GENETICS IN ENDOCRINOLOGY: Approaches to molecular genetic diagnosis in the management of differences/disorders of sex development (DSD): position paper of EU COST Action BM 1303 'DSDnet'

The differential diagnosis of differences or disorders of sex development (DSD) belongs to the most complex fields in medicine. It requires a multidisciplinary team conducting a synoptic and complementary approach consisting of thorough clinical, hormonal and genetic workups. This position paper of EU COST (European Cooperation in Science and Technology) Action BM1303 'DSDnet' was written by leading experts in the field and focuses on current best practice in genetic diagnosis in DSD patients. Ascertainment of the karyotpye defines one of the three major diagnostic DSD subclasses and is therefore the mandatory initial step. Subsequently, further analyses comprise molecular studies of monogenic DSD causes or analysis of copy number variations (CNV) or both. Panels of candidate genes provide rapid and reliable results. Whole exome and genome sequencing (WES and WGS) represent valuable methodological developments that are currently in the transition from basic science to clinical routine service in the field of DSD. However, in addition to covering known DSD candidate genes, WES and WGS help to identify novel genetic causes for DSD. Diagnostic interpretation must be performed with utmost caution and needs careful scientific validation in each DSD case.

Association between Carotid Plaque Features on CTA and Cerebrovascular Ischemia: A Systematic Review and Meta-Analysis.

BACKGROUND: CTA is a widely available imaging examination that may allow the evaluation of high-risk carotid plaque features. PURPOSE: Our aim was to evaluate the association between specific carotid plaque features on CTA and ipsilateral cerebrovascular ischemia. DATA SOURCES: We performed a systematic review of Ovid MEDLINE, Ovid Embase, Scopus, and the Cochrane Library from inception to March 2016 for articles that evaluated the relationship between CTA-detected carotid plaque features and ischemic events, defined as ipsilateral ischemic stroke or transient ischemic attack. STUDY SELECTION: Sixteen studies were ultimately included after screening 12,557. DATA ANALYSIS: Two readers recorded data from each study and assessed the study quality with all disagreements resolved by a third reader. A random-effects OR was used to evaluate the association between cerebrovascular ischemia and each of the evaluated plaque features. DATA SYNTHESIS: We found significant positive relationships with cerebrovascular ischemia for the presence of soft plaque (OR, 2.9; 95% CI, 1.4-6.0), plaque ulceration (OR, 2.2; 95% CI, 1.4-3.4), and increased common carotid artery wall thickness (OR, 6.2; 95% CI, 2.5-15.6). We found a significant negative relationship between calcified plaque and ipsilateral ischemia (OR, 0.5; 95% CI, 0.4-0.7). LIMITATIONS: We found heterogeneity in the existing literature secondary to lack of standardized plaque features and clinical definitions. CONCLUSIONS: Soft plaque, plaque ulceration, and increased common carotid artery wall thickness on CTA are associated with ipsilateral cerebrovascular ischemia, while calcified plaque is negatively associated with downstream ischemic events.

Factors Associated With Nutritional Risk Among Homebound Older Adults With Depressive Symptoms.

