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Stabilisation ponds are the most frequently used wastewater treatment technology in Argentina. This study focuses on the performance of two maturation ponds (MPs) that are part of the full-scale sewage treatment system of Puerto Madryn. Seventy-seven shots of surface water were analysed for organic matter, inorganic nitrogen, phytoplankton dynamics and bacterial removal. The system presented a clear evolution with respect to oxygenation and phytoplankton development. The treated wastewater reached values above 8 mg-O2/L, an important organic matter removal, and this was accompanied by a strong increase in pH. [ABSTRACT FROM AUTHOR]

Thrombocytopenia 4 (THC4) is an autosomal-dominant thrombocytopenia caused by mutations in CYCS, the gene encoding cytochrome c (CYCS), a small haeme protein essential for electron transport in mitochondria and cell apoptosis. THC4 is considered an extremely rare condition since only a few patients have been reported so far. These subjects presented mild thrombocytopenia and no or mild bleeding tendency. In this study, we describe six Italian families with five different heterozygous missense CYCS variants: p.Gly42Ser and p.Tyr49His previously associated with THC4, and three novel variants (p.Ala52Thr, p.Arg92Gly, and p.Leu99Val), which have been classified as pathogenic by bioinformatics and segregation analyses. Moreover, we supported functional effects of p.Ala52Thr and p.Arg92Gly on oxidative growth and respiratory activity in a yeast model. The clinical characterization of the 22 affected individuals, the largest series of THC4 patients ever reported, showed that this disorder is characterized by mild-to-moderate thrombocytopenia, normal platelet size, and function, low risk of bleeding, and no additional clinical phenotypes associated with reduced platelet count. Finally, we describe a significant correlation between the region of CYCS affected by mutations and the extent of thrombocytopenia, which could reflect different degrees of impairment of CYCS functions caused by different pathogenetic variants., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

ACTN1-related thrombocytopenia is a rare disorder caused by heterozygous variants in the ACTN1 gene characterized by macrothrombocytopenia and mild bleeding tendency. We describe for the first time two patients affected with ACTN1-RT caused by a homozygous variant in ACTN1 (c.982G>A) with mild heart valve defects unexplained by any other genetic variants investigated by WES. Within the reported family, the homozygous sisters have moderate thrombocytopenia and marked platelet macrocytosis with giant platelets, revealing a more severe haematological phenotype compared to their heterozygous relatives and highlighting a significant effect of allelic burden on platelet size. Moreover, we hypothesize that some ACTN1 variants, especially when present in the homozygous state, may also contribute to the cardiac abnormalities., (© 2024 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

MECOM-associated syndrome (MECOM-AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B-cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP-1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM-AS and CAMT., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Somatic mosaicism appears as a recurrent phenomenon among patients suffering from Fanconi anemia (FA), but its direct prognostic significance mostly remains an open question. The clinical picture of FA mosaic subjects could indeed vary from just mild features to severe hematologic failure. Here, we illustrate the case of a proband whose FA familiarity, modest signs (absence of hematological anomalies and fertility issues), and chromosome fragility test transition to negative overtime were suggestive of somatic mosaicism. In line with this hypothesis, genetic testing on patient's peripheral blood and buccal swab reported the presence of the only FANCA paternal variant (FANCA:c.2638C>T, p. Arg880*) and of both parental alleles (the additional FANCA:c.3164G>A, p. Arg1055Gln), respectively. Moreover, the SNP analysis performed on the same biological specimens allowed us to attribute the proband's mosaicism status to a possible gene conversion mechanism. Our case clearly depicts the positive association between somatic mosaicism and the proband's favorable clinical course due to the occurrence of the reversion event at the hematopoietic stem cell level. Since this condition concerns only a limited subgroup of FA individuals, the accurate evaluation of the origin and extent of clonality would be key to steer clinicians toward the most appropriate therapeutic decision for their FA mosaic patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Persico, Fiscarelli, Pelle, Faleschini, Pasini, Savoia and Bottega.)

