Lessons from BWS twins: complex maternal and paternal hypomethylation and a common source of haematopoietic stem cells

The Beckwith–Wiedemann syndrome (BWS) is a growth disorder for which an increased frequency of monozygotic (MZ) twinning has been reported. With few exceptions, these twins are discordant for BWS and for females. Here, we describe the molecular and phenotypic analysis of 12 BWS twins and a triplet; seven twins are MZ, monochorionic and diamniotic, three twins are MZ, dichorionic and diamniotic and three twins are dizygotic. Twelve twins are female. In the majority of the twin pairs (11 of 13), the defect on chromosome 11p15 was hypomethylation of the paternal allele of DMR2. In 5 of 10 twins, there was additional hypomethylation of imprinted loci; in most cases, the loci affected were maternally methylated, but in two cases, hypomethylation of the paternally methylated DLK1 and H19 DMRs was detected, a novel finding in BWS. In buccal swabs of the MZ twins who share a placenta, the defect was present only in the affected twin; comparable hypomethylation in lymphocytes was detected in both the twins. The level of hypomethylation reached levels below 25%. The exchange of blood cells through vascular connections cannot fully explain the degree of hypomethylation found in the blood cell of the non-affected twin. We propose an additional mechanism through which sharing of aberrant methylation patterns in discordant twins, limited to blood cells, might occur. In a BWS-discordant MZ triplet, an intermediate level of demethylation was found in one of the non-affected sibs; this child showed mild signs of BWS. This finding supports the theory that a methylation error proceeds and possibly triggers the twinning process.     

The ClinSeq Project: Piloting large-scale genome sequencing for research in genomic medicine

ClinSeq is a pilot project to investigate the use of whole-genome sequencing as a tool for clinical research. By piloting the acquisition of large amounts of DNA sequence data from individual human subjects, we are fostering the development of hypothesis-generating approaches for performing research in genomic medicine, including the exploration of issues related to the genetic architecture of disease, implementation of genomic technology, informed consent, disclosure of genetic information, and archiving, analyzing, and displaying sequence data. In the initial phase of ClinSeq, we are enrolling roughly 1000 participants; the evaluation of each includes obtaining a detailed family and medical history, as well as a clinical evaluation. The participants are being consented broadly for research on many traits and for whole-genome sequencing. Initially, Sanger-based sequencing of 300–400 genes thought to be relevant to atherosclerosis is being performed, with the resulting data analyzed for rare, high-penetrance variants associated with specific clinical traits. The participants are also being consented to allow the contact of family members for additional studies of sequence variants to explore their potential association with specific phenotypes. Here, we present the general considerations in designing ClinSeq, preliminary results based on the generation of an initial 826 Mb of sequence data, the findings for several genes that serve as positive controls for the project, and our views about the potential implications of ClinSeq. The early experiences with ClinSeq illustrate how large-scale medical sequencing can be a practical, productive, and critical component of research in genomic medicine.Elucidating the sequence of the human genome (International Human Genome Sequencing Consortium 2001, 2004) and subsequent advances in DNA sequencing technologies (Mardis 2008) have the potential to dramatically improve the delivery of health care through the acquisition of genomic information about individual patients. However, much research will be needed to develop medical applications of genomics; for example, little is known about how to organize and implement large-scale medical sequencing (LSMS; i.e., systematic resequencing of human DNA) in a clinical context. Other approaches for applying high-throughput genomics to health care (e.g., assaying single-nucleotide polymorphisms and establishing gene-expression profiles) offer diagnostic promise; these are not further considered here, as our focus is on LSMS for studying the relationship of germline genomic variation to health and disease.We recently launched ClinSeq (http://genome.gov/20519355), a project that aims to apply LSMS within a clinical research environment to answer questions about the genetic basis of health, disease, and drug response. The application of genomic approaches (in particular LSMS) in a clinical research context is associated with a number of considerations that define key “dimensions” of any study: the number of subjects, the associated clinical data, and the breadth of genome covered (Fig. 1). Numerous detailed studies of single genes have been carried out; while often performed on many participants with significant amounts of phenotypic information, they are focused on a very small portion of the genome. The flurry of papers that describe recently generated whole-genome sequences (Levy et al. 2007; Bentley et al. 2008; Wang et al. 2008; Wheeler et al. 2008) has provided the first true individual genome sequences, including a modest amount of associated clinical data; however, the number of examples is small to date. Greater numbers are promised by the 1000 Genomes Project (http://www.1000genomes.org/), although no phenotypic information will be available for the individuals being studied. ClinSeq aims to model a more ideal study with respect to these three dimensions (Fig. 1), with the potential to further move toward the ultimate ideal as technology advances.Open in a separate windowFigure 1.A spatial conceptualization of research studies in genomic medicine. There are three key “dimensions” to consider when applying genomics to clinical research: genome breadth (the fraction of the genome that is interrogated), number of subjects or participants, and the associated clinical data about those individuals (including its depth, breadth, and rigor). While the ideal study would acquire whole-genome sequences from large numbers of extensively phenotyped subjects, this is currently impractical. Single-gene studies can involve a few or numerous subjects and extensive clinical data, but by definition involve the examination of only a single gene and thus occupy one wall of this space. The individual genomes that have recently been sequenced (Levy et al. 2007; Bentley et al. 2008; Wang et al. 2008; Wheeler et al. 2008) provide nearly complete genome breadth, but with limited clinical data; further, their limited subject numbers place them on another wall of this space. The 1000 Genomes Project (http://www.1000genomes.org/) is providing large subject numbers and extensive genome breadth, but no clinical data—positioning it on the floor of this space. ClinSeq aims to reside in the center of this space, having attributes of substantial subject size (n = 1000 initially), moderate genome breadth (∼400 genes initially, with plans for expanding this breadth), and substantial clinical data.The general aims of ClinSeq are to: (1) develop the infrastructure and approaches to acquire and analyze genome sequence from individual research participants; (2) pilot the use of LSMS to elucidate the genetic architecture underlying human traits; (3) provide an open, shared resource and environment for basic and clinical researchers to work collaboratively to perform research in genomic medicine; and (4) establish approaches for informed consent and the return of genetic information to subjects participating in LSMS studies. In pursuing these aims, our overriding goals include modeling whole-genome sequence acquisition in a manner that is practical for a clinical research setting, advancing our understanding of the genetic basis of important human diseases and traits, and establishing how to scale LSMS prior to the day when whole-genome sequencing becomes part of routine clinical practice. In this paper, we describe the ClinSeq study design, provide a snapshot of our very early data generation, and discuss the implications of this study for the nascent field of genomic medicine.     

