Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Characterizing intubation practices in response to the COVID-19 pandemic: a survey of the Canadian COVID-19 Emergency Department Rapid Response Network (CCEDRRN) sites.

OBJECTIVE: The risk of occupational exposure during endotracheal intubation has required the global Emergency Medicine (EM), Anesthesia, and Critical Care communities to institute new COVID- protected intubation guidelines, checklists, and protocols. This survey aimed to deepen the understanding of the changes in intubation practices across Canada by evaluating the pre-COVID-19, early-COVID-19, and present-day periods, elucidating facilitators and barriers to implementation, and understanding provider impressions of the effectiveness and safety of the changes made. METHODS: We conducted an electronic, self-administered, cross-sectional survey of EM physician site leads within the Canadian COVID-19 Emergency Department Rapid Response Network (CCEDRRN) to characterize and compare airway management practices in the pre-COVID-19, early-COVID-19, and present-day periods. Ethics approval for this study was obtained from the University of Manitoba Health Research Ethics Board. The electronic platform SurveyMonkey ( www.surveymonkey.com ) was used to collect and store survey tool responses. Categorical item responses, including the primary outcome, are reported as numbers and proportions. Variations in intubation practices over time were evaluated through mixed-effects logistic regression models. RESULTS: Invitations were sent to 33 emergency department (ED) physician site leads in the CCEDRRN. We collected 27 survey responses, 4 were excluded, and 23 analysed. Responses were collected in English (87%) and French (13%), from across Canada and included mainly physicians practicing in mainly Academic and tertiary sites (83%). All respondents reported that the intubation protocols used in their EDs changed in response to the COVID-19 pandemic (100%, n = 23, 95% CI 0.86-1.00). CONCLUSIONS: This study provides a novel summary of changes to airway management practices in response to the evolving COVID-19 pandemic in Canada. Information from this study could help inform a consensus on safe and effective emergent intubation of persons with communicable respiratory infections in the future.

Development of covalent chemogenetic K2P channel activators.

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (Covalent Activation of TREK family K+ channels to cLAmp Membrane Potential) that leverages the discovery of a site in the K2P modulator pocket that reacts with electrophile-bearing derivatives of a TREK subfamily small molecule activator, ML335, to activate the channel irreversibly. We show that the CATKLAMP strategy can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a new means to alter K2P channel activity that should facilitate studies both molecular and systems level studies of K2P function and enable the search for new K2P modulators.

UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron dysfunction and loss. A portion of ALS cases are caused by mutation of the proteasome shuttle factor Ubiquilin 2 (UBQLN2), but the molecular pathway leading from UBQLN2 dysfunction to disease remains unclear. Here, we demonstrate that UBQLN2 regulates the domesticated gag-pol retrotransposon 'paternally expressed gene 10 (PEG10)' in human cells and tissues. In cells, the PEG10 gag-pol protein cleaves itself in a mechanism reminiscent of retrotransposon self-processing to generate a liberated 'nucleocapsid' fragment, which uniquely localizes to the nucleus and changes the expression of genes involved in axon remodeling. In spinal cord tissue from ALS patients, PEG10 gag-pol is elevated compared to healthy controls. These findings implicate the retrotransposon-like activity of PEG10 as a contributing mechanism in ALS through the regulation of gene expression, and restraint of PEG10 as a primary function of UBQLN2.

Emergency airway management in a tertiary trauma centre (AIRMAN): a one-year prospective longitudinal study.

