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.

Mutations in Mismatch Repair Genes and Microsatellite Instability Status in Pancreatic Cancer.

Patients with pancreatic cancer (PC) showing mismatch repair (MMR) deficiency may benefit from immunotherapy. Microsatellite instability (MSI) is a hallmark of MMR deficiency (MMR-D). Here, we estimated the prevalence of MSI in PC, investigated germline and somatic mutations in the three MMR genes (MLH1, MSH2, and MSH6), and assessed the relationship between MMR genes mutations and MSI status in PC. Clinical specimens from PC patients were analyzed using targeted next-generation sequencing, including paired normal and tumor specimens from 155 patients, tumor-only specimens from 86 patients, and normal-only specimens from 379 patients. The MSI status of 235 PCs was assessed via PCR. Pathogenic/likely pathogenic (P/LP) germline variants in the MMR genes were identified in 1.1% of patients, while somatic variants were found in 2.6% of patients. No MSI-H tumors were detected. One patient carried two variants (P (VAF = 0.57) and LP (VAF = 0.25)) simultaneously; however, their germline/somatic status remains unknown due to the investigation focusing solely on the tumor and MSI analysis was not performed for this patient. MSI is rare in PC, even in tumors with MMR genes mutations. Our findings underscore the importance of assessing tumor MMR-D status in PC patients with confirmed Lynch syndrome when deciding whether to prescribe immunotherapy.

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.

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.

A kinetic assay of total lipase activity for detecting lysosomal acid lipase deficiency (LAL-D) and the molecular characterization of 18 LAL-D patients from Russia.

Laboratory diagnostics of lysosomal acid lipase deficiency (LAL-D), a rare disorder associated with LIPA alterations, are based on the evaluation of LAL activity. In dry blood spots (DBS) submitted for LAL-D diagnostics (the screening cohort) over a two-year period or obtained from a cohort of retrospective LAL-D patients, we measured: (1) LAL activity using a two-reaction assay with 4-methylumbelliferone palmitate (4-MU-Palm) and Lalistat-2, a specific LAL inactivator; (2) total lipase (TL) activity by a 1-hour kinetic 4-MU-Palm cleavage reaction (no Lalistat-2). The TL activity was expressed as the area under the kinetic curve after 1 hour (TL-AUC1h) of the reaction and presented as the median (min-max). LAL activity was reduced in 30/537 individuals from the screening cohort, among which LIPA sequencing revealed six patients and one carrier. Overall, 16 (89%) individuals among six novel and 12 retrospective LAL-D patients carried at least one c.894G>A mutation (six were homozygous). The TL-AUC1h in nonLAL-D specimens with normal LAL activity (n = 90) was unambiguously higher (9471 [4015-23 585] RFU*h/punch) compared to LAL-D patients, including six new and nine retrospective patients (1810 [357-2608] RFU*h/punch). Importantly, in 13/15 examined nonLAL-D specimens with reduced LAL activity the TL-AUC1h was above a threshold of 2652 RFU*h/punch. Applying this threshold, the TL-AUC1h index discriminated all LAL-D patients (100% sensitivity) and 103/105 nonLAL-D specimens (98% specificity). Given that there is no need for Lalistat-2 and two parallel enzymatic reactions in conjunction with high sensitivity and specificity, the kinetic assay seems to be practical for LAL-D screening. SYNOPSIS: Lysosomal acid lipase deficiency responsible for Wolman disease and cholesterol ester storage disease could be reliably detected using a kinetic assay of total lipase activity with a fluorogenic substrate.

Genetic analysis of 17 children with Hunter syndrome: identification and functional characterization of four novel mutations in the iduronate-2-sulfatase gene.

Mucopolysaccharidosis type II (MPS II) is a rare X-linked disorder caused by alterations in the iduronate-2-sulfatase (IDS) gene. In this study, IDS activity in peripheral mononuclear blood monocytes (PMBCs) was measured with a fluorimetric enzyme assay. Urinary glycosaminoglycans (GAGs) were quantified using a colorimetric assay. All IDS exons and intronic flanks were bidirectionally sequenced. A total of 15 mutations (all exonic region) were found in 17 MPS II patients. In this cohort of MPS II patients, all alterations in the IDS gene were caused by point nucleotide substitutions or small deletions. Mutations p.Arg88His and p.Arg172* occurred twice. All mutations were inherited except for p.Gly489Alafs*7, a germline mutation. We found four new mutations (p.Ser142Phe, p.Arg233Gly, p.Glu430*, and p.Ile360Tyrfs*31). In Epstein-Barr virus (EBV)-immortalized PMBCs derived from the MPS II patients, no IDS protein was detected in case of the p.Ser142Phe and p.Ile360Tyrfs*31 mutants. For p.Arg233Gly and p.Glu430*, we observed a residual expression of IDS. The p.Arg233Gly and p.Glu430* mutants had a residuary enzymatic activity that was lowered by 14.3 and 76-fold, respectively, compared with healthy controls. This observation may help explain the mild disease phenotype in MPS II patients who had these two mutations whereas the p.Ser142Phe and p.Ile360Tyrfs*31 mutations caused the severe disease manifestation.

Molecular characteristics of patients with glycosaminoglycan storage disorders in Russia.

BACKGROUND: The mucopolysaccharidoses (MPSs) are rare genetic disorders caused by mutations in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). In this study, we analyzed a total of 48 patients including MPSI (n=6), MPSII (n=18), MPSIIIA (n=11), MPSIVA (n=3), and MPSVI (n=10). METHODS: In MPS patients, urinary GAGs were colorimetrically assayed. Enzyme activity was quantified by colorimetric and fluorimetric assays. To find mutations, all IDUA, IDS, SGSH, GALNS, and ARSB exons and intronic flanks were sequenced. New mutations were functionally assessed by reconstructing mutant alleles with site-directed mutagenesis followed with expression of wild-type and mutant genetic variants in CHO cells, measuring enzymatic activity, and Western blot analysis of protein expression of normal and mutated enzymes in cell lysates. RESULTS: A total of five novel mutations were found including p.Asn348Lys (IDUA) in MPSI, p.Tyr240Cys (GALNS) in MPSIVA, and three ARSB mutations (p.Gln110*, p.Asn262Lysfs*14, and pArg315*) in MPSVI patients. In case of mutations p.Asn348Lys, p.Asn262Lysfs*14, and p.Gln110*, no mutant protein was detected while activity of the mutant protein was <1% of that of the normal enzyme. For p.Tyr240Cys, a trace of mutant protein was observed with a remnant activity of 3.6% of the wild-type GALNS activity. For pArg315*, a truncated 30-kDa protein that had 7.9% of activity of the normal ARSB was detected. CONCLUSIONS: These data further enrich our knowledge of the genetic background of MPSs.