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Background Anthropometric z-scores used commonly for diagnosis and determining degree of malnutrition, specifically body mass index (BMIz), weight-for-length (WLz), and mid-upper arm circumference (MUACz), are not wholly concordant, yet the proposed thresholds for classification are identical. This study was designed to critically examine MUACz thresholds and their ability to correctly classify nutrition status. Methods This was a 2-year, prospective single-center study of children ≤18 years seen by registered dietitians within a large pediatric institution. The sensitivity, specificity, and predictive performance of the malnutrition classification thresholds were estimated against clinician-based classification. Results Sixty-one dietitians enrolled 10,401 patients with distributions of z-scores for weight (-0.5 ± 1.9), length (-0.8 ± 1.6), BMI or WL (-0.1 ± 1.8), and MUAC (-0.4 ± 1.5), suggesting participants were smaller and shorter than the reference U.S. Population Distributions of MUACz were broad and overlapped between nutrition classification groups, an observation that extended to BMIz and WLz as well. Consequently, existing thresholds do not accurately classify 100% of children. Misclassification rates increase, with increasing severity ranging from 8% in children with no malnutrition to 71% in children with severe malnutrition. Algorithm- and manually-based refinement of thresholds result in mixed improvements and can be explored by the reader with the associated supplement. Conclusion The sensitivity of proposed MUACz thresholds systematically decreases with increasing severity of malnutrition and will require optimization if we aim to limit the number of children at risk of misclassification. Indicators for overnutrition remain to be addressed but are explored herein.

Background: Rapid reperfusion in ischaemic stroke with emergent large vessel occlusion (ELVO) reduces morbidity and mortality. Limited distribution of endovascular clot retrieval (ECR) capable comprehensive stroke centres (CSCs) necessitates development of pre-hospital models of care to provide equitable and economical access to reperfusion therapy. We examine the time metrics of the traditional secondary transfer strategy in comparison to the direct bypass strategy and the potential utility of the ACT-FAST prehospital triage algorithm on a large volume Melbourne primary stroke centre (PSC).Method: Retrospective analysis of consecutive patients presenting to a PSC from 1 January 2020 to 31 December 2020. Clinical records were interrogated for ACT-FAST positive patients. Time metrics were established using Google Maps traffic modelling and local/published door-to-needle, door-in-door out and door-to-groin data.Results: 88 patients during the study period were ACT-FAST positive. Of these, 49/88 (56%) cases had ELVO ischaemic strokes, 24/88 (27%) cases had intracranial haemorrhages and the remaining 15/88 (17%) had non ELVO ischaemic strokes or mimics (seizure, complex migraine, etc). 28/88 (32%) cases met indication for and were subsequently transferred to a CSC for consideration of ECR. The modelled median scene to groin time for the direct bypass strategy is 94 min whereas the median scene to groin time for the secondary transfer strategy is 109 min, giving a difference of 15 min.Conclusion: Time savings to groin puncture for the direct bypass strategy is substantially less than previous estimates and suggests that the secondary transfer strategy continues to be a viable pathway for a high efficiency PSC.

Broad-range fungal PCR is a powerful tool for identifying pathogens directly from patient specimens; however, reported estimates of clinical utility vary and costs discourage universal testing. We investigated the diagnostic and clinical utility of broad-range fungal PCR by examining 9 years of results from sinonasal specimens, hypothesizing that this anatomic location would identify immunocompromised patients at high risk for invasive fungal disease. We retrospectively identified 644 PCRs and 1,446 fungal cultures from sinus sites. To determine the relative performance of each testing modality, we performed chart review on 52 patients having specimens submitted for culture and PCR on the same day. Positivity rates were significantly higher for PCR (37.1%) than culture (13.7%) but similar for formalin-fixed and fresh tissues (42.3% versus 34.6%). Relative to culture, PCR had significantly faster turnaround time to both preliminary (94.5 versus 108.8 h) and final positive (137.9 versus 278.5 h) results. Among chart-reviewed patients, 88% were immunocompromised, 65% had proven or probable fungal disease, and testing sensitivities for culture and PCR (67.5% and 85.0%) were not statistically different. Nevertheless, PCR identified pathogens not recovered by culture in 14.9% of cases and informed clinical decision-making in 16.7% of all reviewed cases, and sensitivity of PCR combined with culture (90.0%) was higher than that of culture alone. We conclude that broad-range fungal PCR is frequently informative for patients at risk of serious fungal disease and is complementary to and has faster turnaround time than culture. Formalin-fixed tissue does not adversely affect diagnostic yield, but anatomic site may impact assay positivity rates.