PURPOSE: Emergency airway management can be associated with a range of complications including long-term neurologic injury and death. We studied the first-pass success rate with emergency airway management in a tertiary care trauma centre. Secondary outcomes were to identify factors associated with first-pass success and factors associated with adverse events peri-intubation. METHODS: We performed a single-centre, prospective, observational study of patients ≥ 17 yr old who were intubated in the emergency department (ED), surgical intensive care unit (SICU), medical intensive care unit (MICU), and inpatient wards at our institution. Ethics approval was obtained from the local research ethics board. RESULTS: In a seven-month period, there were 416 emergency intubations and a first-pass success rate of 73.1%. The first-pass success rates were 57.5% on the ward, 66.1% in the intensive care units (ICUs) and 84.3% in the ED. Equipment also varied by location; videolaryngoscopy use was 65.1% in the ED and only 10.6% on wards. A multivariate regression model using the least absolute shrinkage and selection algorithm (LASSO) showed that the odds ratios for factors associated with two or more intubation attempts were location (wards, 1.23; MICU, 1.24; SICU, 1.19; reference group, ED), physiologic instability (1.19), an anatomically difficult airway (1.05), hypoxemia (1.98), lack of neuromuscular blocker use (2.28), and intubator inexperience (1.41). CONCLUSIONS: First-pass success rates varied widely between locations within the hospital and were less than those published from similar institutions, except for the ED. We are revamping ICU protocols to improve the first-pass success rate.

RéSUMé: OBJECTIF: La prise en charge d'urgence des voies aériennes peut être associée à une multitude de complications, y compris des lésions neurologiques à long terme et la mort. Nous avons étudié le taux de réussite à la première tentative lors de la prise en charge d'urgence des voies aériennes dans un centre de traumatologie tertiaire. Les critères d'évaluation secondaires étaient l'identification des facteurs associés à la réussite de la première tentative et des facteurs associés aux événements indésirables péri-intubation. MéTHODE: Nous avons réalisé une étude observationnelle prospective monocentrique sur des patients âgés de 17 ans ou plus qui avaient été intubés à l'urgence, à l'unité de soins intensifs chirurgicaux (USIC), à l'unité de soins intensifs médicaux (USIM) et aux étages dans notre établissement. L'approbation a été obtenue du comité d'éthique de la recherche local. RéSULTATS: Au cours d'une période de sept mois, il y a eu 416 intubations d'urgence et un taux de réussite à la première tentative de 73,1 %. Les taux de réussite à la première tentative étaient de 57,5 % aux étages, de 66,1 % dans les unités de soins intensifs (USI) et de 84,3 % à l'urgence. Le matériel variait également selon l'emplacement; l'utilisation de la vidéolaryngoscopie était de 65,1 % à l'urgence et de seulement 10,6 % aux étages. Un modèle de régression multivariée utilisant l'algorithme LASSO (Least Absolute Shrinkage and Selection Algorithm) a montré que les rapports de cotes pour les facteurs associés à deux tentatives d'intubation ou plus étaient l'emplacement (étages, 1,23; USIM, 1,24; USIC, 1,19; groupe de référence, urgence), l'instabilité physiologique (1,19), des voies aériennes présentant des complications anatomiques (1,05), l'hypoxémie (1,98), la non-utilisation de bloqueurs neuromusculaires (2,28) et l'inexpérience de la personne pratiquant l'intubation (1,41). CONCLUSION: Les taux de réussite à la première tentative variaient considérablement d'un emplacement à l'autre au sein de l'hôpital et étaient inférieurs à ceux publiés par des établissements comparables, à l'exception du service des urgences. Nous retravaillons les protocoles des soins intensifs afin d'améliorer le taux de réussite à la première tentative.

A prospective observational study comparing outcomes before and after the introduction of an intubation protocol during the COVID-19 pandemic.

