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Neovascular age-related macular degeneration (nAMD) is a frequent cause of vision loss among the elderly in the Western world. Current disease management with repeated injections of anti-VEGF agents accumulates the risk for adverse events and constitutes a burden for society and the individual patient. Sustained suppression of VEGF using gene therapy is an attractive alternative, which we explored using adeno-associated virus (AAV)-based delivery of novel RNA interference (RNAi) effectors in a porcine model of choroidal neovascularization (CNV). The potency of VEGFA-targeting, Ago2-dependent short hairpin RNAs placed in pri-microRNA scaffolds (miR-agshRNA) was established in vitro and in vivo in mice. Subsequently, AAV serotype 8 (AAV2.8) vectors encoding VEGFA-targeting or irrelevant miR-agshRNAs under the control of a tissue-specific promotor were delivered to the porcine retina via subretinal injection before CNV induction by laser. Notably, VEGFA-targeting miR-agshRNAs resulted in a significant and sizable reduction of CNV compared with the non-targeting control. We also demonstrated that single-stranded and self-complementary AAV2.8 vectors efficiently transduce porcine retinal pigment epithelium cells but differ in their transduction characteristics and retinal safety. Collectively, our data demonstrated a robust anti-angiogenic effect of VEGFA-targeting miR-aghsRNAs in a large translational animal model, thereby suggesting AAV-based delivery of anti-VEGFA RNAi therapeutics as a valuable tool for the management of nAMD.

Purpose: Choroidal neovascularization (CNV) accounts for the majority of severe vision loss in neovascular age-related macular degeneration (AMD). Despite therapies that target VEGF, patients are often under-responsive, require frequent eye injections to control disease, and eventually lose some vision despite chronic therapy implicating a multifactorial etiology in treatment response. Genetic studies implicate systemic immunity in AMD and systemic immune cells accumulate within CNV lesions, yet a role for these cells in anti-VEGF response remains undetermined. The purpose of this study was to identify transcriptional signatures of circulating immune cells that are associated with high anti-VEGF treatment burden. Design: Experimental pilot study. Participants: Patients with neovascular AMD seen at Washington University School of Medicine in St. Louis and BJC Health System. Methods: We profiled by single cell RNA sequencing the peripheral blood mononuclear cells of 27 treatment-experienced patients with wet AMD. We stratified this cohort into 2 groups with low and high treatment burden (≤ 5 or ≥ 6 injections in the past 12 months, respectively). Main Outcome Measures: Identification of immune cells associated with high treatment burden. Results: Gene expression signature of CD16+ monocytes may be associated with high treatment burden. Conclusions: These studies delineate potential signatures of circulating immune cells that may be associated with high treatment burden in neovascular AMD, potentially informing the development of diagnostic predictors of anti-VEGF response and new precision medicine-based approaches to complement anti-VEGF therapies. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Purpose: To report the clinical utility of optical coherence tomography angiography (OCTA) for demonstrating choroidal neovascularization (CNV) associated with Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency (LCHADD) retinopathy. Methods: Thirty-three participants with LCHADD (age 7–36 years; median 17) were imaged with OCTA and the Center for Ophthalmic Optics & Lasers Angiography Reading Toolkit (COOL-ART) software was implemented to process OCTA scans. Results: Seven participants (21 %; age 17–36 years; median 25) with LCHADD retinopathy demonstrated evidence of CNV by retinal examination or presence of CNV within outer retinal tissue on OCTA scans covering 3 × 3 and/or 6 × 6-mm. These sub-clinical CNVs are adjacent to hyperpigmented areas in the posterior pole. CNV presented at stage 2 or later of LCHADD retinopathy prior to the disappearance of RPE pigment in the macula. Conclusion: OCTA can be applied as a non-invasive method to evaluate the retinal and choroidal microvasculature. OCTA can reveal CNV in LCHADD even when the clinical exam is inconclusive. These data suggest that the incidence of CNV is greater than expected and can occur even in the early stages of LCHADD retinopathy.

The presence of oncogene carrying eccDNAs is strongly associated with carcinogenesis and poor patient survival. Tumour biopsies and in vitro cancer cell lines are frequently utilized as models to investigate the role of eccDNA in cancer. However, eccDNAs are often lost during the in vitro growth of cancer cell lines, questioning the reproducibility of studies utilizing cancer cell line models. Here, we conducted a comprehensive analysis of eccDNA variability in seven cancer cell lines (MCA3D, PDV, HaCa4, CarC, MIA-PaCa-2, AsPC-1, and PC-3). We compared the content of unique eccDNAs between triplicates of each cell line and found that the number of unique eccDNA is specific to each cell line, while the eccDNA sequence content varied greatly among triplicates (∼ 0–1% eccDNA coordinate commonality). In the PC-3 cell line, we found that the large eccDNA (ecDNA) with MYC is present in high-copy number in an NCI cell line isolate but not present in ATCC isolates. Together, these results reveal that the sequence content of eccDNA is highly variable in cancer cell lines. This highlights the importance of testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.

Cancer cells have an altered transcriptome, which contributes to their abnormal behavior. Many tumors have high levels of kinetochore genes, which play important roles in genome stability. This overexpression could be utilized to destabilize cancer cell genomes, however this has not been proven specifically. We investigated the link between kinetochore gene overexpression, chromosomal number variations (CNVs) and genomic instability.Data on RNA expression and CNV from 12 different cancer types were evaluated using information theory. In all cancer types, we looked at the relationship between RNA expression and CNVs. Kinetochore gene expression was found to be substantially linked with CNV levels. In all cancer types, with the exception of thyroid cancer, highly expressed kinetochore genes were enriched in the most dominant cancer-specific co-expression subnetworks characterizing the largest patient subgroups. Except for thyroid cancer, kinetochore inner protein CENPA was among the transcripts most strongly associated with CNV values in all cancer types studied, with significantly higher expression levels in patients with high CNVs than in patients with low CNVs. CENPA function was investigated further in cell models by transfecting genomically stable (HCT116) and unstable (MCF7 and HT29) cancer cell lines using CENPA overexpression vectors. This overexpression increased the number of abnormal cell divisions in the stable cancer cell line HCT116 and, to a lesser extent, in the unstable cell lines MCF7 and HT29. Overexpression improved anchorage-independent growth properties of all cell lines.Our findings suggest that overexpression of kinetochore genes in general, and CENPA in particular, can cause genomic instability and cancer progression.