BACKGROUND Hormonal changes influence the composition of vaginal flora, which is directly related to the health of an individual. Transgender men prescribed testosterone experience a vaginal hormone composition that differs from cisgender women. To the author's knowledge, there are no clinical studies evaluating the influence that testosterone administration has on the vaginal microbiome. METHODS Vaginal swabs were self-collected by a cohort of self-identified healthy transgender men prescribed testosterone for at least 1 year (n = 28) and from cisgender women who were used as the comparator (n = 8). Participants completed a questionnaire to indicate the mode and dose of testosterone administration, sexual history, and vaginal health. Serum was collected for hormone analysis. Bacterial community profiles were assessed with broad-range PCR primers targeting the V3–V4 hypervariable region of the 16S bacterial rRNA, next-generation sequencing, and analysis by phylogenetic placement. RESULTS Compared to cisgender women, the vaginal floras of transgender men were less likely to have Lactobacillus as their primary genus. Intravaginal estrogen administration was positively associated with the presence of Lactobacillus in transgender men (P = 0.045). Transgender men had a significantly increased relative abundance of >30 species and a significantly higher α diversity (P = 0.0003). The presence of Lactobacillus was significantly associated with a lower α diversity index (P = 0.017). CONCLUSIONS The vaginal microbiome of transgender men who were assigned a female sex at birth and use testosterone may differ from that of cisgender women. Intravaginal estrogen administration may reduce these differences by promoting colonization with Lactobacillus species and decreasing α diversity.

Background. Mid-upper arm circumference (MUAC) has been extensively used to classify pediatric malnutrition. Recently, MUAC z score was recommended; however, the logistics of implementation were not addressed. This study examines the usability of a device that provides MUAC and corresponding z score range in a single device. Methods. This was a single-center study of nutrition services providers. The device was applied to children aged 2 months to 18 years admitted as inpatients or seen as outpatients. Surveys incorporated benchmarking questions, assessed the ease with which respondents could perform 6 critical tasks, and provided an open-ended question to elicit feedback. Users were surveyed monthly until saturation was reached. Survey data were analyzed in aggregate and cross-tabulated by the respondents’ experience with the device. Thematic analysis of the open-ended responses followed a structured approach. Results. Sixty device users responded to the survey 280 times. Respondents were female (100%) with a mean age of 45.2 ± 13.2 years and 9.6 ± 8.0 years in practice. Increasing device use was accompanied by significantly shortened measuring times ( P < .001) and shifts in ease of performance for 5 of 6 critical tasks ( P < .05). Open-ended response themes related largely to design and materials. These were used to iteratively refine the device. Conclusions. The active engagement of end users in the real-world testing of our nutritional assessment device allowed us to refine the innovation with special attention paid to the needs of dietitians. The result is a device the majority of dietitians found easy to use, efficient, convenient, and preferable to alternative measurement options.

BACKGROUND Mid-upper arm circumference (MUAC) z-score, has recently been listed as an independent indicator for pediatric malnutrition. This investigation examined the relationship between MUAC z-score and the z-scores for conventional indicators (ie, weight-for-length and body mass index) to expand the available evidence for nutrition classification z-score threshold ranges in U.S. practice settings. METHODS This was a single-center study of children through 18 years of age seen between October 2015 and September 2016. Height and weight were obtained on intake. MUAC was measured at midpoint of the humerus, between the acromion and olecranon. Age-specific and gender-specific z-score values were calculated using published λ, μ, and σ values derived from Centers for Disease Control and Prevention reference data. Nutrition status was determined from biochemical data; prior history; anthropometrics; weight gain velocity; weight loss, if present; and nutrient intake. RESULTS 5,004 children (7.5 ± 5.7 years, 53% boys) were evaluated. As expected, MUAC z-scores were significantly correlated with body mass index (r = 0.789, P < .01) and weight-for-length (r = 0.638, P < .01) z-scores. There was a large degree of overlap in z-scores for all indicators between nutrition status groups; however, MUAC z-scores spanned a narrower range of values such that mean MUAC z-scores are lower in children classified as overweight/obese and higher in children who were severely malnourished than the corresponding body mass index or weight-for-length z-scores. CONCLUSION These data are the first to suggest that the z-score ranges used to define various stages of malnutrition may not be the same for all indicators.

Introduction: Rhodococcus spp. have been implicated in a variety of infections in immunocompromised and immunocompetent hosts. Rhodococcus equi is responsible for the majority of reported cases, but Rhodococcus erythropolis, Rhodococcusgordoniae and Rhodococcusruber infections have been described. There are no prior reports of human infection with Rhodococcus fascians. Case presentation: We describe the unexpected finding of R. fascians in liver lesions incidentally noted at autopsy in an immunosuppressed patient status after bone-marrow transplant for acute lymphoblastic leukaemia who died of unrelated causes (septic shock due to Clostridium difficile colitis). At autopsy, an otherwise unremarkable liver contained several dozen well-demarcated sclerotic-appearing lesions measuring 0.1–0.3 cm in size. The absence of other bacterial or fungal DNA in the setting of histologically visible organisms argues against its presence as a contaminant and raises the consideration that R. fascians represents a human pathogen for the immunocompromised. Conclusion: Whether it represents the sole infectious agent responsible for the miliary lesions or a partially treated co-infection is impossible to determine, but our finding continues to reinforce the importance of molecular techniques in associating organisms with sites of infection and optimizing treatment of infectious diseases.