OBJECTIVE: Orotracheal intubation is a life-saving procedure commonly performed in the Intensive Care unit and Emergency Department as a part of emergency airway management. Prior to the COVID-19 pandemic, our center undertook a prospective observational study to characterize emergency intubation performed in the emergency department and critical care settings at Manitoba's largest tertiary hospital. During this study, a natural experiment emerged when a standardized "COVID-Protected Rapid Sequence Intubation Protocol" was implemented in response to the pandemic. The resultant study aimed to answer the question; in adult ED patients undergoing emergent intubation by EM and CCM teams, does the use of a "COVID-Protected Rapid Sequence Intubation Protocol" impact first-pass success or other intubation-related outcomes? METHODS: A single-center prospective quasi-experimental before and after study was conducted. Data were prospectively collected on consecutive emergent intubations. The primary outcome was the difference in first-pass success rates. Secondary outcomes included best Modified Cormack-Lehane view, hypoxemia, hypotension, esophageal intubation, cannot intubate cannot oxygenate scenarios, CPR post intubation, vasopressors required post intubation, Intensive Care Unit (ICU) mortality, ICU length of stay (LOS), and mechanical ventilation days. RESULTS: Data were collected on 630 patients, 416 in the pre-protocol period and 214 in the post-protocol period. First-pass success rates in the pre-protocol period were found to be 73.1% (n = 304). Following the introduction of the protocol, first-pass success rates increased to 82.2% (n = 176, p = 0.0105). There was a statistically significant difference in Modified Cormack-Lehane view favoring the protocol (p = 0.0191). Esophageal intubation rates were found to be 5.1% pre-protocol introduction versus 0.5% following the introduction of the protocol (p = 0.0172). CONCLUSION: A "COVID-Protected Protocol" implemented by Emergency Medicine and Critical Care teams in response to the COVID-19 pandemic was associated with increased first-pass success rates and decreases in adverse events.

RéSUMé: OBJECTIFS: L'intubation orotrachéale est une procédure de sauvetage couramment réalisée dans l'unité de soins intensifs et le service des urgences dans le cadre de la gestion des voies aériennes d'urgence. Avant la pandémie de COVID-19, notre centre a entrepris une étude prospective d'observation pour caractériser l'intubation d'urgence effectuée dans le service des urgences et les établissements de soins intensifs du plus grand hôpital tertiaire du Manitoba. Au cours de cette étude, une expérience naturelle est apparue lorsqu'un " protocole d'intubation à séquence rapide protégé contre le COVID " standardisé a été mis en œuvre en réponse à la pandémie. L'étude qui en a résulté visait à répondre à la question suivante : chez les patients adultes des urgences soumis à une intubation urgente par les équipes de médecine d'urgence et de médecine de soins critiques, l'utilisation d'un " protocole d'intubation à séquence rapide protégé par COVID " a-t-elle un impact sur la réussite du premier passage ou sur d'autres résultats liés à l'intubation ? MéTHODE: Une étude prospective quasi-expérimentale avant et après a été menée dans un seul centre. Les données ont été recueillies prospectivement sur des intubations émergentes consécutives. Le résultat principal était la différence entre les taux de réussite au premier passage. Les résultats secondaires comprenaient la meilleure vue de Cormack­Lehane modifiée, l'hypoxémie, l'hypotension, l'intubation œsophagienne, les scénarios d'impossibilité d'intubation et d'oxygénation, la réanimation cardio-pulmonaire après l'intubation, les vasopresseurs nécessaires après l'intubation, la mortalité en unité de soins intensifs (USI), la durée de séjour en USI et les jours de ventilation mécanique. RéSULTATS: Des données ont été recueillies sur 630 patients, 416 dans la période pré-protocole et 214 dans la période post-protocole. Les taux de réussite au premier passage dans la période pré-protocole se sont avérés être de 73,1 % (n = 304). Suite à l'introduction du protocole, les taux de réussite au premier passage ont augmenté à 82,2 % (n = 176, p = 0,0105). Il y avait une différence statistiquement significative dans la vue Cormack­Lehane modifiée en faveur du protocole (p = 0,0191). Les taux d'intubation œsophagienne se sont avérés être de 5,1 % avant l'introduction du protocole contre 0,5 % après l'introduction du protocole (p = 0,0172). CONCLUSION: Un « protocole protégé contre la COVID ¼ mis en œuvre par les équipes de médecine d'urgence et de médecine de soins critiques en réponse à la pandémie de COVID-19 a été associé à une augmentation des taux de réussite du premier passage et à une diminution des événements indésirables.

