Safety and efficacy of canakinumab treatment for undifferentiated autoinflammatory diseases: the data of a retrospective cohort two-centered study.

Introduction: The blockade of interleukine-1 (anakinra and canakinumab) is a well-known highly effective tool for monogenic autoinflammatory diseases (AIDs), such as familial Mediterranean fever, tumor necrosis factor receptor-associated periodic syndrome, hyperimmunoglobulinaemia D syndrome, and cryopyrin-associated periodic syndrome, but this treatment has not been assessed for patients with undifferentiated AIDs (uAIDs). Our study aimed to assess the safety and efficacy of canakinumab for patients with uAIDs. Methods: Information on 32 patients with uAIDs was retrospectively collected and analyzed. Next-generation sequencing and Federici criteria were used for the exclusion of the known monogenic AID. Results: The median age of the first episode was 2.5 years (IQR: 1.3; 5.5), that of the disease diagnosis was 5.7 years (IQR: 2.5;12.7), and that of diagnostic delay was 1.1 years (IQR: 0.4; 6.1). Patients had variations in the following genes: IL10, NLRP12, STAT2, C8B, LPIN2, NLRC4, PSMB8, PRF1, CARD14, IFIH1, LYST, NFAT5, PLCG2, COPA, IL23R, STXBP2, IL36RN, JAK1, DDX58, LACC1, LRBA, TNFRSF11A, PTHR1, STAT4, TNFRSF1B, TNFAIP3, TREX1, and SLC7A7. The main clinical features were fever (100%), rash (91%; maculopapular predominantly), joint involvement (72%), splenomegaly (66%), hepatomegaly (59%), lymphadenopathy (50%), myalgia (28%), heart involvement (31%), intestinal involvement (19%); eye involvement (9%), pleuritis (16%), ascites (6%), deafness, hydrocephalia (3%), and failure to thrive (25%). Initial treatment before canakinumab consisted of non-biologic therapies: non-steroidal anti-inflammatory drugs (NSAID) (91%), corticosteroids (88%), methotrexate (38%), intravenous immunoglobulin (IVIG) (34%), cyclosporine A (25%), colchicine (6%) cyclophosphamide (6%), sulfasalazine (3%), mycophenolate mofetil (3%), hydroxychloroquine (3%), and biologic drugs: tocilizumab (62%), sarilumab, etanercept, adalimumab, rituximab, and infliximab (all 3%). Canakinumab induced complete remission in 27 patients (84%) and partial remission in one patient (3%). Two patients (6%) were primary non-responders, and two patients (6%) further developed secondary inefficacy. All patients with partial efficacy or inefficacy were switched to tocilizumab (n = 4) and sarilumab (n = 1). The total duration of canakinumab treatment was 3.6 (0.1; 8.7) years. During the study, there were no reported Serious Adverse Events (SAEs). The patients experienced non-frequent mild respiratory infections at a rate that is similar as before canakinumab is administered. Additionally, one patient developed leucopenia, but it was not necessary to stop canakinumab for this patient. Conclusion: The treatment of patients with uAIDs using canakinumab was safe and effective. Further randomized clinical trials are required to confirm the efficacy and safety.

Genotype-Phenotype Correlations in 293 Russian Patients with Causal Fabry Disease Variants.

BACKGROUND: Fabry disease (FD) is a rare hereditary multisystem disease caused by variants of the GLA gene. Determination of GLA gene variants and identification of genotype-phenotype correlations allow us to explain the features of FD associated with predominant damage of one or another system, both in the classical and atypical forms of FD, as well as in cases with late manifestation and involvement of one of the systems. METHODS: The study included 293 Russian patients with pathogenic variants of the GLA gene, which were identified as a result of various selective screening programs. Screening was carried out for 48,428 high-risk patients using a two-step diagnostic algorithm, including the determination of the concentration of the biomarker lyso-Gb3 as a first-tier test. Screening of atypical FD among patients with HCM was carried out via high-throughput sequencing in another 2427 patients. RESULTS: 102 (0.20%) cases of FD were identified among unrelated patients as a result of the study of 50,855 patients. Molecular genetic testing allowed us to reveal the spectrum and frequencies of 104 different pathogenic variants of the GLA gene in 293 examined patients from 133 families. The spectrum and frequencies of clinical manifestations in patients with FD, including 20 pediatric patients, were described. Correlations between the concentration of the lyso-Gb3 biomarker and the type of pathogenic variants of the GLA gene have been established. Variants identified in patients with early stroke were described, and the association of certain variants with the development of stroke was established. CONCLUSIONS: The results of a large-scale selective FD screening, as well as clinical and molecular genetic features, in a cohort of 293 Russian patients with FD are described.

