The authors of this article analyzed the available literature with the results of studying the prevalence of complex alleles of the
gene among patients with cystic fibrosis, and their pathogenicity ...and influence on targeted therapy with CFTR modulators. Cystic fibrosis (CF) is a multisystemic autosomal recessive disease caused by a defect in the expression of the CFTR protein, and more than 2000 genetic variants are known. Clinically significant variants are divided into seven classes. Information about the frequency of complex alleles appears in a number of registers, along with the traditional presentation of data on genetic variants. Complex alleles (those with the presence of more than two nucleotide variants on one allele) can complicate the diagnosis of the disease, and change the clinical manifestations of cystic fibrosis and the response to treatment, since each variant in the complex allele can contribute to the functional activity of the CFTR protein, changing it both in terms of increasing and decreasing function. The role of complex alleles is often underestimated, and their frequency has not been studied. At the moment, characteristic frequently encountered complex alleles have been found for several populations of patients with cystic fibrosis, but the prevalence and pathogenicity of newly detected complex alleles require additional research. In this review, more than 35 complex alleles of the
gene from existing research studies were analyzed, and an analysis of their influence on the manifestations of the disease and the effectiveness of CFTR modulators was also described.
The pathogenic variant E92K (c.274G > A) of the
gene is rare in America and Europe, but it is common for people with cystic fibrosis from Russia and Turkey. We studied the effect of the E92K genetic ...variant on the CFTR function. The function of the CFTR channel was studied using the intestinal current measurements (ICM) method. The effects of CFTR modulators on the restoration of the CFTR function were studied in the model of intestinal organoids. To assess the effect of E92K on pre-mRNA splicing, the RT-PCR products obtained from patients' intestinal organoid cultures were analyzed. Patients with the genetic variant E92K are characterized by an older age of diagnosis compared to homozygotes F508del and a high frequency of pancreatic sufficiency. The results of the sweat test and the ICM method showed partial preservation of the function of the CFTR channel. Functional analysis of
gene expression revealed a weak effect of the E92K variant on mRNA-CFTR splicing. Lumacaftor (VX-809) has been shown to restore CFTR function in an intestinal organoid model, which allows us to consider the E92K variant as a promising target for therapy with CFTR correctors.
The presence of complex alleles in the CFTR gene can lead to difficulties in diagnosing cystic fibrosis and cause resistance to therapy with CFTR modulators. Tezacaftor/ivacaftor therapy for 8 months ...in a patient with the initially established F508del/F508del genotype did not lead to an improvement in her condition—there was no change in spirometry and an increase in the patient’s weight, while there was only a slight decrease in NaCl values, measured by a sweat test. The intestinal current measurements of the patient’s rectal biopsy showed no positive dynamics in the rescue of CFTR function while taking tezacaftor/ivacaftor. The assumption that the patient had an additional mutation in the cis position was confirmed by sequencing the CFTR gene, and the complex allele L467F;F508del was identified. Based on the rescue of CFTR function by elexacaftor/tezacaftor/ivacaftor obtained using forskolin-induced swelling on intestinal organoids, the patient was prescribed therapy with this targeted drug. The use of elexacaftor/tezacaftor/ivacaftor for 7 months resulted in a significant improvement in the patient’s clinical condition.