Establishment of Amblyomma americanum populations and new records of Borrelia burgdorferi-infected Ixodes scapularis in South Dakota.

Tick-borne diseases are an emerging public health threat in the United States, but surveillance is lacking in some regions. To advance current knowledge of the ecology of ticks and tick-borne diseases in South Dakota, we conducted a survey in the summer of 2019, focusing on the eastern counties of the state. We collected and identified 266 ticks and a subset were tested for the presence of Borrelia burgdorferi by polymerase chain reaction (PCR). Dermacentor variabilis, a ubiquitous species in the state, was the most commonly identified tick, present in all counties surveyed. However, we also identified 15 Amblyomma americanum from three different locations, providing the first evidence of established populations in the state and expanding the range of this species. In addition, we identified 22 Ixodes scapularis from five different locations, confirming a previous report of an established population in the state. Two adult I. scapularis from two different sites were found to harbor B. burgdorferi, including an individual from Lincoln County, suggesting the ongoing presence of the pathogen in tick populations in the state and representing its southwestern-most detection in the midwest United States. These findings provide important information for assessing and monitoring the public health risk from tick-borne diseases in an area where surveillance is lacking.

A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection.

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.

Absolute measurement of the tissue origins of cell-free DNA in the healthy state and following paracetamol overdose.

BACKGROUND: Despite the emergence of cell-free DNA (cfDNA) as a clinical biomarker in cancer, the tissue origins of cfDNA in healthy individuals have to date been inferred only by indirect and relative measurement methods, such as tissue-specific methylation and nucleosomal profiling. METHODS: We performed the first direct, absolute measurement of the tissue origins of cfDNA, using tissue-specific knockout mouse strains, in both healthy mice and following paracetamol (APAP) overdose. We then investigated the utility of total cfDNA and the percentage of liver-specific cfDNA as clinical biomarkers in patients presenting with APAP overdose. RESULTS: Analysis of cfDNA from healthy tissue-specific knockout mice showed that cfDNA originates predominantly from white and red blood cell lineages, with minor contribution from hepatocytes, and no detectable contribution from skeletal and cardiac muscle. Following APAP overdose in mice, total plasma cfDNA and the percentage fraction originating from hepatocytes increased by ~ 100 and ~ 19-fold respectively. Total cfDNA increased by an average of more than 236-fold in clinical samples from APAP overdose patients with biochemical evidence of liver injury, and 18-fold in patients without biochemically apparent liver injury. Measurement of liver-specific cfDNA, using droplet digital PCR and methylation analysis, revealed that the contribution of liver to cfDNA was increased by an average of 175-fold in APAP overdose patients with biochemically apparent liver injury compared to healthy subjects, but was not increased in overdose patients with normal liver function tests. CONCLUSIONS: We present a novel method for measurement of the tissue origins of cfDNA in healthy and disease states and demonstrate the potential of cfDNA as a clinical biomarker in APAP overdose.

All-terrain vehicle-related injuries and deaths in Newfoundland and Labrador between 2003 and 2013: a retrospective trauma registry review.

