Next generation sequencing (NGS) to improve the diagnosis and management of patients with disorders of sex development (DSD).

Disorders of sex development (DSDs) are a diverse group of conditions where the chromosomal, gonadal or anatomical sex can be atypical. The highly heterogeneous nature of this group of conditions often makes determining a genetic diagnosis challenging. Prior to next generation sequencing (NGS) technologies, genetic diagnostic tests were only available for a few of the many DSD-associated genes, which consequently had to be tested sequentially. Genetic testing is key in establishing the diagnosis, allowing for personalised management of these patients. Pinpointing the molecular cause of a patient's DSD can significantly impact patient management by informing future development needs, altering management strategies and identifying correct inheritance pattern when counselling family members. We have developed a 30-gene NGS panel, designed to be used as a frontline test for all suspected cases of DSD (both 46,XX and 46,XY cases). We have confirmed a diagnosis in 25 of the 80 patients tested to date. Confirmed diagnoses were linked to mutations in AMH, AMHR2, AR, HSD17B3, HSD3B2, MAMLD1, NR5A1, SRD5A2 and WT1 which have resulted in changes to patient management. The minimum diagnostic yield for patients with 46,XY DSD is 25/73. In 34/80 patients, only benign or likely benign variants were identified, and in 21/80 patients only variants of uncertain significance (VOUS) were identified, resulting in a diagnosis not being confirmed in these individuals. Our data support previous studies that an NGS panel approach is a clinically useful and cost-effective frontline test for patients with DSDs.

A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis.

BACKGROUND: We studied the overlap between the major (epi)genomic events microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) in colorectal cancer (CRC), and whether specific (epi)genotypes were associated with CRC-related deaths. PATIENTS AND METHODS: Molecular analyses using tumor DNA were successful in 509 CRC cases identified within the Netherlands Cohort Study in the period 1989-1993. Follow-up for the vital status until May 2005 was 100%. RESULTS: MSI (12.6%), CIMP-only (5.3%), CIMP + CIN (13.4%), CIN-only (58.2%) and triple-negative tumors (10.6%) differed significantly regarding tumor localization, differentiation grade, initial adjuvant therapy (AT) use and genetic characteristics (P ≤ 0.03). CIMP-only, CIMP + CIN and triple-negative tumors, compared with CIN-only tumors, were significantly associated with a 3.67, 2.44 and 3.78-fold risk of CRC-related deaths after 2-year follow-up (95% confidence intervals, CIs, 1.70-7.91, 1.35-4.41 and 1.97-7.25, respectively), but not after late follow-up. MSI tumors were borderline significantly associated with a 0.40-fold risk of CRC-related deaths after late follow-up (95% CI 0.15-1.03). CONCLUSION(S): This is the first study to show that specific (epi)genotypes may hold a differential prognostic value that may vary over time. Although no specific treatment data were available, an explanation for the differential findings over time might be that (epi)genotypes modify therapy response.

Antimicrobial prescribing practice in UK equine veterinary practice.

REASONS FOR PERFORMING STUDY: Antimicrobial use is known to select for the emergence of resistant strains of bacteria; therefore prudent use in both human and veterinary medicine is essential to preserve their efficacy. OBJECTIVES: To characterise antimicrobial prescribing patterns in UK equine practice and evaluate factors associated with prescribing. METHODS: A postal questionnaire including 4 clinical scenarios was sent to 740 veterinary surgeons that treat horses. Data were collected on the clinician, their practice and sources of information regarding antimicrobials and their use. Multivariable logistic regression analysis was used to ascertain factors associated with 5 outcomes; prescribing off-licence drugs; prescribing at incorrect doses; prescribing 3rd or 4th generation cephalosporins; prescribing fluoroquinolones and prescribing potentiated sulphonamides. RESULTS: Questionnaires were completed by 38% of veterinary surgeons who were sent questionnaires. Less than 1% of practices had antimicrobial use guidelines. Trimethoprim-sulphonamides were most commonly prescribed in each clinical scenario. Eleven percent of prescriptions were for antimicrobial drugs not licensed for use in horses in the UK. Five percent of prescriptions for licensed antimicrobials were under the recommended dose rate and 56% over the recommended dose rate. Fluoroquinolones and 3rd and 4th generation cephalosporins accounted for 1 and 3% of prescriptions, respectively. Veterinary surgeons working at referral practices were more likely to prescribe 3rd and 4th generation cephalosporins and fluoroquinolones and off-licence antimicrobials whereas those working in first-opinion practices were more likely to prescribe potentiated sulphonamides. Sources of information regarding antimicrobials also had an effect on prescribing. CONCLUSIONS AND POTENTIAL RELEVANCE: Prescription of antimicrobials at inappropriate doses was common even when provided with the weight of the animal. It is uncommon for UK equine veterinary practices to have guidelines for antimicrobial use. The introduction of such guidelines could lead to more prudent use of antimicrobials.