GS2 as a retinol transacylase and as a catalytic dyad independent regulator of retinylester accretion

GS2 (PNPLA4; iPLAη) is the smallest member of the patatin-like family of phospholipases (PNPLA). It was initially identified by its ability to hydrolyze retinylesters (RE) in cell homogenates, and was later found to esterify retinol using a variety of acyl donors. In the present study we set out to determine its cellular function and examined its impact on RE status in 293T cells transfected with GS2, GS2-M1 (a non-translatable mutant of GS2) and empty vector, in fibroblasts isolated from normal and GS2-null donors and in SCC12b and in a somatic cell knock-out of GS2 (SCC12b-GS2^neo/−), that we generated by homologous recombination. At 50 nM medium retinol, GS2 had no significant impact on RE accumulation. However, at 2 μM retinol, GS2 promoted a 1.6- to 5-fold increase in RE accumulation. To verify role of GS2 as a catalyst, RE levels were measured in 293T transfected wild type GS2, catalytic dyad mutants devoid of enzymatic activity, or alanine substitution mutants spanning the entire GS2 sequence. Surprisingly, every GS2 mutant promoted RE accumulation. This activity was also observed in the GS2 paralogues and rat orthologue. The data demonstrate that within the context of the cell GS2 promotes RE accumulation and may do so either as a catalyst or as a regulatory protein that enhances RE formation catalyzed by other acyl transferases.     

Predictors of Completion Axillary Lymph Node Dissection in Patients with Positive Sentinel Lymph Nodes

Background  Completion axillary lymph node dissection (CALND) is routinely performed in breast cancer patients with positive sentinel lymph nodes (SLN). We sought to determine the sociodemographic, pathologic, and therapeutic variables that were associated with CALND. Methods  From 7/1997 to 7/2003, 1,470 patients with invasive breast cancer were SLN positive by intraoperative frozen section or final pathologic exam by hematoxylin–eosin and/or immunohistochemistry (IHC). A comorbidity score was assigned using Adult Comorbidity Evaluation-27 system. Fisher’s exact, Wilcoxon tests, and multivariate logistic regression analysis were used. Results  CALND was performed less often in patients with age ≥ 70 years compared with age < 70 years, moderate or severe comorbidities compared with no or mild, IHC-only positive SLN and breast conservation therapy (BCT compared with mastectomy. Patients who did not undergo CALND were less likely than CALND patients to have grade III disease, lymphovascular invasion multifocal disease, tumor size > 2 cm or to receive adjuvant chemotherapy. However, they were more likely to undergo axillary radiotherapy (RT). On multivariate analysis, age ≥ 70 years [odds ratio (OR) 0.4, 95% confidence interval (CI) 0.26–0.63], IHC-only positive SLN (OR 0.13, 95%CI 0.09–0.19), presence of moderate to severe comorbidities (OR 0.64, 95%CI 0.41–0.99), tumor size ≤ 2 cm (OR 0.44, 95%CI 0.29–0.66), axillary RT (OR 0.39, 95%CI 0.20–0.78), and BCT (OR 0.54, 95%CI 0.37–0.79) were all independently associated with lower odds of CALND. Conclusions  The decision to perform CALND following positive SLN biopsy was multifactorial. Patient factors were a primary determinant for the use of CALND in our study. The decreased use of CALND in the BCT patients probably reflects reliance on the radiotherapy tangents to maintain local control in the axilla.     

