138dup, L138ins) mutation and their parents using a previously described procedure . This research project was approved by the Ethics committee of the Research Center for Medical Genetics (Moscow). CF patients, or their fiduciaries, provided written informed consent.
Results and discussion
In 2000, while testing for the 621+1G>T mutation in a Russian CF patient using RFLP test, the abnormal mobility of the exon 4 fragment of the CFTR gene was first observed (Figure 1). Sequencing confirmed the presence of a CFTR mutation (L138ins) not previously found in the Russian population (Figure 2). By 2006, the L138ins mutation was detected in six unrelated CF patients from Moscow and the Moscow region . Subsequently, the L138ins mutation was also identified in two patients from the Krasnodar region . Currently, the L138ins mutation is included in the panel of common CFTR mutations routinely tested for in the Russian population .
Analysis of the chromatogram (Figure 1) shows that the initial sequence of 5'-CACTGCTCCTACACCCAGCC is changed to 5'-CACTGCTCCTACTACACCCAGCC. Formally, four different events can lead to such a rearrangement (Table 1): insertion of the CTA triplet between 411 and 412 (1) or 414 and 415 (4) positions of the coding sequence; a TAC insertion between 412 and 413 (2) and an ACT insertion between 413 and 414 (3) positions. Any of these rearrangements will lead to duplication of the CTA codon without changing the reading frame, leading to duplication of leucine at position 138. According to the current nomenclature, the L138ins mutation should be designated as c.411_412insCTA (p.Leu138dup).
|Sweat chloride (mmol/l)||86.99 ± 17.02||78.29 ± 24.29||103.49 ± 22.89|
|Age at diagnosis (yrs)||6.71 ± 9.73||14.34 ± 8.89||1.99 ± 0.16|
|PI (% of patients)||19.1||33.3||94.1|
|BMI (kg/m2)||21.87 ± 2.58||18.84 ± 2.73||18.64 ± 2.46|
|FEV1 (%)||77.56 ± 29.43||54.52 ± 21.88||72.34 ± 27.21|
|Age of patients with chronic P.aeruginosa (yrs)||26.44 + 10.33||27.67 ± 10.18||16.24 ± 8.13|
This mutation is located in the second membrane penetrating motif of the first transmembrane domain (MSD1) involved in the formation of the pore of the CFTR chloride channel. The likely consequence of this mutation is the impairment of the conductive properties of the chloride channel. The L138ins mutation was first described by Dörk et al. in 1996 in a 34-year-old patient with a congenital bilateral absence of the vas deferens (CBAVD). The patient was pancreatic sufficient, without lung lesions, a sweat chloride level of 53 mmol/l, with the 5T variant in the second allele . The CFTR1 database  describes two insertions of three nucleotides in the region under consideration: in one case the mutation, using the legacy nomenclature referred to as L138ins, is an ACT insertion between nucleotides 412 and 413, which leads to the insertion of histidine between two leucines located at 137 and 138 (c.412_413insACT; p.Leu137_Leu138insHis). In another case, the 546insCTA (c.414_415insCTA) mutation was described, however, the wrong amino acid sequence at the protein level was reported as p.Leu139X (i.e., a premature stop codon at position 139); the correct designation of this mutation should be p.Leu138dup (i.e., leucine duplication at position 138).
Since 31st August 2018, the mutation understudy was included in the CFTR2 database, referred to as c.413_415dupTAC (p.Leu138dup, L138ins, rs397508679) . It is also included in the EXAC database of 1000 genomes  and the ClinVar . The first reference identified was the correct description of the mutation in the work of McGinniss et al. (2011) .
In our previous paper regarding the genotype-phenotype correlation in Russian CF patients with c.411_412insCTA (p.Leu138dup) mutation, we showed that the p.Leu138dup mutation could be considered as a disease-causing mutation, leading to a variable but relatively mild course of CF (Table 1) .
To date, in the Laboratory of Genetic Epidemiology, mutation L138ins (c.411_412insCTA, p.Leu138dup), in trans with other CF-causing mutations, has been detected in 37 Russian CF patients from 33 families. The average patient age is 13.13 ± 11.42 years (1.00–43.00), with a ratio by sex: 0.43 m: 0.57 f (16:21). Eleven different genotypes have been identified and are described in Table 2.
|Genotype||Number of patients|
|L138ins/F508del (c.[411_412insCTA];[1521_1523delCTT], p.[Leu138dup];[Phe508del])||15|
|L138ins/CFTRdele2,3 (c.[411_412insCTA];[54-5940_273+10250del21kb], p.[Leu138dup];[Ser18Arg*fsX16])||4|
|L138ins/2184insA (c.[411_412insCTA];[2052_2053insA], p.[Leu138dup];Gln685ThrfsX4])||3|
|L138ins/2143delT (c.[411_412insCTA];[2012delT], p.[Leu138dup];[Leu671X])||1|
|L138ins/N1303K (c.[411_412insCTA];[3909C>G], p.[Leu138dup];[Asn1303Lys])||1|
|L138ins/R1162X (c.[411_412insCTA];[3484C>T], p.[Leu138dup];[Arg1162X])||4|
|L138ins/E92K (c.[411_412insCTA];[274G>A], p.[Leu138dup];[Glu92Lys])||1|
|L138ins/ 2183AA->G (c.[411_412insCTA];[ 2051_2052delAAinsG], p.[Leu138dup];[ Lys684SerfsX38])||1|
|L138ins/ L138ins (c.[411_412insCTA];[ 411_412insCTA], p.[Leu138dup];[ Leu138dup])||1|
|L138ins/W1282R (c.[411_412insCTA];[3844T>C], p.[Leu138dup];[Trp1282Arg])||1|
|L138ins/not identified (c.[411_412insCTA];[?], p.[Leu138dup];[?])||4|
The most common genotype, L138ins/F508del (c.[411_412insCTA];[1521_1523delCTT], p.[Leu138dup];[Phe508del]), has been found in 15 CF patients (40.5%); the second allele remains unidentified in four patients. The L138ins (c.411_412insCTA, p.Leu138dup) mutation is always linked to one haplotype of the intrinsic marker IVS1CA-IVS6aGATT-IVS8CA-IVS17bCA - 22-7-16-13, suggesting that the L138ins (c.411_412insCTA, p.Leu138dup) variant occurred as the result of a single mutation event.
Most CF patients with the L138ins (c.411_412insCTA, p.Leu138dup) mutation reside in Moscow (17) or the Moscow region (7) and all patients belonged to a Slavic ethnic group (Russians, Ukrainians). The L138ins (c.411_412insCTA, p.Leu138dup) mutation has been detected in six of the nine Federal Regions (predominantly Russian) of the Russian Federation, with a relative proportion ranging from 0.35% to 3.11%. Relative frequencies of the L138ins (c.411_412insCTA, p.Leu138dup) mutation (according to RCF-PR 2017) are given in Table 3 .
|North Western European||0.65|
The L138ins (c.411_412insCTA, p.Leu138dup) mutation identified in Russian CF patients (a Slavic ethnic group) is a duplication of three nucleotides (CTA) in exon 4 of the CFTR gene and is categorised as a small in-frame insertion/deletion.
The work was carried within the state assignment of Ministry of Science and Higher Education of the Russian Federation for Medical Genetics and in part with the financial support of the Russian Fund for Fundamental Research (RFFR) (project No. 20-015-00061) (expeditions, DNA research, writing the manuscript) and Czech Ministry of Health CZ.2.16/3.1.00/24022OPPK; IP00064203/6003 for University Hospital Motol (general management of the project, reviewing the manuscript).
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