The distribution and frequency of the
gene mutations vary considerably between countries and ethnic groups. Russians are an East Slavic ethnic groups are native to Eastern Europe. Russians, the most ...numerous people of the Russian Federation (RF), make about 80% of the population. The aim is to reveal the molecular causes of CF in ethnic Russian patients as comprehensively as possible. The analysis of most common
mutations utilized for CF diagnosis in multiethnic RF population accounts for about 83% of all CF-causing mutations in 1384 ethnic Russian patients. Variants c.1521_1523delCTT (F508del), c.54-5940_273+10250del21kb (CFTRdele2,3), c.2012delT (2143delT), c.2052_2053insA (2184insA), and c.3691delT (3821delT) are most typical for CF patients of Russian origin. DNA of 154 CF patients, Russian by origin, in whom at least one mutant allele was not previously identified (164 CF alleles), was analyzed by Sanger sequencing followed by the multiplex ligase-dependent probe amplification (MLPA) method. In addition to the 29 variants identified during the previous test for common mutations, 91 pathogenic
variants were also revealed: 29 missense, 19 nonsense, 14 frame shift in/del, 17 splicing, 1 in frame ins, and 11 copy number variations (CNV). Each of the 61 variants was revealed once, and 17 twice. Each of the variants c.1209G>C (E403D), c.2128A>T (K710X), c.3883delA (4015delA), and c.3884_3885insT (4016insT) were detected for three, c.1766+1G>A (1898+1G>A) and c.2834C>T (S945L) for four, c.1766+1G>C (1898+1G>C) and c.(743+1_744-1)_(1584+1_1585-1)dup (CFTRdup6b-10) for five, c.2353C>T (R785X) and c.4004T>C (L1335P) for six, c.3929G>A (W1310X) for seven, c.580-1G>T (712-1G>T for eight, and c.1240_1244delCAAAA (1365del5) for 11 unrelated patients. A comprehensive analysis of
mutant alleles with sequencing followed by MLPA, allowed not only the identification of 163 of 164 unknown alleles in our patient sample, but also expansion of the mutation spectrum with novel and additional frequent variants for ethnic Russians.
The intricate nature of complex alleles presents challenges in the classification of CFTR gene mutations, encompassing potential disease-causing, neutral, or treatment-modulating effects. Notably, ...the complex allele E217G;G509D remains absent from international databases, with its pathogenicity yet to be established. Assessing the functionality of apical membrane ion channels in intestinal epithelium employed the intestinal current measurements (ICM) method, using rectal biopsy material. The effectivity of CFTR-targeted therapy was evaluated using a model of intestinal organoids of a patient harboring the genotype F508del/E217G;G509D. ICM analysis revealed diminished chloride channel function. Remarkably, E217G;G509D presence within intestinal organoids correlated with heightened residual CFTR function. Employing CFTR modulators facilitated the restoration of the functional CFTR protein. This multifaceted study intertwines genetic investigations, functional analyses, and therapeutic interventions, shedding light on the intricate interplay of complex alleles within CFTR mutations. The results highlight the potential of targeted CFTR modulators to restore functional integrity, offering promise for advancing precision treatments in cystic fibrosis management.
Background
Juvenile idiopathic arthritis (JIA) is a chronic autoimmune disease characterized by destructive and inflammatory damage to the joints. The aim in this study was to compare vitamin D ...levels between children and adolescents, 1–18 years of age, with juvenile idiopathic arthritis (JIA) and a health control group of peers. We considered effects of endogenous, exogenous, and genetic factors on measured differences in vitamin D levels among children with JIA.
Methods
Our findings are based on a study sample of 150 patients with various variants of JIA and 277 healthy children. The blood level of vitamin D was assessed by calcidiol level. The following factors were included in our analysis: age and sex; level of insolation in three regions of country (center, south, north); assessment of dietary intake of vitamin D; effect of prophylactic doses of cholecalciferol; a relationship between the TaqI, FokI, and BsmI polymorphisms of the
VDR
gene and serum 25(OH)D concentration.
Results
We identified a high frequency of low vitamin D among children with JIA, prevalence of 66%, with the medial level of vitamin D being within the range of “insufficient” vitamin D. We also show that the dietary intake of vitamin D by children with JIA is well below expected norms, and that prophylactic doses of vitamin D supplementation (cholecalciferol) at a dose of 500–1,000 IU/day and 1,500–2,000 IU/day do not meet the vitamin D needs of children with JIA. Of importance, we show that vitamin D levels among children with JIA are not affected by clinical therapies to manage the disease nor by the present of VDR genetic variants.
