Systemic auto-inflammatory disorders (SAIDs) are a heterogeneous group of monogenic diseases sharing a primary dysfunction of the innate immune system. More than 50% of patients with SAID does not ...show any mutation at gene(s) tested because of lack of precise clinical classification criteria and/or incomplete gene screening. To improve the molecular diagnosis and genotype interpretation of SAIDs, we undertook the development of a next-generation sequencing (NGS)-based protocol designed to simultaneous screening of 10 genes.
Fifty patients with SAID, already genotyped for the respective causative gene(s), were massively sequenced for the coding portions of MEFV, MVK, TNFRSF1A, NLRP3, NLRP12, NOD2, PSTPIP1, IL1RN, LPIN2 and PSMB8. Three different bioinformatic pipelines (Ion Reporter, CLC Bio Genomics Workbench, GATK-based in-house workflow) were compared.
Once resulting variants were compared with the expected mutation list, no workflow turned out to be able to detect all the 79 variants known in the 50 DNAs. Additional variants were also detected, validated by Sanger sequencing and compared to assess true and false positive detection rates of the three workflows. Finally, the overall clinical picture of 34 patients was re-evaluated in the light of the new mutations found.
The present gene panel has resulted suitable for molecular diagnosis of SAIDs. Moreover, genotype-phenotype correlation has confirmed that the interpretation of NGS data in patients with an undefined inflammatory phenotype is remarkably difficult, thus supporting the need of evidence-based and validated clinical criteria to be used concurrently with the genetic analysis for the final diagnosis and classification of patients with SAIDs.
Alexander disease is a leukodystrophy caused by heterozygous mutations of
gene. Recurrence in siblings from healthy parents provides a confirmation to the transmission of variants through germinal ...mosaicism. With the use of DNA isolated from peripheral blood, next-generation sequencing (NGS) of
locus was performed with deep coverage (≥500×) in 11 probands and their parents (trios) with probands heterozygous for apparently
mutations. Indeed, one parent had somatic mosaicism, estimated in the range of 8.9%-16%, for the mutant allele transmitted to the affected sibling. Parental germline mosaicism deserves attention, as it is critical in assessing the risk of recurrence in families with Alexander disease.
Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the "dysglycaemia ...panel" composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants.
T-cell-based immunotherapy strategies have profoundly improved the clinical management of several solid tumors and hematological malignancies. A recently developed and promising immunotherapy ...approach is to redirect polyclonal MHC-unrestricted T lymphocytes toward cancer cells by bispecific antibodies (bsAbs) that engage the CD3 complex and a tumor-associated antigen (TAA). The TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) is an attractive immunotherapy target, frequently expressed by neoplastic cells, that we decided to exploit as a TAA. We found that a TRAIL-R2xCD3 bsAb efficiently activates T cells and specifically redirect their cytotoxicity against cancer cells of different origins
, thereby demonstrating its potential as a pan-carcinoma reagent. Moreover, to mimic
conditions, we assessed its ability to retarget T-cell activity in an
model of ovarian cancer patients' ascitic fluids containing both effector and target cells-albeit with a suboptimal effector-to-target ratio-with remarkable results.
Deficiency of the natural antagonist of interleukin-1 was first described in 2009 and so far 20 patients has been reported. In Brazil just two cases have been reported both carrying the same ...homozygous 15 bp deletion. Blocking interleukin-1 has changed rate survival for DIRA patients. The use of anakinra and rilonacept has been reported safe and efficient, whereas the selective blockade of interleukin-1 beta, using the monoclonal antibody canakinumab has been reported in a single case only.
Here we report a case of a 7 years old Brazilian boy that presented with recurrent episodes of systemic inflammation with severe disabling osteomyelitis with mild pustular skin rash. A Next Generation Sequencing gene panel allowed to detect two pathogenic mutations in the IL1RN gene, described in compound heterozygosity. Corticosteroids was effective in controlling inflammation and anti-IL1 beta blocker triggered disease flare. Complete clinical control could be achieved using IL-1 receptor antagonist.
DIRA is a severe, life threatening autoinflammatory condition with low numbers of patients described all over the world. The mutation p.Asp72_Ile76del in IL1RN is presented in all Brazilian DIRA patients already described and p.Q45* (rs1019766125) is a new mutation affecting the IL1RN gene. Following the pathogenesis of DIRA, blocking both subunits of interleukin one as well as antagonizing the receptor using anakinra or rilonacept seems to be effective. There is just one report using canakinumab for the treatment of DIRA and this is the first report of disease flare using this drug.
