Experience-Induced Remodeling of the Hippocampal Post-synaptic Proteome and Phosphoproteome.

The postsynaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is a key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the hippocampal PSD proteome and phosphoproteome in mice following four different types of experience. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated from individual mice to investigate molecular mechanisms underlying experience-dependent remodeling of synapses. We developed a new strategy to identify and quantify the relatively low level of site-specific phosphorylation of PSD proteome from the hippocampus, by using a modified iTRAQ-based TiSH protocol. In the PSD, we identified 3938 proteins and 2761 phosphoproteins in the sequential strategy covering a total of 4968 unique protein groups (at least two peptides including a unique peptide). On the phosphoproteins, we identified a total of 6188 unambiguous phosphosites (75%

Leishmaniinae: Evolutionary inferences based on protein expression profiles (PhyloQuant) congruent with phylogenetic relationships among Leishmania, Endotrypanum, Porcisia, Zelonia, Crithidia, and Leptomonas.

Evolutionary relationships among parasites of the subfamily Leishmaniinae, which comprises pathogen agents of leishmaniasis, were inferred based on differential protein expression profiles from mass spectrometry-based quantitative data using the PhyloQuant method. Evolutionary distances following identification and quantification of protein and peptide abundances using Proteome Discoverer and MaxQuant software were estimated for 11 species from six Leishmaniinae genera. Results clustered all dixenous species of the genus Leishmania, subgenera L. (Leishmania), L. (Viannia), and L. (Mundinia), sister to the dixenous species of genera Endotrypanum and Porcisia. Placed basal to the assemblage formed by all these parasites were the species of genera Zelonia, Crithidia, and Leptomonas, so far described as monoxenous of insects although eventually reported from humans. Inferences based on protein expression profiles were congruent with currently established phylogeny using DNA sequences. Our results reinforce PhyloQuant as a valuable approach to infer evolutionary relationships within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unraveled. In addition to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, tissue tropisms, potential to jump from insects to vertebrates including humans, and targets for species-specific diagnostic and drug development.

Male with an apparently normal phenotype carrying a BRCA1 exon 20 duplication in trans to a BRCA1 frameshift variant.

BACKGROUND: Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype. METHODS: We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells. RESULTS: Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability. CONCLUSIONS: This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

NanoImprint: A DNA methylation tool for clinical interpretation and diagnosis of common imprinting disorders using nanopore long-read sequencing.

INTRODUCTION: Long-read whole genome sequencing like Oxford Nanopore Technology, is increasingly being introduced in clinical settings. With its ability to simultaneously call sequence variation and DNA modifications including 5-methylcytosine, nanopore is a promising technology to improve diagnostics of imprinting disorders. METHODS: Currently, no tools to analyze DNA methylation patterns at known clinically relevant imprinted regions are available. Here we present NanoImprint, which generates an easily interpretable report, based on long-read nanopore sequencing, to use for identifying clinical relevant abnormalities in methylation levels at 14 imprinted regions and diagnosis of common imprinting disorders. RESULTS AND CONCLUSION: NanoImprint outputs a summarizing table and visualization plots displays methylation frequency (%) and chromosomal positions for all regions, with phased data color-coded for the two alleles. We demonstrate the utility of NanoImprint using three imprinting disorder samples from patients with Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS) and Prader-Willi syndrome (PWS). NanoImprint script is available from https://github.com/carolinehey/NanoImprint.

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature.

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Quantitative Non-targeted Screening to Profile Micropollutants in Sewage Sludge Used for Agricultural Field Amendments.

