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During the SARS-CoV-2 pandemic, many countries established wastewater (WW) surveillance to objectively monitor the level of infection within the population. As new variants continue to emerge, it has become clear that WW surveillance is an essential tool for the early detection of variants. The EU Commission published a recommendation suggesting an approach to establish surveillance of SARS-CoV-2 and its variants in WW, besides specifying the methodology for WW concentration and RNA extraction. Therefore, different groups have approached the issue with different strategies, mainly focusing on WW concentration methods, but only a few groups highlighted the importance of prefiltering WW samples and/or purification of RNA samples. Aiming to obtain high-quality sequencing data allowing variants detection, we compared four experimental conditions generated from the treatment of: i) WW samples by WW filtration and ii) the extracted RNA by DNase treatment, purification and concentration of the extracted RNA. To evaluate the best condition, the results were assessed by focusing on several sequencing parameters, as the outcome of SARS-CoV-2 sequencing from WW is crucial for variant detection. Overall, the best sequencing result was obtained by filtering the WW sample. Moreover, the present study provides an overview of some sequencing parameters to consider when optimizing a method for monitoring SARS-CoV-2 variants from WW samples, which can also be applied to any sample preparation methodology. [ABSTRACT FROM AUTHOR]

In genetic diseases, the most prevalent mechanism of pathogenicity is an altered expression of dosage‐sensitive genes. Drugs that restore physiological levels of these genes should be effective in treating the associated conditions. We developed a screening strategy, based on a bicistronic dual‐reporter vector, for identifying compounds that modulate protein levels, and used it in a pharmacological screening approach. To provide a proof‐of‐principle, we chose autosomal dominant leukodystrophy (ADLD), an ultra‐rare adult‐onset neurodegenerative disorder caused by lamin B1 (LMNB1) overexpression. We used a stable Chinese hamster ovary (CHO) cell line that simultaneously expresses an AcGFP reporter fused to LMNB1 and a Ds‐Red normalizer. Using high‐content imaging analysis, we screened a library of 717 biologically active compounds and approved drugs, and identified alvespimycin, an HSP90 inhibitor, as a positive hit. We confirmed that alvespimycin can reduce LMNB1 levels by 30%–80% in five different cell lines (fibroblasts, NIH3T3, CHO, COS‐7, and rat primary glial cells). In ADLD fibroblasts, alvespimycin reduced cytoplasmic LMNB1 by about 50%. We propose this approach for effectively identifying potential drugs for treating genetic diseases associated with deletions/duplications and paving the way toward Phase II clinical trials. [ABSTRACT FROM AUTHOR]

Background More than 100 X-linked intellectual disability (X-LID) genes have been identified to be involved in 10–15% of intellectual disability (ID). Method To identify novel possible candidates, we selected 18 families with a male proband affected by isolated or syndromic ID. Pedigree and/or clinical presentation suggested an X-LID disorder. After exclusion of known genetic diseases, we identified seven cases whose mother showed a skewed X-inactivation (>80%) that underwent whole exome sequencing (WES, 50X average depth). Results WES allowed to solve the genetic basis in four cases, two of which (Coffin-Lowry syndrome, RPS6K3 gene; ATRX syndrome, ATRX gene) had been missed by previous clinical/genetics tests. One further ATRX case showed a complex phenotype including pontocerebellar atrophy (PCA), possibly associated to an unidentified PCA gene mutation. In a case with suspected Lujan-Fryns syndrome, a c.649C>T (p.Pro217Ser) MECP2 missense change was identified, likely explaining the neurological impairment, but not the marfanoid features, which were possibly associated to the p.Thr1020Ala variant in fibrillin 1. Finally, a c.707T>G variant (p.Phe236Cys) in the DMD gene was identified in a patient retrospectively recognized to be affected by Becker muscular dystrophy (BMD, OMIM 300376). Conclusion Overall, our data show that WES may give hints to solve complex ID phenotypes with a likely X-linked transmission, and that a significant proportion of these orphan conditions might result from concomitant mutations affecting different clinically associated genes. [ABSTRACT FROM AUTHOR]