OBJECTIVES: This study used the Evans model of public health determinants to identify factors associated with nutritional risk in older adults. DESIGN: The Evans model domains (physical and mental well-being, social/environmental statuses, individual choice, and economic security) were measured in a sample of homebound older adults. Regularized logistic regression analysis with LASSO penalty function was used to determine the strongest domain of the Evans model. Using traditional logistic regression, individual variables across all domains were compared to identify the significant predictors. SETTING: Older adults receiving home meal services were referred to the study by community program staff. PARTICIPANTS: Participants included 164 homebound older adults (age > 60) who endorsed at least one gateway symptom of depression. MEASUREMENTS: Measurements: Nutritional risk was determined using the Mini Nutritional Assessment. Domains of the Evans model were measured using the MAI Medical Condition Checklist, items from the IADL scale, the Structured Clinical Interview for DSM-IV Axis I Disorders, the Duke Social Support Index, living arrangements, marital status, the Alcohol Use Disorders Identification Test, items from the SCID Screening Module, and a self-report of perceived financial security. RESULTS: Poor mental well-being, defined by a diagnosis of major depressive disorder, was identified as the strongest Evans model domain in the prediction of nutritional risk. When each variable was independently evaluated across domains, instrumental support (Wald's Z=-2.24, p=0.03) and a history of drug use (Wald's Z=-2.40, p=0.02) were significant predictors. CONCLUSIONS: The Evans model is a useful conceptual framework for understanding nutritional health, with the mental domain found to be the strongest domain predictor of nutritional risk. Among individual variables across domains, having someone to help with shopping and food preparation and a history of drug use were associated with lower nutritional risk. These analyses highlight potential targets of intervention for nutritional risk among older adults.

Optimization of normal-phase chromatographic separation of compounds with primary, secondary and tertiary amino groups.

The retention behavior of primary, secondary and tertiary amines was studied using normal-phase-HPLC on silica, diol, and cyano stationary phases. Several classes of amines, including benzylamines, anilines, ephedrines, tryptamines, and azatryptamines were chromatographed using mixtures of hexane and ethoxynonafluorobutane with methylene chloride and methanol. Peak tailing, diminished selectivity and low plate count were minimized by the addition of volatile amines to the mobile phase. The optimal additive was n-propylamine at 0.1% concentration. On diol columns, the elution order of free primary, N-N-methyl, and N,N-dimethylamines was predictable, while the elution order of primary and secondary amines on cyano columns varied depending on the alcohol modifier concentration. The feasibility of preparative normal-phase chromatography was demonstrated by the separation of a mixture of primary, secondary and tertiary amines obtained by direct methylation of norephedrine. The procedures described may provide a practical alternative to traditional methods of analysis and purification of potential drug candidates.

Inferior pancreaticoduodenal artery aneurysms in association with celiac axis stenosis or occlusion.

PURPOSE: To describe the pathophysiology, identification and management of inferior pancreaticoduodenal artery aneurysms in association with celiac axis stenosis or occlusion has been reported. REVIEW FINDINGS: These aneurysms are thought to arise due to increased flow through the pancreaticoduodenal arcades. The arcades first enlarge, and then form focal aneurysms which may rupture. The aneurysms can be treated through endovascular techniques or by surgery, though the former is a preferred approach.

Normal-phase chiral liquid chromatography-mass spectrometry of non-UV-active compounds: applications for pharmaceutically relevant racemates.

Mixtures of hexane-like ethoxynonafluorobutane with alcohols were used as MS-friendly mobile phases for separation and efficient detection of non-UV-active enantiomers and diastereomers using normal-phase HPLC-APCI-MS. Racemic muscone, camphorsulfonamide, camphorsultam, BOC-protected 1-(3-aminopropyl)-2-pipecoline and diastereomeric 2-methylhexanoyl camphorsultams were resolved on Chiralpak AS and AD and achiral Luna CN columns. The responses of UV and APCI-MS detectors were compared under separation conditions studied, with MS detection achieving lowest detectable quantity in the range of 0.5-2 ng per chromatographic peak. The absolute configuration of crystalline derivatives of racemic 2-methylhexanoic acid with (S)-(-)-2,10-camphorsultam was determined by X-ray analysis after their automatic purification by preparative LC-MS. The technique described can be used to purify and determine the absolute stereochemistry of compounds of unknown structure which contain free carboxy group and lack sufficient UV absorbance.

Screening for novel ENU-induced rhythm, entrainment and activity mutants.

