Synaptic Ras GTPase-Activating Protein 1 (SynGAP) is a key regulator of synaptic plasticity, neurodevelopment, and neuronal circuit function. It is encoded by the SYNGAP1 gene, in which de novo dominant pathogenic variants are a major cause of SYNGAP1 Encephalopathy, a rare neurodevelopmental disorder characterised by intellectual disability, epilepsy, autistic traits, and other clinical manifestations. While some genetic studies have reported genotype-phenotype correlations in this condition, our understanding of how specific genetic variants contribute to the heterogeneous clinical symptoms remain limited. Here, we analysed a cohort of 44 cases extensively characterised at the phenotypic level to investigate the impact of genetic variants in SYNGAP1 and in potentially modulatory genes on the clinical features of SYNGAP1 Encephalopathy. Our results include the identification of four previously unreported likely pathogenic SYNGAP1 variants associated with the disease. In our cohort, individuals carrying variants within the PH domain of SynGAP exhibit milder phenotypes compared with other patients. Finally, patients harbouring rare or low-frequency variants in SYNGAP1-related genes tend to present with higher global severity. Taken together, these findings suggest that the location of SYNGAP1 variants, together with additional genetic modifiers, may contribute to variability in clinical presentation and disease severity. Further studies in larger cohorts and functional validation are needed to refine genotype-phenotype correlations and to support the development of personalized management strategies.

Epstein-Barr virus (EBV) is an oncogenic virus ubiquitous in human populations. CD8 T cells play a crucial role in establishing a strong anti-EBV immune response. Among the various inborn errors of immunity (IEI) showing a restricted vulnerability to EBV, TNFRSF9 (CD137, 4-1BB) deficiency was described in 2019 in patients with chronic EBV viremia and EBV-associated lymphoproliferative diseases. We here investigated a patient with a history of chronic EBV infection and CD137 deficiency who had previously undergone transplantation from her HLA-identical brother. We found that the brother was also a homozygous carrier of the same TNFRSF9 variant, explaining the patient's inability to control EBV after transplantation. Remarkably, during a period of spontaneous clinical improvement and EBV control, we detected two somatic variants in the patient, which resulted in the emergence of two independent revertant CD8 T cell clones that accounted for up to 20% of CD8 T cells in peripheral blood. Using single cell RNA sequencing we demonstrated that both revertant clones originated post-transplant from donor-derived cells. We report here the first described case of a somatic reversion phenomenon in TNFRSF9 deficiency, correlating with clinical improvement and paving the way for future gene therapy strategies for this IEI.

NLRP3 mosaicism is a well-established mechanism causing the monogenic autoinflammatory disease named cryopyrin-associated periodic syndromes (CAPS). The number of reported patients with NLRP3 mosaicism is small, and the knowledge about the long-term disease behavior is limited. Herein we assembled the largest cohort of individuals with NLRP3 mosaicism reported to date to obtain additional evidence that strengthens the understanding of this disease. The novel genetic data were obtained by using Sanger and next-generation sequencing methods, whereas in vitro analyses determined the functional consequences of detected variants. A total of seventeen individuals with NLRP3 mosaicism were enrolled, with 16/17 experiencing different CAPS phenotypes. An overrepresentation of late-onset forms was detected (37.5%). Overall, clinical manifestations, analytical results, and outcomes of treatments were markedly similar to those detected in patients with germline variants. A large mutational diversity was identified, with 16 different variants among 17 individuals. Two main patterns of mosaicism (extended vs. myeloid-restricted) were detected, with the last one overrepresented in the late-onset group. The evaluation of mosaicism over time identified three different patterns, being the group with stable mosaicism the largest one. Collected evidence supports the marked similarities among patients carrying somatic or germline NLRP3 variants. The overrepresentation of NLRP3 mosaicism in late-onset forms should be considered in patients with inflammatory manifestations starting in adulthood. Analysis of mosaicism at the biological level confirms the two known patterns of corporal distribution and reveals that mosaicism remains stable over time in most patients, but it may also vary during the course of the disease.

