Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.     

Characterization of urgent versus nonurgent early readmissions (<30 days) following primary bariatric surgery: a single-institution experience

BackgroundStudies on early postoperative readmissions after bariatric surgery (BS) have examined readmissions as a single entity, regardless of urgency. Strategies to lower nonurgent readmissions would reduce unnecessary hospital utilization.ObjectivesTo identify predictors of urgent readmissions (UR) versus nonurgent readmissions (NUR) at 30 days post-BS.SettingSingle academic institution.MethodsPatients undergoing primary BS over 2 years (n = 589) were retrospectively reviewed. Baseline demographic, medical, and hospitalization data were compared between readmitted patients, stratified by urgency, and nonreadmitted patients. Multivariate regression models of UR and NUR were created using variables with a P value ≤ .2 on univariate analyses. A P value ≤ .05 was considered statistically significant.ResultsThere were 39 documented instances of 30-day readmissions, of which 44% (n = 17) were NUR; NUR patients were more likely to be female (100% versus 78.2% male; P = .03) and trended toward being younger, experiencing ≥2 perioperative complications, and having a longer index hospital length of stay (LOS). Patients with URs had a higher baseline BMI (52.5 ± 11.4 kg/m² versus 48.7 ± 8.3 kg/m², respectively; P = .04), were more likely to have sleep apnea (77.3% versus 56.1%, respectively; P = .05), had a longer LOS (3 versus 2 d, respectively; P = .007), and were more likely to have ≥2 postoperative complications (46% versus 17.0%, respectively; P = .003) compared with those with an NUR. Independent predictors of NUR included public insurance (odds ratio [OR] = 3.7; 95% confidence interval [CI], 1.17–11.67; P = .03), younger age (OR = 1.05; 95% CI, 1–1.01; P = .04), and female sex, while URs were independently predicted by LOS (OR = 1.3; 95% CI, 1.04–1.5; P = .02).ConclusionsPublic insurance appears to be associated with NURs, while LOS predicts URs after BS. This suggests an important dichotomy within readmissions based on urgency, which has important implications for targeted quality initiatives.     

Asymptomatic Uterine Torsion in a Pregnant Woman

Some degree rotation of the gravid uterus in the third trimester of pregnancy is not an abnormal finding. However, extreme uterine torsion of 180° around its cervical junction is a relatively rare event in obstetrical practice. We report here such a case that detected at laparotomy for an emergency cesarean section due to rapture of amniotic membrane.     

Proline at the P2 position in protein C is important for calcium- mediated regulation of protein C activation and secretion

Protein C is a vitamin K-dependent plasma serine protease zymogen, which upon activation, functions as an anticoagulant. Protein C activation is catalyzed by a complex of thrombin (T) with thrombomodulin (TM). This activation is Ca(2+)-dependent, but Ca2+ inhibits protein C activation by thrombin alone. In most proteases, specificity is determined primarily by the residues that lie near the scissile bond. In protein C, the P2 position is Pro, whereas in the fibrinogen A chain, P2 is Val. We have expressed a Pro-->Val mutant of protein C (P168V) in mammalian cells. At saturating Ca2+, the P168V and wild-type proteins were activated by the T-TM complex equivalently, but half maximal rates of activation were obtained at 50 mumol/L Ca2+ for wild type and approximately 5 mmol/L Ca2+ for the P168V mutant. In the absence of TM, Ca2+ no longer inhibited the activation of the P168V mutant. These results indicate that Pro168 influences the Ca(2+)- dependent conformational changes in protein C that control activation. Recently, a patient with thrombotic complications has been identified with a Pro168-->Leu substitution. Both the P168V and the P168L mutation lead to impaired secretion caused by retention within the cell.     

Echocardiographic estimation of left ventricular filling pressures in patients with mitral valve stenosis

Mitral stenosis (MS) is prevalent in developing countries. By improving healthcare systems, it could be expected that the incidence of new cases would decrease and therefore the mean age of mitral stenosis patients would increase. This increase in age of MS patients is accompanied by the occurrence of other diseases, such as coronary artery disease, hypertension, diabetes mellitus and chronic obstructive pulmonary disease.In a number of patients with MS, the question arises of the impact of mitral valve disease (MVD) on the presenting symptom. For example, in patients presenting with dyspnea, with both significant MS and hypertension, increased left ventricular (LV) filling pressure due to hypertension could influence assessment of the severity of MS. In these patients, severity of MS could be underestimated because the increased diastolic pressure reduces the mitral valve gradient, and the increased LV stiffness shortens pressure half-time (PHT).Similarly, patients with both pulmonary disease and MS may have dyspnoea because of pulmonary rather than cardiac cause. It is therefore advantageous to assess LV filling pressure in these cases in an attempt to prove or refute a cardiac cause for dyspnoea.Using Doppler measurements to estimate LV filling pressures is desirable. However, conventional Doppler measurements have limitations in the prediction of left ventricular end-diastolic pressure (LVEDP) in this group of patients. For example, in patients with MS, the left atrium (LA) is enlarged to compensate for the increase in LA pressure. Similarly, mitral inflow peak early diastolic velocity (E) is highly dependent on LA pressure1 and also preload.2 Pulmonary venous (PV) flow also has a blunted pattern in most patients with MS.3 Therefore, in MS patients, LA size, mitral inflow pattern and pulmonary venous pattern are all altered, making these measurements unreliable for the estimation of LVEDPHowever, other Doppler and tissue Doppler echocardiographic indices and time intervals, such as peak early diastolic velocity of mitral annulus (Ea), E/Ea ratio, mitral inflow propagation velocity (VP), E/VP, pulmonary vein velocities, Tei index and the ratio of isovolumic relaxation time (IVRT) to interval between the onset of mitral E and annular Ea (TE–Ea), which have shown promising values in the prediction of LV filling pressure in a variety of diseases,4-11 have not been assessed in the setting of mitral stenosis.The aim of this study was to analyse the components of mitral and pulmonary waves in patients with mitral stenosis and to construct a Doppler-derived LVEDP prediction model based on the combined analysis of transmitral and pulmonary venous flow velocity curves.     