Pythium insidiosum is an emerging human pathogen classified among brown algae and diatoms that can cause significant morbidity and mortality in otherwise healthy individuals. Here we describe a pediatric patient with pythiosis acquired in the southern United States, diagnosed by molecular screening and DNA sequencing of internal transcribed spacer region 1.

OBJECTIVE: The Reimbursement and Payment Subcommittee of the National Association of Children's Hospitals and Related Institutions FOCUS on a Fitter Future group sought to guide medical providers, patients, and payers to better serve obese children and adolescents to enable optimum health. Recommendations are provided for the essential components of a stage 3 comprehensive multidisciplinary intervention program as defined by the 2007 Expert Committee recommendations. In addition, suggestions are offered for a stepwise approach to implement these recommendations. METHODS: In 2009, key informant interviews were conducted with 15 children's hospitals participating in FOCUS on a Fitter Future and 1 nonparticipating hospital. Interview transcripts identified 5 financially sustainable stage 3 programs, each funded differently. RESULTS: The stage 3 programs interviewed ranged from being nascent to 21 years old (27%, 6 years). All of them had multidisciplinary teams that delivered services through 1 of 3 institutional structures: 60% freestanding; 7% specialty; and 33% hospital within a hospital. One-third of them had 1 to 2 funding sources, and 67% had ≥3 sources. CONCLUSIONS: The stage 3 programs in this review shared some common strategies for achieving financial stability. All of them followed key strategies of the chronic care model, the details of which led to the following recommendation: stage 3 programs should include a health care team with a medical provider, registered dietitian, physical activity specialist, mental health specialist, and coordinator who, as a team, provide service to overweight and obese children at no less than moderate intensity (26–75 hours).

Learning logs or reflective journals are frequently used in further and higher education to encourage students' reflection on their learning. Such approaches are rare in school. This study employed a learning log over a five-week period, with a class of 14–15 year old students learning about digestion, respiration and breathing at a Suffolk upper school in the UK. The study aimed to establish (1) how the learning log can prompt reflection on the learning benefits of classroom activities and increase cognitive control of learning strategies, and (2) how greater understanding of learning strategies may affects students' motivation to learn. The learning logs themselves formed one source of data. Student reflections on the learning benefits of classroom activities were also assessed through classroom dialogue and semi-structured interviews. Motivation was assessed at the start and at the end of the teaching sequence, using a questionnaire. The learning log did stimulate student reflection, but did not prompt...

With the rapidly increasing prevalence of childhood obesity, it is important for all practitioners to be aware of the rising problem and to be comfortable in beginning a discussion with their patients and families regarding how to address overweight. Each visit to any clinician including cardiologists and cardiac surgeons should include an accurate measure of height and weight with the calculation of body mass index (BMI) and subsequent plotting on the appropriate BMI chart. Body mass index is currently the most accepted measure of assessing overweight status. It correlates with the amount of body fat in children and with the level of health risks, particularly cardiovascular risk factors. When overweight is identified, an assessment of nutrition-related behaviors should be completed and used to help patients and families target behavior changes that are realistic and achievable. New Expert Committee Recommendations regarding the prevention, assessment, and treatment of childhood and adolescent overweight were released in December 2007. These guidelines identified key contributing factors based on current evidence. The nutrition related factors include breast-feeding, consumption of sugar-sweetened drinks, intake of fruits and vegetables, eating breakfast, eating family meals at home, frequency of eating out, intake of energy dense foods and portion sizes. This article will provide information about these contributing factors and practical solutions to discuss with patients and families to begin the process of behavior change.

Plexiform neurofibromas are peripheral nerve sheath tumors that arise frequently in neurofibromatosis type 1 (NF1) and have a risk of malignant progression. Past efforts to establish xenograft models for neurofibroma involved the implantation of tumor fragments or heterogeneous primary cultures, which rarely achieved significant tumor growth. We report a practical and reproducible animal model of plexiform-like neurofibroma by xenograft of an immortal human NF1 tumor-derived Schwann cell line into the peripheral nerve of scid mice. The S100 and p75 positive sNF94.3 cell line was shown to possess a normal karyotype and have apparent full-length neurofibromin by Western blot. These cells were shown to have a constitutional NF1 microdeletion and elevated Ras-GTP activity, however, suggesting loss of normal neurofibromin function. Localized intraneural injection of the cell line sNF94.3 produced consistent and slow growing tumors that infiltrated and disrupted the host nerve. The xenograft tumors resembled plexiform neurofibromas with a low rate of proliferation, abundant extracellular matrix (hypocellularity), basal laminae, high vascularity, and mast cell infiltration. The histologic features of the developed tumors were particularly consistent with those of human plexiform neurofibroma as well. Intraneural xenograft of sNF94.3 cells enables the precise initiation of intraneural, plexiform-like tumors and provides a highly reproducible model for the study of plexiform neurofibroma tumorigenesis. This model facilitates testing of potential therapeutic interventions, including angiogenesis inhibitors, in a relevant cellular environment.

