Genetic variants regulating immune cell levels in health and disease.

The complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on "self" result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease.

Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis.

Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.

Changes in guilt cognitions mediate the effect of trauma-informed guilt reduction therapy on PTSD and depression outcomes.

OBJECTIVE: Trauma-informed guilt reduction therapy (TrIGR), a six-session cognitive behavioral therapy targeting trauma-related guilt and distress, reduces guilt and symptoms of posttraumatic stress disorder (PTSD) and depression, yet little is known regarding how and why TrIGR may be effective. METHOD: This study examined treatment-related changes in avoidant coping and trauma-related guilt cognitions as possible mediators of treatment effects on PTSD and depression outcomes at 3- and 6-month follow-up. Data were from a randomized controlled trial for treatment of trauma-related guilt comparing TrIGR and supportive care therapy among 145 post-9/11 US veterans (Mage = 39.2 [8.1], 93.8% male). RESULTS: At pretreatment, most (86%) met PTSD criteria. Intent to treat analyses using parallel mediation models indicated changes in guilt cognitions, but not avoidant coping, mediated the effect of TrIGR on reducing PTSD severity at 3-month (a × b = -0.15, p < 0.01, 95% CI: [-0.24 to -0.06], p = 0.001) and 6-month (a × b = -0.17, 95% CI: [-0.26 to -0.07], p = 0.001) follow-up. Similarly, changes in guilt cognitions, but not avoidant coping, mediated the effect of TrIGR on reducing depression severity at 3-month (a × b = -0.10, 95% CI: [-0.18 to -0.02], p = 0.02) and 6-month (a × b = -0.11, 95% CI: [-0.20 to -0.03], p = 0.01) follow-up. CONCLUSIONS: Compared to guilt cognitions, changes in avoidant coping were less integral to downstream PTSD and depression symptom reduction. Guilt cognition change may be a salient active ingredient of PTSD and depression treatment for those with trauma-related guilt and a key therapy element to which providers should be attuned.

Estimation of DNA contamination and its sources in genotyped samples.

Array genotyping is a cost-effective and widely used tool that enables assessment of up to millions of genetic markers in hundreds of thousands of individuals. Genotyping array data are typically highly accurate but sensitive to mixing of DNA samples from multiple individuals before or during genotyping. Contaminated samples can lead to genotyping errors and consequently cause false positive signals or reduce power of association analyses. Here, we propose a new method to identify contaminated samples and the sources of contamination within a genotyping batch. Through analysis of array intensity and genotype data from intentionally mixed samples and 22,366 samples of the Michigan Genomics Initiative, an ongoing biobank-based study, we show that our method can reliably estimate contamination. We also show that identifying sources of contamination can implicate problematic sample processing steps and guide process improvements. Compared to existing methods, our approach can estimate the proportion of contaminating DNA more accurately, eliminate the need for external databases of allele frequencies, and provide contamination estimates that are more robust to the ancestral origin of the contaminating sample.

The relationship between negative trauma-related cognitions and psychosocial functioning in veterans with posttraumatic stress disorder and alcohol use disorder.

Background: The comorbidity of posttraumatic stress disorder (PTSD) and alcohol use disorder (AUD) is highly prevalent and associated with especially poor psychosocial functioning. Negative trauma-related cognitions are theoretically proposed to be associated with poor psychosocial functioning in PTSD, but few studies have examined the association between negative trauma-related cognitions and psychosocial functioning in PTSD/AUD. Evaluating this association may provide evidence of a potential treatment target for improving psychosocial functioning in PTSD/AUD. We hypothesized that negative trauma-related cognitions, including cognitions about the self, world, and self-blame, would be independently associated with poor psychosocial functioning in the following domains: vitality, psychosocial well-being, role limitations due to emotional distress, and social functioning. Methods: We examined the relationship between negative trauma-related cognitions and psychosocial functioning in 145 treatment-seeking veterans with PTSD/AUD using multiple linear regression analyses while controlling for PTSD and alcohol abuse and dependence severity. Results: Our hypotheses were partially supported. We found that negative trauma-related cognitions were uniquely associated with greater psychosocial functional impairment, independent of PTSD and alcohol abuse and dependence severity. Specifically, negative trauma-related cognitions about the self were associated with greater psychosocial functional impairment across all domains, cognitions about the world were associated with worse social functioning and psychological well-being, and self-blame was associated with impaired psychological well-being. Conclusions: Given that improvements in negative trauma-related cognitions are a mechanism of trauma-focused treatment, future studies should examine whether changes in negative trauma-related cognitions through trauma-focused treatment are associated with improved psychosocial functioning.

