EBS in Children with De Novo Pathogenic Variants Disturbing Krt14.

Epidermolysis bullosa simplex (EBS) is a dermatological condition marked by skin fragility and blister formation resulting from separation within the basal layer of the epidermis, which can be attributed to various genetic etiologies. This study presents three pathogenic de novo variants in young children, with clinical manifestations appearing as early as the neonatal period. The variants contribute to the EBS phenotype through two distinct mechanisms: direct keratin abnormalities due to pathogenic variants in the Krt14 gene, and indirect effects via pathogenic mutation in the KLHL24 gene, which interfere with the natural proteasome-mediated degradation pathway of KRT14. We report one severe case of EBS with mottled pigmentation arising from the Met119Thr pathogenic variant in KRT14, another case involving a pathogenic KLHL24 Met1Val variant, and a third case featuring the hot spot mutation Arg125His in KRT14, all manifesting within the first few weeks of life. This research underscores the complexity of genetic influences in EBS and highlights the importance of early genetic screening for accurate diagnosis and management.

The Search of Association of HLA Class I and Class II Alleles with COVID-19 Mortality in the Russian Cohort.

HLA genes play a pivotal role in the immune response via presenting the pathogen peptides on the cell surface in a host organism. Here, we studied the association of HLA allele variants of class I (loci A, B, C) and class II (loci DRB1, DQB1, DPB1) genes with the outcome of COVID-19 infection. We performed high-resolution sequencing of class HLA I and class II genes based on the sample population of 157 patients who died from COVID-19 and 76 patients who survived despite severe symptoms. The results were further compared with HLA genotype frequencies in the control population represented by 475 people from the Russian population. Although the obtained data revealed no significant differences between the samples at a locus level, they allowed one to uncover a set of notable alleles potentially contributing to the COVID-19 outcome. Our results did not only confirm the previously discovered fatal role of age or association of DRB1*01:01:01G and DRB1*01:02:01G alleles with severe symptoms and survival, but also allowed us to single out the DQB1*05:03:01G allele and B*14:02:01G~C*08:02:01G haplotype, which were associated with survival. Our findings showed that not only separate allele, but also their haplotype, could serve as potential markers of COVID-19 outcome and be used during triage for hospital admission.

Candidate Genes for IgA Nephropathy in Pediatric Patients: Exome-Wide Association Study.

IgA nephropathy (IgAN) is an autoimmune disorder which is believed to be non-monogenic. We performed an exome-wide association study of 70 children with IgAN and 637 healthy donors. The HLA allele frequencies were compared between the patients and healthy donors from the bone marrow registry of the Pirogov University. We tested 78,020 gene markers for association and performed functional enrichment analysis and transcription factor binding preference detection. We identified 333 genetic variants, employing three inheritance models. The most significant association with the disorder was observed for rs143409664 (PRAG1) in the case of the additive and dominant models (PBONF = 1.808 × 10-15 and PBONF = 1.654 × 10-15, respectively), and for rs13028230 (UBR3) in the case of the recessive model (PBONF = 1.545 × 10-9). Enrichment analysis indicated the strongly overrepresented "immune system" and "kidney development" terms. The HLA-DQA1*01:01:01G allele (p = 0.0076; OR, 2.021 [95% CI, 1.322-3.048]) was significantly the most frequent among IgAN patients. Here, we characterized, for the first time, the genetic background of Russian IgAN patients, identifying the risk alleles typical of the population. The most important signals were detected in previously undescribed loci.

Performance comparison of Agilent new SureSelect All Exon v8 probes with v7 probes for exome sequencing.