A considerable number of micropollutants from human activities enter the wastewater network for removal. However, at the wastewater treatment plant (WWTP), some proportion of these compounds is retained in the sewage sludge (biosolids), and due to its high content of nutrients, sludge is widely applied as an agricultural fertilizer and becomes a means for the micropollutants to be introduced to the environment. Accordingly, a holistic semiquantitative nontarget screening was performed on sewage sludges from five different WWTPs using nanoflow liquid chromatography coupled to high-resolution Orbitrap mass spectrometry. Sixty-one inorganic elements were measured using inductively coupled plasma mass spectrometry. Across all sludges, the nontarget analysis workflow annotated >21,000 features with chemical structures, and after strict prioritization and filtering, 120 organic micropollutants with diverse chemical structures and applications such as pharmaceuticals, pesticides, flame retardants, and industrial and natural compounds were identified. None of the tested sludges were free from organic micropollutants. Pharmaceuticals contributed the largest share followed by pesticides and natural products. The predicted concentration of identified contaminants ranged between 0.2 and 10,881 ng/g dry matter. Through quantitative nontarget analysis, this study comprehensively demonstrated the occurrence of cocktails of micropollutants in sewage sludges.

Associations of dexamethasone's effect on morphine consumption after total knee arthroplasty-Subgroup analyses.

The DEXamethasone twice for pain treatment after Total Knee Arthroplasty (DEX-2-TKA) trial showed that adding one and two doses of 24 mg intravenous dexamethasone to paracetamol, ibuprofen and local infiltration analgesia, reduced morphine consumption (primary outcome) within 48 h after TKA. We aimed to explore the differences in the effect of dexamethasone on morphine consumption in different subgroups. Quantile regression adjusted for site was used to test for significant interaction between the predefined dichotomised subgroups and treatment group. The subgroups were defined based on baseline data: sex (male/female), age (≤65 years/>65 years), American Society of Anaesthesiologists (ASA)-score (ASA I + II/III), visual analogue score of preoperative pain at rest (≤30 mm/>30 mm), pain during mobilisation (≤30 mm/>30 mm), type of anaesthesia (spinal anaesthesia/general anaesthesia and spinal converted to general anaesthesia), and prior daily use of analgesics (either paracetamol and/or NSAID/neither). These analyses were supplemented with post hoc multivariate linear regression analyses. Test of interaction comparing sex in the pairwise comparison between DX2 (dexamethasone [24 mg] + dexamethasone [24 mg]) versus placebo (p = .02), showed a larger effect of dexamethasone on morphine consumption in male patients compared to females. Test of interaction comparing age in the pairwise comparison between DX1 (dexamethasone [24 mg] + placebo) versus placebo (p = .04), showed a larger effect of dexamethasone on morphine consumption in younger patients (≤65 years) compared to older. All remaining subgroup analyses showed no evidence of a difference. The supplemental multivariate analyses did not support any significant interaction for sex (p = .256) or age (p = .730) but supported a significant interaction with the type of anaesthesia (p < .001). Our results from the quantile regression analyses indicate that the male sex and younger age (≤65 years) may be associated with a larger analgesic effect of dexamethasone than the effects in other types of patients. However, this is not supported by post-hoc multivariate linear regression analyses. The two types of analyses both supported a possible interaction with the type of anaesthesia.

Prolonged effects of dexamethasone following total knee arthroplasty: A pre-planned sub-study of the DEX-2-TKA trial.

OBJECTIVES: The DEX-2-TKA trial demonstrated that one and two doses of 24 mg intravenous dexamethasone reduced opioid consumption and pain after total knee arthroplasty (TKA). We aimed to investigate the prolonged effects of dexamethasone after the 48-h intervention period. DESIGN: This was a prospective, pre-planned questionnaire follow-up on postoperative days 3-7 of patients in the DEX-2-TKA trial that randomly received: DX1 (dexamethasone 24 mg + placebo), DX2 (dexamethasone 24 mg + dexamethasone 24 mg), and placebo (placebo + placebo) perioperatively and 24 h later. SETTING: A multicenter trial performed at five Danish hospitals. PARTICIPANTS: We analyzed 434 of 485 adult participants enrolled in the DEX-2-TKA trial. OUTCOME MEASURES: Primary outcome was difference between groups in average of all numerical rating scale (NRS) pain scores reported in the morning, at bedtime, and the daily average pain on postoperative days 3-7. Secondary outcomes were sleep quality and patient satisfaction. RESULTS: The median (interquartile range) pain intensity levels for postoperative days 3-7 were: DX2 3.2 (2.1-4.3); DX1 3.3 (2.3-4.1); and placebo 3.3 (2.5-4.7). Hodges-Lehmann median differences between groups were: 0 (95% confidence interval - 0.54 to 0.2), P = 0.38 between DX1 and placebo; 0.1 (-0.47 to 0.33), p = .87 between DX1 and DX2; and 0.1 (-0.6 to 0.13), p = .20 between DX2 and placebo. We found no relevant differences between groups on sleep quality on postoperative days 3-7 nor for patient satisfaction with the analgesic treatment. CONCLUSIONS: We found that neither one nor two doses of 24 mg intravenous dexamethasone demonstrated prolonged effects on overall pain or sleep quality on postoperative days 3-7 after total knee arthroplasty. We also found that dexamethasone had no effect on patient satisfaction. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov NCT03506789 (main result trial).