To identify the mechanism of loss of heterozygosity (LOH) and potential modifier gene(s), we investigated the molecular basis of somatic NF1 inactivation in myeloid malignancies from 10 children with neurofibromatosis type 1. Loci across a minimal 50-Mb region of primarily the long arm of chromosome 17 showed LOH in 8 cases, whereas a less than 9-Mb region of loci flanking NF1 had LOH in the remaining 2 cases. Two complementary techniques, quantitative polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), were used to determine whether the copy number at loci that showed LOH was 1 or 2 (ie, deleted or isodisomic). The 2 cases with LOH limited to less than 9 Mb were intrachromosomal deletions. Among the 8 leukemias with 50-Mb LOH segments, 4 had partial uniparental isodisomy and 4 had interstitial uniparental isodisomy. These isodisomic cases showed clustering of the centromeric and telomeric LOH breakpoints. This suggests that the cases with interstitial uniparental isodisomy arose in a leukemia-initiating cell by double-homologous recombination events at intervals of preferred mitotic recombination. Homozygous inactivation of NF1 favored outgrowth of the leukemia-initiating cell. Our studies demonstrate that LOH analyses of loci distributed along the chromosomal length along with copy-number analysis can reveal novel mechanisms of LOH that may potentially identify regions harboring “cryptic” tumor suppressor or modifier genes whose inactivation contributes to tumorigenesis.

Recombination between paralogs that flank the NF1 gene at 17q11.2 typically results in a 1.5-Mb microdeletion that includes NF1 and at least 13 other genes. We show that the principal sequences responsible are two 51-kb blocks with 97.5% sequence identity (NF1REP-P1-51 and NF1REP-M-51). These blocks belong to a complex group of paralogs with three components on 17q11.2 and another on 19p13.13. Breakpoint sequencing of deleted chromosomes from multiple patients revealed two paralogous recombination hot spots within the 51-kb blocks. Lack of sequence similarity between these sites failed to suggest or corroborate any putative cis-acting recombinogenic motifs. However, the NF1REPs showed relatively high alignment mismatch between recombining paralogs, and we note that the NF1REP hot spots were regions of good alignment bordered by relatively large alignment gaps. Statistical tests for gene conversion detected a single significant tract of perfect match between the NF1REPs that was 700 bp long and coincided with PRS2, the predominant recombination hot spot. Tracts of perfect match occurring by chance may contribute to breakpoint localization, but our result suggests that perfect tracts at recombination hot spots may be a result of gene conversion at sites at which preferential pairing occurs for other, as-yet-unknown reasons. © 2004 Wiley-Liss, Inc.

The neurofibromatoses are a group of neurocutaneous disorders that show extreme clinical heterogeneity and are characterised by growth abnormalities in tissues derived from the embryonic neural crest.1,2 Two main clinical forms exist, type 1 (NF1) and type 2 (NF2), as well as several alternate and related forms.2,3 NF1 and NF2 are the only clinically well defined disorders and both genes have been identified.4–8 The NIH diagnostic criteria for NF1, as defined by the conference statement,9 are met if two or more of the following are found: six or more CAL spots; two or more neurofibromas of any type or one plexiform neurofibroma; axillary or inguinal freckling; optic glioma; two or more Lisch nodules; a distinct osseous lesion; a first degree relative (parent, sib, or offspring) with NF1 according to the above criteria. Spinal nerve sheath tumours are described as symptomatic findings in only 5% of NF1 patients,10 although they can be observed by MRI in up to 36% of patients.11–13 The presence of a wide, symmetrical distribution of spinal neurofibromas, occurring in all adult affected members of the same family and segregating in an autosomal dominant fashion, is however extremely rare. This form, familial spinal NF (FSNF), has been considered an alternate form of NF since patients generally lack dermal neurofibromas and Lisch nodules, both typical hallmarks of NF1, and since symptomatic and generalised spinal neurofibromas are uncommon in classical NF1. FSNF has been reported in only four families.12,14,15 Three multigenerational families with spinal neurofibromas and CAL spots were shown to be linked to markers surrounding the NF1 locus.12,14,15 In the fourth family, presenting with spinal neurofibromas without CAL spots, linkage to the NF1 locus was excluded.14 Only in one FSNF family has …