Clinical features of pediatric Danon disease and the importance of early diagnosis.

Successful therapy in a cohort with early onset Danon disease (DD) highlights the potential importance of earlier disease recognition. We present experience from the largest National Pediatric Center in Russia for cardiomyopathy patients. This report focuses on identification of early clinical features of DD in the pediatric population by detailed pedigree analysis and review of medical records. RESULTS: Nine patients (3 females) were identified with DD at the Russian National Medical Research Center of Children's Health ("National Pediatric Center") aged birth to 16 years. At presentation/evaluation: all patients had left ventricular hypertrophy (LVH), ECG features of Wolff-Parkinson-White (WPW), and an increase in hepatic enzymes (particularly lactate dehydrogenase (LDH)); three had marked increase in NT-proBNP; two had HCM with impaired LV function; one had LVH with LV noncompaction; five had arrhythmia with paroxysmal supraventricular and/or ventricular tachycardia. Two teenagers died at ages 16-17 from refractory heart failure and two underwent heart transplantation. All patients were found to have a pathogenic/likely pathogenic variant in the LAMP2 gene, six patients had no family history and a de novo evolvement was documented in 4/6 of those available for genetic tested. Retrospective review related to family background and earlier clinical evaluations revealed a definitive or highly suspicious family history of DD in 3, early clinical presentation with cardiac abnormalities (ECG, echo) in 3, and cerebral, hepatic and/or neuromuscular symptoms in 5. Abnormalities were detected 9,5 months to 5,8 years, median 3,5 years prior to referral to the National Pediatric Center. CONCLUSION: The earliest clinical manifestations of Danon disease occur in the first 12 years of life with symptoms of skeletal muscle and cerebral disease, raised hepatic enzymes, and evidence of cardiac disease on ECG/echo.

Comparison of disease phenotypes and mechanistic insight on causal variants in patients with DADA2.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) results in heterogeneous manifestations including systemic vasculitis and red cell aplasia. The basis of different disease phenotypes remains incompletely defined. OBJECTIVE: We sought to further delineate disease phenotypes in DADA2 and define the mechanistic basis of ADA2 variants. METHODS: We analyzed the clinical features and ADA2 variants in 33 patients with DADA2. We compared the transcriptomic profile of 14 patients by bulk RNA sequencing. ADA2 variants were expressed experimentally to determine impact on protein production, trafficking, release, and enzymatic function. RESULTS: Transcriptomic analysis of PBMCs from DADA2 patients with the vasculitis phenotype or pure red cell aplasia phenotype exhibited similar upregulation of TNF, type I interferon, and type II interferon signaling pathways compared with healthy controls. These pathways were also activated in 3 asymptomatic individuals with DADA2. Analysis of ADA2 variants, including 7 novel variants, showed different mechanisms of functional disruption including (1) unstable transcript leading to RNA degradation; (2) impairment of ADA2 secretion because of retention in the endoplasmic reticulum; (3) normal expression and secretion of ADA2 that lacks enzymatic function; and (4) disruption of the N-terminal signal peptide leading to cytoplasmic localization of unglycosylated protein. CONCLUSIONS: Transcriptomic signatures of inflammation are observed in patients with different disease phenotypes, including some asymptomatic individuals. Disease-associated ADA2 variants affect protein function by multiple mechanisms, which may contribute to the clinical heterogeneity of DADA2.

Targeted NGS in Diagnostics of Genodermatosis Characterized by the Epidermolysis Bullosa Symptom Complex in 268 Russian Children.