Conclusion
Prophylactic administration of cholecalciferol and season of year play a determining role in the development of vitamin D deficiency and insufficiency.
Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease, associated with pathogenic variants in the CFTR gene. The splicing variant c.3140-16T>A (3272-16T>A) has been described ...previously and, according to the Russian CF Patients Registry, occurs with a frequency of 0.34%. The phenotypic features of CF patients with the c.3140-16T>A variant were compared with those of patients with the genotype F508del/F508del. Patients with the allele c.3140-16T>A had higher average age and age at diagnosis, and the allele was present in a greater proportion of adults. Patients carrying the c.3140-16T>A allele were characterised by better physical development indicators, both in adults and in children, had preserved pancreatic function, as well as the absence of a number of complications, and required pancreatic enzyme replacement therapy less often than patients with the F508del/F508del genotype. Sweat test values also were lower in patients with the c.3140-16T>A genotype. According to the results of clinical and laboratory studies, the phenotype of patients with the genetic variant c.3140-16T>A can be considered “mild”. Functional CFTR protein activity in the presence of c.3140-16T>A was evaluated using intestinal current measurements (ICM) and the forskolin-induced swelling assay on organoids obtained from patients’ rectal biopsies. c.3140-16T>A had high residual CFTR channel activity and was amenable to effective pharmacological correction with thea VX-770 potentiator. To evaluate the effect of the variant on CFTR pre-mRNA splicing we performed a minigene assay, as well as RT-PCR analysis of RNA isolated from the nasal epithelium and rectal biopsy of patients. We showed that the c.3140-16T>A variant creates a novel acceptor AG dinucleotide within CFTR intron 19, resulting in a 14-nucleotide extension of exon 20. This frameshift produces a premature termination codon and triggers mRNA degradation by the nonsense-mediated decay (NMD) mechanism. Moreover, we observed that the c.3140-16T>A allele could produce a residual amount of normally spliced transcript, thus explaining the patient’s mild phenotype.
In order to assess the effectiveness of the detection of cystic fibrosis (CF) patients by screening compared with diagnoses based on clinical manifestations, the data of the National CF Patient ...Registry (NCFPR) from the year 2012 (group I: children aged 6–9 years, diagnosed prior to the start of screening) were compared with the data in the NCFPR from the year 2015 (group II: children 6–9 years after the start of screening) for CF patients from the Moscow region. Homozygotes for c.1521_1523delCTT (F508del) were separately compared in both groups. The average diagnosis age, genotype, body mass index, spirometry data, pulmonary infection, medications, and presence of complications were analyzed. This study demonstrated that in the c.1521_1523delCTT (F508del) homozygote group, the patients diagnosed by screening had significant advantages over the patients born before the start of newborn screening in the diagnosis age, the number of patients with chronic Pseudomonas aeruginosa infection, the pulmonary function, and the growth in the percentiles. Newborn screening (NBS) detects nearly twice as many CF patients as the diagnostics based on clinical symptoms during the same time period. Importantly, patients will benefit from the early diagnosis of the disease and the early start of therapy.
The problem of vitamin D deficiency is particularly relevant for the entire territory of Russia, since most parts of the country are located above the 42nd geographical latitude and the residents are ...therefore at risk of vitamin D deficiency. Despite the urgency of the problem, a comprehensive study of the molecular and genetic mechanisms and exogenous factors of vitamin D deficiency in children living in various geographical areas of the Russian Federation has not been conducted. Different variants in the loci of the genes responsible for the synthesis, hydroxylation, and transport of vitamin D (such as
, and
), as well as
gene polymorphisms may also be associated with the risk of vitamin D deficiency. The aim of this study was to analyze the influence of exogenous factors on the blood levels of 25-hydroxyvitamin D (25(OH)D) in children of three regions of the Russian Federation, as well as the relationship of blood 25(OH)D levels with polymorphic variants of cytochrome P450 genes and
gene.