The autosomal-dominant spinocerebellar ataxias (ADCA) are a heterogeneous group of neurodegenerative disorders with variable expression and phenotypic overlap. An accurate diagnosis relies on detection of a mutation in a specific causative gene, which is typically an abnormal number of CAG trinucleotide repeats. To streamline testing in a clinical setting, we converted our current panel of tests for the spinocerebellar ataxias (SCA) types SCA1, SCA2, SCA3, SCA6, and SCA7 from five independent amplification reactions analyzed by polyacrylamide gel electrophoresis (PAGE) to a single multiplex amplification reaction analyzed by capillary electrophoresis (CE). Multiplex amplification was facilitated by the use of chimeric primers; different lengths and fluorochromes distinguished the amplicons. During CE with commercially available molecular weight standards, the SCA amplicons migrated faster than predicted, thereby underestimating their length compared to that determined previously by PAGE. This was observed to varying degrees for each of the five loci, with the greatest size differential occurring in amplicons with greater (CAG)(n). To determine accurate amplicon length, and therefore an accurate number of CAG repeats, a size correction formula was calculated for each locus. This multiplex semi-automated assay has been reliable during 1 year of use in a clinical setting during which 57 samples were tested and five positive samples were detected.

Some bacterial infections involve potentially complex mixtures of species that can now be distinguished using next-generation DNA sequencing. We present a case of mastoiditis where Gram stain, culture, and molecular diagnosis were nondiagnostic or discrepant. Next-generation sequencing implicated coinfection of Fusobacterium nucleatum and Actinomyces israelii , resolving these diagnostic discrepancies.

Epidermolysis bullosa simplex are dominant disorders of skin fragility characterized by intraepidermal blistering upon mild mechanical trauma. Skin fragility is caused by expression of either an abnormal keratin 5 or an abnormal keratin 14 protein, which compromises the structure and function of the keratin cytoskeleton of basal cells. We report an epidermolysis bullosa simplex patient with a novel single base substitution (A--T1414) that changes the lysine residue at amino acid 472 to a non-sense codon (K472X). This change predicts the synthesis of a truncated keratin 5, missing 119 amino acids, including the entire tail domain and the highly conserved KLLEGE motif at the carboxy terminus of the 2B domain of the central rod. Expression of an altered keratin 5, of predicted mass and pI for the product of the K472X allele, was documented by one- and two-dimensional western blots of protein extracts from patient skin. Ultrastructural analysis of the patient's nonhyperkeratotic skin was remarkable for basal keratinocytes with dense and irregular keratin filaments proximal to the basement membrane. Keratinocytes, transfected with a cDNA carrying the A--T1414 non-sense mutation, overexpressed a truncated keratin 5, and showed a disorganized and collapsed keratin filament cytoskeleton. This is the second epidermolysis bullosa simplex patient reported with a premature termination mutation in the KLLEGE motif. The remarkable occurrence of severe palmar--plantar hyperkeratosis in both patients suggests that the keratin 5 tail domain may have unrecognized, but important, normal functions in palmar-plantar tissues.

Children with neurofibromatosis type 1 (NF1) carry germline mutations in one allele of the NF1 gene and are predisposed to myeloid malignancies, particularly juvenile myelomonocytic leukemia (JMML). Disruption of the remaining NF1 allele can be found in malignant cells. Flow cytometric cell sorting techniques to isolate the malignant cell populations and molecular genetic methods to assay for somatic loss of the normal NF1 allele were used to study an unusual child with NF1 and JMML who subsequently had T-cell lymphoma. The data show that malignant JMML and lymphoma cells share a common loss of genetic material involving the normal NF1gene and approximately 50 Mb of flanking sequence, suggesting that the abnormal T-lymphoid and myeloid populations were derived from a common precursor cell. These data support the hypothesis that JMML can arise in a pluripotent hematopoietic cell.

The SLC6A4 gene encodes the serotonin transporter, the target of an important class of antidepressant drugs (serotonin selective reuptake inhibitors). Polymorphisms in the SLC6A4 gene have been reported to be associated with susceptibility to depression and other psychiatric disorders. We have constructed a 1 Mb YAC and PAC contig which harbours both the SLC6A4 and the carboxypeptidase D (CPD) genes. The order of loci within the contig was cen-D17S975-D17S1549-24R-D17S1294-SLC6A4-28L+ ++-(CPD, D17S2009, D17S2004)-D17S2120-ter. Both genes were deleted in one of 17 neurofibromatosis type 1 (NF1) patients carrying submicroscopic NF1 contiguous gene deletions.