Negative Affect Mediates the Association between Posttraumatic Cognitions and Craving in Veterans with Posttraumatic Stress Disorder and Alcohol Use Disorder.

Objective: The co-occurrence of posttraumatic stress disorder (PTSD) and alcohol use disorder (AUD) is common. Individuals with PTSD/AUD commonly drink to cope with PTSD symptoms, which maintains PTSD/AUD, and may result in increased craving for alcohol. Negative affect is implicated in negative reinforcement models of craving. Further, Emotional Processing Theory posits that posttraumatic cognitions lead to the experience of negative affect, which may result in increased craving in PTSD/AUD. The current study aims to advance the understanding of craving in PTSD/AUD by evaluating if specific posttraumatic cognitions (e.g., cognitions about the self, world, and self-blame) are associated with increased negative affect, and whether higher negative affect is associated with heightened craving. Methods: Three separate simple mediation models were utilized to test if negative affect mediated the relationship between each specific posttraumatic cognition type and craving among 136 treatment-seeking veterans with PTSD/AUD. Results: We found that negative affect mediated the association between all posttraumatic cognition types and craving. Specifically, viewing oneself as being unable to handle PTSD-related distress, viewing the world as very dangerous, and blaming oneself for one's role in a traumatic event were all associated with increased negative affect, which was related to higher craving. Conclusions: Given that posttraumatic cognitions improve via trauma-focused treatment for PTSD, future work should evaluate whether improvements in posttraumatic cognitions via trauma-focused treatment lead to decreased negative affect and craving in PTSD/AUD.

Impact of New-Onset Postoperative Depression on Readmission Outcomes After Surgical Coronary Revascularization.

BACKGROUND: Depression affects between 10% and 40% of cardiac surgery patients and is associated with significantly worse outcomes. The incidence and impact of new-onset depression beyond acute follow-up remain ill-defined. The present study aimed to evaluate the incidence, risk factors, and prognostic implication of depression on 90-d readmission rates after coronary artery bypass grafting (CABG) surgery. METHODS: A retrospective cohort study was performed identifying adult patients without prior depression who underwent CABG surgery using the 2010-2014 National Readmissions Database. CABG patients who were readmitted more than 2 wk but within 90 d of discharge were categorized based on the presence of new-onset depression. Association between the development of new-onset depression and rehospitalization were morbidity, mortality, costs, and length of stay (LOS) and were examined using multivariable regression. RESULTS: During the study period, 1,001,945 patients underwent CABG. Of these, 11.7% of patients were readmitted after 14 d but within 90 d of discharge with 5.1% of these patients having a diagnosis of new-onset depression. Postoperative new-onset depression was not associated with increased readmission morbidity, costs, or LOS. Mortality in new-onset depression readmissions was 1.2%, compared with 2.3% in all readmitted patients (P = 0.014). Depression was associated with lower odds of mortality (OR = 0.56, P = 0.02). CONCLUSIONS: New-onset depression following CABG discharge was not associated with increased odds of mortality, morbidity, costs, or increased LOS on readmission. Rather, new-onset depression is associated with decreased odds of readmission mortality. Overall, CABG readmissions are decreasing, whereas the rate of new-onset depression is slightly increasing. Implementation of routine depression screening tools in postoperative CABG care may aid in early detection and management of depression to enhance postoperative recovery and quality of life.

FOXP3 orchestrates H4K16 acetylation and H3K4 trimethylation for activation of multiple genes by recruiting MOF and causing displacement of PLU-1.

Both H4K16 acetylation and H3K4 trimethylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here, we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 trimethylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial cells disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells.

The Association Between Negative Trauma-Related Cognitions and Pain-Related Functional Status Among Veterans With Posttraumatic Stress Disorder and Alcohol Use Disorder.