BACKGROUND: Injury and death involving all-terrain vehicles (ATV) has been reported in a number of Canadian provinces. The objective of this study is to describe the frequency, nature, severity, population affected, immediate health costs, efficacy of related legislation, and helmet use in ATV related injuries and deaths in Newfoundland and Labrador (NL). METHODS: A retrospective review of injured or deceased ATV riders of all ages entered in the Newfoundland and Labrador Trauma Registry from 2003 to 2013 was conducted. Variables studied included demographics, injury type and severity, use of helmets, admission/discharge dates, and referring/receiving institution. Data was also obtained from the Newfoundland and Labrador Center for Health Information (NLCHI) and included all in-hospital deaths and hospitalizations due to ATVs between 1995 and 2013. RESULTS: There were a total of 298 patients registered in the trauma registry, resulting in 2759 admission days, nine deaths, and a total estimated immediate healthcare system cost in excess of $1.6 million. More males (N=253, 84.9%) than females (N=45, 15.1%) were injured in ATV related incidents, t(20)=7.12, p<.0001. Head and thorax injuries were the most serious. 38.6% of patients were confirmed to be wearing helmets. Mean injury severity scores are as follows: head injury (M=11, SD=9.51), thorax (M=10, SD=8.3), abdominal/pelvis (M=9, SD=7.62), upper extremity (M=9, SD=8.53), other injuries (M=9, SD=10.56) lower extremity (M=8, SD=8.34), and spine (M=8, SD=6.52). CONCLUSIONS: This study describes ATV related injuries and deaths in NL. Information from this study may guide physician practice, public education, and future legislation.

Developing an Undergraduate Ultrasound Curriculum: A Needs Assessment.

Background The introduction of ultrasound into the undergraduate medical school curriculum is gaining momentum in North America. At present, many institutions are teaching ultrasound to undergraduate medical students using a traditional framework designed to instruct practicing clinicians, or have modeled the curriculum on other universities. This approach is not based on educational needs or supported by evidence. Methods Using a descriptive, cross-sectional survey of stakeholder groups, we assessed the perceived relevance of various ultrasound skills and the attitude towards implementing an undergraduate ultrasound curriculum at our university. Results One hundred and fifty survey respondents representing all major stakeholder groups participated. All medical students, 97% of residents and 82% of educators agreed that the introduction of an ultrasound curriculum would enhance medical students' understanding of anatomy and physiology. All clinical medical students and residents, 92% of preclinical medical students, and 82% of educators agreed that the curriculum should also include clinical applications of ultrasound. Participants also indicated their preferences for specific curriculum content based on their perceived needs. Conclusion An integrated undergraduate ultrasound curriculum composed of specific preclinical and clinical applications was deemed appropriate for our university following a comprehensive needs assessment. Other universities planning such curricula should consider employing a needs assessment to provide direction for curriculum need and content.

Genetic epidemiology of motor neuron disease-associated variants in the Scottish population.

Genetic understanding of motor neuron disease (MND) has evolved greatly in the past 10 years, including the recent identification of association between MND and variants in TBK1 and NEK1. Our aim was to determine the frequency of pathogenic variants in known MND genes and to assess whether variants in TBK1 and NEK1 contribute to the burden of MND in the Scottish population. SOD1, TARDBP, OPTN, TBK1, and NEK1 were sequenced in 441 cases and 400 controls. In addition to 44 cases known to carry a C9orf72 hexanucleotide repeat expansion, we identified 31 cases and 2 controls that carried a loss-of-function or pathogenic variant. Loss-of-function variants were found in TBK1 in 3 cases and no controls and, separately, in NEK1 in 3 cases and no controls. This study provides an accurate description of the genetic epidemiology of MND in Scotland and provides support for the contribution of both TBK1 and NEK1 to MND susceptibility in the Scottish population.

Can We CanMEDS? Intangible Learning Through Tangible Simulation Case Development.

The Royal College CanMEDS framework has become a guide for medical school curricula. This framework aims to improve patient care by identifying and explaining seven key roles that physicians must fulfill in order to deliver high-quality healthcare to their patients. While medical schools incorporate these roles in their teaching processes, students can also apply them outside the classroom. Here, we describe a unique model developed at Memorial University of Newfoundland's Tuckamore Simulation Research Collaborative (TSRC), where students develop simulation cases with the guidance of expert mentors and apply the Royal College CanMEDS framework to writing clinical simulations.

Expert Facilitated Development of an Objective Assessment Tool for Point-of-Care Ultrasound Performance in Undergraduate Medical Education.