Label free quantitative proteomic profiling of serum samples of intellectually disabled young patients revealed dysregulation of complement coagulation and cholesterol cascade systems.

Intellectual disability is a heterogeneous disorder, diagnosed using intelligence quotient (IQ) score criteria. Currently, no specific clinical test is available to diagnose the disease and its subgroups due to inadequate understanding of the pathophysiology. Therefore, current study was designed to explore the molecular mechanisms involved in disease perturbation, and to identify potential biomarkers for disease diagnosis and prognosis. A total of 250 participants were enrolled in this study, including 200 intellectually disabled (ID) subjects from the subgroups (mild, moderate, and severe) with age and gender matched healthy controls (n = 50). Initially, IQ testing score and biochemical profile of each subject was generated, followed by label-free quantitative proteomics of subgroups of IQ and healthy control group through nano-LC/MS- mass spectrometry. A total of 310 proteins were identified, among them198 proteins were common among all groups. Statistical analysis (ANOVA) of the subgroups of ID showed 142 differentially expressed proteins, in comparison to healthy control group. From these, 120 proteins were found to be common among all subgroups. The remaining 22 proteins were categorized as exclusive proteins found only in disease subgroups. Furthermore, the hierarchical cluster analysis (HCL) of common significant proteins was also performed, followed by PANTHER protein classification and GO functional enrichment analysis. Results provides that the datasets of differentially expressed proteins, belong to the categories of immune / defense proteins, transfer carrier proteins, apolipoproteins, complement proteins, protease inhibitors, hemoglobin proteins etc., they are known to involvein immune system, and complement and coagulation pathway cascade and cholesterol metabolism pathway. Exclusively expressed 22 proteins were found to be disease stage specific and strong PPI network specifically those that have significant role in platelets activation and degranulation, such as Filamin A (FLNA). Furthermore, to validate the mass spectrometric findings, four highly significant proteins (APOA4, SAP, FLNA, and SERPING) were quantified by ELISA in all the study subjects. AUROC analysis showed a significant association of APOA4 (0.830), FLNA (0.958), SAP (0.754) and SERPING (0.600) with the disease. Apolipoprotein A4 (APOA4) has a significant role in cholesterol transport, and in modulation of glucose and lipid metabolism in the CNS. Similarly, FLNA has a crucial role in the nervous system, especially in the functioning of synaptic network. Therefore, both APOA4, and FLNA proteins represent good potential for candidate biomarkers for the diagnosis and prognosis of the intellectual disability. Overall, serum proteome of ID patients provides valuable information of proteins/pathways that are altered during ID progression.

Risk and Epidemiology of Periprosthetic Knee Fractures After Primary Total Knee Arthroplasty: A Nationwide Cohort Study.