Exome sequencing is becoming a routine in health care, because it increases the chance of pinpointing the genetic cause of an individual patient's condition and thus making an accurate diagnosis. It is important for facilities providing genetic services to keep track of changes in the technology of exome capture in order to maximize throughput while reducing cost per sample. In this study, we focused on comparing the newly released exome probe set Agilent SureSelect Human All Exon v8 and the previous probe set v7. In preparation for higher throughput of exome sequencing using the DNBSEQ-G400, we evaluated target design, coverage statistics, and variants across these two different exome capture products. Although the target size of the v8 design has not changed much compared to the v7 design (35.24 Mb vs 35.8 Mb), the v8 probe design allows you to call more of SNVs (+ 3.06%) and indels (+ 8.49%) with the same number of raw reads per sample on the common target regions (34.84 Mb). Our results suggest that the new Agilent v8 probe set for exome sequencing yields better data quality than the current Agilent v7 set.

Diplocloster agilis gen. nov., sp. nov. and Diplocloster modestus sp. nov., two novel anaerobic fermentative members of Lachnospiraceae isolated from human faeces.

Three novel strains of Gram-stain-negative, obligately anaerobic, spore-forming straight or slightly curved rods with pointed ends occurring singly or in pairs were isolated from the faeces of healthy human children. The strains were characterized by mesophilic fermentative metabolism and production of acetate, ethanol and H2 as the end metabolic products. Strains ASD3451 and ASD5720T were motile, fermented lactose and raffinose, and weakly fermented maltose. Strain ASD4241T was non-motile and did not ferment the carbohydrates listed above but fermented starch. Strains ASD3451 and ASD5720T shared average nucleotide identity higher than 98.5 % with each other, while ASD4241T had only 88.5-89 % identity to them. Based on phylogenetic and chemotaxonomic analyses, we propose Diplocloster agilis gen. nov., sp. nov. (ASD5720T=JCM 34353T=VKM B-3497T) and Diplocloster modestus sp. nov. (ASD4241T=JCM 34351T=VKM B-3498T) within the family Lachnospiraceae.

The newborns Torque teno virus dynamics depending on the term, feeding type and maternal viral load.

The first weeks of life are extremely important for the development of the immunity-virome interaction that affects human health in adulthood. In this study we analyzed Torque teno virus (TTV) dynamics during the first weeks of life in the full-term/premature infants in relation with the maternal TTV load and the type of feeding. 152 infants aged 1-14 weeks (63 full-term and 89 premature) and 33 mother-child pairs were analyzed for the whole blood TTV load by qPCR with test sensitivity of 1000 viral copies/ml. 50 infants were retested (at 2-11 time points) for TTV dynamics data. All one-week babies (n = 71) from TTV-positive mothers were TTV-negative, consistently with the previous findings of the lack of transplacental transmission of the virus. TTV was not detectable in newborns under two weeks of age. Most infants are TTV-positive by 14 weeks of age. Whole blood TTV load does not show significant correlation with full-term/prematurity, maternal TTV load, or feeding type. Keywords: Torque teno virus; transfusion-transmitted virus; commensal virus; TTV; viral load dynamics; TORCH infections; full-term and premature babies; breastfeeding; virome.

Constant companion: clinical and developmental aspects of torque teno virus infections.

Torque teno virus (TTV) is a commensal human virus observed as a circular single-negative-strand DNA molecule in various tissues and biological samples, notably in blood serum and lymphocytes. TTV has no apparent clinical significance, although it might be very useful as a prospective tool for gene delivery or as an epidemiological marker. Human populations are ubiquitously infected with TTV; the prevalence may reach 100%. The majority of babies become spontaneously infected with TTV, so that by the end of the first year of life, the prevalence reaches 'adult' values. TTV positivity in healthy early infancy and the presence of TTV in umbilical cord blood samples have been reported. The mechanism of infection and the dynamics of TTV prevalence in infants with age remain understudied. Meanwhile, the potential diagnostic and prognostic value of TTV as a marker deserves special attention and study, along with the possibility, causes and consequences of placental transmission of TTV under normal or pathological conditions.

Oxysterol/chitotriosidase based selective screening for Niemann-Pick type C in infantile cholestasis syndrome patients.