We conducted blood 25(OH)D level analysis in 333 healthy children and adolescents in three regions located in different geographical zones of the Russian Federation. We studied the polymorphic variants c.1075A>C (I359L, rs1057910,
) and c.430C>T (R144C, rs1799853,
) in the
gene, c.1334T>C (M445T, rs4986910,
), and
(c.-392C>T, rs2740574) in the
gene, 1846G>A, (rs3892097,
) in the
gene,
I (NM_000376.2: c.1056T>C; rs731236),
I (NM_000376.2:c.2T>C; (rs2228570), and
I (NM_000376.2: c.1024+283G>A; rs1544410) in the
gene. We also analyzed the influence of exogenous factors on the level of 25(OH)D in children of the three study regions, as well as the relationship of the level of 25(OH)D with variants
(c.430C>T; R144C),
(c,1075A>C; I359L),
(1846G>A),
(c.1334T>C), and
(c.-392C>T) and rs731236, rs2228570 and rs1544410 in the
gene.
We found that the blood level of 25(OH)D depended on the geographical location and the number of sunny days per year. The average blood level of 25(OH)D in adolescent boys was statistically significantly lower than in girls of this age group. The level of 25(OH)D also significantly depended on the prophylactic dose of cholecalciferol administered to the subjects. In the study, it was shown that a dose of cholecalciferol ≥1,000 IU per day can achieve a normal level of 25(OH)D in healthy children. We found no statistically significant association between single-nucleotide polymorphic variants of cytochrome P450 genes (
, and
) and blood level of 25(OH)D in the subjects. We also did not find a relationship between the
I,
I, and
I polymorphisms of the
gene and serum 25(OH)D concentration.
Exogenous factors (time of year, place of residence, and prophylactic administration of cholecalciferol), as well as endogenous factors (age and sex), play a determining role in the development of vitamin D deficiency and insufficiency; in contrast to genetic factors-polymorphic variants of the genes of xenobiotic phase 1 enzymes (
, and
) and the
gene-which do not play such role. This study shows the need to create a diagnostic algorithm for Vitamin D deficiency based on the age, season of the year, and prophylactic dose of cholecalciferol.
Aimto evaluate the impact of Gilbert’s Syndrome (GS) on the course of the disease and the development of complications in cystic fibrosis (CF)Material and MethodPolymorphic variants of the UGT1A1 ...gene were identified in 54 patients (11.3 ±4 years, 28 boys and 26 girls) with CF by PCR method. Hyperbilirubinemia was detected in all 54 patients during receiving targeted drug therapy of CF elexacaftor/tezacaftor/ivacaftor. The analysis of the clinical course and complications was carried out on the basis of data to the national CF registry 2021 (2826 children).ResultsCF patients with GS more often had severe genotype (91.8%) against 80.3% CF patients without GS (p=0.044); more often had cystic fibrosis liver disease (CFLD) - 44.2% against 25% CF patients without GS (p=0.022), including cirrhosis with portal hypertension – 5.8% against 3.2% CF patients without GS (p<0.05) and without portal hypertension – 9.6% against 3.7% CF patients without GS (p<0.05); more often had chronic rhinosinusitis – 86.3% against 60.9% CF patients without GS (p=0.001). There were no significant differences in the microbial status of the respiratory tract, as well as in the nutritional status. Lung function (FEV1 pred,%) tended to be higher in CF patients with GS – 98% against 89% CF patients without GS (p=0.06). During targeted therapy with the elexacaftor/tezacaftor/ivacaftor, all these patients had a persistent increase in total and unconjugated bilirubin of 1.5–2.5 normal values.ConclusionsCF patients with GS are a higher risk group for the development of liver diseases and chronic rhinosinusitis. Further monitoring of CF patients with GS is necessary.
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