Objective: To evaluate outcome in the pregnancy following a stillbirth (SB) by a placental vascular disorders. Study Design: A prospective, observational, multicenter study was conducted in woman with a history of stillbirth (> 22 weeks) between 2005 and June 2013, in 3 Italian University Hospitals. Causes of SB were previously identified after extensive investigations. Pregnant women were enrolled within the first trimester. The main outcome was “adverse neonatal outcome”, including perinatal death, fetal growth restriction, early preterm birth <33+6 weeks, hypoxic-ischemic encephalopathy, intracranial hemorrhage or respiratory distress. Results: Out of 364 index pregnancies, 320 women (87.9%) had a subsequent pregnancy during the study period. Forty-seven had an early pregnancy loss. Out of 273 babies, 67 (24.5%) had an adverse perinatal outcome, including 1 SB and 1 early neonatal death (3.7/1000). Women who had a SB related to placental vascular disorders (39.6%), were at higher risk of an adverse neonatal outcome compared with women whose SB was unexplained or resulted from other causes (Adj. OR = 2.1, 95%CI: 1.2–3.8). Moreover, also obesity independently predicts an adverse perinatal outcome (Adj OR = 2.1, 95%CI: 1.1–4.3). Conclusion: When previous SB is related to placental vascular disorders there is a high risk for adverse neonatal outcomes in the subsequent pregnancy. Maternal obesity is an additional risk factor. [ABSTRACT FROM AUTHOR]

Data on the outcome of trisomy T18 (T18) when diagnosed during pregnancy are lacking. We performed a retrospective study of pregnancies complicated by T18 diagnosed at our center and a literature search for publications on the topic, with pooled estimates of survival rates at different gestational and post-natal ages. In our series, all the 60 patients included in the analysis had prenatally detected ultrasound anomalies, which were evidenced in the first trimester or at the second trimester scan in 73% of cases. In the continued pregnancies, ultrasound findings did not correlate with prenatal or post-natal outcome. A meta-analysis of available literature and our data showed that 48% [37-60%] of fetuses were live born, and among these 39% [11-72%] survived beyond 48 hr and 11% [3-21%] beyond 1 month. Our results confirm that prenatal ultrasound has high sensitivity in detection of T18 but is not predictive of the outcome of the continued pregnancies. The data on survival support that T18, even when antenatally diagnosed, cannot be considered as a uniformly lethal syndrome. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: Hereditary ataxias are a heterogeneous group of neurodegenerative disorders, where exome sequencing may become an important diagnostic tool to solve clinically or genetically complex cases. Methods: We describe an Italian family in which three sisters were affected by ataxia with postural/intentional myoclonus and involuntary movements at onset, which persisted during the disease. Oculomotor apraxia was absent. Clinical and genetic data did not allow us to exclude autosomal dominant or recessive inheritance and suggest a disease gene. Results: Exome sequencing identified a homozygous c.6292C > T (p.Arg2098*) mutation in SETX and a heterozygous c.346G > A (p.Gly116Arg) mutation in AFG3L2 shared by all three affected individuals. A fourth sister (II.7) had subclinical myoclonic jerks at proximal upper limbs and perioral district, confirmed by electrophysiology, and carried the p.Gly116Arg change. Three siblings were healthy. Pathogenicity prediction and a yeast-functional assay suggested p.Gly116Arg impaired m-AAA (ATPases associated with various cellular activities) complex function. Conclusions: Exome sequencing is a powerful tool in identifying disease genes. We identified an atypical form of Ataxia with Oculoapraxia type 2 (AOA2) with myoclonus at onset associated with the c.6292C > T (p.Arg2098*) homozygous mutation. Because the same genotype was described in six cases from a Tunisian family with a typical AOA2 without myoclonus, we speculate this latter feature is associated with a second mutated gene, namely AFG3L2 (p.Gly116Arg variant). We suggest that variant phenotypes may be due to the combined effect of different mutated genes associated to ataxia or related disorders, that will become more apparent as the costs of exome sequencing progressively will reduce, amplifying its diagnostics use, and meanwhile proposing significant challenges in the interpretation of the data. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: To estimate the risk of stillbirth in dichorionic and monochorionic twins compared with singletons, and to evaluate the relevant causes of stillbirth in each group. Study design: A retrospective cohort analysis of all pregnancies ≥22 weeks of gestation was performed at a tertiary care center from January 1995 to June 2011. The overall fetal survival and the prospective risk of stillbirth were compared in monochorionic diamniotic (MCDA) twins, dichorionic diamniotic (DCDA) twins, and singletons. Causes of stillbirth were classified using the ReCoDe classification and were compared among the three study groups. Results: A total of 46,200 singletons, 462 MCDA twins and 1108 DCDA twins were included in the study. Both Kaplan–Meier analysis and prospective risk calculation showed that MCDA twins had the highest risk of stillbirth (OR ranging between 13.5 95% CI 8.7–20.7 at 22.0–24.6 weeks and 4.0 95% CI 1.1–13.1 at 31.0–33.6 weeks, compared to singletons), while singletons had the lowest. Main causes of stillbirth were major congenital malformations in singletons (25.1%) and in DCDA twins (75%), and twin–twin transfusion syndrome in MCDA twins (81.5%). When excluding fetuses affected by major congenital anomalies, MCDA twins (p <0.001) but not DCDA twins (p =0.2) remained at increased risk for stillbirth compared with singletons. Conclusion: The risk of stillbirth is significantly higher both in MCDA and DCDA twins compared with singletons. Stillbirths are mainly due to twin–twin transfusion syndrome in MCDA twins and major congenital anomalies in DCDA twins. When major congenital anomalies are excluded, DCDA twins have a similar in utero mortality to singletons. [Copyright &y& Elsevier]

