Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing

Currently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ∼100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.Worldwide, more than 5 million babies (Ferraretti et al. 2013) have been born through in vitro fertilization (IVF) since the birth of the first in 1978 (Steptoe and Edwards 1978). Exact numbers are difficult to determine, but it has been estimated that currently 350,000 babies are born yearly through IVF (de Mouzon et al. 2009, 2012; Centers for Disease Control and Prevention 2011; Ferraretti et al. 2013). That number is expected to rise, as advanced maternal age is associated with decreased fertility rates and women in developed countries continue to delay childbirth to later ages. In 95% of IVF procedures, no diagnostic testing of the embryos is performed (https://www.sartcorsonline.com/rptCSR_PublicMultYear.aspx?ClinicPKID=0). Couples with prior difficulties conceiving or those wishing to avoid the transmission of highly penetrant heritable diseases often choose to perform preimplantation genetic diagnosis (PGD). PGD involves the biopsy of one cell from a 3-d embryo or the recently more preferred method, due to improved implantation success rates (Scott et al. 2013b), of up to 10 cells from a 5- to 6-d blastocyst-stage embryo. Following biopsy, genetic analysis is performed on the isolated cell(s). Currently this is an assay for translocations and the correct chromosome copy number (Hodes-Wertz et al. 2012; Munne 2012; Yang et al. 2012; Scott et al. 2013a; Yin et al. 2013), a unique test designed and validated for each specific heritable disease (Gutierrez-Mateo et al. 2009), or a combination of both (Treff et al. 2013). Importantly, none of these approaches can detect de novo mutations.Advanced maternal age has long been associated with an increased risk of producing aneuploid embryos (Munne et al. 1995; Crow 2000; Hassold and Hunt 2009) and giving birth to a child afflicted with Down syndrome or other diseases resulting from chromosomal copy number alterations. Conversely, children of older fathers have been shown to have an increase in single base and short multibase insertion/deletion (indels) de novo mutations (Kong et al. 2012). Many recent large-scale sequencing studies have found that de novo variations spread across many different genes are likely to be the cause of a large fraction of autism cases (Michaelson et al. 2012; O’Roak et al. 2012; Sanders et al. 2012; De Rubeis et al. 2014; Iossifov et al. 2014), severe intellectual disability (Gilissen et al. 2014), epileptic encephalopathies (Epi4K Consortium and Epilepsy Phenome/Genome Project 2013), and many other congenital disorders (de Ligt et al. 2012; Veltman and Brunner 2012; Yang et al. 2013; Al Turki et al. 2014). Additionally rare and de novo variations have been suggested to be prevalent in patients with schizophrenia (Fromer et al. 2014; Purcell et al. 2014), and Michaelson et al. (2012) found that single base de novo mutations affect conserved regions of the genome and essential genes more often than regions of unknown function. Current targeted approaches to PGD would miss many of these important functional changes within the embryonic DNA sequence, and even a whole-genome sequencing (WGS)–based carrier screen of both parents would not enable comprehensive preimplantation or prenatal diagnoses due to de novo mutations. As more parents delay childbirth into their mid-30s and later, these studies suggest we should try to provide better diagnostic tests for improving the health of newborns. In this study, we demonstrate the use of an advanced WGS process that provides an accurate and phased genome sequence from about 10 cells, allowing highly sensitive and specific detection of single base de novo mutations from IVF blastocyst biopsies.     

Accelerated Return to Sport After Anterior Cruciate Ligament Reconstruction and Early Knee Osteoarthritis Features at 1 Year: An Exploratory Study

Background

A timely return to competitive sport is a primary goal of anterior cruciate ligament reconstruction (ACLR). It is not known whether an accelerated return to sport increases the risk of early-onset knee osteoarthritis (KOA).

Does the impact of intensive lifestyle intervention on cognitive function vary depending baseline level of frailty? An ancillary study to the Action for Health in Diabetes (Look AHEAD) Trial

AimsTo assess whether there is an opportune window when intensive lifestyle intervention (ILI) benefits cognitive function.MethodsStandardized cognitive assessments were collected following ≥8 years of either ILI or a control condition of diabetes support and education (DSE) in 3708 individuals, ages 45-76 years at enrollment, with type 2 diabetes and overweight or obesity. Frailty index (FI) scores were used to group individuals at baseline into tertiles according to their age-related health status. Linear models were used to describe intervention adherence and cognitive function, with interaction terms to examine the consistency of relationships among tertiles.ResultsWorse baseline FI scores were associated with poorer subsequent performance in tests of attention, processing speed, and executive function. No differences in any measure of cognitive function were observed between intervention groups within any FI tertile (all p > 0.10). Among individuals with worse baseline FI scores, weight gain was associated with poorer global cognitive function among participants assigned to DSE. There was no association between weight changes and cognitive function among participants assigned to ILI.ConclusionsAmong adults with type 2 diabetes and overweight/obesity, we found no evidence that there is a window of opportunity based on FI when ILI benefits cognitive function.     

