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.     

Menetrier's disease associated with ulcerative colitis

In this paper a 21 year old patient was presented with Ménétrier's disease, associated with ulcerative colitis. The first symptoms of ulcerative colitis occurred at the age of eleven, since when the patient has been conservatively treated several times because of the exacerbations of the disease. During control examinations presence of polyploid changes in stomach was discovered by upper endoscopy. Gastrectomy was suggested because the patient had excessive anemic syndrome which required weekly substitutional therapy with deplasmatic eritrocytes, as well as hypoproteinemia, while multiple polyploid changes suspect for malignancy were gastroscopically identified. Patient accepted surgical treatment, and was transferred to the Clinic of Surgery. Total gastrectomy was performed, and patohistological finding confirmed Ménétrier's disease. After two weeks, the patient was released from the hospital in good general condition, with regular clinical and laboratory findings.     

Capecitabine cardiotoxicity--case reports and literature review

This study presents 3 case reports of patients who experienced anginous pain during treatment with capecitabine. The interruption of capecitabine and sublingual or intravenous nitroglycerine treatment lead to recovery. Rechallenge of capecitabine with dose reduction of 30% lead to repeated anginous pain in 2 patients. Treatment with capecitabine had been replaced with weekly bolus 5FU-LV, without further cardiotoxicity. The literature contains data from about 50 patients who experienced cardiotoxicity during capecitabine treatment. The most frequent manifestations of capecitabine cardiotoxicity included: anginous pain in 38/53 (71.7%), arrhythmia in 6/53 (11.3%), myocardial infarction in 6/53 (11.3%). Cardiotoxicity of capecitabine lead to death in 6/53 (11.3%) patients. Risk factors for cardiotoxicity are associated with the grade 4 and the fatal outcome of cardiotoxicity (p = 0.035, p = 0.015), but not with the symptom recurrence upon capecitabine rechallenge (p = 0.18). The combination chemotherapy regimens are associated with the grade 4 of cardiotoxicity (p = 0.048), but not with the fatal outcome (p = 0.3). Rechallenge of capecitabine lead to symptoms recurrence in 10/16 patients. Neither the dose reduction of capecitabine (p = 0.18) nor the additional medical prophylaxis (p = 0.37) were important for the outcome of capecitabine rechallenge.     

Abdominal neurenteric cyst

Neurenteric cysts are extremely rare congenital anomalies, often presenting in the first 5 years of life, and are caused by an incomplete separation of the notochord from the foregut during the third week of embryogenesis. They are frequently accompanied with spinal or gastrointestinal abnormalities, but the latter may be absent in adults. Although usually located in the thorax, neurenteric cysts may be found along the entire spine. We present a 24-year-old woman admitted for epigastric pain, nausea, vomiting, low grade fever and leucocytosis. She underwent cystgastrostomy for a Ioculated cyst of the distal pancreas at the age of 4 years, which recurred when she was at the age of 11 years. Ultrasound and computer tomograghy （CT） scan revealed a 16 cmx 15 cm cystic mass in the body and tail of pancreas, with a 6-7 mm thickened wall. Laboratory data and chest X-ray were normal and spinal radiographs did not show any structural abnormalities. The patient underwent a complete cyst excision, and after an uneventful recovery, remained symptom-free without recurrence during the 5-year follow-up. The cyst was found to contain 1200 mL of pale viscous fluid. It was covered by a primitive singlelayered cuboidal epithelium, along with specialized antral glandular parenchyma and hypoplastic primitive gastric mucosa. Focal glandular groups resembling those of the body of the stomach were also seen. In addition, ciliary respiratory epithelium, foci of squamous metaplasia and mucinous glands were present. The wall of the cyst contained a muscular layer, neuroglial tissue with plexogenic nerve fascicles, Paccini corpuscle-like structures, hyperplastic neuroganglionar elements and occasional psammomatous bodies, as well as fibroblast-like areas of surrounding stroma. Cartilagenous tissue was not found in any part of the cyst. Immunohistochemistry confirmed the presence of neurogenic elements marked by S-100, GFAP, NF and NSE. The gastric epithelium showed mostly CK7 and EMA immunoexpression, and the respiratory epithelium reve     

Mining injuries in Serbian underground coal mines - A 10-year study

Mining, especially underground coal mining, has always been a dangerous occupation. Injuries, unfortunately, even those resulting in death, are one of the major occupational risks that all miners live with. Despite the fact that all workers are aware of the risk, efforts must be and are being made to increase the safety of mines. Injury monitoring and data analysis can provide us with valuable data on the causes of accidents and enable us to establish a correlation between the conditions in the work environment and the number of injuries, which can further lead to proper preventive measures. This article presents the data on the injuries in Serbian coal mines during a 10-year period (2000-2009). The presented results are only part of an ongoing study whose aim is to assess the safety conditions in Serbian coal mines and classify them according to that assessment.     

Haematological and iron-related parameters in male and female athletes according to different metabolic energy demands

We investigated the iron-related haematological parameters in both male and female athletes participating in different sporting disciplines necessitating different metabolic energy demands. A total of 873 athletes (514 males, mean age: 22.08 ± 4.95 years and 359 females, mean age: 21.38 ± 3.88 years) were divided according to gender and to the predominant energy system required for participation in sport (aerobic, anaerobic or mixed) and haematological and iron-related parameters were measured. For both male and female athletes, significant differences related to the predominant energy system were found at a general level: male (Wilks’ λ = 0.798, F = 3.047, p < 0.001) and female (Wilks’ λ = 0.762, F = 2.591, p < 0.001). According to the ferritin cutoff value of 35 μg/L, whole body iron and sTfR significantly differed in all three groups of male and female athletes (p < 0.001). The percentage of hypochromic erythrocytes in male athletes was significantly higher only in those who required an anaerobic energy source (p < 0.001), whilst in the females hypochromic erythrocytes (p < 0.001) and haemoglobin (anaerobic, p = 0.042; mixed, p = 0.006) were significantly different only in anaerobic and mixed energy source athletes. According to the ferritin cutoff value of 22 μg/L, in females, whole body iron, sTfR and hypochromic erythrocytes were significantly higher in all three groups of athletes than those below the aforementioned cutoff value (p < 0.001). We conclude that the predominant energy system required for participation in sport affects haematological parameters. sTfR and body iron proved to be reliable parameters for monitoring the dynamics of iron metabolism and could contribute to successful iron-deficiency prevention.     

Use of free microvascular flaps in the management of the head and neck defects

BACKGROUND/AIM: In the field of contemporary head and neck reconstructive surgery, free vascularized tissue transfer is becoming a gold standard. The aim of this study was to review our clinical results and experience, with use of free microvascular flaps and compare them with the recently published patient series. METHODS: During the period from 2001 to 2005, 37 patients underwent microsurgical reconstruction after the tumor ablation in the region of head and neck. Flap viability was monitored intraoperatively with the Ackland test and postoperatively by the clinical observation and mini-Doppler test. RESULTS: The overall success rate was 83.8%. The complications that appeared were: one complete flap necrosis due to venous thrombosis, and five late flap ischemic necroses, in the period from the 10th to 14th postoperative day. CONCLUSION: Free flap reconstruction of the head and neck is a surgical technique that provides the reconstruction of complex and extensive defects, that could not be performed by using local or regional flaps.