Ultrasonographic findings of experimental glaucoma in rabbits

Ocular ultrasonography is a valid and non-invasive diagnostic method used to evaluate ocular and retrobulbar structures, especially when opacity of the anterior segments precludes ophthalmic examination of deeper structures of the eye or when exophthalmos is present. This study describes the B-mode ultrasonographic findings of the globe in 10 rabbits with experimental glaucoma. Ultrasonography of the eyes, using Titan TM machine and 7 MHz linear array transducer, was performed transpalpebrally. Ocular ultrasonographic findings revealed two cases of increased corneal thickness, seven cases of change in the anterior chamber depth, one case of increased echogenicity in the anterior chamber, three cases of retinal detachment, one case of increased iris and ciliary body thickness and six cases of change in axial globe length. Increased echogenicity of the capsule, cortex and nucleus was found in two cases. Six cases showed change in lens diameter. Increased echogenicity and altered lens diameter are typical of cataract. Two cases of point-like lesions, mass and/or linear echodensities in mild extent were observed within the vitreous representing haemorrhage, vitreous degeneration or detachment. The B-mode ultrasonographic imaging technique has become an essential diagnostic tool in most ocular disease. This method provides additional information allowing the clinician to offer more accurate diagnosis, treatment and prognosis in glaucoma.     

Metabolite Profiles During Oral Glucose Challenge

To identify distinct biological pathways of glucose metabolism, we conducted a systematic evaluation of biochemical changes after an oral glucose tolerance test (OGTT) in a community-based population. Metabolic profiling was performed on 377 nondiabetic Framingham Offspring cohort participants (mean age 57 years, 42% women, BMI 30 kg/m²) before and after OGTT. Changes in metabolite levels were evaluated with paired Student t tests, cluster-based analyses, and multivariable linear regression to examine differences associated with insulin resistance. Of 110 metabolites tested, 91 significantly changed with OGTT (P ≤ 0.0005 for all). Amino acids, β-hydroxybutyrate, and tricarboxylic acid cycle intermediates decreased after OGTT, and glycolysis products increased, consistent with physiological insulin actions. Other pathways affected by OGTT included decreases in serotonin derivatives, urea cycle metabolites, and B vitamins. We also observed an increase in conjugated, and a decrease in unconjugated, bile acids. Changes in β-hydroxybutyrate, isoleucine, lactate, and pyridoxate were blunted in those with insulin resistance. Our findings demonstrate changes in 91 metabolites representing distinct biological pathways that are perturbed in response to an OGTT. We also identify metabolite responses that distinguish individuals with and without insulin resistance. These findings suggest that unique metabolic phenotypes can be unmasked by OGTT in the prediabetic state.Diabetes affects >1 in 10 adults 20 years of age or older in the U.S., and more than one-third of all adults have prediabetes (1). Changes in traditional measures of glucose and insulin metabolism are known to occur years before the diagnosis of diabetes is made (2). Using high-throughput profiling of metabolic status, we have shown that elevations in plasma branched-chain and aromatic amino acids are also able to predict future diabetes in otherwise normoglycemic, healthy adults (3). Similarly, lipid profiling has demonstrated novel perturbations in triacylglycerol distribution that signal future diabetes risk (4). These findings highlight how emerging technologies are able to broaden our perspective on early disease states, potentially lending insights into biological mechanisms that underlie diabetes and metabolic disease. Characterizing early metabolic changes may also lead to the early identification of at-risk individuals and may prompt the initiation of proven preventive strategies (5).The oral glucose tolerance test (OGTT) provides a dynamic view of glucose and insulin physiology and has been widely used for decades to diagnose diabetes (6,7). Therefore, we conducted a systematic evaluation of biochemical changes after OGTT in a community-based population, with the goal of providing a broad view of the metabolic response to a glucose challenge. An important advantage of profiling plasma samples before and after glucose ingestion is that each individual is able to serve as their own biological control. In addition to attenuating noise attributable to interindividual variation, this approach limits confounding effects of diet, medications, and other inputs that impact the human metabolome. We used a liquid chromatography/mass spectrometry (LC/MS)–based platform that allowed highly specific identification of small molecules in a targeted manner. In prior pilot studies, our group has shown that metabolite excursions with OGTT revealed a switch from catabolism to anabolism, largely attributable to insulin actions (8). In the current study, we sought to evaluate perturbations with OGTT in an expanded panel of metabolites and in a more comprehensive population-based sample with a high propensity for the development of diabetes, and to investigate these changes in individuals with and without insulin resistance.