Multi-locus sequence typing of Salmonella enterica serovar Typhimurium isolates from wild birds in northern England suggests host-adapted strain.

AIMS: Recent studies have suggested that Salmonella Typhimurium strains associated with mortality in UK garden birds are significantly different from strains that cause disease in humans and livestock and that wild bird strains may be host adapted. However, without further genomic characterization of these strains, it is not possible to determine whether they are host adapted. The aim of this study was to characterize a representative sample of Salm. Typhimurium strains detected in wild garden birds using multi-locus sequence typing (MLST)to investigate evolutionary relationships between them. METHODS AND RESULTS: Multi-locus sequence typing was performed on nine Salm. Typhimurium strains isolated from wild garden birds. Two sequence types were identified, the most common of which was ST568. Examination of the public Salmonella enterica MLST database revealed that only three other ST568 isolates had been cultured from a human in Scotland. Two further isolates of Salm. Typhimurium were determined to be ST19. CONCLUSIONS: Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, which is rarely detected in other species, supporting the hypothesis that this strain is host adapted. SIGNIFICANCE AND IMPACT OF THE STUDY: Host-pathogen evolution is often assumed to lead to pathogens becoming less virulent to avoid the death of their host; however, infection with ST568 led to high mortality rates among the wild birds examined, which were all found dead at wild bird-feeding stations. We hypothesize that by attracting unnaturally high densities of birds, wild bird-feeding stations may facilitate the transmission of ST568 between wild birds, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a host-adapted strain with increased virulence.

Risk factors for the occurrence of Escherichia coli virulence genes eae, stx1 and stx2 in wild bird populations.

Shiga toxin-producing Escherichia coli (STEC) can cause serious disease in human beings. Ruminants are considered to be the main reservoir of human STEC infections. However, STEC have also been isolated from other domestic animals, wild mammals and birds. We describe a cross-sectional study of wild birds in northern England to determine the prevalence of E. coli-containing genes that encode Shiga toxins (stx1 and stx2) and intimin (eae), important virulence determinants of STEC associated with human disease. Multivariable logistic regression analysis identified unique risk factors for the occurrence of each virulence gene in wild bird populations. The results of our study indicate that while wild birds are unlikely to be direct sources of STEC infections, they do represent a potential reservoir of virulence genes. This, coupled with their ability to act as long-distance vectors of STEC, means that wild birds have the potential to influence the spread and evolution of STEC.

Motor, cognitive, and behavioural disorders in children born very preterm.