BACKGROUND: Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disease (1 per 100 000 newborns) caused by NPC proteins impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. To date the NP-C diagnostics is usually based on cholesterol detection in fibroblasts using an invasive and time-consuming Filipin staining and we need more arguments to widely introduce oxysterols as a biomarkers in NP-C. METHODS: Insofar as NP-C represents about 8% of all infant cholestases, in this prospective observational study we tried to re-assess the specificity plasma oxysterol and chitotriosidase as a biochemical screening markers of NP-C in children with cholestasis syndrome of unknown origin. For 108 patients (aged from 2 weeks to 7 years) the levels of cholestane-3ß,5α,6ß-triol (C-triol) and chitotriosidase (ChT) were measured. For patients with elevated C-triol and/or ChT the NPC1 and NPC2 genes were Sanger-sequenced and 47 additional genes (from the custom liver damage panel) were NGS-sequenced. RESULTS: Increased C-triol level (> 50 ng/ml) was detected in 4 (of 108) infants with cholestasis syndrome of unknown origin, with following molecular genetic NP-C diagnosis for one patient. Plasma cholesterol significantly correlates with C-triol (p < 0.05). NGS of high C-triol infants identified three patients with mutations in JAG1 (Alagille syndrome) and ABCB11 (Byler disease) genes. Increased ChT activity was detected in 8 (of 108) patients with various aetiologies, including NP-C, Byler disease and biliary atresia. CONCLUSION: Combined analysis of ChT activity and C-triol levels is an effective method for identifying NP-C.

CD8+ T cells with characteristic T cell receptor beta motif are detected in blood and expanded in synovial fluid of ankylosing spondylitis patients.

Objective: The risk of AS is associated with genomic variants related to antigen presentation and specific cytokine signalling pathways, suggesting the involvement of cellular immunity in disease initiation/progression. The aim of the present study was to explore the repertoire of TCR sequences in healthy donors and AS patients to uncover AS-linked TCR variants. Methods: Using quantitative molecular-barcoded 5'-RACE, we performed deep TCR ß repertoire profiling of peripheral blood (PB) and SF samples for 25 AS patients and 108 healthy donors. AS-linked TCR variants were identified using a new computational approach that relies on a probabilistic model of the VDJ rearrangement process. Results: Using the donor-agnostic probabilistic model, we reveal a TCR ß motif characteristic for PB of AS patients, represented by eight highly homologous amino acid sequence variants. Some of these variants were previously reported in SF and PB of patients with ReA and in PB of AS patients. We demonstrate that identified AS-linked clones have a CD8+ phenotype, present at relatively low frequencies in PB, and are significantly enriched in matched SF samples of AS patients. Conclusion: Our results suggest the involvement of a particular antigen-specific subset of CD8+ T cells in AS pathogenesis, confirming and expanding earlier findings. The high similarity of the clonotypes with the ones found in ReA implies common mechanisms for the initiation of the diseases.

Torque teno virus dynamics during the first year of life.

BACKGROUND: Torque teno virus is a small chronically persisting circular negative ssDNA virus reaching near 100% prevalence. It is reported to be a marker for immune function in immunocompromised patients. The possibility of vertical maternal-fetal transmission remains controversial but incidence rate of TTV DNA in children increased with age. TTV dynamics well studied for allogeneic hematopoietic stem cell transplantation as a predictor of post-transplant complications but there is no viral proliferation kinetics data for other patient groups or healthy individuals. The aim of this study was to determine TTV dynamics during the first year of life of healthy infants. METHODS: Ninety eight clinically healthy breastfeeding infants (1-12 months of age) were analyzed by quantitative PCR for the whole blood TTV load with the test sensitivity of about 1000 viral copies per milliliter of blood (total number of samples including repeatedly tested infants was 109). RESULTS: 67% of all analyzed samples were TTV-positive demonstrating significant positive correlation between age and TTV load (r = 0.81, p < 0.01). CONCLUSIONS: This is the first study to suggest that viral load increases during the first year of life reaching a plateau after 6 months with strong proliferation for the first 60 days. Our data well correlates with TTV dynamics in patients following allogeneic hematopoietic stem cell transplantation.