BACKGROUND: With the various applications of point-of-care ultrasound (PoCUS) steadily increasing, many medical schools across North America are incorporating PoCUS training into their undergraduate curricula. The Faculty of Medicine at Memorial University also intends to introduce PoCUS training into its own undergraduate medical program. The proposed approach is to introduce a PoCUS curriculum focusing on anatomy and physiology while developing cognitive and psychomotor skills that are later transferred into clinical applications. This has been the common approach taken by most undergraduate ultrasound programs in the United States. This project highlights the development and the challenges involved in creating an objective assessment tool that meets the unique needs of this proposed undergraduate ultrasound curriculum. METHODS: After a thorough review of existing literature and input from experts in PoCUS, a prototype global rating scale (GRS) and three exam-specific checklists were created by researchers. The exam-specific checklists include aorta exam, subxiphoid cardiac exam, and focused abdominal exam. A panel of 18 emergency room physicians certified in PoCUS were recruited to evaluate the GRS and three checklists. This was accomplished using a modified Delphi technique. The items were rated on a 5-point Likert scale. If an item received a mean score of less than 4, it was deemed unimportant for the assessment of PoCUS performance in undergraduate medical learners and was excluded. Experts were also encouraged to provide comments and suggest further items to be added to the GRS or checklists. Items were modified according to these comments. All of the edits were then sent back to the experts for revisions. RESULTS: A consensus was achieved after three rounds of surveys, with the final GRS containing nine items. The final aorta checklist contained nine items, and the subxiphoid cardiac and focused abdominal checklists each contained 11 items. CONCLUSION: By using a modified Delphi technique, we developed a single GRS and three checklists. A panel of independent PoCUS practitioners supports the content validity of these tools. Research is currently ongoing to evaluate their validity for assessing PoCUS competency in undergraduate medical students.

Targeted next-generation sequencing makes new molecular diagnoses and expands genotype-phenotype relationship in Ehlers-Danlos syndrome.

PURPOSE: Ehlers-Danlos syndrome (EDS) comprises a group of overlapping hereditary disorders of connective tissue with significant morbidity and mortality, including major vascular complications. We sought to identify the diagnostic utility of a next-generation sequencing (NGS) panel in a mixed EDS cohort. METHODS: We developed and applied PCR-based NGS assays for targeted, unbiased sequencing of 12 collagen and aortopathy genes to a cohort of 177 unrelated EDS patients. Variants were scored blind to previous genetic testing and then compared with results of previous Sanger sequencing. RESULTS: Twenty-eight pathogenic variants in COL5A1/2, COL3A1, FBN1, and COL1A1 and four likely pathogenic variants in COL1A1, TGFBR1/2, and SMAD3 were identified by the NGS assays. These included all previously detected single-nucleotide and other short pathogenic variants in these genes, and seven newly detected pathogenic or likely pathogenic variants leading to clinically significant diagnostic revisions. Twenty-two variants of uncertain significance were identified, seven of which were in aortopathy genes and required clinical follow-up. CONCLUSION: Unbiased NGS-based sequencing made new molecular diagnoses outside the expected EDS genotype-phenotype relationship and identified previously undetected clinically actionable variants in aortopathy susceptibility genes. These data may be of value in guiding future clinical pathways for genetic diagnosis in EDS.Genet Med 18 11, 1119-1127.

Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus.