Children born preterm have been shown to exhibit poor motor function and behaviour that is associated with school failure in the presence of average intelligence. A geographically determined cohort of two-hundred and eighty preterm children (151 males, 129 females) born before 32 weeks' gestation and attending mainstream schools were examined at 7 to 8 years of age together with 210 (112 males, 98 females) age- and sex-matched control participants were tested for motor, cognitive, and behavioural problems. Tests applied were the Movement Assessment Battery for Children (MABC), Clinical Observations of Motor and Postural Skills (COMPS), Developmental Test of Visual-Motor Integration (VMI), Wechsler Intelligence Scale for Children, and Connors' Teacher Rating Scale for attention-deficit-hyperactivity disorder (ADHD). Control children scored significantly better than the preterm group on all motor, cognitive, and behavioural measures. The lowest birthweight and most preterm individuals tended to score the lowest. Motor impairment was diagnosed in 86 (30.7%) of the preterm group and 14 (6.7%) of the control children using the MABC; 97 (42.7%) and 18 (10.2%) using the COMPS; and 68 (24.3%) and 17 (8.1%) respectively using the VMI. Each test of motor function identified different children with disability, although 23 preterm children were identified as having motor disability by all three tests. Preterm children were more likely to have signs of inattention and impulsivity and have a diagnosis of ADHD. Minor motor disabilities persist in survivors of preterm birth despite improvements in care and are not confined to the smallest or most preterm infants. They may exist independently of cognitive and behavioural deficits, although they often co-exist. The condition is heterogeneous and may require more than one test to identify all children with potential learning problems.

Effect of skin pigmentation on pulse oximetry accuracy in the emergency department.

OBJECTIVE: To determine whether pulse oximeter (PO) accuracy and signal quality are affected by level of skin pigmentation. METHODS: Observational study in a community hospital ED. Consecutive adult patients undergoing arterial blood gas determination were enrolled into the study. Skin pigmentation was determined by comparison with standardized color swatches under controlled lighting; assigned values were used to stratify patients into 3 groups (light, intermediate, and dark) using predetermined criteria. Simultaneous with arterial blood sampling, staff recorded PO reading of O2 saturation using the Nellcor D-25 oximeter. PO values were compared with criterion standard values measured using a 4-wavelength spectrophotometer or co-oximeter. PO signal quality also was recorded. Bias (the mean difference between PO and co-oximeter-measured values of hemoglobin saturation) and precision (the standard deviation of the bias) were calculated. Groups were compared using one-way ANOVA, Bartlett's test for variances, and chi2 test. RESULTS: O2 saturation data were obtained for 284 patients. Bias values did not differ between the 3 skin pigment groups (p = 0.79). Precision was of borderline significance (p = 0.05), but there was no dose-response relation between skin pigmentation and precision. Study personnel reported suboptimal PO function most often among patients in the dark group (p = 0.003), but this finding was of no clinical significance. PO signal failure was rare (<1% of all patients). CONCLUSIONS: Although several prior studies suggest the contrary, this study found that skin pigmentation does not affect the bias or precision of pulse oximetry. Furthermore, skin pigmentation has no clinically significant effect on PO signal quality.

A continuum of care model.

An interdisciplinary documentation tool provides a composite of all of the planning activities that occur relative to each individual patient and his/her family before discharge. In response to the Joint Commission on Accreditation of Healthcare Organization's mandate, this tool evolved into a "Continuum of Care" model. Now, all disciplines maintain a patient's continuity of care.

Safety of MR imaging in patients who have retained metallic materials after cardiac surgery.

OBJECTIVE: Epicardial pacing wires retained in patients who undergo cardiac surgery are thought to be a relative contraindication to MR imaging. However, to our knowledge no published evidence supports this belief. Because other metallic materials retained after cardiac surgery might represent a hazard to patients who undergo MR imaging, we sought to determine the safety of such imaging. SUBJECTS AND METHODS: We examined 200 patients who underwent MR imaging at 1 or 1.5 T after cardiac surgery. Eighty-one were examined with ECG monitoring. The presence of temporary epicardial pacing wires, prosthetic valves, and other metal materials was confirmed by chest radiography. RESULTS: Of the 200 patients reviewed, all had postoperative metallic material visible on chest radiographs. Temporary epicardial pacing wire, cut short at the skin, was seen in 51 patients. Of the 81 patients examined with ECG monitoring, we found that MR imaging produced no changes from baseline ECG rhythms. None of the 200 patients reported symptoms suggesting arrhythmia or other cardiac dysfunction during MR imaging. CONCLUSION: MR imaging can be performed safely in patients who have undergone cardiac surgery and have retained metallic material, including valve replacements and temporary epicardial pacing wires cut short at the skin. MR imaging of patients with pacemakers was not evaluated, and we recommend that pacemakers remain a contraindication to MR imaging.