Parkinson's disease (PD), a neurodegenerative disease characterized by both motor neuron and non-motor neuron symptoms, is the most frequent neurodegenerative disease after Alzheimer's disease. Both genetic and environmental factors take part in disease etiology. Most cases are considered complex multifactorial diseases. About 15% of PD appear in the familial form, and about 5% of all cases arise from a single gene mutation. Among Mendelian causes of PD, PARK7 is one of the autosomal recessive forms due to loss-of-function mutations in both gene alleles. Both single nucleotide variants (SNVs) and copy number variations (CNVs) are observed in PARK7. This study presents an Iranian family with familial PD where some relatives had psychiatric disorders. A homozygous 1617 bp deletion in a female with early-onset PD was detected through copy-number analysis from whole-exome sequencing (WES) data in this consanguineous family. Further investigation by surveying microhomology revealed that the actual size of the deletion is 3,625 bp. This novel CNV that was in the PARK7gene is supposed to co-relation with early-onset PD and infertility in this family.

Objective: To explore the characteristic changes of multiple ERP components associated with attention impairments in patients with temporal lobe epilepsy (TLE)., Methods: A total of 92 patients diagnosed with TLE at Xiangya Hospital during May 2022 and January 2023 and 85 healthy controls were included in this study. Participants were asked to complete attention network test with recording of electroencephalogram., Results: Compared with healthy controls, significant lower amplitudes (cue-related N1, N2 and CNV) and longer latencies (target-related N2) were found in TLE patients. Besides classical components, other components could also reveal the impairments of attention function. Cue-related N1 (p ≤ 0.007) and N2 (p ≤ 0.01) components indicated impaired alerting and orienting network in TLE. And cue-related CNV-E component (p = 0.003) promoted the alerting network was damaged and target-related N2 component (p = 0.008) indicated the executive control network was impaired., Conclusion: These findings consummate the non-classical ERP features of attention impairments in TLE patients., Significance: The above findings have strong clinical guiding significance for early identification and intervention., Competing Interests: Conflicts of interest All authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: This study aimed to investigate the genetic etiology of male infertility patients., Method: A total of 1600 male patients with infertility, including 1300 cases of azoospermia and 300 cases of severe oligozoospermia, underwent routine semen analysis, chromosomal karyotype analysis and sex hormone level testing. The Azoospermia factor (AZF) on the Y chromosome was detected using the multiple fluorescence quantitative PCR technique. Additionally, copy number variation (CNV) analysis was performed on patients with Sertoli-cell-only syndrome who had a normal karyotype and AZF., Result: Chromosomal abnormalities were found in 334 cases (20.88 %) of the 1600 male infertility patients. The most common type of abnormality was sex chromosome abnormalities (18.94 %), with 47, XXY being the most frequent abnormal karyotype. The rates of chromosomal abnormalities were significantly different between the azoospermia group and the severe oligospermia group (23.69 % and 8.67 %, respectively; P<0.05). AZF microdeletions were detected in 155 cases (9.69 %), with various deletion types and AZFc region microdeletion being the most prevalent. The rates of AZF microdeletions were not significantly different between the azoospermia group and the severe oligospermia group (9.15 % and 12 %, respectively; P=0.133). In 92 patients with Sertoli-cell-only syndrome who had a normal karyotype and AZF, the detection rate of CNV was 16.3 %. Compared to the severe oligospermia group, the azoospermia group had higher levels of FSH and LH and lower levels of T and E2, and the differences were statistically significant (P<0.05)., Conclusions: Male infertility is a complex multifactorial disease, with chromosomal abnormalities and Y chromosome microdeletions being important genetic factors leading to the disease. Initial genetic testing of infertile men should include karyotyping and Y chromosome microdeletions. If necessary, CNV testing should be performed to establish a clinical diagnosis and provide individualized treatment for male infertility., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Discuss evidence referring to the genetic role in congenital heart diseases, whether chromosomic alterations or monogenic diseases. Data source: LILACS, PubMed, MEDLINE, SciELO, Google Scholar, and references of the articles found. Review articles, case reports, book chapters, master's theses, and doctoral dissertations were included. Summary of findings: Congenital heart diseases are among the most common type of birth defects, afflicting up to 1% of the liveborn. Traditionally, the etiology was defined as a multifactorial model, with both genetic and external contribution, and the genetic role was less recognized. Recently, however, as the natural evolution and epidemiology of congenital heart diseases change, the identification of genetic factors has an expanding significance in the clinical and surgical management of syndromic or non-syndromic heart defects, providing tools for the understanding of heart development. Conclusions: Concrete knowledge of congenital heart disease etiology and recognition of the genetic alterations may be helpful in the bedside management, defining prognosis and anticipating complications. Resumo: Objetivo: Discutir as evidências referentes ao papel genético em cardiopatias congênitas, sejam alterações cromossômicas ou doenças monogênicas. Fonte de dados: Lilacs, PubMed, Medline, SciELO, Google Scholar e referências dos artigos encontrados. Artigos de revisão, relatos de casos, capítulos de livros, dissertações de mestrado e teses de doutorado foram incluídos. Síntese dos dados: As cardiopatias congênitas estão entre os tipos mais comuns de defeitos congênitos, afetando até 1% dos nascidos vivos. Tradicionalmente, a etiologia era definida como um modelo multifatorial, com contribuição tanto genética quanto externa, sendo o papel genético menos reconhecido. Recentemente, no entanto, à medida que a evolução natural e a epidemiologia das cardiopatias congênitas mudaram, a identificação de fatores genéticos tem adquirido importância crescente no tratamento clínico e cirúrgico de defeitos cardíacos sindrômicos e não-sindrômicos, fornecendo ferramentas para a compreensão do desenvolvimento do coração. Conclusões: O conhecimento concreto da etiologia das cardiopatias congênitas e o reconhecimento das alterações genéticas podem ser úteis no tratamento à beira do leito, definindo o prognóstico e antecipando as complicações.

While 9p deletion and duplication syndromes have been studied for several years, small sample sizes and minimal high-resolution data have limited a comprehensive delineation of genotypic and phenotypic characteristics. In this study, we examined genetic data from 719 individuals in the worldwide 9p Network Cohort: a cohort seven to nine times larger than any previous study of 9p. Most breakpoints occur in bands 9p22 and 9p24, accounting for 35% and 38% of all breakpoints, respectively. Bands 9p11 and 9p12 have the fewest breakpoints, with each accounting for 0.6% of all breakpoints. The most common phenotype in 9p deletion and duplication syndromes is developmental delay, and we identified eight known neurodevelopmental disorder genes in 9p22 and 9p24. Since it has been previously reported that some individuals have a secondary structural variant related to the 9p variant, we examined our cohort for these variants and found 97 events. The top secondary variant involved 9q in 14 individuals (1.9%), including ring chromosomes and inversions. We identified a gender bias with significant enrichment for females (p = 0.0006) that may arise from a sex reversal in some individuals with 9p deletions. Genes on 9p were characterized regarding function, constraint metrics, and protein-protein interactions, resulting in a prioritized set of genes for further study. Finally, we achieved precision genomics in one child with a complex 9p structural variation using modern genomic technologies, demonstrating that long-read sequencing will be integral for some cases. Our study is the largest ever on 9p-related syndromes and provides key insights into genetic factors involved in these syndromes.