Neurofibromatosis type 1 (NF1) is a proteiform genetic condition caused by pathogenic variants in
and characterized by a heterogeneous phenotypic presentation. Relevant genotype-phenotype ...correlations have recently emerged, but only few pertinent studies are available. We retrospectively reviewed clinical, instrumental, and genetic data from a cohort of 583 individuals meeting at least 1 diagnostic National Institutes of Health (NIH) criterion for NF1. Of these, 365 subjects fulfilled ≥2 NIH criteria, including 235 pediatric patients. Genetic testing was performed through cDNA-based sequencing, Next Generation Sequencing (NGS), and Multiplex Ligation-dependent Probe Amplification (MLPA). Uni- and multivariate statistical analysis was used to investigate genotype-phenotype correlations. Among patients fulfilling ≥ 2 NIH criteria, causative single nucleotide variants (SNVs) and copy number variations (CNVs) were detected in 267/365 (73.2%) and 20/365 (5.5%) cases. Missense variants negatively correlated with neurofibromas (
= 0.005). Skeletal abnormalities were associated with whole gene deletions (
= 0.05) and frameshift variants (
= 0.006). The c.3721C>T; p.(R1241*) variant positively correlated with structural brain alterations (
= 0.031), whereas Lisch nodules (
= 0.05) and endocrinological disorders (
= 0.043) were associated with the c.6855C>A; p.(Y2285*) variant. We identified novel NF1 genotype-phenotype correlations and provided an overview of known associations, supporting their potential relevance in the implementation of patient management.
Inborn errors of immunity (IEI) include a large group of inherited diseases sharing either poor, dysregulated, or absent and/or acquired function in one or more components of the immune system. ...Next-generation sequencing (NGS) has driven a rapid increase in the recognition of such defects, though the wide heterogeneity of genetically diverse but phenotypically overlapping diseases has often prevented the molecular characterization of the most complex patients. Two hundred and seventy-two patients were submitted to three successive NGS-based gene panels composed of 58, 146, and 312 genes. Along with pathogenic and likely pathogenic causative gene variants, accounting for the corresponding disorders (37/272 patients, 13.6%), a number of either rare (probably) damaging variants in genes unrelated to patients' phenotype, variants of unknown significance (VUS) in genes consistent with their clinics, or apparently inconsistent benign, likely benign, or VUS variants were also detected. Finally, a remarkable amount of yet unreported variants of unknown significance were also found, often recurring in our dataset. The NGS approach demonstrated an expected IEI diagnostic rate. However, defining the appropriate list of genes for these panels may not be straightforward, and the application of unbiased approaches should be taken into consideration, especially when patients show atypical clinical pictures.
Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder caused by mutations in
gene, coding for neurofibromin 1. NF1 can be associated with Moyamoya disease (MMD), and this association, typical ...of paediatric patients, is referred to as Moyamoya syndrome (MMS). MMD is a cerebral arteriopathy characterized by the occlusion of intracranial arteries and collateral vessel formation, which increase the risk of ischemic and hemorrhagic events.
gene mutations have been associated with MMD, so we investigated whether rare variants of
could act as genetic modifiers of MMS phenotype in a pediatric cohort of 20 MMS children, 25 children affected by isolated MMD and 47 affected only by isolated NF1. By next-generation re-sequencing (NGS) of patients' DNA and gene burden tests, we found that
seems to play a role only for MMD occurrence, while it does not appear to be involved in the increased risk of Moyamoya for MMS patients. We postulated that the loss of neurofibromin 1 can be enough for the excessive proliferation of vascular smooth muscle cells, causing Moyamoya arteriopathy associated with NF1. Further studies will be crucial to support these findings and to elucidate the possible role of other genes, enhancing our knowledge about pathogenesis and treatment of MMS.
Global warming is facilitating the poleward range expansion of plant and animal species. In the Mediterranean Sea, the concurrent temperature increase and abundance of (sub)tropical non-indigenous ...species (NIS) is leading to the so-called ‘tropicalization’ of the Mediterranean Sea, which is dramatically evident in the south-eastern sectors of the basin. At the same time, the colder north-western sectors of the basin have been said to undergo a process of ‘meridionalization’, that is the establishment of warm-water native species (WWN) previously restricted to the southern sectors. The Gulf of Genoa (Ligurian Sea) is the north-western reach for southern species of whatever origin in the Mediterranean. Recent (up to 2015) observations of NIS and WWN by diving have been collated to update previous similar inventories. In addition, the relative occurrences of both groups of southern species have been monitored by snorkelling between 2009 and 2015 in shallow rocky reefs at Genoa, and compared with the trend in air and sea surface temperatures. A total of 20 southern species (11 NIS and 9 WWN) was found. Two WWN (the zebra seabream Diplodus cervinus and the parrotfish Sparisoma cretense) and three NIS (the SW Atlantic sponge Paraleucilla magna, the Red Sea polychaete Branchiomma luctuosum, and the amphi-American and amphi-Atlantic crab Percnon gibbesi) are new records for the Ligurian Sea, whereas juveniles of the Indo-Pacific bluespotted cornetfish Fistularia commersonii have been found for the first time. While temperature has kept on increasing for the whole period, with 2014 and 2015 being the warmest years since at least 1950, the number of WWN increased linearly, that of NIS increased exponentially, contradicting the idea of meridionalization and supporting that of tropicalization even in the northern sectors of the Mediterranean basin.
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