Recent development of next-generation DNA sequencing (NGS) techniques is changing the approach to search for mutations in human genetic diseases. We applied NGS to study an A-T patient in which one of the two expected mutations was not found after DHPLC, cDNA sequencing and MLPA screening. The 160-kb ATM genomic region was divided into 31 partially overlapping fragments of 4-6 kb and amplified by long-range PCR in the patient and mother, who carried the same mutation by segregation. We identified six intronic variants that were shared by the two genomes and not reported in the dbSNP(132) database. Among these, c.1236-405C>T located in IVS11 was predicted to be pathogenic because it affected splicing. This mutation creates a cryptic novel donor (5′) splice site (score 1.00) 405 bp upstream of the exon 12 acceptor (3′) splice site. cDNA analysis showed the inclusion of a 212-bp non-coding 'pseudoexon' with a premature stop codon. We validated the functional effect of the splicing mutation using a minigene assay. Using antisense morpholino oligonucleotides, designed to mask the cryptic donor splice-site created by the c.1236-405C>T mutation, we abrogated the aberrant splicing product to a wild-type ATM transcript, and in vitro reverted the functional ATM kinase impairment of the patients' lymphoblasts. Resequencing is an effective strategy for identifying rare splicing mutations in patients for whom other mutation analyses have failed (DHPLC, MLPA, or cDNA sequencing). This is especially important because many of these patients will carry rare splicing variants that are amenable to antisense-based correction. [ABSTRACT FROM AUTHOR]

Objective: This study was undertaken to establish the optimal threshold of birth weight discordance for prediction of adverse outcome in liveborn, nonmalformed preterm twins. Study design: We accessed a cohort of twin gestations for the period 1990 through 2000 delivered at less than 37.0 weeks' gestation. Adverse neonatal outcome was defined as stillbirth or occurrence of major morbidities. Results: A total of 335 twin gestations (670 twins) were included, of which 104 (31%) experienced adverse neonatal outcome. The average birth weight discordance was 12.4% ± 10.6% versus 19.4% ± 14.8% in those with good versus adverse outcome (P < .001). Birth weight discordance was a significant predictor of adverse neonatal outcome that was independent of gestational age at delivery, small for gestational age, and chorionicity. Receiver operating characteristic curve analysis suggested that optimal thresholds of birth weight discordance for clinical use should take into account gestational age. Conclusion: Birth weight discordance is an independent predictor of adverse neonatal outcome in liveborn, nonmalformed preterm twins. [ABSTRACT FROM AUTHOR]