Similarity in facial characteristics between relatives suggests a strong genetic component underlies facial variation. While there have been numerous studies of the genetics of facial abnormalities and, more recently, single nucleotide polymorphism (SNP) genome-wide association studies (GWASs) of normal facial variation, little is known about the role of genetic structural variation in determining facial shape. In a sample of Bantu African children, we found that only 9% of common copy number variants (CNVs) and 10-kb CNV analysis windows are well tagged by SNPs (r2 ≥ 0.8), indicating that associations with our internally called CNVs were not captured by previous SNP-based GWASs. Here, we present a GWAS and gene set analysis of the relationship between normal facial variation and CNVs in a sample of Bantu African children. We report the top five regions, which had p values ≤ 9.35 × 10−6 and find nominal evidence of independent CNV association (p < 0.05) in three regions previously identified in SNP-based GWASs. The CNV region with strongest association (p = 1.16 × 10−6, 55 losses and seven gains) contains NFATC1, which has been linked to facial morphogenesis and Cherubism, a syndrome involving abnormal lower facial development. Genomic loss in the region is associated with smaller average lower facial depth. Importantly, new loci identified here were not identified in a SNP-based GWAS, suggesting that CNVs are likely involved in determining facial shape variation. Given the plethora of SNP-based GWASs, calling CNVs from existing data may be a relatively inexpensive way to aid in the study of complex traits.

Because of their high specificity and low side effects, protein drugs possess a substantial global market. However, the low bioavailability of protein is still a major obstacle to their expanded applications, which is expected to be answered with proper protein formulations. Taking corneal neovascularization (CNV) as an example, we demonstrated a co-assembled system of hexa-histidine and Ava (Avastin) with metal ions (HmA@Ava) could cross the cornea, the most important bio-barrier during the treatment of most diseases of the anterior segment in clinics. We found that the nanosized HmA@Ava efficiently encapsulated Ava with impressive loading capacity without destroying the bioactivity of Ava and assisted Ava penetration through the corneal barriers to effectively inhibit CNV development in an alkali burn rat model with sustained and pH-dependent Ava release. Our results suggested that the co-assembled strategy of protein and HmA is a proper formulation to protein drugs, with promising penetration ability to deliver protein across bio-barriers, which could open a path for topical administration of protein drugs for treatment of various ocular diseases and hold enormous potential for delivery of therapeutic proteins not only for ocular diseases but also for other diseases that require protein treatment.

Background: Previous studies have implicated both rare copy number variations (CNVs) and common variants in liability for attention-deficit/hyperactivity disorder (ADHD). However, how common and rare genetic variants jointly contribute to individual liability requires further investigation in larger cohorts., Methods: This study comprises 9385 participants of European descent and 7810 participants of African American ancestry who were recruited from the greater Philadelphia area by the Children's Hospital of Philadelphia. The polygenic risk score (PRS) of each participant was estimated by linkage disequilibrium pruning and p-value thresholding (P + T) methods using PRSice-2. We investigated whether the risk of ADHD follows a polygenic liability threshold model wherein 1) the risk of ADHD requires less contribution from common variants in the presence of a rare CNV, and 2) control carriers of ADHD-associated CNVs have lower common risk allele burden than noncarriers., Results: CNVs previously reported in ADHD cases were significantly associated with ADHD risk in both the European American cohort and the African American cohort. Healthy control participants carrying those same risk CNVs had lower PRSs than those without risk CNVs in the European American cohort. This result was replicated in the African American cohort. However, PRSs were not significantly different in case participants carrying risk CNVs versus those without risk CNVs., Conclusions: These findings provide evidence in support of interactive effects of PRS and ADHD-associated CNVs on disease risk and add novel insights into the genetic basis of ADHD by highlighting a protective role of low PRS in ADHD., (Copyright © 2023. Published by Elsevier Inc.)

The importance of structural genetic variants, such as copy number variations (CNVs), in modulating human disease is being increasingly recognized. Several clinical conditions require investigation of human neutrophil antigen (HNA-1), which is encoded by the Fc gamma receptor IIIb gene (FCGR3B), including suspicion of neutropenia, infections, and proactive testing of blood component donors to reduce the potential risk in transfusion. In this study, we compared real-time quantitative polymerase chain reaction (qPCR) with two digital PCR (dPCR) platforms, namely droplet digital PCR and an array-based platform, to determine copy numbers (CNs) in FCGR3B. We initially tested 400 anonymous blood donors with qPCR using a commercially available TaqMan probe assay (Applied Biosystems) on a Quant Studio 12 Flex. CNs was determined for all 400 tested individuals with CNs ranging from zero to four. Zero copies were detected in 0.2% (1/400), one copy was detected in 3.8% (15/400), two copies were detected in 87.8% (351/400), three copies were detected in 8.0% (32/400), and four copies were detected in 0.2% (1/400) of tested individuals. From this cohort, we selected 32 donors with CNs from zero to four for analyses with Digital Real-Time PCR (dPCR) using Lab on an array (LOAA) on an On-Point analyzer from Optolane Technologies Inc. and the Droplet Digital PCR (ddPCR) platform from Bio-Rad Laboratories. We compared the obtained CNs of FCGR3B on the three platforms and found full concordance between the CNs obtained. We therefore conclude that all three platforms can be used for quantification of CNs for FCGR3B, and although dPCR has some advantages over qPCR, it was not necessary for reliably estimating CNs of the FCGR3B gene., Competing Interests: Declaration of competing interest All authors declare that there are no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Pathological neovascularization of the outer retina is the hallmark of neovascular age-related macular degeneration (nAMD). Building on our previous observations that semaphorin 3F (Sema3f) is expressed in the outer retina and demonstrates anti-angiogenic potential, we have investigated whether Sema3f can be used to protect against subretinal neovascularization in two mouse models. Both in the very low-density lipid-receptor knockout (Vldlr−/−) model of spontaneous subretinal neovascularization as well as in the mouse model of laser-induced choroidal neovascularization (CNV), we found protective effects of Sema3f against the formation of pathologic neovascularization. In the Vldlr−/− model, AAV-induced overexpression of Sema3f reduced the size of pathologic neovascularization by 56%. In the laser-induced CNV model, intravitreally injected Sema3f reduced pathologic neovascularization by 30%. Combined, these results provide the first evidence from two distinct in vivo models for a use of Sema3f in protecting the outer retina against subretinal neovascularization.