BACKGROUND: A stroke’s functional outcome presents vast variability among patients, which is influenced by age, sex, characteristics of the lesion, and genetic factors. However, there is little knowledge about stroke recovery genetics. Recently, some GWAS (Genome-Wide Association Studies) have highlighted the involvement of common or low-frequency variants near or within PATJ , PPP1R21 , PTCH1 , NTN4 , and TEK genes , whereas the role of rare variants is still unclear. This study aims to identify the genetic contributions to differences in stroke outcomes by analyzing the effect of rare variants. METHODS: We performed a pilot study analyzing 90 exomes of extreme good and bad recovery (modified Rankin Scale score at 3 months, 0–1 versus 4–5) to select target genes involved in stroke recovery. To expand this study, 702 additional samples were sequenced by targeted next-generation sequencing capturing loci selected from the pilot study, GWASs, and literature input. Here, we performed continuous (modified Rankin Scale score, 0–6) and dichotomous (modified Rankin Scale score, 0–1 versus 3–6) analyses, yielding 1 candidate gene. All samples were selected by a retrospective cohort study from incidental stroke cases collected at Spanish Hospitals between 2000 and 2018. The identified VNN2 variants were assessed for protein structure and stability analysis, and an analysis of their effect on basal inflammation levels was performed using UK Biobank data. RESULTS: Our work identified rare coding variants in VNN2 associated with patients with better stroke recovery (∆ deviance information criterion >10, equivalent to P <0.001). Six rare variants were predicted to significantly affect protein stability (∆∆G >1.6 kcal/mol); meanwhile, another variant, located in the active site, could affect the electrostatic surface. CONCLUSIONS: We propose that VNN2 might play a role in stroke outcomes by modulating poststroke inflammation. A potentially affected function would be neutrophil cell adhesion and migration.

AbstractObjectiveNLRP3mosaicism is a well-established mechanism causing cryopyrin-associated periodic syndromes (CAPS). The number of reported patients with mosaicism is small, and the knowledge about the long-term disease behavior is limited. Herein we have assembled the largest cohort of individuals withNLRP3mosaicism reported to date to obtain additional evidence that strengthens the understanding of this disease.MethodsPatients’ data were collected from their medical charts. Genetic analyses were performed using Sanger and next-generation sequencing.In vitroanalyses determined the functional consequences of detected variants.ResultsSeventeen individuals withNLRP3mosaicism were enrolled, with 16/17 experiencing different CAPS phenotypes. An overrepresentation of late-onset forms was detected (37.5%). Overall, clinical manifestations, analytical results, and outcomes of treatments were markedly similar to those detected in patients with germline variants. A large mutational diversity was identified, with 16 different variants among 17 individuals. Two main patterns of mosaicism (extended vs myeloid-restricted) were detected, with the last one overrepresented in the late-onset group. The evaluation of mosaicism over time identified three different patterns, being the group with stable mosaicism the largest one.ConclusionsCollected evidence supports the marked similarities among patients carrying somatic or germlineNLRP3variants. The overrepresentation ofNLRP3mosaicism in late-onset forms should be considered in patients with inflammatory manifestations starting in adulthood. Analysis of mosaicism at the biological level confirms the two known patterns of corporal distribution and reveals that mosaicism remains stable over time in most patients, but it may also vary during the course of the disease.Key MessagesWhat is already known about this subject?Cryopyrin-associated periodic syndromes (CAPS) is a dominantly-inherited autoinflammatory disease with three different phenotypes of increasing severity across a continuous spectrum.The disease is a consequence of monoallelic, gain-of-functionNLRP3variants leading to a constitutive hyperactivation of the NLRP3-inflammasome, with the subsequent IL-1β overproduction.Recent investigations have established the key role ofNLRP3mosaicism in the pathogenesis of CAPS. However, the number of individuals withNLRP3mosaicism reported to date is small, and the data on the long-term behavior of the disease at clinical and biological levels are scarce.What does this study add?We have assembled the largest cohort of individuals withNLRP3mosaicism reported to date to obtain novel evidence that strengthens the understanding of this syndrome.The clinical manifestations, the results of analytical tests, and the outcomes of treatments in patients withNLRP3mosaicism were in line with previous results collected from patients with germlineNLRP3variants.We have detected a previously unidentified overrepresentation of late-onset forms among patients withNLRP3mosaicism.Genetic evidence supports for a large mutational diversity among individuals withNLRP3mosaicism. Despite the fact that, theoretically, any amino acid residue in the protein may be involved in mosaicism, the regions spanning the amino acid residues 300-310 and 560-570 concentrate the larger proportion of post-zygotic variants.Additional experiments confirmed the two main patterns of mosaicism distribution (myeloid-restricted vs extended) and that the degree of mosaicism remains stable in most patients but can also change over time.How might this impact on clinical practice?Considering that genetic analyses represent the unique way to establish a CAPS diagnosis, the data presented here strongly suggest for an adequate analysis of the sequence reads of theNLRP3gene in candidate patients, which should include differential analyses for either germline and post-zygotic variants.The overrepresentation of late-onset forms among patients withNLRP3mosaicism should be taken into consideration by clinicians treating adult patients in the differential diagnosis of inflammatory diseases starting during adulthood.The evidence presented here support periodic evaluation of circulatingNLRP3mosaicism to evaluate the disease behavior at biological level over time.