Objective: The study was undertaken to assess whether prenatal Doppler variables can identify cases of fetal growth restriction (FGR) approaching term who are at risk for adverse neonatal outcome.Study Design: From a cohort of FGR cases delivered at >or=34 weeks, fetal biometry and pulsatility indices (PI) of fetal arteries obtained less than 2 weeks before delivery were related to adverse neonatal outcome, defined as admission to the neonatal intensive care unit (NICU) for indications other than low birth weight alone.Results: Stepwise regression analysis showed that after controlling for gestational age at delivery and fetal biometry, only the last umbilical artery (UA) PI percentile was significantly predictive of adverse neonatal outcome (odds ratio=1.02, 95% CI 1.01-1.03, P=.02). Receiver operating characteristic curve analysis identified a UA PI at the 65th percentile as optimal predictor of adverse neonatal outcome (sensitivity=60%, false-positive rate=30%).Conclusion: In FGR cases delivered at >/=34 weeks' gestation, Doppler PI at the UA independently predicts the likelihood of admission to the NICU for reasons other than low birth weight alone. [ABSTRACT FROM AUTHOR]

Abstract Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m -AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development. Graphical abstract Unlabelled Image Highlights • We generated a knock-in mouse model of SCA28 harbouring a p.Met665Arg -Afg3l2 allele. • Homozygous mutants died perinatally with evidence of cardiac atrophy. • Heterozygous mice developed normally but presented late-onset cerebellar ataxia. • MEFs showed altered mitochondrial bioenergetics and dynamics. • Proteotoxicity is suggested to be responsible for SCA28 pathogenesis. [ABSTRACT FROM AUTHOR]

Transition metals are cofactors for a wide range of vital enzymes and are directly or indirectly involved in the response against reactive oxygen species (ROS), which can damage cellular components. Their altered homeostasis has been studied in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), but no data are available on rarer conditions. We aimed at studying the role of essential trace elements in ataxia telangiectasia (A-T), a rare form of pediatric autosomal recessive cerebellar ataxia with altered antioxidant response. We found an increased level of copper (Cu, p = 0.0002) and a reduced level of zinc (Zn, p = 0.0002) in the blood of patients (n. 16) compared to controls, using inductively coupled plasma mass spectrometry (ICP-MS). Other trace elements involved in the oxidative stress response, such as manganese (Mn) and selenium (Se), were unaltered. Cu/Zn-dependent superoxide dismutase (SOD1) was shown to have a 30% reduction in gene expression and 40% reduction in enzyme activity upon analysis of lymphoblastoid cell lines of patients (Student's t -test, p = 0.0075). We also found a 30% reduction of Mn-SOD (SOD2; Student's t -test, p = 0.02), probably due to a feedback regulatory loop between the two enzymes. The expression of antioxidant enzymes, such as erythrocyte glutathione peroxidase ( GPX1 ), and SOD2 was unaltered, whereas catalase ( CAT ) was increased in A-T cells, both at the mRNA level and in terms of enzyme activity (~ 25%). Enhanced CAT expression can be attributed to the high ROS status, which induces CAT transcription. These results suggest that alterations in essential trace elements and their related enzymes may play a role in the pathogenesis of A-T, although we cannot conclude if altered homeostasis is a direct effect of A-T mutated genes ( ATM ). Altered homeostasis of trace elements may be more prevalent in neurodegenerative diseases than previously thought, and it may represent both a biomarker and a generic therapeutic target for different disorders with the common theme of altered antioxidant enzyme responses associated with an unbalance of metals. [ABSTRACT FROM AUTHOR]