Neurofibromatosis type 1 patients with a submicroscopic deletion spanning the NF1 tumor suppressor gene are remarkable for an early age at onset of cutaneous neurofibromas, suggesting the deletion of an additional locus that potentiates neurofibromagenesis. Construction of a 3.5 Mb BAC/PAC/YAC contig at chromosome 17q11.2 and analysis of somatic cell hybrids from microdeletion patients showed that 14 of 17 cases had deletions of 1.5 Mb in length. The deletions encompassed the entire 350 kb NF1 gene, three additional genes, one pseudogene and 16 expressed sequence tags (ESTs). In these cases, both proximal and distal breakpoints mapped at chromosomal regions of high identity, termed NF1REPs. These REPs, or clusters of paralogous loci, are 15-100 kb and harbor at least four ESTs and an expressed SH3GL pseudogene. The remaining three patients had at least one breakpoint outside an NF1REP element; one had a smaller deletion thereby narrowing the critical region harboring the putative locus that exacerbates neurofibroma development to 1 Mb. These data show that the likely mechanism of NF1 microdeletion is homologous recombination between NF1REPs on sister chromatids. NF1 microdeletion is the first REP-mediated rearrangement identified that results in loss of a tumor suppressor gene. Therefore, in addition to the germline rearrangements reported here, NF1REP-mediated somatic recombination could be an important mechanism for the loss of heterozygosity at NF1 in tumors of NF1 patients.

There is accumulating evidence for the importance of small, dense low-density lipoprotein (LDL), the defining feature of the atherogenic lipoprotein phenotype, as a risk factor for coronary heart disease. Although both family studies and twin studies have demonstrated genetic influences on this phenotype, the specific gene(s) involved remain to be identified. The purpose of this study was to determine whether there was evidence for genetic linkage between small, dense LDL (LDL subclass phenotype B), as determined by gradient gel electrophoresis, and selected candidate genes known to be involved in lipid metabolism. The linkage analyses were based on a sample of 19 families, including 142 individual family members, using a lod score linkage analysis approach. Nine candidate genes were examined, including loci for manganese superoxide dismutase (Mn SOD2), apolipoproteins CIII, AII, and apo CII, lipoprotein lipase, hepatic lipase, microsomal triglyceride transport protein, the insulin receptor and the LDL receptor. The analyses did not provide significant evidence for genetic linkage between markers for any of these genes and LDL subclass phenotype B, nor did it confirm previous reports of linkage between the LDL receptor gene and LDL subclass phenotype B. Using three closely linked markers for the Mn SOD2 locus excluded close linkage between this candidate gene region and LDL subclass phenotype B. These findings demonstrate the complexity of genetically mapping risk factor phenotypes, and emphasize the necessity of identifying new genetic loci, other than known candidate genes, involved in susceptibility to atherosclerosis.

The KRT5 and KRT14 genes encode the proteins keratin 5 and 14, respectively, which are the primary structural components of the 10-nm intermediate filaments of the mitotic epidermal basal cells. A single mutation in either gene can disrupt the keratin intermediate filament cytoskeleton, resulting in the skin fragility and blistering that is characteristic of the group of inherited disorders known as epidermolysis bullosa simplex. We have established a mutation detection system that facilitates KRT5 gene analysis from leukocyte genomic DNA, obviating the need for a skin sample or keratinocyte culture for cDNA synthesis. KRT5 intronic regions that flanked each exon were sequenced and sets of facing intronic primers were designed for specific amplification of each of the nine KRT5 exons. Direct sequencing of KRT5-amplified exons identified three novel missense mutations. One mutation recurred in two unrelated patients with sporadic EBS. This glutamate to lysine substitution (E477K), located in the highly conserved KLLEGE motif at the end of the central rod domain, is the third recurrent mutation identified in dominant epidermolysis bullosa simplex disease. The corresponding glutamate in keratin 2e was previously reported to be frequently mutated in ichthyosis bullosa of Siemens, suggesting that this highly conserved residue may be a potential mutational hot spot in other type II keratins or nonkeratin intermediate filament proteins.

Cyclic hematopoiesis, (CH, or cyclic neutropenia) is a rare disease manifested by transient severe neutropenia that recurs approximately every 21 days. The hematologic profile of families with the autosomal dominant form (ADCH) has not been well characterized, and it is unknown if the phenotype is distinct from the more common sporadic congenital or acquired forms of CH. We studied nine ADCH families whose children displayed typical CH blood patterns. Pedigrees confirmed dominant inheritance without evidence of heterogeneity or decreased penetrance; three pedigrees suggested new mutations. Families were Caucasian with exception of one with a Cherokee Native American founder: A wide spectrum of symptom severity, ranging from asymptomatic to life-threatening illness, was observed within families. The phenotype changed with age. Children displayed typical neutrophil cycles with symptoms of mucosal ulceration, lymphadenopathy, and infections. Adults often had fewer and milder chronic neutropenia without distinct cycles. While CH is commonly described as "benign", four children in three of the nine families died of Clostridium or E. coli colitis, documenting the need for urgent evaluation of abdominal pain. Misdiagnosis with other neutropenias was common but can be avoided by serial blood counts in index cases. Genetic counseling requires specific histories and complete blood counts in relatives at risk to assess status regardless of symptoms, especially to determine individuals with new mutations. We propose diagnostic criteria for ADCH in affected children and adults. Recombinant human granulocyte colony-stimulating factor treatment resulted in dramatic improvement of neutropenia and morbidity. The differential diagnosis from other forms of familial neutropenia is reviewed.