BACKGROUND: The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a region of high linkage disequilibrium, despite its high variability in copy number (n = 3-16); hence, the mechanisms responsible for changes in copy number at this locus are unclear. RESULTS: In this study, a region flanking the DEFA1A3 locus was sequenced across 120 independent haplotypes with European ancestry, identifying five common classes of DEFA1A3 haplotype. Assigning DEFA1A3 class to haplotypes within the 1000 Genomes project highlights a significant difference in DEFA1A3 class frequencies between populations with different ancestry. The features of each DEFA1A3 class, for example, the associated DEFA1A3 copy numbers, were initially assessed in a European cohort (n = 599) and replicated in the 1000 Genomes samples, showing within-class similarity, but between-class and between-population differences in the features of the DEFA1A3 locus. Emulsion haplotype fusion-PCR was used to generate 61 structural haplotypes at the DEFA1A3 locus, showing a high within-class similarity in structure. CONCLUSIONS: Structural haplotypes across the DEFA1A3 locus indicate that intra-allelic rearrangement is the predominant mechanism responsible for changes in DEFA1A3 copy number, explaining the conservation of linkage disequilibrium across the locus. The identification of common structural haplotypes at the DEFA1A3 locus could aid studies into how DEFA1A3 copy number influences expression, which is currently unclear.

Accurate measurement of gene copy number for human alpha-defensin DEFA1A3.

BACKGROUND: Multi-allelic copy number variants include examples of extensive variation between individuals in the copy number of important genes, most notably genes involved in immune function. The definition of this variation, and analysis of its impact on function, has been hampered by the technical difficulty of large-scale but accurate typing of genomic copy number. The copy-variable alpha-defensin locus DEFA1A3 on human chromosome 8 commonly varies between 4 and 10 copies per diploid genome, and presents considerable challenges for accurate high-throughput typing. RESULTS: In this study, we developed two paralogue ratio tests and three allelic ratio measurements that, in combination, provide an accurate and scalable method for measurement of DEFA1A3 gene number. We combined information from different measurements in a maximum-likelihood framework which suggests that most samples can be assigned to an integer copy number with high confidence, and applied it to typing 589 unrelated European DNA samples. Typing the members of three-generation pedigrees provided further reassurance that correct integer copy numbers had been assigned. Our results have allowed us to discover that the SNP rs4300027 is strongly associated with DEFA1A3 gene copy number in European samples. CONCLUSIONS: We have developed an accurate and robust method for measurement of DEFA1A3 copy number. Interrogation of rs4300027 and associated SNPs in Genome-Wide Association Study SNP data provides no evidence that alpha-defensin copy number is a strong risk factor for phenotypes such as Crohn's disease, type I diabetes, HIV progression and multiple sclerosis.

Using In silico LD clumping and meta-analysis of genome-wide datasets as a complementary tool to investigate and validate new candidate biomarkers in Alzheimer's disease.

Despite the recent wealth of genome-wide association studies, insufficient power may explain why much of the heritable contribution to common diseases remains hidden. As different SNP panels are genotyped by commercial chips, increasing study power through meta-analysis is made problematic. To address these power issues we suggest an approach which permits meta-analysis of candidate SNPs from multiple GWAS. By identifying correlated SNPs from different platforms (r(2)=1), using PLINK's 'clumping' method, we generated combined p-values (using Fisher's combined and random effects meta-analysis) for each clump. P-values were corrected for the number of clumps (representing the number of independent tests). We also explored to what extent commercial platforms tag HapMap SNPs within these candidate genes. To illustrate this approach, and to serve as 'proof-of-principle', we used 3 late-onset Alzheimer's disease GWAS datasets to explore SNP-disease associations in 4 new candidate genes encoding cerebro-spinal fluid biomarkers for Alzheimer's disease; Fibrinogen γ-chain (FGG), SPARC-like1 (SPARCL1), Contactin-1 (CNTN1) and Contactin-2 (CNTN2). Genes encoding current Alzheimer's biomarkers; APP (Aß), MAPT (Tau) and APOE were also included. This method identified two SNP 'clumps'; one clump in APOE (rs4420638) and one downstream of CNTN2 (which harboured rs7523477 and rs4951168) which were significant following random effects meta-analysis (P < 0.05). The latter was linked to three conserved SNPs in the 3'-UTR of CNTN2. We cannot rule out that this result is a false positive due to the large number of statistical tests applied; nevertheless this approach is easily applied and might well have utility in future '-omics' studies.