Chronic myeloproliferative neoplasm in adulthood in CBL syndrome harboring a splice-site CBL variant alongside a novel constitutional CSF3R variant.

Casitas B-cell lineage (CBL) syndrome is a rare RASopathy known to predispose to CBL-mutated juvenile myelomonocytic leukemia (JMML) in childhood. Adulthood acute myeloid leukemia arising out of a genetic aberrancies consistent with prior CBL-mutated JMML has been twice previously described, but chronic myeloproliferative neoplasia has not. We present a case of progressive myeloproliferative neoplasm in adulthood in the context of CBL syndrome alongside a novel CSF3R variant. We also review pathogenic splice-site mutations in CBL-mutated JMML.

Whole exome and genome sequencing in mendelian disorders: a diagnostic and health economic analysis.

Whole genome sequencing (WGS) improves Mendelian disorder diagnosis over whole exome sequencing (WES); however, additional diagnostic yields and costs remain undefined. We investigated differences between diagnostic and cost outcomes of WGS and WES in a cohort with suspected Mendelian disorders. WGS was performed in 38 WES-negative families derived from a 64 family Mendelian cohort that previously underwent WES. For new WGS diagnoses, contemporary WES reanalysis determined whether variants were diagnosable by original WES or unique to WGS. Diagnostic rates were estimated for WES and WGS to simulate outcomes if both had been applied to the 64 families. Diagnostic costs were calculated for various genomic testing scenarios. WGS diagnosed 34% (13/38) of WES-negative families. However, contemporary WES reanalysis on average 2 years later would have diagnosed 18% (7/38 families) resulting in a WGS-specific diagnostic yield of 19% (6/31 remaining families). In WES-negative families, the incremental cost per additional diagnosis using WGS following WES reanalysis was AU$36,710 (£19,407;US$23,727) and WGS alone was AU$41,916 (£22,159;US$27,093) compared to WES-reanalysis. When we simulated the use of WGS alone as an initial genomic test, the incremental cost for each additional diagnosis was AU$29,708 (£15,705;US$19,201) whereas contemporary WES followed by WGS was AU$36,710 (£19,407;US$23,727) compared to contemporary WES. Our findings confirm that WGS is the optimal genomic test choice for maximal diagnosis in Mendelian disorders. However, accepting a small reduction in diagnostic yield, WES with subsequent reanalysis confers the lowest costs. Whether WES or WGS is utilised will depend on clinical scenario and local resourcing and availability.

Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants.

PURPOSE: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). METHODS: A total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. RESULTS: Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from ≥2 affected individuals or heterozygotes and 10 cases with ≥2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. CONCLUSION: RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.

Effectiveness of a Pre-emptive Preoperative Belladonna and Opium Suppository on Postoperative Urgency and Pain After Ureteroscopy.

PURPOSE: Postoperative ureteroscopy patients can develop bladder spasms, complaints of pain, and the urgent need to void during emergence from anesthesia. Discomfort leads to patient agitation, resulting in a risk to patient safety. The purpose of this study was to determine the effectiveness of a preemptive preoperative belladonna and opium (B + O) suppository on postoperative bladder comfort, narcotic requirements, and length of stay of ureteroscopy patients. DESIGN: A prospective double-blind study was conducted. METHODS: Fifty adult outpatients scheduled for ureteroscopy were assigned to routine care or a B + O suppository immediately after anesthesia induction. Urinary urgency and pain were assessed every 15 minutes. FINDINGS: Urgency significantly decreased in the B+O group, with less than half reporting urgency at discharge. CONCLUSIONS: Pre-emptive preoperative administration of a B + O suppository before ureteroscopy results in decreased urinary urgency during the postoperative recovery. Pre-emptive preoperative interventions can result in positive outcomes before discharge.

KBG syndrome: An Australian experience.

In 2011, heterozygous mutations in the ANKRD11 gene were identified in patients with KBG syndrome. Since then, 100 cases have been described with the expansion of the clinical phenotype. Here we present 18 KBG affected individuals from 13 unrelated families, 16 with pathogenic mutations in the ANKRD11 gene. Consistent features included intellectual disability, macrodontia, and the characteristic broad forehead with hypertelorism, and a prominent nasal bridge. Common features included hand anomalies, cryptorchidism, and a large number of palate abnormalities. Distinctive findings in this series included malrotation of the abdominal viscera, bilateral inguinal herniae in two patients, basal ganglia calcification and the finding of osteopenia in three patients. Nine novel heterozygous variants were found and the genotype-phenotype correlation was explored. This report highlights the need for thorough examination and investigation of the dental and skeletal systems. The results confirm the specificity of ANKRD11 mutations in KBG and further evidence for this transcription repressor in neural, cardiac, and skeletal development. The description of further cases of KBG syndrome is needed to further delineate this condition, in particular the specific neurological and behavioral phenotype.