Alterations in the reproductive patterns of female mice exposed to xenobiotics.

Chemicals, by virtue of their varied interactions with biological molecules, are expected to differ in the way they may alter female reproduction. Reproductive toxicity may reflect effects either on the female germ cells or on various maternal processes such as ovulation, implantation, pregnancy, and parturition. In either case, the ultimate manifestation of chemical toxicity on female reproduction is a decrease in the number of normal young born. Very little information is available on the effects of chemicals that are nonhormonal in nature on the long-term ability of treated females to produce offspring. This report presents the results of long-term female total reproductive capacity (TRC) tests on 29 chemicals, including pharmaceuticals, pesticides, and alkylating and industrial agents. For each chemical, the minimum test involved an evaluation of the maximum tolerated dose administered as a single intraperitoneal injection. Females were single-pair mated with an untreated male for most of the female's reproductive life span (a minimum of 347 days posttreatment) and scored for the number of live births produced during this period. Confirmatory dominant lethal experiments or histological examinations for numbers of small follicles were carried out when mutagenic effects or cytotoxicity, respectively, were suspected as the basis for reduced fertility. Of the 29 chemicals studied, 17 had reproductive effects which may be grouped into one of three classes: (1) those that reduced the total number of young and litters per female, (2) those that reduced the total number of young but not of litters, and (3) those that had no significant effect on the total number of young produced but reduced the size of the first and/or second litters. The TRC provides a capacity for detecting a range of toxic insults upon female reproduction. Many of the chemicals were indeed shown to affect the reproductive performance of females through mutagenic and/or cytotoxic effects on follicles. In some cases, however, no causative mechanism could be identified for the observed reduction in reproductive performance. Nevertheless, with this report the number of chemicals tested by this TRC procedure has been quadrupled and the categories of chemicals tested have been substantially broadened.

Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

Magnetic resonance imaging of pericardial constriction: comparison of cine MR angiography and spin-echo techniques.

AIM: Spin-echo (SE) MRI detects pericardial thickening in pericardial constriction but the validity of extrapolating SE criteria to cine MRA imaging has not been tested. Pericardial thickness measured by SE and cine MRA was compared in patients with and without pericardial thickening to determine if the range of pericardial thickness measured by the two techniques is the same. PATIENT AND METHODS: Fourteen patients, investigated for possible pericardial constriction (PC), were compared with 24 subjects without evidence of pericardial disease (controls). Images were acquired using SE and cine MRA. Pericardial thickness was compared with final diagnosis. RESULTS: Pericardial thickening ( > 3.5 mm) by SE detected pericardial constriction: sensitivity = 100% specificity = 96%, kappa = 0.91. Cine MRA had a sensitivity = 86%, specificity = 63%, kappa = 0.33. Maximum differences between SE and cine MRA pericardial thickness ranged from +2.5 mm to -2/7 mm. CONCLUSIONS: Spin-echo identifies pericardial thickening with little overlap between measurements in patients with and without pericardial constriction. Pericardial thickness on cine MRA usually exceeds SE thickness, but with considerable overlap of thickness measurements in patients with and without pericardial constriction. Cine MRA cannot be used alone to diagnose pericardial thickening.

Time-of-flight MR angiography of the portal venous system: value compared with other imaging procedures.

OBJECTIVE: The purpose of this study was to retrospectively compare two-dimensional time-of-flight MR angiography with other imaging procedures in the evaluation of the portal venous system in 152 consecutive patients. MATERIALS AND METHODS: The findings on MR angiography performed on 152 patients to depict breath-hold, two-dimensional time of flight MR angiography. Selective arterial presaturation, bolus tracking, and three-dimensional reconstruction were used routinely. Findings were correlated with findings on sonography (104 patients), CT (8 patients), and conventional digital subtraction angiography (19 patients) as well as surgery (23 patients). RESULTS: Agreement between results of MR angiography and alternative types of imaging was excellent (99%). Agreement with sonography (100 of 104), CT (8 of 8), conventional angiography (18 of 19), and surgery (23 of 23) was good. Visualization of varices and spontaneous shunts by MR angiography was superior to that by other imaging techniques. CONCLUSION: Our experience shows that time-of-flight MR angiography is reliable and accurate for depicting portal venous anatomy. MR angiography shows vessels that are not visible with sonography. Complicated pathology is clearly visualized in a way that is not possible with other techniques.