Among veterans with posttraumatic stress disorder (PTSD), alcohol use disorders (AUDs) are highly prevalent. Furthermore, PTSD frequently co-occurs with chronic pain (CP), and CP is associated with an increased risk of AUD. Pain-related beliefs and appraisals are significantly associated with poorer pain-related functional status, yet few studies have examined negative trauma-related cognitions and their impact on pain-related functional disability in veterans with co-occurring PTSD and AUD. Accordingly, we examined the association between negative trauma-related cognitions and pain severity and pain disability in 137 veterans seeking treatment for PTSD and AUD. Using hierarchical multiple linear regression, we found that higher levels of negative trauma-related cognitions (e.g., "I am completely incompetent") were associated with a higher level of pain severity, after controlling for PTSD symptom severity and frequency of alcohol use, total R2 = .07, ΔR2 = .06. Additionally, as hypothesized, we found that higher levels of negative trauma-related cognitions were associated with higher levels of pain disability, after controlling for PTSD symptom severity, frequency of alcohol use, and pain severity, total R2 = .46, ΔR2 = .03. Given that negative trauma-related cognitions contributed to pain severity and pain disability, even when controlling for PTSD severity and frequency of alcohol use, future studies should explore the potential impact of interventions that address negative trauma-related cognitions (e.g., prolonged exposure or cognitive processing therapy) on pain severity and disability.

Spanish Abstracts by Asociación Chilena de Estrés Traumático (ACET) La asociación entre las cogniciones negativas relacionadas con el trauma y el estado funcional relacionado con el dolor entre los veteranos con trastorno por estrés postraumático y trastorno por uso de alcohol COGNICIONES NEGATIVAS DE TRAUMA Y DOLOR Entre los veteranos con trastorno por estrés postraumático (TEPT), los trastornos por consumo de alcohol (AUD en su sigla en inglés) son altamente prevalentes. Además, el TEPT con frecuencia coexiste con el dolor crónico (DC), y el DC se asocia con un mayor riesgo de AUD. Las creencias y evaluaciones relacionadas con el dolor se asocian significativamente con un estado funcional más pobre relacionado con el dolor, sin embargo, pocos estudios han examinado las cogniciones negativas relacionadas con el trauma y su impacto en la discapacidad funcional relacionada con el dolor en veteranos con coexistencia de TEPT y AUD. En consecuencia, examinamos la asociación entre las cogniciones negativas relacionadas con el trauma y la gravedad del dolor y la discapacidad del dolor en 137 veteranos que buscaban tratamiento para TEPT y AUD. Al utilizar la regresión lineal múltiple jerárquica, encontramos que los niveles más altos de cogniciones negativas relacionadas con el trauma (por ejemplo "Soy completamente incompetente") se asociaron con un mayor nivel de severidad del dolor, después de controlar la severidad de los síntomas de TEPT y la frecuencia del consumo de alcohol, total R2 = .07, ΔR2 = .06. Además, como hipotetizamos, encontramos que los niveles más altos de cogniciones negativas relacionadas con el trauma se asociaron con niveles más altos de discapacidad del dolor, después de controlar la gravedad de los síntomas de TEPT, la frecuencia del consumo de alcohol y la gravedad del dolor, R2 total = .46, ΔR2 = . 03. Dado que las cogniciones negativas relacionadas con el trauma contribuyeron a la severidad del dolor y la discapacidad del dolor, incluso cuando se controla la gravedad y la frecuencia del consumo de alcohol, los estudios futuros deben explorar el impacto potencial de las intervenciones que abordan las cogniciones negativas relacionadas con el trauma (por ejemplo, la terapia de exposición prolongada o la terapia de procesamiento cognitivo) sobre la severidad del dolor y la discapacidad.

Continuous 1.3-million-year record of East African hydroclimate, and implications for patterns of evolution and biodiversity.

The transport of moisture in the tropics is a critical process for the global energy budget and on geologic timescales, has markedly influenced continental landscapes, migratory pathways, and biological evolution. Here we present a continuous, first-of-its-kind 1.3-My record of continental hydroclimate and lake-level variability derived from drill core data from Lake Malawi, East Africa (9-15° S). Over the Quaternary, we observe dramatic shifts in effective moisture, resulting in large-scale changes in one of the world's largest lakes and most diverse freshwater ecosystems. Results show evidence for 24 lake level drops of more than 200 m during the Late Quaternary, including 15 lowstands when water levels were more than 400 m lower than modern. A dramatic shift is observed at the Mid-Pleistocene Transition (MPT), consistent with far-field climate forcing, which separates vastly different hydroclimate regimes before and after ∼800,000 years ago. Before 800 ka, lake levels were lower, indicating a climate drier than today, and water levels changed frequently. Following the MPT high-amplitude lake level variations dominate the record. From 800 to 100 ka, a deep, often overfilled lake occupied the basin, indicating a wetter climate, but these highstands were interrupted by prolonged intervals of extreme drought. Periods of high lake level are observed during times of high eccentricity. The extreme hydroclimate variability exerted a profound influence on the Lake Malawi endemic cichlid fish species flock; the geographically extensive habitat reconfiguration provided novel ecological opportunities, enabling new populations to differentiate rapidly to distinct species.