Chemical mutagenesis has provided an opportunity to develop and expand the repertoire of behavioural mutants for gene function studies. With this in mind, we have established a screen in mice for mutations affecting circadian rhythms, entrainment to light and other wheel-running parameters. The screen consists of an assessment of mouse wheel-running activity in a 12:12 h light/dark cycle for 7-10 days followed by assessment in constant darkness for up to 20 days. Responses to light are assessed using two protocols; a 15 minute light pulse given at circadian time 16 on the tenth day in constant darkness and an additional 12 h of light upon transition from light/dark conditions to constant darkness. To date, approximately 1300 progeny of chemically mutagenised mice have been screened. Computer-aided assessment of wheel-running parameters has helped in identifying abnormal phenotypes in approximately 5% of all animals screened. Inheritance testing of mice with abnormal phenotypes has confirmed the number of robustly inherited mutant phenotypes to be 1% of the total screened. Confirmed mutants including those affecting free-running period, light-responsiveness and wheel-running endurance have been identified. Thus far, low-resolution map positions have been established for four mutants by completing genome scans in backcross progeny. Mutant loci do not correspond with those previously associated with wheel-running behaviour. This result confirms that phenotype-driven approaches such as this should continue to provide material for mammalian gene function studies.

Molecular phenotyping of the mouse ky mutant reveals UCP1 upregulation at the neuromuscular junctions of dystrophic soleus muscle.

The ky mutant mouse displays a muscular dystrophy that affects almost exclusively slow type muscles in which persistent muscle regeneration, neuromuscular junction instability and an absence of the hypertrophic response are prominent features. In order to gain insights into the pathogenesis of this muscular dystrophy we have undertaken RNA profiling of the extensor digitorum longus, a fast unaffected muscle, and the highly pathological soleus slow muscle, followed by further expression studies to validate the results. In dystrophic soleus, there is a coordinated change in the expression level of genes encoding energy transducing mitochondrial proteins and an increase in the expression of stretch response genes. Upregulation of uncoupling proteins 1 and 2 is a unique molecular signature of the ky muscular dystrophy and was further characterised at the protein level. Our results show a spatial and temporal association between disorganisation of acetylcholine receptor clusters and upregulation of uncoupling protein 1. There is also evidence of a breakdown of neuromuscular junction muscle-specific kinase-dependent signalling in adult mutant soleus. Sarcolemma-associated proteins implicated in muscular dystrophies revealed no differences on microarrays and were confirmed as normally distributed by immunofluorescence. Altogether, the data presented suggest that the ky muscular dystrophy develops by a distinctive pathogenic mechanism.

Estrogen induces phosphorylation of cyclic AMP response element binding (pCREB) in primary hippocampal cells in a time-dependent manner.

Using hippocampal primary cell cultures at 14 days in vitro (div), we have investigated actions of 17-beta estradiol (E; 10 nM) on the phosphorylation of CREB and on signaling pathways that regulate CREB phosphorylation. After demonstrating that 14 div is optimal for these studies, we examined the time course of E induction of CREB phosphorylation (pCREB) at serine residue 133. The induction of pCREB occurs as early as 1 h following E treatment, presumably via a mechanism involving an E-stimulated signal transduction system, which is sustained for at least 24 h but inhibited by 48 h. The early activity may represent an initial signal required for events leading to phosphorylation of CREB while the sustained signal may lead to CREB-mediated gene expression for cell survival and synapse formation. Furthermore, we examined the pathways for E action preceding pCREB induction by blocking three major kinases (protein kinase; mitogen activated protein kinase, MAPK; and calcium-calmodulin kinase II, CaMKII) upstream of pCREB. We found that E stimulates each pathway at 24 h and that phosphorylation of CREB is dependent on both MAPK and CaMK activities, but less dependent on the Akt pathway. Because CREB has been linked to E induction of excitatory spine synapses, we used a spine marker, spinophilin, to establish E effects on spine formation. Spinophilin expression was up-regulated in response to E and this effect was blocked by an inhibitor of (CaMKII). These studies demonstrate the central role played by CaMKII pathway in the actions of E on both transcriptional regulation and structural reorganization in neurons.