Body magnetic resonance angiography and its effect on the use of alternative imaging--experience in 1026 patients.

Time-of-flight magnetic resonance (MR) angiography has been described for numerous applications but the number of patients described in published reports is usually relatively small. There is little information concerning the general utility of MR angiography in the body, outside the cranial circulation, or its effect on use of other imaging studies. Utilization of two-dimensional (2D) time-of-flight MR angiography over a 3 year period was reviewed to determine its accuracy and impact on other imaging modalities. Cranial and cardiac studies were excluded. Correlation was made with alternative imaging and surgical findings. Between January 1990 and December 1992 2D time-of-flight MR angiography was used to examine 1026 patients. MR angiography was used most frequently to examine the venous system. There was a slight reduction in the use of alternative imaging with two exceptions. When chest MR venography was performed, alternative imaging was completely abolished. Also, contrast portal venography was virtually eliminated. Five errors (0.2% of total MR angiography examinations) were recorded. Time-of-flight MR angiography, especially MR venography, is accurate and can replace contrast venography for abdominal and thoracic applications. Cost and limited availability means that other imaging, usually ultrasound, often precedes MR angiography.

Magnetic resonance angiography of the central chest veins. A new gold standard?

PURPOSE: The systemic chest veins may be difficult to show comprehensively by contrast venography, especially if there is limited venous access or contraindications to intravenous contrast. As an alternative, can magnetic resonance angiography (MRA) reliably detect occluded chest veins and predict suitable sites for central venous access? PATIENTS AND METHODS: Eighty-four patients were examined using breath-hold time-of-flight MRA and three-dimensional image reconstruction. Thirty-three were evaluated to identify possible central venous access. Fifty-seven patients were examined to diagnose and stage central venous occlusion. RESULTS: The associated diagnoses were malignancy 46, parenteral nutrition 21, hemodialysis 6, chemotherapy 4, and other long-term venous access 7. Of the 28 patients in whom MRA predicted a patent site for central venous access, satisfactory access was achieved. In two patients, cannulation of veins shown to be occluded on MRA was attempted unsuccessfully. Correlation with contrast venography was available in 17. There was agreement with MRA concerning the level of occluded veins in all cases. Contrast venography did not show all patent veins, including some accessed during surgical line placement. CONCLUSION: Compared with surgical line placement or contrast venography, MRA of the systemic chest veins is accurate. Patent and occluded chest veins are reliably defined, including potential sites for central line placement, in a way that is not possible with other techniques. MRA may be the new "gold standard" for defining systemic venous anatomy in the chest.

Normal arborization of the deep branch of the ulnar nerve into the interossei and lumbricals.

Ten adult hands were dissected to reexamine in new detail the distribution of the deep branch of the ulnar nerve from its origin in the ulnar tunnel to its termination in the first dorsal interosseous muscle. The relationships of the subdividing branches to each other and to the muscle bellies were clarified. The palmar interossei along with the first and second dorsal interossei were all innervated within the middle third of their corresponding metacarpal. The third and fourth dorsal interossei were innervated within the proximal third of their corresponding metacarpals, and the third lumbrical was innervated within the middle third whereas the fourth lumbrical was innervated along its distal third.

Comprehension of discharge instructions by patients in an urban emergency department.