Children with neurofibromatosis type 1 (NF1) are at increased risk of developing malignant myeloid disorders, particularly juvenile chronic myelogenous leukemia/juvenile myelomonocytic leukemia (JCML/JMML). We investigated bone marrows from 11 such patients (8 boys and 3 girls) and detected allelic losses at the NF1 locus in 4 of them and probable losses in 2 others. To determine which hematopoietic cell lineages were derived from the abnormal clones, Epstein-Barr virus (EBV)-transformed cell lines and CD34+ cells were analyzed from 3 children with JCML with allelic losses in unfractionated marrow. CD34 cells from these 3 patients lacked the normal NF1 allele, whereas EBV cell lines retained it. Erythroblasts plucked from the burst-forming unit-erythroid colonies of one of these children lacked the normal NF1 allele. We also studied a 10-month-old boy with NF1 who developed an unusual myeloproliferative syndrome. His bone marrow and EBV cell line both showed loss of the normal NF1 allele. In our series and in the literature, male sex and maternal transmission of NF1 were associated with the highest risk of myeloid leukemia. These data (1) provide strong genetic evidence that NF1 functions as a tumor-suppressor in early myelopoiesis, (2) confirm the clonal nature of JCML/JMML, (3) suggest that the elevation in fetal hemoglobin seen in JCML/JMML is a result of primary involvement of erythroid progenitors in the malignant clone, (4) show consistent loss of NF1 in the CD34 cells of affected children and show that the malignant clone may also give rise to pre-B cells in some cases, and (5) implicate epigenetic factors in the development of leukemia in children with NF1.

Neurofibromatosis type 1 (NFl) is a common genetic disorder characterized primarily by the development of multiple neurofibromas and pigmentary changes. The recent identification of contiguous gene deletions in NF1 a previously unrecognized molecular basis for this disorder, raises important questions regarding deletion frequency in the patient population and the role that contiguous genes may play in the physical manifestations of NF1 patients. To facilitate the identification of patients with large NFl deletions, we have isolated clones carrying large genomic segments from the NF1 locus and tested their efficacy as probes for fluorescence in situ hybridization (FISH). Clone P1-9 spans approximately 65 kb of the NFl gene, including exons 2–11, and clone P1-12 carries ∼55 kb of NFl intron 27B. FISH studies performed with P1-9, P1-12, and a set of overlapping 1F10 cosmid clones mapping telomeric to the NF1 locus identified large deletions in two new neurofibromatosis type 1 patients who, like previously characterized deletion patients, had mildly dysmorphic facial features and large numbers of cutaneous neurofibromas.

Recently, two patients with the Dowling-Meara subtype of epidermolysis bullosa simplex (EBS-DM) were reported with different mutations in codon 125 of the keratin 14 gene. To determine whether these are common mutations, we screened ten EBS-DM patients and their families using single nucleotide primer extension. Four of ten unrelated EBS-DM patients had a G→-A substitution at base pair 434 of codon 125, whereas one case out of ten had a C→-T substitution at position 433 of the same codon. The G434A alteration cosegregated with the disorder in two multigenerational families; no recombination events were detected. In these two families, linkage analysis provided significant evidence in favor of linkage between G434A and the EBS-DM phenotype, with a LOD score of 3.29 at a recombination rate of 0%. Codon 125 substitutions identified in three unrelated sporadic EBS-DM patients were not found in their clinically unaffected parents. Together, these data provide compelling genetic evidence that the codon 125 substitutions are causal for EBS-DM. The high frequency of mutation at this site in individuals with EBS-DM now makes DNA-based diagnosis of this disorder feasible.

Fault Management is a critical aspect of deep-space missions. For the purposes of this paper, fault management is defined as the ability of a system to detect, isolate, and mitigate events that impact, or have the potential to impact, nominal mission operations. The fault management capabilities are commonly distributed across flight and ground subsystems, impacting hardware, software, and mission operations designs. The National Aeronautics and Space Administration (NASA) Discovery & New Frontiers (DN unclear ownership of end-to-end fault management engineering; inadequate understanding of the impact of mission-level requirements on fault management complexity; and practices, processes, and tools that have not kept pace with the increasing complexity of mission requirements and spacecraft systems. This paper summarizes the findings and recommendations from that workshop, as well as opportunities identified for future investment in tools, processes, and products to facilitate the development of space flight fault management capabilities.