A study of the clinical and radiological features in a cohort of 93 patients with a COL2A1 mutation causing spondyloepiphyseal dysplasia congenita or a related phenotype.

Type 2 collagen disorders encompass a diverse group of skeletal dysplasias that are commonly associated with orthopedic, ocular, and hearing problems. However, the frequency of many clinical features has never been determined. We retrospectively investigated the clinical, radiological, and genotypic data in a group of 93 patients with molecularly confirmed SEDC or a related disorder. The majority of the patients (80/93) had short stature, with radiological features of SEDC (n = 64), others having SEMD (n = 5), Kniest dysplasia (n = 7), spondyloperipheral dysplasia (n = 2), or Torrance-like dysplasia (n = 2). The remaining 13 patients had normal stature with mild SED, Stickler-like syndrome or multiple epiphyseal dysplasia. Over 50% of the patients had undergone orthopedic surgery, usually for scoliosis, femoral osteotomy or hip replacement. Odontoid hypoplasia was present in 56% (95% CI 38-74) and a correlation between odontoid hypoplasia and short stature was observed. Atlanto-axial instability, was observed in 5 of the 18 patients (28%, 95% CI 10-54) in whom flexion-extension films of the cervical spine were available; however, it was rarely accompanied by myelopathy. Myopia was found in 45% (95% CI 35-56), and retinal detachment had occurred in 12% (95% CI 6-21; median age 14 years; youngest age 3.5 years). Thirty-two patients complained of hearing loss (37%, 95% CI 27-48) of whom 17 required hearing aids. The ophthalmological features and possibly also hearing loss are often relatively frequent and severe in patients with splicing mutations. Based on clinical findings, age at onset and genotype-phenotype correlations in this cohort, we propose guidelines for the management and follow-up in this group of disorders.

Value of whole exome sequencing for syndromic retinal dystrophy diagnosis in young patients.

BACKGROUND: Several retinal dystrophies are associated with syndromic features including such conditions as Bardet-Biedl and Joubert syndromes. Cohen syndrome is an autosomal recessive disorder associated with multiple clinical manifestations including developmental delay, acquired microcephaly, myopia, pigmentary retinopathy, joint hypermobility, truncal obesity, friendly disposition and intermittent neutropenia. In young patients, diagnosis is difficult, because several of the characteristic features may not be present until school age or later years and the intermittent neutropenia is not always detectable. DESIGN: This was a prospective study using whole exome sequencing in syndromic retinal dystrophy. It was undertaken in a hospital and research institute setting. PARTICIPANTS: Participants in this study were members of a consanguineous Australian family of Lebanese ethnicity with two siblings with retinal dystrophy, microcephaly and developmental delay. METHODS: Detailed clinical evaluation was undertaken. Whole exome capture and sequencing of patient genomic DNA samples was followed by sequence alignment, variant detection, comparison and prioritization. MAIN OUTCOME MEASURES: Pathogenic variant identification in the disease-causing gene in affected individuals. RESULTS: We identified a novel homozygous deletion leading to a frameshift mutation in VPS13B, c.11327del, p.(Asn3776Thrfs*102), the disease gene associated with Cohen syndrome. CONCLUSIONS: This report emphasizes the value of a broad-based whole exome sequencing approach in disease gene identification in the syndromic retinal dystrophies, where all disease characteristics may not be present in young patients to allow a clinical diagnosis. This facilitates improved prognostic and genetic information for patients and families.

ZEB2 zinc-finger missense mutations lead to hypomorphic alleles and a mild Mowat-Wilson syndrome.