Expression of a novel mammalian epidermal growth factor-related gene during mouse neural development.

We have recently reported the preliminary characterisation of a novel EGF-related gene, Scube1 (signal peptide-CUB domain-EGF-related, gene 1), that is expressed prominently in the developing gonad, nervous system, somites, surface ectoderm and limb buds of the mouse. Here we describe the expression pattern of a closely related gene, Scube2 (also known as Cegp1), which maps to the distal region of mouse chromosome 7. Scube2 transcription is restricted to the embryonic neurectoderm but is also detectable in the adult heart, lung and testis.

Cloning, mapping, and expression analysis of a gene encoding a novel mammalian EGF-related protein (SCUBE1).

The epidermal growth factor (EGF) superfamily comprises a diverse group of proteins that function as secreted signaling molecules, growth factors, and components of the extracellular matrix, many with a role in vertebrate development. We have isolated a novel mammalian gene encoding an EGF-related protein with a CUB (C1s-like) domain that defines a new mammalian gene family. The Scube1 (signal peptide-CUB domain-EGF-related 1) gene was isolated from a developing mouse urogenital ridge cDNA library and is expressed prominently in the developing gonad, nervous system, somites, surface ectoderm, and limb buds. We have mapped Scube1 to mouse chromosome 15 and show that it is orthologous to a human gene in the syntenic region of chromosome 22q13. We discuss the possible functions of this novel gene and its role in heritable disease in light of these data.

Longitudinal effects of integrated treatment on alcohol use for persons with serious mental illness and substance use disorders.

A randomized experimental design was used to assign participants to an integrated mental health and substance use treatment program or to standard hospital treatment. A multilevel, nonlinear model was used to estimate hospital treatment effects on days of alcohol use for persons with serious mental illness and substance use disorders over 18 months. The integrated treatment program had a significant effect on the rate of alcohol use at 2 months postdischarge, reducing the rate of use by 54%. Motivation for sobriety at hospital discharge, posttreatment self-help attendance, and social support for sobriety were also found to reduce the rate of use during the follow-up period. Implications for mental health treatment and aftercare support are discussed.

Sexually dimorphic expression of protease nexin-1 and vanin-1 in the developing mouse gonad prior to overt differentiation suggests a role in mammalian sexual development.

The mammalian sex-determining pathway is controlled by the presence or absence of SRY expression in the embryonic gonad. Expression of SRY in males is believed to initiate a pathway of gene expression resulting in testis development. In the absence of SRY, ovary development ensues. Several genes have now been placed in this pathway but our understanding of it is far from complete and several functional classes of protein appear to be absent. Sex-determining genes frequently exhibit sexually dimorphic patterns of expression in the developing gonad both before and after overt differentiation of the testis or ovary. In order to identify additional sex-determining or gonadal differentiation genes we have examined gene expression in the developing gonads of the mouse using cDNA microarrays constructed from a normalized urogenital ridge library. We screened for genes exhibiting sexually dimorphic patterns of expression in the gonad at 12.5 and 13.5 days post-coitum, after overt gonad differentiation, by comparing complex cDNA probes derived from male and female gonadal tissue at these stages on micro-arrays. Using in situ hybridization analysis we show here that two genes identified by this screen, protease nexin-1 (Pn-1) and vanin-1 (Vnn1), exhibit male-specific expression prior to overt gonadal differentiation and are detected in the somatic portion of the developing gonad, suggesting a possible direct link to the testis-determining pathway for both genes.

Groucho/transducin-like enhancer of split (TLE) family members interact with the yeast transcriptional co-repressor SSN6 and mammalian SSN6-related proteins: implications for evolutionary conservation of transcription repression mechanisms.