The pathogenic variants of genes encoding proteins, participating in the formation and functioning of epidermis and dermo-epidermal junctions, create a large variety of clinical phenotypes from: small localized to severe generalized dermatitis, as well as early, or even, prenatal death due to extensive epidermis loss. The diagnostic panel in this study was developed for the purposes of identifying these pathogenic genetic variants in 268 Russian children, who possessed the epidermolysis bullosa symptom complex in a selection of 247 families. This panel included the targeted areas of 33 genes, which are genetic variants that can lead to the development of the phenotype mentioned above. The usage of next generation sequencing allowed the revelation of 192 various altered alleles (of which 109 alleles were novel, i.e., had not been described previously). In addition, it allowed the definition of the genetic variants that are both typical for most of the examined children and for the separate ethnic groups inhabiting modern Russia. We found that the most characteristic mutations for the Dargin and Chechen ethnic groups are the c.3577del deletion in the COL7A1 gene and the c.2488G>A missense mutation in the COL17A1 gene, respectively. In addition, the study of haplotypes of microsatellite markers, which we managed to conduct in the Dargin population, confirmed the presence of the founder effect.

Clinical and Genetic Characteristics of Pediatric Patients with Hypophosphatasia in the Russian Population.

(1) Hypophosphatasia (HPP) is a rare inherited disease caused by mutations (pathogenic variants) in the ALPL gene which encodes tissue-nonspecific alkaline phosphatase (TNSALP). HPP is characterized by impaired bone mineral metabolism due to the low enzymatic activity of TNSALP. Knowledge about the structure of the gene and the features and functions of various ALPL gene variants, taking into account population specificity, gives an understanding of the hereditary nature of the disease, and contributes to the diagnosis, prevention, and treatment of the disease. The purpose of the study was to describe the spectrum and analyze the functional features of the ALPL gene variants, considering various HPP subtypes and clinical symptoms in Russian children. (2) From 2014−2021, the study included the blood samples obtained from 1612 patients with reduced alkaline phosphatase activity. The patients underwent an examination with an assessment of their clinical symptoms and biochemical levels of TNSALP. DNA was isolated from dried blood spots (DBSs) or blood from the patients to search for mutations in the exons of the ALPL gene using Sanger sequencing. The PCR products were sequenced using a reagent BigDye Terminator 3.1 kit (Applied Biosystems). Statistical analysis was performed using the GraphPad Prism 8.01 software. (3) The most common clinical symptoms in Russian patients with HPP and two of its variants (n = 22) were bone disorders (75%), hypomyotonia (50%), and respiratory failure (50%). The heterozygous carriage of the causal variants of the ALPL gene was detected in 225 patients. A total of 2 variants were found in 27 patients. In this group (n = 27), we identified 28 unique variants of the ALPL gene, of which 75.0% were missense, 17.9% were frameshift, 3.6% were splicing variants, and 3.6% were duplications. A total of 39.3% (11/28) of the variants were pathogenic, with two variants being probably pathogenic, and 15 variants had unknown clinical significance (VUS). Among the VUS group, 28.6% of the variants (7/28) were discovered by us for the first time. The most common variants were c.571G > A (p.Glu191Lys) and c.1171del (Arg391Valfs*12), with frequencies of 48.2% (13/28) and 11% (3/28), respectively. It was found that the frequency of nonsense variants of the ALPL gene was higher (p < 0.0001) in patients with the perinatal form compared to the infantile and childhood forms of HPP. Additionally, the number of homozygotes in patients with the perinatal form exceeded (p < 0.01) the frequencies of these genotypes in children with infantile and childhood forms of HPP. On the contrary, the frequencies of the compound-heterozygous and heterozygous genotypes were higher (p < 0.01) in patients with infantile childhood HPP than in perinatal HPP. In the perinatal form, residual TNSALP activity was lower (p < 0.0005) in comparison to the infantile and childhood (p < 0.05) forms of HPP. At the same time, patients with the heterozygous and compound-heterozygous genotypes (mainly missense variants) of the ALPL gene had greater residual activity (of the TNSALP protein) regarding those homozygous patients who were carriers of the nonsense variants (deletions and duplications) of the ALPL gene. Residual TNSALP activity was lower (p < 0.0001) in patients with pathogenic variants encoding the amino acids from the active site and the calcium and crown domains in comparison with the nonspecific region of the protein.