Dynamics of Individual T Cell Repertoires: From Cord Blood to Centenarians.

The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRß repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.

Transplacental transmission of torque teno virus.

BACKGROUND: TTV has been detected in almost every human tissue type or body fluid reaching near 100% prevalence. Several studies report mother-to-child postnatal transmission of TTV in infancy but the risk of transplacental transmission of TTV is still unclear. METHODS: The blood and plasma collected postpartum from 100 mother-child pairs were analyzed using TTV-specific qPCR. Samples were collected from the peripheral vein of the mother and the umbilical cord. RESULTS: Eighty four percent of pregnant women were TTV positive (median titers: 8 × 104 copies/mL; range: 103 - 3 × 107). The TTV load in plasma was approximately 100 times lower than in whole blood. TTV was not detected in any of cord blood samples. CONCLUSIONS: Our data demonstrate the lack of transplacental transmission of TTV (or effective prenatal inhibition of viral proliferation). The presence of the virus in infants may be associated with mother-to-child transmission through breast feeding or other routes of transmission.

Microbiome markers in HPV-positive and HPV-negative women of reproductive age with ASCUS and SIL determined by V4 region of 16S rRNA gene sequencing.

Introduction: Human papilloma virus (HPV) is the most common sexually transmitted infection worldwide. Cervicovaginal microbiota plays an important role in HPV infection and is associated with the development of squamous intraepithelial lesions (SIL). The natural history of cervical cancer involves reversible changes in the cervical tissue from a normal state, in which no neoplastic changes are detected in the squamous epithelium, to varying states of cellular abnormalities that ultimately lead to cervical cancer. Low-grade SIL (LSIL), like another cytological category - atypical squamous cells of undetermined significance (ASCUS), may progress to high-grade SIL (HSIL) and invasive cervical cancer or may regress to a normal state. Methods: In this work, we studied cervical canal microbiome in 165 HPV-positive and HPV-negative women of a reproductive age with ASCUS [HPV(+) n = 29; HPV(-) n = 11], LSIL [HPV(+) n = 32; HPV(-) n = 25], HSIL [HPV(+) n = 46], and the control group with negative for intraepithelial lesion malignancy (NILM) [HPV(-) n = 22]. Results and Discussion: HPV16 is the most prevalent HPV type. We have not found any differences between diversity in studied groups, but several genus [like Prevotella (p-value = 0.026), Gardnerella (p-value = 0.003), Fannyhessea (p-value = 0.024)] more often occurred in HSIL group compared by NILM or LSIL regardless of HPV. We have found statistically significant difference in occurrence or proportion of bacterial genus in studied groups. We also identified that increasing of the ratio of Lactobacillus iners or age of patient lead to higher chance to HSIL, while increasing of the ratio of Lactobacillus crispatus lead to higher chance to LSIL. Patients with a moderate dysbiosis equally often had either of three types of vaginal microbial communities (CST, Community State Type) with the prevalence of Lactobacillus crispatus (CST I), Lactobacillus gasseri (CST II), and Lactobacillus iners (CST III); whereas severe dysbiosis is linked with CST IV involving the microorganisms genera associated with bacterial vaginosis and aerobic vaginitis: Gardnerella, Fannyhessea, Dialister, Sneathia, Anaerococcus, Megasphaera, Prevotella, Finegoldia, Peptoniphilus, Porphyromonas, Parvimonas, and Streptococcus.

Combined Predictors of Long-Term Outcomes of Kasai Surgery in Infants with Biliary Atresia.