Working memory (WM) plays an important role in daily life and is known to correlated with aerobic fitness. However, whether the relationship between aerobic fitness and WM is dependent on the stimulus modality or is associated with one or multiple subprocesses involved in WM remains unknown. Accordingly, this study utilized event-related potentials (ERPs) to comprehensively examine the encoding, preparation, and retrieval processes during verbal and spatial WM performance. Eighty-eight young adults aged 18-30years were recruited to participate in two laboratory visits on separate days. On day 1, aerobic fitness was assessed by maximum oxygen consumption (V˙O 2max ) during a treadmill-based graded exercise test. On day 2, participants completed verbal and spatial WM tasks while P2, contingent negative voltage (CNV), and P3 components of ERP were recorded during the encoding, preparatory, and retrieval stages of WM, respectively. Results of hierarchical regression analysis showed that V˙O 2max was positively correlated with response accuracy during the high-demanding condition of spatial WM after controlling for age, sex, and self-reported physical activity. Additionally, a higher level of V˙O 2max was associated with larger terminal CNV amplitude at the Cz electrode during the high-demanding condition of spatial WM. These findings suggest that aerobic fitness may have selective beneficial associations with the motor preparatory process and subsequent task performance requiring a greater amount of spatial information but not the encoding and retrieval stages nor the verbal modality of WM., (Copyright © 2024 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Objectives: Photodynamic therapy (PDT) is a vaso-occlusive treatment for a number of chorioretinal vascular pathologies. We aimed to retrospectively analyse efficiency and safety of PDT for different conditions (central serous retinopathy (CSR), age-related macular degeneration (AMD), macular telangiectasia type 2 and choroidal hemangioma) and with different verteporfin parameters., Methods: Clinical parameters were ascertained from the medical records of patients undergoing PDT over a 6-year period. This included indications for PDT, dosing regimens of verteporfin PDT (which includes treatment dose of verteporfin and fluence). Response to treatment was measured by best corrected visual acuity (BCVA) and central foveal thickness (CFT) on ocular coherence tomography. Complications and side effects were recorded., Results: 67.4 % (31/46) of PDT treatments performed over the last six years were for CSR. In the CSR cohort, there were significant improvements in BCVA (0.47 ± 0.24 to 0.29 ± 0.27, p < 0.05) and CFT (350.2μm ± 66.9 μm to 286.1μm ± 60.6 μm. In the AMD cohort, there was no change in BCVA (1.08 ± 0.52 to 1.07 ± 0.53, p = 0.96) but significant improvement in CFT (488.2μm ± 164.6 μm to 348.7μm ± 65.7 μm, p < 0.05). There was no significant difference in BCVA or CFT for macular telangiectasia type 2 and choroidal hemangioma., Conclusions: PDT continues to have a role in the management of medical retina conditions. Our results show PDT is most effective in improving and stabilizing visual acuity in CSR, with earlier intervention resulting in better outcomes., Competing Interests: Declaration of Competing Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Temporal orienting (TO) is the allocation of attentional resources in time based on the a priori generation of temporal expectancy of relevant stimuli as well as the a posteriori updating of this expectancy as a function of both sensory-based evidence and elapsing time. These processes rely on dissociable cognitive mechanisms and neural networks. Yet, although there is evidence that TO may be a core mechanism for cognitive functioning in childhood, the developmental spatiotemporal neural dynamics of this mechanism are little understood. In this study we employed a combined approach based on the application of distributed source reconstruction on a high spatial resolution ERP data array obtained from eighteen 8- to 12-year-old children completing a TO paradigm in which both the cue (Temporal vs. Neutral) and the SOA (Short vs. Long) were manipulated. Results show both cue (N1) and SOA (CNV, Omission Detection Potential and Anterior Anticipatory Index) ERP effects, which were associated with expectancy generation and updating, respectively. Only cue-related effects were correlated with age, as revealed by a reduction of the N1 delta effect with increasing age. Our data suggest that the neural correlates underlying TO are already established at least from 8 to 12 years of age.

Purpose: HNRNPU haploinsufficiency is associated with developmental and epileptic encephalopathy 54. This neurodevelopmental disorder is characterized by developmental delay, intellectual disability, speech impairment, and early-onset epilepsy. We performed genome-wide DNA methylation (DNAm) analysis in a cohort of individuals to develop a diagnostic biomarker and gain functional insights into the molecular pathophysiology of HNRNPU-related disorder., Methods: DNAm profiles of individuals carrying pathogenic HNRNPU variants, identified through an international multicenter collaboration, were assessed using Infinium Methylation EPIC arrays. Statistical and functional correlation analyses were performed comparing the HNRNPU cohort with 56 previously reported DNAm episignatures., Results: A robust and reproducible DNAm episignature and global DNAm profile were identified. Correlation analysis identified partial overlap and similarity of the global HNRNPU DNAm profile to several other rare disorders., Conclusion: This study demonstrates new evidence of a specific and sensitive DNAm episignature associated with pathogenic heterozygous HNRNPU variants, establishing its utility as a clinical biomarker for the expansion of the EpiSign diagnostic test., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2023 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: Schizophrenia (SCZ) is a debilitating psychiatric disorder with a large genetic contribution; however, its neurodevelopmental substrates remain largely unknown. Modeling pathogenic processes in SCZ using human induced pluripotent stem cell-derived neurons (iNs) has emerged as a promising strategy. Copy number variants confer high genetic risk for SCZ, with duplication of the 16p11.2 locus increasing the risk 14.5-fold., Methods: To dissect the contribution of induced excitatory neurons (iENs) versus GABAergic (gamma-aminobutyric acidergic) neurons (iGNs) to SCZ pathophysiology, we induced iNs from CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 isogenic and SCZ patient-derived induced pluripotent stem cells and analyzed SCZ-related phenotypes in iEN monocultures and iEN/iGN cocultures., Results: In iEN/iGN cocultures, neuronal firing and synchrony were reduced at later, but not earlier, stages of in vitro development. These were fully recapitulated in iEN monocultures, indicating a primary role for iENs. Moreover, isogenic iENs showed reduced dendrite length and deficits in calcium handling. iENs from 16p11.2 duplication-carrying patients with SCZ displayed overlapping deficits in network synchrony, dendrite outgrowth, and calcium handling. Transcriptomic analysis of both iEN cohorts revealed molecular markers of disease related to the glutamatergic synapse, neuroarchitecture, and calcium regulation., Conclusions: Our results indicate the presence of 16p11.2 duplication-dependent alterations in SCZ patient-derived iENs. Transcriptomics and cellular phenotyping reveal overlap between isogenic and patient-derived iENs, suggesting a central role of glutamatergic, morphological, and calcium dysregulation in 16p11.2 duplication-mediated pathogenesis. Moreover, excitatory dysfunction during early neurodevelopment is implicated as the basis of SCZ pathogenesis in 16p11.2 duplication carriers. Our results support network synchrony and calcium handling as outcomes directly linked to this genetic risk variant., (Published by Elsevier Inc.)