BACKGROUND: Periprosthetic knee fractures (PPKFs) following total knee arthroplasty (TKA) are uncommon, but potentially serious injuries. We analyze the risk and risk factors for a PPKF in standard primary TKA patients who have osteoarthritis and a minimally (cruciate-retaining TKAs without a femoral box cut) or posterior-stabilized TKA. In addition, we report the risk for patients who have other underlying knee disorders and/or a higher level of TKA constraint. METHODS: All primary TKAs were identified from the Danish National Patient Register and the Danish Knee Arthroplasty Register using data between 1997 and 2022. Subsequent fractures were identified through the International Classification of Diseases diagnosis code, Nordic Medico-Statistical Committee procedure code, or indication for revision TKA. RESULTS: We included 120,642 standard primary TKA patients who had 1,434 PPKFs. The cumulated proportions were 0.4% (95% confidence interval (CI) 0.3 to 0.4) at 2 years 0.8% (0.7 to 0.8) at 5 years. At 10 years, the cumulated proportion was 1.7% (1.6 to 1.8), with 1.3% in the femur, 0.2% in the patella, and 0.2% in the tibia. Significant risk factors were (hazard ratio [HR] [95% CI]); ipsilateral hip arthroplasty (2.3 [2.0 to 2.6]); women (2.1 [1.8 to 2.4]), osteoporosis (1.4 [1.2 to 1.7]); age 80+ (1.4 [1.3 to 1.6]), uncemented TKA (1.3 (1.1 to 1.5) and Charlson Comorbidity Index score 3+ (1.4 [1.1 to 1.8]). An additional 22,624 primary TKA patients who had other underlying knee disorders and/or a higher level of implant constraint were included with 485 PPKFs. The 10-year cumulated proportions were 8.3% (95% CI 6.9 to 9.8) when the underlying disorder was a previous fracture, 2.8% (2.2 to 3.5) for rheumatic disorders, and 5.2% (2.6 to 10.6) for osteonecrosis. In patients who had condylar constrained knees, it was 6.9% (5.1 to 9.4), and 12.4% (8.0 to 16.04) for hinges. CONCLUSIONS: In standard primary TKA patients, the 10-year cumulated proportion of PPKFs was 1.7%, and ipsilateral hip arthroplasty, women, osteoporosis, advanced age, uncemented TKA and higher Charlson Comorbidity Index increased the risk. Higher risks were observed in non-osteoarthritis patients and/or patients who had a higher level of TKA constraint.

Mortality after major lower extremity amputation and association with index level: a cohort study based on 11,205 first-time amputations from nationwide Danish databases.

BACKGROUND AND PURPOSE: Mortality after major lower extremity amputations is high and may depend on amputation level. We aimed to examine the mortality risk in the first year after major lower extremity amputation divided into transtibial and transfemoral amputations. METHODS: This observational cohort study used data from the Danish Nationwide Health registers. 11,205 first-time major lower extremity amputations were included from January 1, 2010, to December 31, 2021, comprising 3,921 transtibial amputations and 7,284 transfemoral amputations. RESULTS: The 30-day mortality after transtibial amputation was overall 11%, 95% confidence interval (CI) 10-12 (440/3,921) during the study period, but declined from 10%, CI 7-13 (37/381) in 2010 to 7%, CI 4-11 (15/220) in 2021. The 1-year mortality was 29% overall, CI 28-30 (1,140 /3,921), with a decline from 31%, CI 21-36 (117/381) to 20%, CI 15-26 (45/220) during the study period. For initial transfemoral amputation, the 30-day mortality was overall 23%, CI 22-23 (1,673/7,284) and declined from 27%, CI 23-31 (138/509) to 22%, CI 19-25 (148/683) during the study period. The 1-year mortality was 48% overall, CI 46-49 (3,466/7,284) and declined from 55%, CI 50-59 (279/509) to 46%, CI 42-50 (315/638). CONCLUSION: The mortality after major lower extremity amputation declined in the 12-year study period; however, the 1-year mortality remained high after both transtibial and transfemoral amputations (20% and 46% in 2021). Hence, major lower extremity amputation patients constitute one of the most fragile orthopedic patient groups, emphasizing an increased need for attention in the pre-, peri-, and postoperative setting.

Major lower extremity amputations - risk of re-amputation, time to re-amputation, and risk factors: a nationwide cohort study from Denmark.