Undiagnosed monogenic diseases represent a challenging group of human conditions highly suspicious to have a genetic origin, but without conclusive evidences about it. We identified two brothers born prematurely from a non-consanguineous healthy couple, with a neonatal-onset, chronic disease characterized by severe skin and bone inflammatory manifestations and a fatal outcome in infancy. We conducted DNA and mRNA analyses in the patients’ healthy relatives to identify the genetic cause of the patients’ disease. DNA analyses were performed by both Sanger and next-generation sequencing, which identified two novel heterozygous IL1RN variants: the intronic c.318 + 2T>G variant in the father and a ≈2,600-bp intragenic deletion in the mother. IL1RN mRNA production was markedly decreased in both progenitors when compared with healthy subjects. The mRNA sequencing performed in each parent identified two novel, truncated IL1RN transcripts. Additional experiments revealed a perfect intrafamilial phenotype–genotype segregation following an autosomal recessive inheritance pattern. The evidences shown here supported for the presence of two novel loss-of-function (LoF) IL1RN pathogenic variants in the analyzed family. Biallelic LoF variants at the IL1RN gene cause the deficiency of interleukin-1 receptor antagonist (DIRA), a monogenic autoinflammatory disease with marked similarities with the patients described here. Despite the non-availability of the patients’ samples representing the main limitation of this study, the collected evidences strongly suggest that the patients described here suffered from a lethal form of DIRA likely due to a compound heterozygous genotype at IL1RN, thus providing a reliable genetic diagnosis based on the integration of old medical information with currently obtained genetic data.

OBJECTIVE:Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is a complex immune disorder consequence of somatic UBA1 variants. Most reported pathogenic UBA1 variants are missense or splice site mutations directly impairing the translational start site at p.Met41, with recent studies showing that these variants are frequent causes of recurrent inflammation in older individuals. Here we aimed to characterize a novel UBA1 variant found in two patients clinically presenting with VEXAS syndrome. METHODS:Patients' data were collected from direct assessments and from their medical charts. Genomics analyses were undertaken by both Sanger and amplicon-based deep sequencing, and mRNA studies were undertaken by both cDNA subcloning and mRNA sequencing. RESULTS:We report a novel, somatic variant in a canonical splice site of the UBA1 gene (c.346-2A>G), which was identified in two unrelated adult male patients with late-onset, unexplained inflammatory manifestations including recurrent fever, Sweet syndrome-like neutrophilic dermatosis, and lung inflammation responsive only to glucocorticoids. RNA analysis of the patients' samples indicated aberrant mRNA splicing leading to multiple in-frame transcripts, including a transcript retaining the full sequence of intron 4 and a different transcript with the deletion of the first 15 nucleotides of exon 5. CONCLUSION:Here we describe abnormal UBA1 transcription as a consequence of the novel c.346-2A>G variant, identified in two patients with clinical features compatible with VEXAS syndrome. Overall, these results further demonstrate the expanding spectrum of variants in UBA1 leading to pathology and provide support for a complete gene evaluation in those patients considered candidates for VEXAS syndrome.

Functional annotation and interpretation of genetic variants are a critical step in genetic diagnosis, as it may lead to personalized therapeutic options and genetic counseling. While the number of confirmed pathogenic genetic variants in an individual is relatively low, the number of variants of uncertain significance (VOUS) can be considerably higher, increasing the number of potential carriers of genetic disorders. Thus, reducing uncertainty and assessing the real effect of VOUS are crucial for clinical and medical genetics. In this study, we evaluated the efficacy of genetic screening technologies in accurately predicting pathogenic variants and their corresponding disease prevalence in a cohort of over 6000 healthy individuals involved in assisted reproduction programs. Using data from 305 genes associated with recessive disorders, we determined the frequency of carriers of pathogenic variants and VOUS in our dataset and compared the predicted prevalence based on this information with reported population prevalence data. The higher predicted prevalence in some disorders when considering VOUS suggests a mostly benign effect.