Differences of sex development (DSDs) are a group of congenital conditions characterized by a discrepancy between chromosomal, gonadal, and genital sex development of an individual, with significant impact on medical, psychological and reproductive life. The genetic heterogeneity of DSDs complicates the diagnosis and almost half of the patients remains undiagnosed. In this context, chromosomal imbalances in syndromic DSD patients may help to identify new genes implicated in DSDs. In this study, we aimed at describing the burden of chromosomal imbalances including submicroscopic ones (copy number variants or CNVs) in a cohort of prenatal syndromic DSD patients, and review their role in DSDs. Our patients carried at least one pathogenic or likely pathogenic chromosomal imbalance/CNV or low-level mosaicism for aneuploidy. Almost half of the cases resulted from an unbalanced chromosomal rearrangement. Chromosome 9p/q, 4p/q, 3q and 11q anomalies were more frequently observed. Review of the literature confirmed the causative role of CNVs in DSDs, either in disruption of known DSD-causing genes (SOX9, NR0B1, NR5A1, AR, ATRX, …) or as a tool to suspect new genes in DSDs (HOXD cluster, ADCY2, EMX2, CAMK1D, …). Recurrent CNVs of regulatory elements without coding sequence content (i.e. duplications/deletions upstream of SOX3 or SOX9) confirm detection of CNVs as a mean to explore our non-coding genome. Thus, CNV detection remains a powerful tool to explore undiagnosed DSDs, either through routine techniques or through emerging technologies such as long-read whole genome sequencing or optical genome mapping., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Copy-number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are associated with schizophrenia. Model mice (Arhgap10 S490P/NHEJ mice) that carry "double-hit" mutations in the Arhgap10 gene mimic the schizophrenia in a Japanese patient, exhibiting altered spine density, methamphetamine-induced cognitive dysfunction, and activation of RhoA/Rho-kinase signaling. However, it remains unclear whether the activation of RhoA/Rho-kinase signaling due to schizophrenia-associated Arhgap10 mutations causes the phenotypes of these model mice. Here, we investigated the effects of fasudil, a brain permeable Rho-kinase inhibitor, on altered spine density in the medial prefrontal cortex (mPFC) and on methamphetamine-induced cognitive impairment in a touchscreen‑based visual discrimination task in Arhgap10 S490P/NHEJ mice. Fasudil (20 mg/kg, intraperitoneal) suppressed the increased phosphorylation of myosin phosphatase-targeting subunit 1, a substrate of Rho-kinase, in the striatum and mPFC of Arhgap10 S490P/NHEJ mice. In addition, daily oral administration of fasudil (20 mg/kg/day) for 7 days ameliorated the reduced spine density of layer 2/3 pyramidal neurons in the mPFC. Moreover, fasudil (3-20 mg/kg, intraperitoneal) rescued the methamphetamine (0.3 mg/kg)-induced cognitive impairment of visual discrimination in Arhgap10 S490P/NHEJ mice. Our results suggest that Rho-kinase plays significant roles in the neuropathological changes in spine morphology and in the vulnerability of cognition to methamphetamine in mice with schizophrenia-associated Arhgap10 mutations., Competing Interests: Declaration of Competing Interest This study was funded in part by Sumitomo Pharma Co., Ltd., (Copyright © 2022. Published by Elsevier Ltd.)

Background: A number of rare copy number variants (CNVs) have been linked to neurodevelopmental disorders. However, because CNVs encompass many genes, it is often difficult to identify the mechanisms that lead to developmental perturbations., Methods: We used 15q13.3 microdeletion to propose and validate a novel strategy to predict the impact of CNV genes on brain development that could further guide functional studies. We analyzed single-cell transcriptomics datasets containing cortical interneurons to identify their developmental vulnerability to 15q13.3 microdeletion, which was validated in mouse models., Results: We found that Klf13-but not other 15q13.3 genes-is expressed by precursors and neuroblasts in the medial and caudal ganglionic eminences during development, with a peak of expression at embryonic day (E)13.5 and E18.5, respectively. In contrast, in the adult mouse brain, Klf13 expression is negligible. Using Df(h15q13.3)/+ and Klf13 +/- embryos, we observed a precursor subtype-specific impairment in proliferation in the medial ganglionic eminence and caudal ganglionic eminence at E13.5 and E17.5, respectively, corresponding to vulnerability predicted by Klf13 expression patterns. Finally, Klf13 +/- mice showed a layer-specific decrease in parvalbumin and somatostatin cortical interneurons accompanied by changes in locomotor and anxiety-related behavior., Conclusions: We show that the impact of 15q13.3 microdeletion on precursor proliferation is grounded in a reduction in Klf13 expression. The lack of Klf13 in Df(h15q13.3)/+ cortex might be the major reason for perturbed density of cortical interneurons. Thus, the behavioral defects seen in 15q13.3 microdeletion could stem from a developmental perturbation owing to selective vulnerability of cortical interneurons during sensitive stages of their development., (Copyright © 2021 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: Visual outcome of patients with neovascular age-related macular degeneration has significantly improved during the last years following the introduction of anti-vascular endothelial growth factor (VEGF) therapy. However, about one third of patients show persistent exudation and decreasing visual acuity despite recurrent anti-VEGF treatment, which implies a role of other, still unknown proangiogenic mediators., Methods: The present study applied transcriptional profiling of human and mouse (C57BL/6J wildtype) choroidal neovascularization (CNV) membranes each with reference to healthy control tissue to identify yet unrecognized mediators of CNV formation. Key factors were further investigated by immunohistochemistry as well as by intravitreal inhibition experiments and multiplex protein assays in the laser-induced CNV mouse model., Findings: Transcriptional profiles of CNV membranes were characterized by enhanced activation of blood vessel development, cytoskeletal organization, and cytokine production, with angiogenesis and wound healing processes predominating in humans and activation of immune processes in mice. Besides several species-specific factors, 95 phylogenetically conserved CNV-associated genes were detected, among which fibroblast growth factor inducible-14 (FN14), a member of the tumor necrosis factor (TNF) receptor family, was identified as a key player of CNV formation. Blocking the pathway by intravitreal injection of a FN14 decoy receptor modulated the cytokine profile - most notably IL-6 - and led to a significant reduction of CNV size in vivo., Interpretation: This study characterizes the transcriptome of human and mouse CNV membranes in an unprejudiced manner and identifies FN14 as a phylogenetically conserved mediator of CNV formation and a promising new therapeutic target for neovascular AMD., Funding: This study was funded by the Helmut Ecker Foundation and the Volker Homann Foundation., (Copyright © 2022. Published by Elsevier B.V.)