Degree of correspondence between daily monitoring and retrospective recall of alcohol use among men and women with comorbid AUD and PTSD.

BACKGROUND AND OBJECTIVES: The majority of studies that have identified good correspondence between daily monitoring and retrospective recall of alcohol use have included participants who are relatively stable, are moderate drinkers, report abstinence, and are not diagnosed with comorbid disorders. The current study examined degree of correspondence between alcohol use that was reported daily via interactive voice response (IVR) telephone monitoring and retrospectively using an abbreviated Form-90 (Form-35) covering the same 35-day time period. METHODS: Participants were 54 men and women with comorbid alcohol dependence and posttraumatic stress disorder (PTSD) who reported drinking during the time period. RESULTS: Results indicated that participants reported more drinking days via IVR. Correspondence was strong between the reporting methods for aggregate-level alcohol use variables, including presence/absence of drinking days and heavy drinking days and standard drinks, and associations increased for weeks closer to the assessment date for drinking days and heavy drinking days. Day-to-day agreement was moderate for drinking days and heavy drinking days, though there was large between-person variability in correspondence between reporting methods. Post-hoc analyzes suggested that men and participants who drink more tend to have lower correspondence between assessment methods. DISCUSSION AND CONCLUSIONS: Overall, findings partially replicated previous research and extend our knowledge of alcohol assessment in a comorbid sample. SCIENTIFIC SIGNIFICANCE: Findings highlight the importance of considering the influence that moderating variables have on reporting of alcohol use.

Next-generation sequencing identifies the Danforth's short tail mouse mutation as a retrotransposon insertion affecting Ptf1a expression.

The semidominant Danforth's short tail (Sd) mutation arose spontaneously in the 1920s. The homozygous Sd phenotype includes severe malformations of the axial skeleton with an absent tail, kidney agenesis, anal atresia, and persistent cloaca. The Sd mutant phenotype mirrors features seen in human caudal malformation syndromes including urorectal septum malformation, caudal regression, VACTERL association, and persistent cloaca. The Sd mutation was previously mapped to a 0.9 cM region on mouse chromosome 2qA3. We performed Sanger sequencing of exons and intron/exon boundaries mapping to the Sd critical region and did not identify any mutations. We then performed DNA enrichment/capture followed by next-generation sequencing (NGS) of the critical genomic region. Standard bioinformatic analysis of paired-end sequence data did not reveal any causative mutations. Interrogation of reads that had been discarded because only a single end mapped correctly to the Sd locus identified an early transposon (ETn) retroviral insertion at the Sd locus, located 12.5 kb upstream of the Ptf1a gene. We show that Ptf1a expression is significantly upregulated in Sd mutant embryos at E9.5. The identification of the Sd mutation will lead to improved understanding of the developmental pathways that are misregulated in human caudal malformation syndromes.

Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response.

Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.

Evaluation of portability and cost of a fluorescent PCR ribotyping protocol for Clostridium difficile epidemiology.

Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.

A pilot trial of prazosin, an alpha-1 adrenergic antagonist, for comorbid alcohol dependence and posttraumatic stress disorder.

BACKGROUND: Posttraumatic stress disorder (PTSD) and alcohol dependence (AD) commonly co-occur and are associated with greater symptom severity and costs than either disorder alone. No pharmacologic interventions have been found to decrease both alcohol use and PTSD symptom severity relative to matched placebo. Prazosin, an alpha-1 adrenoreceptor antagonist, has demonstrated the efficacy of reducing PTSD and AD symptoms among individuals with one or the other disorder and may be useful in addressing comorbid PTSD/AD. METHODS: Prazosin and matched placebo were compared in the context of an outpatient 6-week double-blind randomized controlled pilot trial involving 30 individuals with comorbid PTSD/AD. Medication was titrated to 4 mg q am, 4 mg q pm and 8 mg qhs by the end of week 2. Participants in both conditions received 5 medical management sessions. Information regarding alcohol use, craving, and PTSD was gathered daily using a telephone interactive voice response system. RESULTS: Participants randomized to prazosin had a greater reduction in percent days drinking per week and percent days heavy drinking per week between baseline and week 6 than did placebo participants. No significant differences were detected within or between groups in change from weeks 1 to 6 in total PTSD symptoms. Participants in the prazosin condition reported drowsiness on significantly more days than those in the placebo condition. CONCLUSIONS: Consistent with the extant research evaluating medications for comorbid PTSD/AD, the current evaluation of prazosin also found decreased alcohol consumption but no medication effect on PTSD symptomatology.

