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.

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.

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.

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.

Somatic genetic variants have been studied for several years mostly concerning cancer, where they contribute to its origin and development. It is also clear that the somatic variants load is greater in aged individuals in comparison to younger ones, pointing to a cause/consequence of the senescence process. More recently, researchers have focused on the role of this type of variation in healthy tissue and its dynamics in cell lineages and different organs. In addition, somatic variants have been described to contribute to monogenic diseases, and the number of evidences of their role in complex disorders is also increasing. Thanks to recent advances in next-generation sequencing technologies, this type of genetic variation can be now more easily studied than in the past, although we still face some important limitations. Novel strategies for sampling, sequencing and filtering are being investigated to detect these variants, although validating them with an orthogonal approach will most likely still be needed. In this review, we aim to update our knowledge of somatic variation detection and its relation to healthy tissue and non-cancer diseases.

OBJECTIVES:Peripheral neuropathy is a relevant dose-limiting adverse event that can affect up to 90% of oncologic patients with colorectal cancer receiving oxaliplatin treatment. The severity of neurotoxicity often leads to dose reduction or even premature cessation of chemotherapy. Unfortunately, the limited knowledge about the molecular mechanisms related to oxaliplatin neurotoxicity leads to a lack of effective treatments to prevent the development of this clinical condition. In this context, the present work aimed to determine the exact molecular mechanisms involved in the development of oxaliplatin neurotoxicity in a murine model to try to find new therapeutical targets.METHODS:By single-cell RNA sequencing (scRNA-seq), we studied the transcriptomic profile of sensory neurons and satellite glial cells (SGC) of the Dorsal Root Ganglia (DRG) from a well-characterized mouse model of oxaliplatin neurotoxicity.RESULTS:Analysis of scRNA-seq data pointed to modulation of inflammatory processes in response to oxaliplatin treatment. In this line, we observed increased levels of NF-kB p65 protein, pro-inflammatory cytokines, and immune cell infiltration in DRGs and peripheral nerves of oxaliplatin-treated mice, which was accompanied by mechanical allodynia and decrease in sensory nerve amplitudes.INTERPRETATION:Our data show that, in addition to the well-described DNA damage, oxaliplatin neurotoxicity is related to an exacerbated pro-inflammatory response in DRG and peripheral nerves, and open new insights in the development of anti-inflammatory strategies as a treatment for preventing peripheral neuropathy induced by oxaliplatin.

We have genotyped the 58 STRs (27 autosomal, 24 Y-STRs and 7 X-STRs) and 94 autosomal SNPs in Illumina ForenSeq (TM) Primer Mix A in a sample of 248 men and 143 women from El Salvador, Central America. Regional division (Centro, Oriente, Occidente) showed in almost all cases F-ST values not significantly different from 0, and further analyses were applied only to the undivided, country-wide population. The overall random match probability (RMP) decreased from 6.79 x 10(-31) in length-based genotypes in the 27 autosomal STRs to 1.47 x 10(-34) in repeat-sequence based genotypes. Combining the autosomal loci in this set, RMP reaches 2.97 x 10(-70). In a population genetic analysis, El Salvador showed the lowest F-ST values with US Hispanics both for autosomal and X-STRs; however, it was much closer to Native Americans for the latter than for the former, in accordance with the well-known gender-biased admixture that created most Latin American populations.

BackgroundCardiopulmonary bypass is a specialized technique requiring high technical skills and generic competencies.MethodsThe Virtual Perfusionist is a 3-dimensional serious game for training nurses in cardiopulmonary bypass management. The game design and the results of a preliminary usability analysis with a group of 28 perfusion nurse students are described. The games played, the errors, and their causes are analyzed. Students’ opinions are measured with an extended System Usability Scale.ResultsThe game was well praised by attendees who could complete the learning objectives after a short period of familiarization with the technology.ConclusionsThe game was found useable and satisfying. Further studies will analyze its effectiveness in cardiovascular perfusion education.

There are increasing evidences showing the contribution of somatic genetic variants to non-cancer diseases. However, their detection using massive parallel sequencing methods still has important limitations. In addition, the relative importance and dynamics of somatic variation in healthy tissues are not fully understood. We performed high-depth whole-exome sequencing in 16 samples from patients with a previously determined pathogenic somatic variant for a primary immunodeficiency and tested different variant callers detection ability. Subsequently, we explored the load of somatic variants in the whole blood of these individuals and validated it by amplicon-based deep sequencing. Variant callers allowing low frequency read thresholds were able to detect most of the variants, even at very low frequencies in the tissue. The genetic load of somatic coding variants detectable in whole blood is low, ranging from 1 to 2 variants in our dataset, except for one case with 17 variants compatible with clonal haematopoiesis under genetic drift. Because of the ability we demonstrated to detect this type of genetic variation, and its relevant role in disorders such as primary immunodeficiencies, we suggest considering this model of gene mosaicism in future genetic studies and considering revisiting previous massive parallel sequencing data in patients with negative results.