Mowat-Wilson syndrome (MWS) is a severe intellectual disability (ID)-distinctive facial gestalt-multiple congenital anomaly syndrome, commonly associating microcephaly, epilepsy, corpus callosum agenesis, conotruncal heart defects, urogenital malformations and Hirschsprung disease (HSCR). MWS is caused by de novo heterozygous mutations in the ZEB2 gene. The majority of mutations lead to haplo-insufficiency through premature stop codons or large gene deletions. Only three missense mutations have been reported so far; none of which resides in a known functional domain of ZEB2. In this study, we report and analyze the functional consequences of three novel missense mutations, p.Tyr1055Cys, p.Ser1071Pro and p.His1045Arg, identified in the highly conserved C-zinc-finger (C-ZF) domain of ZEB2. Patients' phenotype included the facial gestalt of MWS and moderate ID, but no microcephaly, heart defects or HSCR. In vitro studies showed that all the three mutations prevented binding and repression of the E-cadherin promoter, a characterized ZEB2 target gene. Taking advantage of the zebrafish morphant technology, we performed rescue experiments using wild-type (WT) and mutant human ZEB2 mRNAs. Variable, mutation-dependent, embryo rescue, correlating with the severity of patients' phenotype, was observed. Our data provide evidence that these missense mutations cause a partial loss of function of ZEB2, suggesting that its role is not restricted to repression of E-cadherin. Functional domains other than C-ZF may play a role in early embryonic development. Finally, these findings broaden the clinical spectrum of ZEB2 mutations, indicating that MWS ought to be considered in patients with lesser degrees of ID and a suggestive facial gestalt, even in the absence of congenital malformation.

Mutation-based growth charts for SEDC and other COL2A1 related dysplasias.

From data collected via a large international collaborative study, we have constructed a growth chart for patients with molecularly confirmed congenital spondylo-epiphyseal dysplasia (SEDC) and other COL2A1 related dysplasias. The growth chart is based on longitudinal height measurements of 79 patients with glycine substitutions in the triple-helical domain of COL2A1. In addition, measurements of 27 patients with other molecular defects, such as arginine to cysteine substitutions, splice mutations, and mutations in the C-terminal propeptide have been plotted on the chart. Height of the patients progressively deviate from that of normal children: compared to normal WHO charts, the mean length/height is -2.6 SD at birth, -4.2 SD at 5 years, and -5.8 SD in adulthood. The mean adult height (male and female combined) of patients with glycine substitutions in the triple-helical region is 138.2 cm but there is a large variation. Patients with glycine to cysteine substitutions tend to cluster within the upper part of the chart, while patients with glycine to serine or valine substitutions are situated between +1 SD and -1 SD. Patients with carboxy-terminal glycine substitutions tend to be shorter than patients with amino-terminal substitutions, while patients with splice mutations are relatively tall. However, there are exceptions and specific mutations can have a strong or a relatively mild negative effect on growth. The observation of significant difference in adult height between affected members of the same family indicates that height remains a multifactorial trait even in the presence of a mutation with a strong dominant effect.

Cooperative m-learning with nurse practitioner students.

New technologies give nurse academicians the opportunity to incorporate innovative teaching-learning strategies into the nursing curricula. Mobile technology for learning, or m-learning, has considerable potential for the nursing classroom but lacks sufficient empirical evidence to support its use. Based on Mayer's multimedia learning theory, the effect of using cooperative and interactive m-learning techniques in enhancing classroom and clinical learning was explored. The relationship between m-learning and students' learning styles was determined through a multimethod educational research study involving nurse practitioner students at two mid-Atlantic universities. During the 16-month period, nurse practitioner students and their faculty used personal digital assistants (PDAs) to participate in various m-learning activities. Findings from focus group and survey responses concluded that PDAs, specifically the Pocket PC, are useful reference tools in the clinical setting and that all students, regardless of learning style, benefited from using PDAs. It was also demonstrated that connecting students with classmates and other nurse practitioner students at distant universities created a cooperative learning community providing additional support and knowledge acquisition. The authors concluded that in order to successfully prepare nurse practitioner graduates with the skills necessary to function in the present and future health care system, nurse practitioner faculty must be creative and innovative, incorporating various revolutionary technologies into their nurse practitioner curricula.

Identification of meningococcal genes necessary for colonization of human upper airway tissue.