BACKGROUND AND PURPOSE: Re-amputation after lower extremity amputation is frequent. The primary aim of our study was to investigate cumulative re-amputation risk after transtibial amputation (TTA), knee disarticulation (KD), and transfemoral amputation (TFA) and secondarily to investigate time to re-amputation, and risk factors. METHODS: This observational cohort study was based on data from the Danish Nationwide Health registers. The population included first-time major lower extremity amputations (MLEA) performed in patients ≥ 50 years between 2010 and 2021. Both left and right sided MLEA from the same patient were included as index procedures. RESULTS: 11,743 index MLEAs on 10,052 patients were included. The overall cumulative risks for re-amputation were 29% (95% confidence interval [CI] 27-30), 30% (CI 26-35), and 11% (CI 10-12) for TTA, KD, and TFA, respectively. 58% of re-amputations were performed within 30 days after index MLEA. Risk factors for re-amputation within 30 days were dyslipidemia (hazard ratio [HR] 1.2, CI 1.0-1.3), renal insufficiency (HR 1.2, CI 1.1-1.4), and prior vascular surgery (HR 1.3, CI 1.2-1.5). CONCLUSION: The risk of re-amputation was more than twice as high after TTA (29%) and KD (30%) compared with TFA (11%). Most re-amputations were conducted within 30 days of the index MLEA. Dyslipidemia, renal insufficiency, and prior vascular surgery were associated with higher risk of re-amputation.

Half of all hip and knee arthroplasty patients may be potential day-case candidates: a nationwide register study of 166,730 procedures.

BACKGROUND AND PURPOSE: The overall potential pool of day-case candidates on a national level in hip and knee arthroplasty is unknown. We aimed to estimate the proportion of hip and knee arthroplasty patients eligible for day-case surgery based on contemporary widely used criteria and determine whether there has been a change in the proportion of eligible patients over time and, secondarily, to investigate the proportion of eligible patients discharged on the day of surgery. METHODS: Based on data from the Danish National Patient Register, we identified all patients undergoing primary unilateral hip or knee arthroplasty from January 2010 to March 2020. Using a modification of day-case eligibility criteria proposed by a national multicenter collaboration, we sorted patients into either day-case eligible or ineligible. A day-case procedure was defined as discharge on the day of surgery. RESULTS: We included patients comprising a total of 166,730 primary total hip (THA), total knee (TKA), and unicompartmental knee arthroplasty (UKA). 48% (95% confidence interval [CI] 48-49) were eligible for day-case surgery, with a decline from 50% (CI 49-51) in 2010 to 46% (CI 46-47) eligible in 2019. More UKA patients were day-case eligible (55%, CI 54-56) than THA (47%, CI 47-48) and TKA patients (49%, CI 48-49). A maximum of 8.0% (CI 7.4-8.5) of eligible patients were discharged on the day of surgery in 2019. CONCLUSION: 48% of the Danish hip and knee arthroplasty patients were potential day-case candidates, with a small decline in eligibility from 50% in 2010 to 46% in 2019. Day of surgery discharge among day-case eligible patients peaked at 8% in 2019. Thus, the potential for more day-case surgery seems large.

The impact of comorbidity status on knee function 1 year after total knee arthroplasty: a population-based cohort study.

BACKGROUND AND PURPOSE: Few studies have examined the impact of comorbidity on functional and clinical knee scores after primary total knee arthroplasty (TKA). We compared the effect of having a high Charlson Comorbidity Index (CCI), relative to a low CCI, on changes in the American Knee Society Score (AKSS) functional and clinical scores from baseline to week 52 after TKA in patients with knee osteoarthritis (OA). METHODS: This population-based cohort study included 22,533 patients identified in the Danish Knee Arthroplasty Register from 1997 to 2021. Patients were classified as having low, medium, or high comorbidity based on CCI. The outcome was defined as the mean change (from preoperative to 1-year post-TKA) in functional and clinical knee scores measured by the AKSS (0-100). The association was analyzed using multiple linear regression by calculating mean change scores adjusting for sex, age, weight, cohabiting status, and baseline AKSS. RESULTS: The prevalence of patients with low, medium, and high comorbidity was 75%, 21%, and 4%, respectively. The mean change score in functional AKSS for patients with high comorbidity was -6 points (95% confidence interval [CI] -7 to -5) compared with low comorbidity. The mean change score in clinical AKSS for patients with high comorbidity was -1 point (CI -2 to 0) compared with low comorbidity. CONCLUSION: Patients with knee OA and medium or high comorbidity can expect similar improvements in functional and clinical AKSS after TKA to patients with low comorbidity.