The fetal inflammatory response (FIR) increases the risk of perinatal brain injury, particularly in extremely low gestational age newborns (ELGANs, < 28 weeks of gestation). One of the mechanisms contributing to such a risk is a postnatal intermittent or sustained systemic inflammation (ISSI) following FIR. The link between prenatal and postnatal systemic inflammation is supported by the presence of well‐established inflammatory biomarkers in the umbilical cord and peripheral blood. However, the extent of molecular changes contributing to this association is unknown. Using RNA sequencing and mass spectrometry proteomics, we profiled the transcriptome and proteome of archived neonatal dried blood spot (DBS) specimens from 21 ELGANs. Comparing FIR‐affected and unaffected ELGANs, we identified 782 gene and 27 protein expression changes of 50% magnitude or more, and an experiment‐wide significance level below 5% false discovery rate. These expression changes confirm the robust postnatal activation of the innate immune system in FIR‐affected ELGANs and reveal for the first time an impairment of their adaptive immunity. In turn, the altered pathways provide clues about the molecular mechanisms triggering ISSI after FIR, and the onset of perinatal brain injury.DatabasesEGAS00001003635 (EGA); PXD011626 (PRIDE).

We have genotyped the 58 STRs (27 autosomal, 24 Y-STRs and 7 X-STRs) and 94 autosomal SNPs in Illumina ForenSeq™ Primer Mix A in 88 Spanish Roma (Gypsy) samples and 143 Catalans. Since this platform is based in massive parallel sequencing, we have used simple R scripts to uncover the sequence variation in the repeat region. Thus, we have found, across 58 STRs, 541 length-based alleles, which, after considering repeat-sequence variation, became 804 different alleles. All loci in both populations were in Hardy-Weinberg equilibrium. FST between both populations was 0.0178 for autosomal SNPs, 0.0146 for autosomal STRs, 0.0101 for X-STRs and 0.1866 for Y-STRs. Combined a priori statistics showed quite large; for instance, pooling all the autosomal loci, the a priori probabilities of discriminating a suspect become 1−(2.3×10−70) and 1−(5.9×10−73), for Roma and Catalans respectively, and the chances of excluding a false father in a trio are 1−(2.6×10−20) and 1−(2.0×10−21).

Next‐generation sequencing technologies have opened new opportunities in forensic genetics. Here, we assess the applicability and performance of the MiSeq FGx™ & ForenSeq™ DNA Signature Prep Kit (Illumina) for the identification of individuals from the mass graves of the Spanish Civil War (1936–1939). The main limitations for individual identification are the low number of possible first‐degree living relatives and the high levels of DNA degradation reported in previous studies. Massively parallel sequencing technologies enabling the analysis of hundreds of regions and prioritizing short length amplicons constitute a promising tool for this kind of approaches. In this study, we first explore the power of this new technology to detect first‐ and second‐degree kinship given different scenarios of DNA degradation. Second, we specifically assess its performance in a set of low DNA input samples previously analyzed with CE technologies. We conclude that this methodology will allow identification of up to second‐degree relatives, even in situations with low sequencing performance and important levels of allele drop‐out; it is thus a technology that resolves previous drawbacks and that will allow a successful approximation to the identification of remains.

Essential trace elements possess vital functions at molecular, cellular, and physiological levels in health and disease, and they are tightly regulated in the human body. In order to assess variability and potential adaptive evolution of trace element homeostasis, we quantified 18 trace elements in 150 liver samples, together with the expression levels of 90 genes and abundances of 40 proteins involved in their homeostasis. Additionally, we genotyped 169 single nucleotide polymorphism (SNPs) in the same sample set. We detected significant associations for 8 protein quantitative trait loci (pQTL), 10 expression quantitative trait loci (eQTLs), and 15 micronutrient quantitative trait loci (nutriQTL). Six of these exceeded the false discovery rate cutoff and were related to essential trace elements: 1) one pQTL for GPX2 (rs10133290); 2) two previously described eQTLs for HFE (rs12346) and SELO (rs4838862) expression; and 3) three nutriQTLs: The pathogenic C282Y mutation at HFE affecting iron (rs1800562), and two SNPs within several clustered metallothionein genes determining selenium concentration (rs1811322 and rs904773). Within the complete set of significant QTLs (which involved 30 SNPs and 20 gene regions), we identified 12 SNPs with extreme patterns of population differentiation (FST values in the top 5% percentile in at least one HapMap population pair) and significant evidence for selective sweeps involving QTLs at GPX1, SELENBP1, GPX3, SLC30A9, and SLC39A8. Overall, this detailed study of various molecular phenotypes illustrates the role of regulatory variants in explaining differences in trace element homeostasis among populations and in the human adaptive response to environmental pressures related to micronutrients.

While most patients affected by the influenza A(H1N1) pandemic experienced mild symptoms, a small fraction required hospitalization, often without concomitant factors that could explain such a severe course. We hypothesize that host genetic factors could contribute to aggravate the disease. To test this hypothesis, we compared the allele frequencies of 547,296 genome-wide single nucleotide polymorphisms (SNPs) between 49 severe and 107 mild confirmed influenza A cases, as well as against a general population sample of 549 individuals. When comparing severe vs. mild influenza A cases, only one SNP was close to the conventional p = 5×10-8. This SNP, rs28454025, sits in an intron of the GSK233 gene, which is involved in a neural development, but seems not to have any connections with immunological or inflammatory functions. Indirectly, a previous association reported with CD55 was replicated. Although sample sizes are low, we show that the statistical power in our design was sufficient to detect highly-penetrant, quasi-Mendelian genetic factors. Hence, and assuming that rs28454025 is likely to be a false positive, no major genetic factor was detected that could explain poor influenza A course.