Real-world glycaemic outcomes in patients with type 1 diabetes using glucose sensors-Experience from a single centre in Dublin.

AIMS: To evaluate changes in glycated haemoglobin (HbA1 c) and sensor-based glycaemic metrics after glucose sensor commencement in adults with T1D. METHODS: We performed a retrospective observational single-centre study on HbA1 c, and sensor-based glycaemic data following the initiation of continuous glucose monitoring (CGM) in adults with T1D (n = 209). RESULTS: We observed an overall improvement in HbA1 c from 66 (59-78) mmol/mol [8.2 (7.5-9.3)%] pre-sensor to 60 (53-71) mmol/mol [7.6 (7.0-8.6)%] on-sensor (p < .001). The pre-sensor HbA1 c improved from 66 (57-74) mmol/mol [8.2 (7.4-8.9)%] to 62 (54-71) mmol/mol [7.8 (7.1-8.7)%] within the first year of usage to 60 (53-69) mmol/mol [7.6 (7.0-8.4)%] in the following year (n = 121, p < .001). RT-CGM-user had a significant improvement in HbA1 c (Dexcom G6; p < .001, r = 0.33 and Guardian 3; p < .001, r = 0.59) while a non-significant reduction was seen in FGM-user (Libre 1; p = .279). Both MDI (p < .001, r = 0.33) and CSII group (p < .001, r = 0.41) also demonstrated significant HbA1 c improvement. Patients with pre-sensor HbA1 c of ≥64 mmol/mol [8.0%] (n = 125), had attenuation of pre-sensor HbA1 c from 75 (68-83) mmol/mol [9.0 (8.4-9.7)%] to 67 (59-75) mmol/mol [8.2 (7.6-9.0)%] (p < .001, r = 0.44). Altogether, 25.8% of patients achieved the recommended HbA1 c goal of ≤53 mmol/mol and 16.7% attained the recommended ≥70% time in range (3.9-10.0 mmol/L). CONCLUSIONS: Our study demonstrated that minimally invasive glucose sensor technology in adults with T1D is associated with improvement in glycaemic outcomes. However, despite significant improvements in HbA1 c, achieving the recommended goals for all glycaemic metrics remained challenging.

High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing.

OBJECTIVE: To identify disease-causing mutations within coding regions of 11 known NPHP genes (NPHP1-NPHP11) in a cohort of 192 patients diagnosed with a nephronophthisis-associated ciliopathy, at low cost. METHODS: Mutation analysis was carried out using PCR-based 48.48 Access Array microfluidic technology (Fluidigm) with consecutive next-generation sequencing. We applied a 10-fold primer multiplexing approach allowing PCR-based amplification of 475 amplicons (251 exons) for 48 DNA samples simultaneously. After four rounds of amplification followed by indexing all of 192 patient-derived products with different barcodes in a subsequent PCR, 2 × 100 paired-end sequencing was performed on one lane of a HiSeq2000 instrument (Illumina). Bioinformatics analysis was performed using 'CLC Genomics Workbench' software. Potential mutations were confirmed by Sanger sequencing and shown to segregate. RESULTS: Bioinformatics analysis revealed sufficient coverage of 30 × for 168/192 (87.5%) DNA samples (median 449 ×) and of 234 out of 251 targeted coding exons (sensitivity: 93.2%). For proof-of-principle, we analysed 20 known mutations and identified 18 of them in the correct zygosity state (90%). Likewise, we identified pathogenic mutations in 34/192 patients (18%) and discovered 23 novel mutations in the genes NPHP3 (7), NPHP4 (3), IQCB1 (4), CEP290 (7), RPGRIP1L (1), and TMEM67 (1). Additionally, we found 40 different single heterozygous missense variants of unknown significance. CONCLUSIONS: We conclude that the combined approach of array-based multiplexed PCR-amplification on a Fluidigm Access Array platform followed by next-generation sequencing is highly cost-efficient and strongly facilitates diagnostic mutation analysis in broadly heterogeneous Mendelian disorders.

Urethral reconstruction using a prefabricated pedicled gracilis flap.

This case study describes a patient who experienced an iatrogenic urethral injury because of a Fournier gangrene debridement. Because of the extent of the debridement, which resected all penile and scrotal dartos tissue, no local flaps that would typically be used to reconstruct a urethral disruption were possible. The authors chose to use a prefabricated pedicled gracilis flap to restore urethral continuity.