Signatures of Dermal Fibroblasts from RDEB Pediatric Patients.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a debilitating disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Mutations in the COL7A1 gene induce multiple abnormalities, including chronic inflammation and profibrotic changes in the skin. However, the correlations between the specific mutations in COL7A1 and their phenotypic output remain largely unexplored. The mutations in the COL7A1 gene, described here, were found in the DEB register. Among them, two homozygous mutations and two cases of compound heterozygous mutations were identified. We created the panel of primary patient-specific RDEB fibroblast lines (FEB) and compared it with control fibroblasts from healthy donors (FHC). The set of morphological features and the contraction capacity of the cells distinguished FEB from FHC. We also report the relationships between the mutations and several phenotypic traits of the FEB. Based on the analysis of the available RNA-seq data of RDEB fibroblasts, we performed an RT-qPCR gene expression analysis of our cell lines, confirming the differential status of multiple genes while uncovering the new ones. We anticipate that our panels of cell lines will be useful not only for studying RDEB signatures but also for investigating the overall mechanisms involved in disease progression.

The Prevalence and Clinical Features of Fabry Disease in Hemodialysis Patients: Russian Nationwide Fabry Dialysis Screening Program.

AIM: To evaluate the prevalence and clinical features of Fabry disease in patients with end-stage renal disease (ESRD) undergoing chronic hemodialysis. METHODS: α-Galactosidase A activity was measured in the dried blood spots by tandem mass spectrometry in 5,572 dialysis patients (63.7% males). Diagnosis of Fabry disease was confirmed by sequencing of the GLA gene and by evaluating the globotriaosylsphingosine level in the dried blood spots. RESULTS: Fabry disease was diagnosed in 20 (0.36%) patients at the median age of 43 years (28; 58). There were 19 males and 1 female. The prevalence of Fabry disease in dialysis patients was 0.53% in males and 0.05% in females. However, it was higher in males aged 30-49 years. Seventeen different GLA mutations were identified; 5 of them were novel. The median age at the initiation of hemodialysis was similar between patients with missense and nonsense mutations. Sixteen patients (80.0%) presented with typical symptoms of Fabry disease from childhood (neuropathic pain in 16, angiokeratoma in 7 and hypohidrosis/anhidrosis in 16). All patients had left ventricular hypertrophy, and 8 patients (40%) had a history of ischemic stroke. Two patients died (recurrent stroke in one and sudden cardiac death in another patient). CONCLUSIONS: Screening in at-risk patients remains the feasible approach to diagnose Fabry disease in patients with ESRD and their family members, given a low awareness of Fabry disease among the Russian nephrologists.

Fabry disease in children: a federal screening programme in Russia.

Our objective was to examine the prevalence of Fabry disease in Russian children with chronic pain in the distal limbs. This non-interventional, multi-centre study included children 2-18 years of age with chronic recurrent unilateral or bilateral pain, burning, or acroparesthesia in the hands or feet. The presence of Fabry disease was defined by abnormal alpha-galactosidase A activity in males or alpha-galactosidase gene (GLA) mutation in females. Among 214 patients (110 males), 84.1% had bilateral limb pain and 31.8% had unilateral limb pain recorded at some time point; 61 (28.5%) patients had a positive family history possibly associated with Fabry disease. Alpha-galactosidase A activity was within the normal range in all 109 of the male patients tested. One female patient had a GLA mutation (C937G > T) and alpha-galactosidase A activity within the normal range. CONCLUSION: We did not find definitive evidence of Fabry disease in these children with a history of chronic recurrent unilateral or bilateral limb pain or acroparesthesia. The presence of chronic limb pain does not appear to be highly predictive of a diagnosis of Fabry disease in Russian children and adolescents, suggesting that key early signs and symptoms of Fabry disease are not specific to the disease. What is Known: • Signs and symptoms of Fabry disease are seen in children < 10 years of age; pain in the distal limbs is a common early symptom. What is New: • Fabry disease was not diagnosed in this population of Russian children with a history of chronic limb pain. • The presence of acroparesthesia or chronic limb pain does not appear to be highly predictive of a diagnosis of Fabry disease in Russian children and adolescents, suggesting that these early symptoms of Fabry disease are not specific to the disease.