Purpose: Biliary atresia (BA) is the leading cause of neonatal cholestasis (25-45%). The primary treatment is hepatic portoenterostomy (Kasai procedure), but only 20-40% provide long-term benefits. This study aimed to develop a predictive model for surgical efficacy by comparing preoperative and early postoperative indicators in infants with different outcomes. Methods: We enrolled 166 infants with BA (93 girls, 73 boys) who underwent the Kasai procedure between September 2002 and December 2021, dividing them into favorable or adverse outcome groups. Over 40 parameters were measured, and the diagnostic significance of the prognostic model was evaluated. Results: Kasai surgery was efficacious in 69 patients (42%) and non-efficacious in 97 (58%). Our model assesses efficacy by day 14 after surgery, improving on the <34 µmol/L direct bilirubin threshold established for 3-6 months after the procedure. Including the Desmet fibrosis score refined the model. Conclusion: Blood cholesterol below 5.41 mmol/L, direct bilirubin below 56.3 µmol/L on postoperative days 14±3, and a low Desmet score indicate a high probability of efficacious Kasai surgery in infants with BA.

Expanding phenotype of MED13-associated syndrome presenting novel de novo missense variant in a patient with multiple congenital anomalies.

BACKGROUND: Whole exome sequencing allows rapid identification of causative single nucleotide variants and short insertions/deletions in children with congenital anomalies and/or intellectual disability, which aids in accurate diagnosis, prognosis, appropriate therapeutic interventions, and family counselling. Recently, de novo variants in the MED13 gene were described in patients with an intellectual developmental disorder that included global developmental delay, mild congenital heart anomalies, and hearing and vision problems in some patients. RESULTS: Here we describe an infant who carried a de novo p.Pro835Ser missense variant in the MED13 gene, according to whole exome trio sequencing. He presented with congenital heart anomalies, dysmorphic features, hydrocephalic changes, hypoplastic corpus callosum, bilateral optic nerve atrophy, optic chiasm atrophy, brain stem atrophy, and overall a more severe condition compared to previously described patients. CONCLUSIONS: Therefore, we propose to expand the MED13-associated phenotype to include severe complications that could end up with multiple organ failure and neonatal death.

WES-based screening of 7,000 newborns: A pilot study in Russia.

The effective implementation of whole-exome sequencing- and whole-genome sequencing-based diagnostics in the management of children affected with genetic diseases and the rapid decrease in the cost of next-generation sequencing (NGS) enables the expansion of this method to newborn genetic screening programs. Such NGS-based screening greatly increases the number of diseases that can be detected compared to conventional newborn screening, as the latter is aimed at early detection of a limited number of inborn diseases. Moreover, genetic testing provides new possibilities for family members of the proband, as many variants responsible for adult-onset conditions are inherited from the parents. However, the idea of NGS-based screening in healthy children raises issues of medical and ethical integrity as well as technical questions, including interpretation of the observed variants. Pilot studies have shown that both parents and medical professionals have moved forward and are enthused about these new possibilities. However, either the number of participants or the number of genes studied in previous investigations thus far has been limited to a few hundred, restricting the scope of potential findings. Our current study (NCT05325749) includes 7,000 apparently healthy infants born at our center between February 2021 and May 2023, who were screened for pathogenic variants in 2,350 genes. Clinically significant variants associated with early-onset diseases that can be treated, prevented, or where symptoms can be alleviated with timely introduced symptomatic therapy, were observed in 0.9% of phenotypically normal infants, 2.1% of the screened newborns were found to carry variants associated with reduced penetrance or monogenic diseases of adult-onset and/or variable expressivity, and 0.3% had chromosomal abnormalities. Here, we report our results and address questions regarding the interpretation of variants in newborns who were presumed to be healthy.

Description of the novel HLA-C allele, HLA-C*12:376, identified in a deceased COVID-19 patient.

The new allele HLA-C*12:376 showed one nonsynonymous nucleotide difference compared with the C*12:03:01:01 allele in codon 30.