Early years practitioners can do much to help children living amid domestic violence to develop resilience and see that there are other ways for people to relate to each other, writes Karen Stephens.

Recently the M17S1 gene, encoding an epidermal antigen thought to play a role in cell adhesion, was mapped to chromosome bands 17q11–q12, placing it in the vicinity of the gene for the genetic disorder neurofi-bromatosis 1 (NF1). The pleomorphic cutaneous lesions of NF1 and the precedent for other genes being embedded within the NF1 gene prompted us to investigate whether the M17S1 gene mapped near, or within, the NF1 gene. Genetic linkage analyses revealed that M17S1 was tightly linked to NF1 and mapped within the interval bounded by D17S58 and D17S54. Physical mapping of an M17S1 cDNA on somatic cell hybrids, yeast artificial chromosomes, and an NF1 patient with a deletion involving an entire NF1 allele demonstrated that M17S1 is located at least 180 kb centromeric to the NF1 gene. The distance between the genes suggests that M17S1 is unlikely to contribute to the NF1 phenotype since a gross chromosomal rearrangement would be required to disrupt expression of both genes.

Skin neurofibromas are the most common tumors of patients with neurofibromatosis type 1 (NF1), yet we know very little about how they develop. We are studying subjects with microdeletions because they are predisposed to large numbers of neurofibromas. We propose that deletion of NF1 gene and at least one of the 15 adjacent genes favors the development of skin neurofibromas, and probably other NF1-related tumors. We are evaluating NF1 subjects to identify those with deletions and determine when and what kind of tumors and other clinical features they develop. We have developed rapid and sensitive assays for the detection and mapping of NF1 microdeletions. Using these assays, we screened 242 patients and identified 56 carriers of the recurrent 1.4Mb NF1 microdeletions and 16 carriers of novel deletions. Clinical evaluation of these patients, along with age/sex matched NF1 patients that are not NF1 deletion carriers, i& in progress to delineate the clinical manifestations most prevalent in deletion patients. We have collected tumors from NF1 microdeletion carriers, which are now being examined for mutations and genomic instability. We began a new collaboration and generated preliminary data that identified an apparent new function of neurofibromin as a caspase target. Since this work is outside the scope of this grant, we have submitted a new application for investigating the role of caspase in NF1 disease and tumorigenesis. Therefore, our progress on genotype/phenotype correlations in carriers of NF1 microdeletions, along with our new work identifying neurofibromin as a caspase target, will provide clinically applicable information for patient diagnosis and management and basic knowledge about neurofibromin function in disease pathogenesis.

The proper prioritization of technologies within a technology development program is critical for success in a budget constrained environment. A large portfolio of diverse products at differing states of development all competing for program resources presents a need for a process by which a realistic need date can guide the development strategy for each technology. This paper will document an exercise to identify "mission pull" for each technology that can be used to identify the best projected need date for each. This date is then used to back out a development schedule with corresponding funding profile needed to meet the flight opportunity.

Several large-scale studies of human genetic variation have provided insights into processes such as recombination that have shaped human diversity. However, regions such as low-copy repeats (LCRs) have proven difficult to characterize, hindering efforts to understand the processes operating in these regions. We present a detailed study of genetic variation and underlying recombination processes in two copies of an LCR (NF1REPa and NF1REPc) on chromosome 17 involved in the generation of NF1 microdeletions and in a third copy (REP19) on chromosome 19 from which the others originated over 6.7 million years ago. We find evidence for shared hotspots of recombination among the LCRs. REP19 seems to contain hotspots in the same place as the nonallelic recombination hotspots in NF1REPa and NF1REPc. This apparent conservation of patterns of recombination hotspots in moderately diverged paralogous regions contrasts with recent evidence that these patterns are not conserved in less-diverged orthologous regions of chimpanzees.

Harlequin ichthyosis (HI) is the most severe and frequently lethal form of recessive congenital ichthyosis. Although defects in lipid transport, protein phosphatase activity, and differentiation have been described, the genetic basis underlying the clinical and cellular phenotypes of HI has yet to be determined. By use of single-nucleotide–polymorphism chip technology and homozygosity mapping, a common region of homozygosity was observed in five patients with HI in the chromosomal region 2q35. Sequencing of the ABCA12 gene, which maps within the minimal region defined by homozygosity mapping, revealed disease-associated mutations, including large intragenic deletions and frameshift deletions in 11 of the 12 screened individuals with HI. Since HI epidermis displays abnormal lamellar granule formation, ABCA12 may play a critical role in the formation of lamellar granules and the discharge of lipids into the intercellular spaces, which would explain the epidermal barrier defect seen in this disorder. This finding paves the way for early prenatal diagnosis. In addition, functional studies of ABCA12 will lead to a better understanding of epidermal differentiation and barrier formation.