Background The vacuoles, E1-enzyme, X linked, autoinflammatory and somatic (VEXAS) syndrome is an adult-onset autoinflammatory disease (AID) due to postzygotic UBA1 variants. Objectives To investigate the presence of VEXAS syndrome among patients with adult-onset undiagnosed AID. Additional studies evaluated the mosaicism distribution and the circulating cytokines. Methods Gene analyses were performed by both Sanger and amplicon-based deep sequencing. Patients' data were collected from their medical charts. Cytokines were quantified by Luminex. Results Genetic analyses of enrolled patients (n=42) identified 30 patients carrying UBA1 pathogenic variants, with frequencies compatible for postzygotic variants. All patients were male individuals who presented with a late-onset disease (mean 67.5 years; median 67.0 years) characterised by cutaneous lesions (90%), fever (66.7%), pulmonary manifestations (66.7%) and arthritis (53.3%). Macrocytic anaemia and increased erythrocyte sedimentation rate and ferritin were the most relevant analytical abnormalities. Glucocorticoids ameliorated the inflammatory manifestations, but most patients became glucocorticoid-dependent. Positive responses were obtained when targeting the haematopoietic component of the disease with either decitabine or allogeneic haematopoietic stem cell transplantation. Additional analyses detected the UBA1 variants in both haematopoietic and non-haematopoietic tissues. Finally, analysis of circulating cytokines did not identify inflammatory mediators of the disease. Conclusion Thirty patients with adult-onset AID were definitively diagnosed with VEXAS syndrome through genetic analyses. Despite minor interindividual differences, their main characteristics were in concordance with previous reports. We detected for the first time the UBA1 mosaicism in non-haematopoietic tissue, which questions the previous concept of myeloid-restricted mosaicism and may have conceptual consequences for the disease mechanisms.

The use of mitochondrial DNA (mtDNA) in the field of forensic genetics is widely spread mainly due to its advantages when identifying highly degraded samples. In this sense, massive parallel sequencing has made the analysis of the whole mitogenome more accessible, noticeably increasing the informativeness of mtDNA haplotypes. The civil war (1980-1992) in El Salvador caused many deaths and disappearances (including children) all across the country and the economic and social instability after the war forced many people to emigration. For this reason, different organizations have collected DNA samples from relatives with the aim of identifying missing people. Thus, we present a dataset containing 334 complete mitogenomes from the Salvadoran general population. To the best of our knowledge, this is the first publication of a nationwide forensic-quality complete mitogenome database of any Latin American country. We found 293 different haplotypes, with a random match probability of 0.0041 and 26.6 mean pairwise differences, which is similar to other Latin American populations, and which represent a marked improvement from the values obtained with just control region sequences. These haplotypes belong to 54 different haplogroups, being 91% of them of Native American origin. Over a third (35.9%) of the individuals carried at least a heteroplasmic site (excluding length heteroplasmies). Ultimately, the present database aims to represent mtDNA haplotype diversity in the general Salvadoran populations as a basis for the identification of people that disappeared during or after the civil war.

Abstract Undiagnosed monogenic diseases represent a challenging group of human conditions highly suspicious to have a genetic origin, but with not conclusive evidences about it. We identified two brothers born prematurely from a non-consanguineous healthy couple, with a neonatal-onset, chronic disease characterized by severe skin and bone inflammatory manifestations, and a fatal outcome in infancy. We conducted DNA and mRNA analyses in patients’ healthy relatives to identify the genetic basis of the patients’ disease and to characterize the detected gene variants. DNA analyses were performed by both Sanger and whole genome sequencing, and detected two novel heterozygous IL1RN variants: the intronic c.318 + 2T > G variant in the patients’ father, and a ≈2600bp intragenic deletion in the patients’ mother. IL1RN mRNA production was markedly decreased in both progenitors when compared with healthy subjects. mRNA sequencing identified two novel, truncated IL1RN mRNA transcripts. Additional experiments displayed a perfect intrafamilial phenotype-genotype segregation following an autosomal recessive inheritance pattern. The experimental evidences here shown supported for the presence of two novel loss-of-function (LoF) IL1RN variants in the analyzed family. Biallelic LoF variants at IL1RN gene cause the deficiency of interleukin-1 receptor antagonist (DIRA), a ultra-rare autoinflammatory disease with enormous clinical and analytical similarities with the patients here described. All these evidences strongly suggest that patients suffered from a lethal form of DIRA due to a compound heterozygous IL1RN genotype. However, the main limitation of this study is the non-availability of the patients’ samples, which prevented us from establishing unequivocally their definitive diagnosis.