Introduction: Fanconi anemia (FA) is a genome instability condition that drives somatic mosaicism in up to 25% of all patients, a phenomenon now acknowledged as a good prognostic factor. Herein, we describe the case of P1, a FA proband carrying a splicing variant, molecularly compensated by a de novo insertion. Methods and Results: Targeted next-generation sequencing on P1's peripheral blood DNA detected the known FANCA c.2778 + 83C > G intronic mutation and suggested the presence of a large deletion on the other allele, which was then assessed by MLPA and RT-PCR. To determine the c.2778 + 83C > G splicing effect, we performed a RT-PCR on P1's lymphoblastoid cell line (LCL) and on the LCL of another patient (P2) carrying the same variant. Although we confirmed the expected alternative spliced form with a partial intronic retention in P2, we detected no aberrant products in P1's sample. Sequencing of P1's LCL DNA allowed identification of the de novo c.2778 + 86insT variant, predicted to compensate 2778 + 83C > G impact. Albeit not found in P1's bone marrow (BM) DNA, c.2778 + 86insT was detected in a second P1's LCL established afterward, suggesting its occurrence at a low level in vivo . Minigene assay recapitulated the c.2778 + 83C > G effect on splicing and the compensatory role of c.2778 + 86insT in re-establishing the physiological mechanism. Accordingly, P1's LCL under mitomycin C selection preserved the FA pathway activity in terms of FANCD2 monoubiquitination and cell survival. Discussion: Our findings prove the role of c.2778 + 86insT as a second-site variant capable of rescuing c.2778 + 83C > G pathogenicity in vitro , which might contribute to a slow hematopoietic deterioration and a mild hematologic evolution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Persico, Fontana, Faleschini, Zanchetta, Ammeti, Cappelli, Corsolini, Mosa, Guarina, Bogliolo, Surrallés, Dufour, Farruggia, Savoia and Bottega.)

Inherited thrombocytopenias (IT) are genetic diseases characterized by low platelet count, sometimes associated with congenital defects or a predisposition to develop additional conditions. Next-generation sequencing has substantially improved our knowledge of IT, with more than 40 genes identified so far, but obtaining a molecular diagnosis remains a challenge especially for patients with non-syndromic forms, having no clinical or functional phenotypes that raise suspicion about specific genes. We performed exome sequencing (ES) in a cohort of 116 IT patients (89 families), still undiagnosed after a previously validated phenotype-driven diagnostic algorithm including a targeted analysis of suspected genes. ES achieved a diagnostic yield of 36%, with a gain of 16% over the diagnostic algorithm. This can be explained by genetic heterogeneity and unspecific genotype-phenotype relationships that make the simultaneous analysis of all the genes, enabled by ES, the most reasonable strategy. Furthermore, ES disentangled situations that had been puzzling because of atypical inheritance, sex-related effects or false negative laboratory results. Finally, ES-based copy number variant analysis disclosed an unexpectedly high prevalence of RUNX1 deletions, predisposing to hematologic malignancies. Our findings demonstrate that ES, including copy number variant analysis, can substantially contribute to the diagnosis of IT and can solve diagnostic problems that would otherwise remain a challenge.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a recessive disorder characterized by severe reduction of megakaryocytes and platelets at birth, which evolves toward bone marrow aplasia in childhood. CAMT is mostly caused by mutations in MPL (CAMT-MPL), the gene encoding the receptor of thrombopoietin (THPO), a crucial cytokine regulating hematopoiesis. CAMT can be also due to mutations affecting the THPO coding region (CAMT-THPO). In a child with the clinical picture of CAMT, we identified the homozygous c.-323C>T substitution, affecting a potential regulatory region of THPO. Although mechanisms controlling THPO transcription are not characterized, bioinformatics and in vitro analysis showed that c.-323C>T prevents the binding of transcription factors ETS1 and STAT4 to the putative THPO promoter, impairing THPO expression. Accordingly, in the proband the serum THPO concentration indicates defective THPO production. Based on these findings, the patient was treated with the THPO-mimetic agent eltrombopag, which induced a significant increase in platelet count and stable remission of bleeding symptoms. Herein, we report a novel pathogenic variant responsible for CAMT and provide new insights into the mechanisms regulating transcription of the THPO gene.

Background: Inherited thrombocytopenias (ITs) are rare congenital bleeding disorders characterized by different clinical expression and variable prognosis. ITs are poorly known by clinicians and often misdiagnosed with most common forms of thrombocytopenia., Material and Methods: "CHildren with Inherited Platelet disorders Surveillance" study (CHIPS) is a retrospective - prospective observational cohort study conducted between January 2003 and January 2022 in 17 centers affiliated to the Italian Association of Pediatric Hematology and Oncology (AIEOP). The primary objective of this study was to collect clinical and laboratory data on Italian pediatric patients with inherited thrombocytopenias. Secondary objectives were to calculate prevalence of ITs in Italian pediatric population and to assess frequency and genotype-phenotype correlation of different types of mutations in our study cohort., Results: A total of 139 children, with ITs (82 male - 57 female) were enrolled. ITs prevalence in Italy ranged from 0.7 per 100,000 children during 2010 to 2 per 100,000 children during 2022. The median time between the onset of thrombocytopenia and the diagnosis of ITs was 1 years (range 0 - 18 years). A family history of thrombocytopenia has been reported in 90 patients (65%). Among 139 children with ITs, in 73 (53%) children almost one defective gene has been identified. In 61 patients a pathogenic mutation has been identified. Among them, 2 patients also carry a variant of uncertain significance (VUS), and 4 others harbour 2 VUS variants. VUS variants were identified in further 8 patients (6%), 4 of which carry more than one variant VUS. Three patients (2%) had a likely pathogenic variant while in 1 patient (1%) a variant was identified that was initially given an uncertain significance but was later classified as benign. In addition, in 17 patients the genetic diagnosis is not available, but their family history and clinical/laboratory features strongly suggest the presence of a specific genetic cause. In 49 children (35%) no genetic defect were identified. In ninetyseven patients (70%), thrombocytopenia was not associated with other clinically apparent disorders. However, 42 children (30%) had one or more additional clinical alterations., Conclusion: Our study provides a descriptive collection of ITs in the pediatric Italian population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Lassandro, Palladino, Faleschini, Barone, Boscarol, Cesaro, Chiocca, Farruggia, Giona, Gorio, Maggio, Marinoni, Marzollo, Palumbo, Russo, Saracco, Spinelli, Verzegnassi, Morga, Savoia and Giordano.)