Implementation of outpatient hip and knee arthroplasty in a multicenter public healthcare setting.

BACKGROUND AND PURPOSE: Length of hospital stay after hip and knee arthroplasty is about 1 day in Denmark with few patients discharged on the day of surgery. Hence, a protocol for multicenter implementation of discharge on day of surgery has been instituted. We aimed to describe the implementation of outpatient hip and knee arthroplasty in a multicenter public healthcare setting. METHODS: We performed a prospective multicenter study from 7 public hospitals across Denmark. Patients were screened using well-defined in- and exclusion criteria and were discharged on day of surgery when fulfilling functional discharge criteria. The study period was from September 2022 to February 2023 with variable start of implementation. Data from the same centers in a 6-month period before the COVID pandemic from July 2019 to December 2019 was used for baseline control. RESULTS: Of 2,756 primary hip and knee arthroplasties, 37% (95% confidence interval [CI] 35-39) were eligible (range 21-50% in centers) and 52% (range 24-62%) of these were discharged on day of surgery. 21% (CI 20-23) of all patients (eligible and non-eligible) were discharged on day of surgery with a range of 10-31% within centers. This was an additional 15% (CI 13-17, P < 0.001) compared with patients discharged in the control period (6% in 2019). CONCLUSION: We found it possible to perform outpatient hip and knee replacement in 21% of patients in a public healthcare setting, probably to be increased with further center experience.

Non-BRCA1/BRCA2 high-risk familial breast cancers are not associated with a high prevalence of BRCAness.

BACKGROUND: Familial breast cancer is in most cases unexplained due to the lack of identifiable pathogenic variants in the BRCA1 and BRCA2 genes. The somatic mutational landscape and in particular the extent of BRCA-like tumour features (BRCAness) in these familial breast cancers where germline BRCA1 or BRCA2 mutations have not been identified is to a large extent unknown. METHODS: We performed whole-genome sequencing on matched tumour and normal samples from high-risk non-BRCA1/BRCA2 breast cancer families to understand the germline and somatic mutational landscape and mutational signatures. We measured BRCAness using HRDetect. As a comparator, we also analysed samples from BRCA1 and BRCA2 germline mutation carriers. RESULTS: We noted for non-BRCA1/BRCA2 tumours, only a small proportion displayed high HRDetect scores and were characterized by concomitant promoter hypermethylation or in one case a RAD51D splice variant previously reported as having unknown significance to potentially explain their BRCAness. Another small proportion showed no features of BRCAness but had mutationally active tumours. The remaining tumours lacked features of BRCAness and were mutationally quiescent. CONCLUSIONS: A limited fraction of high-risk familial non-BRCA1/BRCA2 breast cancer patients is expected to benefit from treatment strategies against homologue repair deficient cancer cells.

Ceruloplasmin-deficient mice show changes in PTM profiles of proteins involved in messenger RNA processing and neuronal projections and synaptic processes.