Neisseria meningitidis is an exclusively human pathogen that has evolved primarily to colonize the nasopharynx rather than to cause systemic disease. Colonization is the most frequent outcome following meningococcal infection and a prerequisite for invasive disease. The mechanism of colonization involves attachment of the organism to epithelial cells via bacterial type IV pili (Tfp), but subsequent events during colonization remain largely unknown. We analyzed 576 N. meningitidis mutants for their capacity to colonize human nasopharyngeal tissue in an organ culture model to identify bacterial genes required for colonization. Eight colonization-defective mutants were isolated. Two mutants were unable to express Tfp and were defective for adhesion to epithelial cells, which is likely to be the basis of their attenuation in nasopharyngeal tissue. Three other mutants are predicted to have lost previously uncharacterized surface molecules, while the remaining mutants have transposon insertions in genes of unknown function. We have identified novel meningococcal colonization factors, and this should provide insights into the survival of this important pathogen in its natural habitat.

A rubric for improving the quality of online courses.

All of the graduate students in the School of Nursing take some of their Master of Science courses online. A group of six School of Nursing faculty members and a graduate student received funding to determine best practices in online courses. The group developed an evaluation rubric to measure quality in the graduate online curriculum. They then applied the rubric to the core courses which are primarily offered online and are required for all graduate nursing students. The project had a positive impact on faculty by offering a tool useful for online course evaluation and development. Additionally it brought to attention the needs of faculty member development in online education.

Predictors of preterm birth in birth certificate data.

Demographic factors have been shown to be moderate predictors of preterm birth in prior studies which used hospital databases and epidemiologic sample surveys. This retrospective study used de-identified 2003 North Carolina birth certificate data (n=73,040) and replicated the statistical and computational methods used in a prior study of an academic medical center's data warehouse. Receiver Operating Characteristics (ROC) curves were used to compare results across methods. Due to differences between the data collected for birth certificates and the original clinical database, five of the seven demographic variables in the clinical database model were available for model testing (maternal age, marital status, race/ethnicity, education and county). Even with a reduced model, multiple methods of statistical and computational modeling supported the earlier findings of demographic predictors for preterm birth. The reduced model AUC results were acceptable (logistic regression = 0.605, neural networks = 0.57, SVM = 0.57, Bayesian classifiers = 0.59, and CART = 0.56), but lower than in the prior study as might be expected for a reduced model. On a population level, these results support a prior demographic predictor preterm birth model generated from a clinical database and the use of computational methods for model formation. Additional testing for stronger predictor models within birth certificate data is suggested as birth certificate data is a parsimonious population dataset already routinely collected.

An innocuous duplication of 11.2 Mb at 13q21 is gene poor: sub-bands of gene paucity and pervasive CNV characterize the chromosome anomalies.

A boy with autistic spectrum disorder without dysmorphisms was found to have a chromosome duplication of part of band 13q21. His mother and grandfather both of normal intellect had the same chromosomal duplication. Comparison was made with the Chromosome anomaly database www.som.soton.ac.uk/research/geneticsdiv/anomaly%20register which revealed similar cases. Mapping on DNA microarray for the proband and mother showed the duplication to be of length 11.2 Mb, encompassing the 13q21.1-13q21.32 region. The duplicated region is profoundly gene poor, with a mean gene density of 0.45 genes/Mb. We estimate, that the mean gene density in the sub-bands of the chromosome anomalies is 2.4-2.5 genes/Mb. In addition the percentages of the sub-bands reported as copy number variants (CNV) was estimated from the Database of Chromosome Genomic Variants (http://projects.tcag.ca/variation/). It was found that for some of these sub-bands, gene paucity was likely to be a major contributor to their innocuous phenotypic effect, for example, the gene densities were for: 1p31.2 (1.25 genes/Mb); 2p12 (1.7); 4p15.31 (1.3); 5p14.1 (0.22); 5p14.3 (0.8); 5q21.2 (0.6); 5q21.3 (1.2); 8p23.2 (0.25); 13q21.1 (0.9); 14q31.1 (1.4); 18q22.1 (1.4); 21q21.1 (1.2); and 21q21.2 (0.7). For other sub-bands the percentage of the band in which CNV have been reported was found to be markedly increased, for example, 8p23.2 (94.7% of the band is defined by reported CNV); 3p26.3 (81.6); 5p14.3 (59.3); 8p22 (48.8); 2p12 (44.0); 5q21.1 (43.6); 6q24.2 (41.4); 9p23 (38.8); 10q21.1 (36.5); 5q21.2 (35.4), and 11q14.3 (33.8). We argue that both gene paucity and pervasive CNV are major indicators of bands conforming to the Chromosome Anomaly phenomenon.