Background: Despite consolidated guidelines, the clinical diagnosis and prognosis of cystic fibrosis (CF) is still challenging mainly because of the extensive phenotypic heterogeneity and the high number of CFTR variants, including their combinations as complex alleles., Results: We report a family with a complicated syndromic phenotype, which led to the suspicion not only of CF, but of a dominantly inherited skeletal dysplasia (SD). Whereas the molecular basis of the SD was not clarified, segregation analysis was central to make a correct molecular diagnosis of CF, as it allowed to identify three CFTR variants encompassing two known maternal mutations and a novel paternal microdeletion., Conclusion: This case well illustrates possible pitfalls in the clinical and molecular diagnosis of CF; presence of complex phenotypes deflecting clinicians from appropriate CF recognition, and/or identification of two mutations assumed to be in trans but with an unconfirmed status, which underline the importance of an in-depth molecular CFTR analysis., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

GFI1B is a transcription factor essential for the regulation of erythropoiesis and megakaryopoiesis, and pathogenic variants have been associated with thrombocytopenia and bleeding. Analysing thrombocytopenic families by whole exome sequencing, we identified a novel GFI1B variant (c.648+5G>A), which causes exon 9 skipping and overexpression of a shorter p32 isoform. We report the clinical data of our patients and critically review the phenotype observed in individuals with different GFI1B variants leading to the same effect on the p32 expression. Since p32 is increased in acute and chronic leukemia cells, we tested the expression level of genes playing a role in various type of cancers, including hematological tumors and found that they are significantly dysregulated, suggesting a potential role for GFI1B in carcinogenesis regulation. Increasing the detection of individuals with GFI1B variants will allow us to better characterize this rare disease and determine whether it is associated with an increased risk of developing malignancies.

Warsaw breakage syndrome (WABS), is caused by biallelic mutations of DDX11, a gene coding a DNA helicase. We have recently reported two affected sisters, compound heterozygous for a missense (p.Leu836Pro) and a frameshift (p.Lys303Glufs*22) variant. By investigating the pathogenic mechanism, we demonstrate the inability of the DDX11 p.Leu836Pro mutant to unwind forked DNA substrates, while retaining DNA binding activity. We observed the accumulation of patient-derived cells at the G2/M phase and increased chromosomal fragmentation after mitomycin C treatment. The phenotype partially overlaps with features of the Fanconi anemia cells, which shows not only genomic instability but also defective mitochondria. This prompted us to examine mitochondrial functionality in WABS cells and revealed an altered aerobic metabolism. This opens the door to the further elucidation of the molecular and cellular basis of an impaired mitochondrial phenotype and sheds light on this fundamental process in cell physiology and the pathogenesis of these diseases., (© 2021 Wiley Periodicals LLC.)

A relevant and current aspect of wastewater treatment systems is related to the processes of the nitrogen cycle that results in its elimination in gaseous forms. In the present study, we report the first measurements of nitrate-reducing rate (NRR) at lab-scale, using the flow-through reactor technique with sludge of a sewage stabilization pond system located in Patagonia (Argentina). Sludge was collected from Inlet and Outlet areas, in winter and summer. The sludge was characterized by having high moisture content (>94%) and organic matter concentration greater than 37%. The nitrate reduction experimental dates fitted significantly to the Michaelis-Menten model, allowing the estimation of the parameters that regulate the NR kinetics. The maximum potential nitrate reduction rate (R max ) showed great variability, registering a maximum of 131.6 μmol-N·gdw -1 ·h -1 (Outlet-Summer) and a minimum of 4.1 μmol-N·gdw -1 ·h -1 (Inlet-Winter). The lowest half saturation constant (K m ) was recorded in the Inlet sludge during the winter (6.1 mg N-NO 3 - ·L -1 ), which indicates a greater affinity for nitrate of this bacterial consortium. An unusually high activity of NR was registered, being higher with sludge from the Outlet zone and with summer temperature. In full-scale ponds, the NR activity could explain a relevant part of the nitrogen removal that involves the escape of gaseous forms.

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ . This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ ) in zebrafish, which induced a significantly decreased number of CD41 + thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis., (© 2019 by The American Society of Hematology.)