STUDY OBJECTIVE: To assess patients' comprehension of their emergency department discharge instructions and to determine if inner-city patients' literacy levels are adequate to comprehend written discharge instructions. DESIGN: Prospective, observational study. SETTING: The ED of an inner-city university hospital. PARTICIPANTS: Two hundred seventeen patients consecutively discharged from the ED during 12 separate time slots. INTERVENTIONS: Patients were interviewed after discharge from the ED and asked to state their diagnosis, medication instructions, and follow-up instructions. Comparisons between patient recall and instructions as written in the chart were assessed by independent raters and scored from poor to excellent. Patients were administered a standardized test of reading ability. RESULTS: Overall comprehension rates were judged to be good, although 23% of patients exhibited no understanding of at least one component of their discharge instructions. Mean reading ability of the patients was at the sixth-grade level. The ED's printed discharge instructions were written at an 11th-grade reading level. Patients with low literacy scores were more likely to have poor comprehension of instructions. CONCLUSION: Overall comprehension rates in this population were good despite the fact that ED instruction sheets were written at an inappropriately high reading level. Verbal instructions given by the discharging physician likely have a significant effect on patients' comprehension of instructions.

Studies on the expression of intestinal lactase in different individuals.

Sixty one duodenal biopsy specimens were examined for the expression of lactase at the level of enzyme activity, protein, and messenger RNA. Of the 51 samples with normal villous architecture, 39 were lactase persistent, 11 were nonpersistent (adult type hypolactasia), and one was of indeterminate status. All the lactase persistent individuals showed high mRNA and a high level of the lactase protein as detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis. All the 11 non-persistent individuals tested showed a low level of lactase protein. Nine of the 10 samples tested showed low mRNA and one high mRNA. These results suggest that the lactase persistence polymorphism is controlled at the level of the expression of the lactase gene, though there may be some heterogeneity of the lactase non-persistence phenotype.

MR angiography as the sole method in evaluating abdominal aortic aneurysms: correlation with conventional techniques and surgery.

PURPOSE: To compare magnetic resonance (MR) angiography with conventional preoperative imaging techniques and surgical findings in the evaluation of abdominal aortic aneurysms (AAAs). MATERIALS AND METHODS: MR angiography was performed in 40 patients with an AAA. Two-dimensional time-of-flight MR angiography with maximum intensity projections was compared with conventional angiography, ultrasound, computed tomography, and surgery. RESULTS: In 18 of 20 patients, MR angiography demonstrated more extensive disease than did angiography. MR angiography depicted 41 of 43 renal arteries and seven of eight renal artery stenoses (one false-negative finding of mild stenosis) identified at angiography. When iliac arteries were imaged (30 patients), good correlation with angiography was seen in all but one patient (resulting from surgical clip artifact). Ten iliac stenoses were seen at both studies. Angiography caused underestimation of the extent of seven iliac aneurysms. CONCLUSION: MR angiography can provide all of the necessary preoperative information for evaluation of AAA and can replace conventional angiography in many cases.

Mosaic regulation of lactase in human adult-type hypolactasia.

BACKGROUND/AIMS: We have previously shown that in the proximal-jejunum of hypolactasic humans, just a few villus enterocytes express lactase protein and activity. In the present study, we compared the distribution of lactase messenger RNA (mRNA), protein, and activity in villus enterocytes in tissues obtained from subjects with persistent high lactase activity and those with hypolactasia. METHODS: Immunohistochemical and enzymohistochemical staining was performed on closely adjacent sections of human proximal jejunum from 5 individuals with persistent high lactase activity and 32 with hypolactasia. In all the persistent and in 9 hypolactasic samples, in situ hybridization was also performed using a digoxygenin-labeled RNA probe. RESULTS: In persistent tissues, lactase mRNA, protein, and activity were present in all villus enterocytes. In hypolactasic tissues, lactase mRNA was detected only in some villus enterocytes; some of them also expressed protein and activity, whereas others did not. In 8 of these hypolactasic samples, a variable number of villus enterocytes with lactase mRNA and protein did not express lactase activity. CONCLUSIONS: Various types of enterocytes are present even on a single villus from individuals with adult-type hypolactasia. These results show that different mechanisms control lactase expression in enterocytes on the same villus.