Purpose: Monogenic disorders can present clinically heterogeneous symptoms. We hypothesized that in patients with a monogenic disorder caused by a large deletion, frequently additional loss-of-function (LOF)-intolerant genes are affected, potentially contributing to the phenotype., Methods: We investigated the LOF-intolerant gene distribution across the genome and its association with benign population and pathogenic classified deletions from individuals with presumably monogenic disorders. For people with presumably monogenic epilepsy, we compared Human Phenotype Ontology terms in people with large and small deletions., Results: We identified LOF-intolerant gene dense regions that were enriched for ClinVar and depleted for population copy number variants. Analysis of data from >143,000 individuals with a suspected monogenic disorder showed that 2.5% of haploinsufficiency disorder-associated deletions can affect at least 1 other LOF-intolerant gene. Focusing on epilepsy, we observed that 13.1% of pathogenic and likely pathogenic ClinVar deletions <3 megabase pair, covering the diagnostically most relevant genes, affected at least 1 additional LOF-intolerant gene. Those patients have potentially more complex phenotypes with increasing deletion size., Conclusion: We could systematically show that large deletions frequently affected admditional LOF-intolerant genes in addition to the established disease gene. Further research is needed to understand how additional potential disease-relevant genes influence monogenic disorders to improve clinical care and the efficacy of targeted therapies., Competing Interests: Conflict of Interest D.L. consults for Encoded Therapeutics Inc. All other authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cognitive impairment in schizophrenia is a major contributor to poor outcomes, yet its causes are poorly understood. Some rare copy number variants (CNVs) are associated with schizophrenia risk and affect cognition in healthy populations, but their contribution to cognitive impairment in schizophrenia has not been investigated. We examined the effect of 12 schizophrenia CNVs on cognition in those with schizophrenia., Methods: General cognitive ability was measured using the Measurement and Treatment Research to Improve Cognition in Schizophrenia composite z score in 875 patients with schizophrenia and in a replication sample of 519 patients with schizophrenia using Wechsler Adult Intelligence Scale Full Scale IQ. Using linear regression, we tested for association between cognition and schizophrenia CNV status, covarying for age and sex. In addition, we tested whether CNVs hitting genes in schizophrenia-enriched gene sets (loss-of-function intolerant and synaptic gene sets) were associated with cognitive impairment., Results: A total of 23 schizophrenia CNV carriers were identified. Schizophrenia CNV carriers had lower general cognitive ability than nonschizophrenia CNV carriers in discovery (β = -0.66, 95% confidence interval [CI] = -1.31 to -0.01) and replication samples (β = -0.91, 95% CI = -1.71 to -0.11) and after meta-analysis (β = -0.76, 95% CI = -1.26 to -0.25, p = .003). CNVs hitting loss-of-function intolerant genes were associated with lower cognition (β = -0.15, 95% CI = -0.29 to -0.001, p = .048)., Conclusions: In those with schizophrenia, cognitive ability in schizophrenia CNV carriers is 0.5-1.0 standard deviations below non-CNV carriers, which may have implications for clinical assessment and management. We also demonstrate that rare CNVs hitting genes intolerant to loss-of-function variation lead to more severe cognitive impairment, above and beyond the effect of known schizophrenia CNVs., (Copyright © 2021. Published by Elsevier Inc.)