Effect on iron deficiency anemia of laparoscopic repair of large paraesophageal hernias

Patients with iron deficiency anemia sometimes have a large paraesophageal hernia and no other explanation for their chronic blood loss. The management of these patients can be a dilemma, especially when the hernia is otherwise asymptomatic. We aimed to determine whether a laparoscopic repair of the hernia could cure the anemia. We reviewed a consecutive series of 11 cases of iron deficiency anemia associated with a large paraesophageal hernia, many without associated linear gastric erosions, managed by laparoscopic repair and fundoplication. There was one conversion in a patient with dense adhesions from previous upper abdominal surgery. Another patient required a laparoscopic reoperation for an early recurrence. Major morbidity occurred in three patients and there was no mortality. There was no recurrence of anemia after a median follow-up of more than 2 years. Iron deficiency anemia in association with a large paraesophageal hernia can be treated by laparoscopic repair with acceptable morbidity and minimal mortality. The complications of a large paraesophageal hernia are also prevented.     

Disseminated herpes zoster in the immunocompromised host: a comparative trial of acyclovir and vidarabine. The NIAID Collaborative Antiviral Study Group.

Seventy-three immunocompromised patients with disseminated herpes zoster were evaluated in a double-blind controlled trial of acyclovir (n = 37) versus vidarabine (n = 36) therapy. Acyclovir was administered at 30 mg/kg/day at 8-h intervals and vidarabine was given as a continuous 12-h infusion at 10 mg/kg/day for 7 days (longer if resolution of cutaneous or visceral disease was incomplete). No demographic differences existed between treatment groups. No deaths attributable to varicella-zoster virus infection occurred within 1 month of treatment. Neither rates of cutaneous healing, resolution of acute neuritis, and frequency of postherpetic neuralgia nor adverse clinical and laboratory events differed between treatment groups. Acyclovir recipients were discharged from the hospital more promptly than vidarabine recipients (P = .04, log rank test). These data indicate that disseminated herpes zoster is amenable to therapy with either acyclovir or vidarabine; resultant mortality is low.     

Integrating the Accreditation Council for Graduate Medical Education Core competencies into the model of the clinical practice of emergency medicine

In response to public pressure for greater accountability from the medical profession, a transformation is occurring in the approach to medical education and assessment of physician competency. Over the past 5 years, the Accreditation Council for Graduate Medical Education (ACGME) has implemented the Outcomes and General Competencies projects to better ensure that physicians are appropriately trained in the knowledge and skills of their specialties. Concurrently, the American Board of Medical Specialties, including the American Board of Emergency Medicine (ABEM), has embraced the competency concept. The core competencies have been integral in ABEM's development of Emergency Medicine Continuous Certification and the development of the Model of Clinical Practice of Emergency Medicine (Model).¹ ABEM has used the Model as a significant part of its blueprint for the written and oral certification examinations in emergency medicine and is fully supportive of the effort to more fully define and integrate the ACGME core competencies into training emergency medicine specialists.To incorporate these competencies into our specialty, an Emergency Medicine Competency Taskforce (Taskforce) was formed by the Residency Review Committee–Emergency Medicine to determine how these general competencies fit in the Model.¹ This article represents a consensus of the Taskforce with the input of multiple organizations in emergency medicine. It provides a framework for organizations such as the Council of Emergency Medicine Residency Directors (CORD) and the Society for Academic Emergency Medicine to develop a curriculum in emergency medicine and program requirement revisions by the Residency Review Committee–Emergency Medicine. In this report, we describe the approach taken by the Taskforce to integrate the ACGME core competencies into the Model. Ultimately, as competency-based assessment is implemented in emergency medicine training, program directors, governing bodies such as the ACGME, and individual patients can be assured that physicians are competent in emergency medicine.