Ceruloplasmin (Cp) is a multicopper oxidase with ferroxidase properties being of importance to the mobilisation and export of iron from cells and its ability to bind copper. In ageing humans, Cp deficiency is known to result in aceruloplasminemia, which among other is characterised by neurological symptoms. To obtain novel information about the functions of Cp in the central nervous system (CNS) we compared the brain proteome in forebrains from asymptomatic 4-6-month-old Cp-deficient (B6N(Cg)-Cptm1b(KOMP)Wtsi /J) and wild-type mice. Of more than 5600 quantified proteins, 23 proteins, were regulated, whereas more than 1200 proteins had regulated post-translational modifications (PTMs). The genes of the regulated proteins, glycoproteins and phosphoproteins appeared mostly to be located to neurons and oligodendrocyte precursor cells. Cp deficiency especially affected the function of proteins involved in the extension of neuronal projections, synaptic signalling and cellular mRNA processing and affected the expression of proteins involved in neurodegenerative disease and diabetes. Iron concentration and transferrin saturation were reduced in the blood of even younger, 3- to 5-month-old, Cp-deficient mice. Iron act as cofactor in many enzymatic processes and reactions. Changes in iron availability and oxidation as consequence of Cp deficiency could therefore affect the synthesis of proteins and lipids. This proteomic characterisation is to our knowledge the first to document the changes taking place in the CNS-proteome and its phosphorylation and glycosylation state in Cp-deficient mice.

Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.

Bi-allelic Loss-of-Function Variants in NUP188 Cause a Recognizable Syndrome Characterized by Neurologic, Ocular, and Cardiac Abnormalities.

Nucleoporins (NUPs) are an essential component of the nuclear-pore complex, which regulates nucleocytoplasmic transport of macromolecules. Pathogenic variants in NUP genes have been linked to several inherited human diseases, including a number with progressive neurological degeneration. We present six affected individuals with bi-allelic truncating variants in NUP188 and strikingly similar phenotypes and clinical courses, representing a recognizable genetic syndrome; the individuals are from four unrelated families. Key clinical features include congenital cataracts, hypotonia, prenatal-onset ventriculomegaly, white-matter abnormalities, hypoplastic corpus callosum, congenital heart defects, and central hypoventilation. Characteristic dysmorphic features include small palpebral fissures, a wide nasal bridge and nose, micrognathia, and digital anomalies. All affected individuals died as a result of respiratory failure, and five of them died within the first year of life. Nuclear import of proteins was decreased in affected individuals' fibroblasts, supporting a possible disease mechanism. CRISPR-mediated knockout of NUP188 in Drosophila revealed motor deficits and seizure susceptibility, partially recapitulating the neurological phenotype seen in affected individuals. Removal of NUP188 also resulted in aberrant dendrite tiling, suggesting a potential role of NUP188 in dendritic development. Two of the NUP188 pathogenic variants are enriched in the Ashkenazi Jewish population in gnomAD, a finding we confirmed with a separate targeted population screen of an international sampling of 3,225 healthy Ashkenazi Jewish individuals. Taken together, our results implicate bi-allelic loss-of-function NUP188 variants in a recessive syndrome characterized by a distinct neurologic, ophthalmologic, and facial phenotype.

Whole exome sequencing of 28 families of Danish descent reveals novel candidate genes and pathways in developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is a common condition involving instability of the hip with multifactorial etiology. Early diagnosis and treatment are critical as undetected DDH is an important cause of long-term hip complications. Better diagnostics may be achieved through genetic methods, especially for patients with positive family history. Several candidate genes have been reported but the exact molecular etiology of the disease is yet unknown. In the present study, we performed whole exome sequencing of DDH patients from 28 families with at least two affected first-degree relatives. Four genes previously not associated with DDH (METTL21B, DIS3L2, PPP6R2, and TM4SF19) were identified with the same variants shared among affected family members, in more than two families. Among known association genes, we found damaging variants in DACH1, MYH10, NOTCH2, TBX4, EVC2, OTOG, and SHC3. Mutational burden analysis across the families identified 322 candidate genes, and enriched pathways include the extracellular matrix, cytoskeleton, ion-binding, and detection of mechanical stimulus. Taken altogether, our data suggest a polygenic mode of inheritance for DDH, and we propose that an impaired transduction of the mechanical stimulus is involved in the etiopathological mechanism. Our findings refine our current understanding of candidate causal genes in DDH, and provide a foundation for downstream functional studies.