Background: Next-generation sequencing (NGS) is increasingly used for clinical evaluation of cardiomyopathy patients as it allows for simultaneous screening of multiple cardiomyopathy-associated genes. Adding copy number variant (CNV) analysis of NGS data is not routine yet and may contribute to the diagnostic yield., Objectives: Determine the diagnostic yield of our targeted NGS gene panel in routine clinical diagnostics of Dutch cardiomyopathy patients and explore the impact of exon CNVs on diagnostic yield., Methods: Patients (N = 2002) referred for clinical genetic analysis underwent diagnostic testing of 55-61 genes associated with cardiomyopathies. Samples were analyzed and evaluated for single nucleotide variants (SNVs), indels and CNVs. CNVs identified in the NGS data and suspected of being pathogenic based on type, size and location were confirmed by additional molecular tests., Results: A (likely) pathogenic (L)P variant was detected in 22.7% of patients, including 3 with CNVs and 25 where a variant was identified in a gene currently not associated with the patient's cardiomyopathy subtype. Only 15 out of 2002 patients (0.8%) were found to carry two (L)P variants., Conclusion: The yield of routine clinical diagnostics of cardiomyopathies was relatively low when compared to literature. This is likely due to the fact that our study reports the outcome of patients in daily routine diagnostics, therefore also including patients not fully fulfilling (subtype specific) cardiomyopathy criteria. This may also explain why (L)P variants were identified in genes not associated with the reported subtype. The added value of CNV analysis was shown to be limited but not negligible., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Congenital anomalies of the kidney and urinary tract (CAKUTs) are the most common cause of chronic kidney disease in children. Human 16p11.2 deletions have been associated with CAKUT, but the responsible molecular mechanism remains to be illuminated. To explore this, we investigated 102 carriers of 16p11.2 deletion from multi-center cohorts, among which we retrospectively ascertained kidney morphologic and functional data from 37 individuals (12 Chinese and 25 Caucasian/Hispanic). Significantly higher CAKUT rates were observed in 16p11.2 deletion carriers (about 25% in Chinese and 16% in Caucasian/Hispanic) than those found in the non-clinically ascertained general populations (about 1/1000 found at autopsy). Furthermore, we identified seven additional individuals with heterozygous loss-of-function variants in TBX6, a gene that maps to the 16p11.2 region. Four of these seven cases showed obvious CAKUT. To further investigate the role of TBX6 in kidney development, we engineered mice with mutated Tbx6 alleles. The Tbx6 heterozygous null (i.e., loss-of-function) mutant (Tbx6 +/‒ ) resulted in 13% solitary kidneys. Remarkably, this incidence increased to 29% in a compound heterozygous model (Tbx6 mh/‒ ) that reduced Tbx6 gene dosage to below haploinsufficiency, by combining the null allele with a novel mild hypomorphic allele (mh). Renal hypoplasia was also frequently observed in these Tbx6-mutated mouse models. Thus, our findings in patients and mice establish TBX6 as a novel gene involved in CAKUT and its gene dosage insufficiency as a potential driver for kidney defects observed in the 16p11.2 microdeletion syndrome., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: 22q11.2 deletions and duplications are copy number variations (CNVs) that predispose to developmental neuropsychiatric disorders. Both CNVs are associated with autism spectrum disorder (ASD), while the deletion confers disproportionate risk for schizophrenia. Neurobehavioral profiles associated with these reciprocal CNVs in conjunction with brain imaging measures have not been reported., Methods: We profiled the impact of 22q11.2 CNVs on neurobehavioral measures relevant to ASD and psychosis in 106 22q11.2 deletion carriers, 38 22q11.2 duplication carriers, and 82 demographically matched healthy control subjects. To determine whether brain-behavior relationships were altered in CNV carriers, we further tested for interactions between group and regional brain structure on neurobehavioral domains., Results: Cognitive deficits were observed in both CNV groups, with the lowest IQs in deletion carriers. ASD and dimensionally measured ASD traits were elevated in both CNV groups; however, duplication carriers exhibited increased stereotypies compared to deletion carriers. Moreover, discriminant analysis using ASD subdomains distinguished between CNV cases with 76% accuracy. Both psychotic disorder diagnosis and dimensionally measured positive and negative symptoms were elevated in deletion carriers. Finally, healthy control subjects showed an inverse relationship between processing speed and cortical thickness in heteromodal association areas, which was absent in both CNV groups., Conclusions: 22q11.2 CNVs differentially modulate intellectual functioning and psychosis-related symptomatology but converge on broad ASD-related symptomatology. However, subtle differences in ASD profiles distinguish CNV groups. Processing speed impairments, coupled with the lack of normative relationship between processing speed and cortical thickness in CNV carriers, implicate aberrant development of the cortical mantle in the pathology underlying impaired processing speed ability., (Copyright © 2020 Society of Biological Psychiatry. All rights reserved.)

Background and Aims: A 42 year-old male with premature atherosclerosis, severe dyslipidemia and resistance to treatment with high dose statin and a recommended dose of a PCSK9 inhibitor, was found to have a duplication of the PCSK9 gene. However, the clinical phenotype, which included a more than 15-fold elevation in circulating PCSK9, was unexpected given that he had one additional gene copy., Methods: Here we have carried out whole genome sequencing and transcriptional reporter assays to investigate the molecular mechanism leading to this unusual FH phenotype., Results: The PCSK9 duplication was found to contain the full coding sequence but with an 829 bp shorter 3'-untranslated region (UTR) sequence. All possible rearrangements include a head-to-head fusion between a completely duplicated PCSK9 and a chromosomal region, normally situated ~80 kb away, that includes HNF4 and USF1 binding sites that could promote transcription of the PCSK9 gene. Transcriptional reporter assays demonstrated that a construct harboring the HNF4 binding site significantly increased the promoter activity by 2.5-fold with a smaller effect noted for a USF1 construct., Conclusions: Here we describe, in a patient with resistant hypercholesterolemia, a novel PCSK9 gene rearrangement that enables upregulation of PCSK9 expression by allowing proximity to an active enhancer binding to HNF4A., Competing Interests: Declaration of competing interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Myeloproliferative neoplasms (MPN) are clonal hematological malignancies that lead to overproduction of mature myeloid cells. They are due to acquired mutations in genes encoding for AK2, MPL and CALR that result in the activation of the cytokine receptor/JAK2 signaling pathway. In addition, it exists germline variants that can favor the initiation of the disease or may affect its phenotype. First, they can be common risk alleles, which correspond to frequent single nucleotide variants present in control population and that contribute to the development of either sporadic or familial MPN. Second, some variants predispose to the onset of MPN with a higher penetrance and lead to familial clustering of MPN. Finally, some extremely rare genetic variants can induce MPN-like hereditary disease. We will review these different subtypes of germline genetic variants and discuss how they impact the initiation and/or development of the MPN disease., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Autism is a frequent, precocious behavioral constellation of social and communicative atypicalities associated with apparently restricted interests and repetitive behavior and paired with an uneven ability profile. Its definition has constantly broadened in the past 75 years, introducing phenotypes increasingly distant from its initial description, heterogeneous in intelligence and speech level, and associated conditions. When it is unassociated with other conditions, its origin is mostly genetic, transmissible, and favored by frequent polymorphisms with small effects present in the general population. Identified de novo rare mutations with large deleterious effects produce phenotypes only loosely related to nonsyndromic autism. Autism is associated with brain reorganization at multiple levels, and with a variant of typical information processing, i.e., the way humans perceive, memorize, manipulate, and attribute emotional value to available information. Its phenotype evolves over the span of life, with an overall reduction of autistic signs, but it still requires some level of support. There is no treatment for this condition; however, it is compatible with high levels of integration into society., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although mounting evidence has shown that reward can improve conflict control in the visual domain, little is known about whether and how reward affects conflict processing in the auditory domain. In the present study, we adopted an auditory Stroop task in which the meaning of a sound word ('male' or 'female') could be either congruent or incongruent with the gender of the voice (male or female speaker), and the participants were asked to discriminate the gender of the voice (the phonetic task) or the meaning of the word (the semantic task). Importantly, an auditory cue signalling a potential reward or no-reward for the current trial was presented prior to the sound word. In both tasks, relative to the congruent sound word, response to the incongruent sound word was delayed, i.e., an auditory Stroop effect. However, this auditory Stroop effect was reduced following a reward cue relative to a no-reward cue. Event-related potentials (ERPs) showed a stronger contingent negativity variation (CNV, 1000-1500 ms) for the reward cue than for the no-reward cue. The conflict negativity N inc (300-400 ms) was more negative-going for the incongruent word than for the congruent word, but this effect was significantly reduced in the reward condition. However, the late positive complex (LPC) showed at most a weak reward modulation. These findings suggest that reward expectation improves auditory conflict control by modulating different stages of conflict processing: promoting better attentional preparation for the upcoming target (CNV), and facilitating conflict detection (N inc ) on the presentation of the target., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: To provide a validated method to confidently identify exon-containing copy-number variants (CNVs), with a low false discovery rate (FDR), in targeted sequencing data from a clinical laboratory with particular focus on single-exon CNVs., Methods: DNA sequence coverage data are normalized within each sample and subsequently exonic CNVs are identified in a batch of samples, when the target log 2 ratio of the sample to the batch median exceeds defined thresholds. The quality of exonic CNV calls is assessed by C-scores (Z-like scores) using thresholds derived from gold standard samples and simulation studies. We integrate an ExonQC threshold to lower FDR and compare performance with alternate software (VisCap)., Results: Thirteen CNVs were used as a truth set to validate Atlas-CNV and compared with VisCap. We demonstrated FDR reduction in validation, simulation, and 10,926 eMERGESeq samples without sensitivity loss. Sixty-four multiexon and 29 single-exon CNVs with high C-scores were assessed by Multiplex Ligation-dependent Probe Amplification (MLPA)., Conclusion: Atlas-CNV is validated as a method to identify exonic CNVs in targeted sequencing data generated in the clinical laboratory. The ExonQC and C-score assignment can reduce FDR (identification of targets with high variance) and improve calling accuracy of single-exon CNVs respectively. We propose guidelines and criteria to identify high confidence single-exon CNVs.