CAMK2D De Novo Missense Variant in Patient with Syndromic Neurodevelopmental Disorder: A Case Report.

BACKGROUND: Intellectual disability with developmental delay is the most common developmental disorder. However, this diagnosis is rarely associated with congenital cardiomyopathy. In the current report, we present the case of a patient suffering from dilated cardiomyopathy and developmental delay. METHODS: Neurological pathology in a newborn was diagnosed immediately after birth, and the acquisition of psychomotor skills lagged behind by 3-4 months during the first year of life. WES analysis of the proband did not reveal a causal variant, so the search was extended to trio. RESULTS: Trio sequencing revealed a de novo missense variant in the CAMK2D gene (p.Arg275His), that is, according to the OMIM database and available literature, not currently associated with any specific inborn disease. The expression of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) protein is known to be increased in the heart tissues from patients with dilated cardiomyopathy. The functional effect of the CaMKIIδ Arg275His mutant was recently reported; however, no specific mechanism of its pathogenicity was proposed. A structural analysis and comparison of available three-dimensional structures of CaMKIIδ confirmed the probable pathogenicity of the observed missense variant. CONCLUSIONS: We suggest that the CaMKIIδ Arg275His variant is highly likely the cause of dilated cardiomyopathy and neurodevelopmental disorders.

De Novo Variant in the KCNJ9 Gene as a Possible Cause of Neonatal Seizures.

BACKGROUND: The reduction in next-generation sequencing (NGS) costs allows for using this method for newborn screening for monogenic diseases (MDs). In this report, we describe a clinical case of a newborn participating in the EXAMEN project (ClinicalTrials.gov Identifier: NCT05325749). METHODS: The child presented with convulsive syndrome on the third day of life. Generalized convulsive seizures were accompanied by electroencephalographic patterns corresponding to epileptiform activity. Proband WES expanded to trio sequencing was performed. RESULTS: A differential diagnosis was made between symptomatic (dysmetabolic, structural, infectious) neonatal seizures and benign neonatal seizures. There were no data in favor of the dysmetabolic, structural, or infectious nature of seizures. Molecular karyotyping and whole exome sequencing were not informative. Trio WES revealed a de novo variant in the KCNJ9 gene (1:160087612T > C, p.Phe326Ser, NM_004983), for which, according to the OMIM database, no association with the disease has been described to date. Three-dimensional modeling was used to predict the structure of the KCNJ9 protein using the known structure of its homologs. According to the predictions, Phe326Ser change possibly disrupts the hydrophobic contacts with the valine side chain. Destabilization of the neighboring structures may undermine the formation of GIRK2/GIRK3 tetramers necessary for their proper functioning. CONCLUSIONS: We believe that the identified variant may be the cause of the disease in this patient but further studies, including the search for other patients with the KCNJ9 variants, are needed.

System analysis of the sequencing quality of human whole exome samples on BGI NGS platform.

Human exome sequencing is a classical method used in most medical genetic applications. The leaders in the field are the manufacturers of enrichment kits based on hybridization of cRNA or cDNA biotinylated probes specific for a genomic region of interest. Recently, the platforms manufactured by the Chinese company MGI Tech have become widespread in Europe and Asia. The reliability and quality of the obtained data are already beyond any doubt. However, only a few kits compatible with these sequencers can be used for such specific tasks as exome sequencing. We developed our own solution for library pre-capture pooling and exome enrichment with Agilent probes. In this work, using a set of the standard benchmark samples from the Platinum Genome collection, we demonstrate that the qualitative and quantitative parameters of our protocol which we called "RSMU_exome" exceed those of the MGI Tech kit. Our protocol allows for identifying more SNV and indels, generates fewer PCR duplicates, enables pooling of more samples in a single enrichment procedure, and requires less raw data to obtain results comparable with the MGI Tech's protocol. The cost of our protocol is also lower than that of MGI Tech's solution.