The yeast proteins TUP1 and SSN6 form a transcription repressor complex that is recruited to different promoters via pathway-specific DNA-binding proteins and regulates the expression of a variety of genes. TUP1 is functionally related to invertebrate and vertebrate transcriptional repressors of the Groucho/transducin-like Enhancer of split (TLE) family. The aim was to examine whether similar mechanisms underlie the transcription repression functions of TUP1 and Groucho/TLEs by determining whether TLE family members can interact with yeast SSN6 and mammalian SSN6-like proteins. It is shown in the present work that SSN6 binds to TLE1 and mediates transcriptional repression when expressed in mammalian cells. Moreover, TLE1 and TLE2 interact with two mammalian proteins related to SSN6, designated as the products of the ubiquitously transcribed tetratricopeptide-repeat genes on the Y (or X) chromosomes (UTY/X). These findings suggest that mammalian TLE and UTY/X proteins may mediate repression mechanisms similar to those performed by TUP1-SSN6 in yeast.

The mouse Y chromosome interval necessary for spermatogonial proliferation is gene dense with syntenic homology to the human AZFa region.

The Delta Sxrb deletion interval of the mouse Y chromosome contains Spy, a spermatogenesis factor gene(s) whose expression is essential for the postnatal development of the mitotic germ cells, spermatogonia. The boundaries of Delta Sxrb are defined by the duplicated genes Zfy1 and Zfy2 and four further genes have previously been mapped within the interval: Ube1y and Smcy, linked with Zfy1 on a contig of 250 kb, and Dffry and Uty, which were unanchored. The interval was estimated to be >450 kb. In order to identify any further gene(s) that may underlie Spy, systematic exon trapping was performed on an extended contig, anchored on Zfy1, which covers 750 kb of the Delta Sxrb interval. Exons from two novel genes were isolated and placed together with Dffry and Uty on the contig in the order Dffry-Dby-Uty-Tspy-Eif2gammay-Smcy- Ube1y-Zfy1. All the genes, with the double exception of Tspy, are X-Y homologous and produce putatively functional, spliced transcripts. The tight linkage and order of Dffry, Dby and Uty was shown to be conserved in deletion intervals 5C/5D of the human Y chromosome by the construction of a contig of human PAC and YAC clones; this represents the first example of syntenic homology between Y chromosomes from two distinct mammalian orders. Interval 5C/5D contains the distal boundary of the AZFa interval, which, like Delta Sxrb, is believed to be necessary for spermatogonial development in the prepubertal testis. Our results therefore show that AZFa and Spy may be encoded by homologous genes.

A novel psychophysical illusion resulting from interaction between horizontal vestibular and vertical pursuit stimulation.

HYPOTHESIS: The hypothesis was tested that the perception of an object's motion is made in relation to an internal reference center (IRC), which is under the influence of vestibular receptors. Experiments were designed to evaluate whether induced vestibular self-motion will interact linearly with the perception of a visual target (VT). BACKGROUND: The major complaint of vestibular patients is vertigo, an illusion of motion. However, there is as yet no objective method to measure this illusion, which is produced by vestibular stimulation. METHODS: Six subjects were instructed to track a vertically moving VT while sinusoidally rotating (0.2 Hz) in the yaw plane at 14, 28, and 42 degrees/s. Eye movements were monitored by electro-oculographic electrodes. RESULTS: During visual-vestibular interaction, all subjects perceived a VT moving obliquely while the eyes moved in the vertical plane. The subject then tilted the VT trajectory until vertical was perceived. At this time, the eye had an oblique vectorial trajectory. Interactive horizontal eye velocities, both vertical and horizontal components, were compared with those from rotation in the dark, showing a strong positive linear relationship (slope = 0.96, r = 0.84, n = 18). CONCLUSIONS: Results support the hypothesis of an egocentric sense of orientation whereby velocity of external objects is evaluated in relation to an IRC that is dependent on the status of the vestibular system. These methods may lead to new techniques for clinical evaluation of vestibular patients.