Background: Therapy targeting human epidermal growth factor receptor 2 (HER2, also known as ERBB2) is an effective approach for HER2-positive metastatic colorectal cancer (mCRC). HER2 status is typically determined using immunohistochemistry and fluorescence in situ hybridization. Circulating tumor DNA (ctDNA) enables noninvasive detection of gene mutations and copy number alterations including HER2 amplification., Materials and Methods: We screened 351 patients with mCRC and studied the clinicopathologic characteristics of HER2-positive mCRC. HER2 expression in tumor samples measured with immunohistochemistry and fluorescence in situ hybridization was compared with HER2 copy number variation in plasma ctDNA detected by targeted sequence capture covering exons of 170 genes. We also examined the correlation between changes in tumor burden in ctDNA and antitumor response by imaging evaluation during the treatment course., Results: Positive HER2 status was observed in 12 (3.4%) patients (7 males and 5 females), with a median age of 56 years. The HER2 concordance rate between tumor samples and ctDNA was 66.7% (20/30). Changes in tumor burden in ctDNA during the treatment course correlated with responses on imaging., Conclusions: Detection of HER2 copy number variation in ctDNA may be an alternative option for noninvasive determination of HER2 status. Tumor burden changes in ctDNA were consistent with imaging evaluation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Structural variation (SV) is associated with inherited diseases. Next-generation sequencing (NGS) is an efficient method for SV detection because of its high-throughput, low cost, and base-pair resolution. However, due to lack of standard NGS protocols and a limited number of clinical samples with pathogenic SVs, comprehensive standards for SV detection, interpretation, and reporting are to be established., Methods: We performed SV assessment on 60,000 clinical samples tested with hereditary cancer NGS panels spanning 48 genes. To evaluate NGS results, NGS and orthogonal methods were used separately in a blinded fashion for SV detection in all samples., Results: A total of 1,037 SVs in coding sequence (CDS) or untranslated regions (UTRs) and 30,847 SVs in introns were detected and validated. Across all variant types, NGS shows 100% sensitivity and 99.9% specificity. Overall, 64% of CDS/UTR SVs were classified as pathogenic/likely pathogenic, and five deletions/duplications were reclassified as pathogenic using breakpoint information from NGS., Conclusion: The SVs presented here can be used as a valuable resource for clinical research and diagnostics. The data illustrate NGS as a powerful tool for SV detection. Application of NGS and confirmation technologies in genetic testing ensures delivering accurate and reliable results for diagnosis and patient care.

Background: Many etiological factors have been related to prostate cancer (CaP) development, progression, and survival, such as age, population origin, geographic area, occupational exposures, and nutrition and lifestyle factors. However, physical activity affords health benefits to cancer patients, including those with CaP. Glutathione S-Transferases enzymes have been linked to CaP because of their role in the detoxification of a wide variety of potential carcinogens, steroid hormones and xenobiotics. Among the different glutathione S-transferases isoforms, null genotype for GSTM1 has been associated with an increased risk of CaP, although data are controversial. As the relationship between copy number variation and gene expression of GSTM1 in CaP remains unexplored, this study analyzed GSTM1 gene expression and/or dosage effect on CaP risk and aggressiveness. The potential protective role of physical activity was also explored., Methods: Three hundred and seventeen patients (159 non-CaP and 158 CaP) were recruited from the Service of Urology (Hospital Virgen de las Nieves, Granada, Spain) over the period 2012 to 2014 and were followed-up until January 2018 to ensure a correct classification of control and patients. Individuals were classified in each group based on histological analysis of tissue biopsy, along with data on PSA level, Gleason score and T stage in patients with biopsies positive for CaP. Individuals with a negative biopsy were considered as controls. All controls underwent a systematic 20-core ultrasound guided biopsy in order to limit the false negative rate. Genomic DNA was extracted from peripheral blood to determine the exact copy numbers of GSTM1, and RNA was extracted from prostate tissue samples to determine GSTM1 gene expression. Both analyses were performed using the qPCR method. A questionnaire was administered to all patients to assess environmental exposures, lifestyle, and physical activity. The association of GSTM1 copy number variation and expression with the rest of variables was assessed by chi-square test and the Mann-Whitney test. Multiple logistic regression was used to assess which factors were associated with the risk of CaP., Results: The presence of 1 or 2 copies of the GSTM1 gene was not less prevalent in CaP compared to non-CaP patients; however, a significant decreased GSTM1 gene expression was observed in CaP tissue relative to non-CaP tissue (P = 0.003). CaP patients with environmental exposure to dust and smoke, and smoking habit had a significantly decreased GSTM1 gene expression (and near-significantly decreased for living in urban areas) as compared to non-CaP patients with the same exposures. In addition, physical activity was significantly associated with a lower risk of CaP (P = 0.006) and with increased GSTM1 gene expression (P = 0.002)., Conclusions: A reduced GSTM1 gene expression in prostate tissue was observed in CaP patients with some environmental chemical exposures. Intriguingly, physical activity might play a protective role against CaP development, possibly as a result of increasing GSTM1 gene expression in prostate tissue. However, this observation warrants further confirmation., (Copyright © 2018 Elsevier Inc. All rights reserved.)