Neuroanatomical markers of speaking Chinese

The aim of this study was to identify regional structural differences in the brains of native speakers of a tonal language (Chinese) compared to nontonal (European) language speakers. Our expectation was that there would be differences in regions implicated in pitch perception and production. We therefore compared structural brain images in three groups of participants: 31 who were native Chinese speakers; 7 who were native English speakers who had learnt Chinese in adulthood; and 21 European multilinguals who did not speak Chinese. The results identified two brain regions in the vicinity of the right anterior temporal lobe and the left insula where speakers of Chinese had significantly greater gray and white matter density compared with those who did not speak Chinese. Importantly, the effects were found in both native Chinese speakers and European subjects who learnt Chinese as a non‐native language, illustrating that they were language related and not ethnicity effects. On the basis of prior studies, we suggest that the locations of these gray and white matter changes in speakers of a tonal language are consistent with a role in linking the pitch of words to their meaning. Hum Brain Mapp, 2009. © 2009 Wiley‐Liss, Inc.     

Tests of platelet function

PURPOSE OF REVIEW: Platelets play a vital role in the normal hemostasis, and derangements of their function can lead to hemorrhage or thrombosis. While we have made progress in elucidating the molecular mechanisms leading to platelet adhesion, aggregation, shape change, and secretion, clinically useful tests of platelet function have lagged behind. The following is a review of some of the currently available tests of platelet function, their advantages and drawbacks, as well as the clinical scenarios in which they are likely to be useful. RECENT FINDINGS: Attention is now being paid to standardization and optimization of older tests such as platelet aggregometry, in addition to better defining the role of newer tests such as the platelet function analyzer and thromboelastography in diagnosing and managing disorders of primary hemostasis and platelet function. SUMMARY: Platelet function is complex and may be disrupted at any of a number of steps, including adhesion, aggregation, shape change and secretion. We are better defining the role of the currently available tests, while identifying gaps in our ability to diagnose disorders of platelet function.     

Representation of capsaicin-evoked urge-to-cough in the human brain using functional magnetic resonance imaging

RATIONALE: Coughing in humans is typically preceded by a desire (or urge) to cough. The neural circuitry involved in sensing airway irritation and generating the urge-to-cough in humans is essentially unknown. OBJECTIVES: The aim of the present study was to use functional brain imaging to describe the supramedullary regions that are activated in humans during capsaicin inhalation. METHODS: Experiments were performed on 10 healthy subjects (5 males, 5 females). Capsaicin doses were individually tailored to evoke a transient and reversible urge-to-cough. Blood oxygen level-dependent (BOLD) functional magnetic resonance measures were collected during repeated 24-second challenges with capsaicin or saline inhalation and subjects were asked to rate the urge-to-cough intensity of each challenge. MEASUREMENTS AND MAIN RESULTS: Capsaicin inhalation reliably evoked an urge-to-cough, which was associated with activations in a variety of brain regions, including the insula cortex, anterior midcingulate cortex, primary sensory cortex, orbitofrontal cortex, supplementary motor area, and cerebellum. CONCLUSIONS: These data provide the first insights into the cortical neuronal network involved in sensing airway irritation and modulating coughing in humans.     

Effect of lung volume reduction surgery on resting pulmonary hemodynamics in severe emphysema

RATIONALE: To determine the effect of medical treatment versus lung volume reduction surgery (LVRS) on pulmonary hemodynamics. METHODS: Three clinical centers of the National Emphysema Treatment Trial (NETT) screened patients for additional inclusion into a cardiovascular (CV) substudy. Demographics were determined, and lung function testing, six-minute-walk distance, and maximum cardiopulmonary exercise testing were done at baseline and 6 months after medical therapy or LVRS. CV substudy patients underwent right heart catheterization at rest prerandomization (baseline) and 6 months after treatment. MEASUREMENTS AND MAIN RESULTS: A total of 110 of the 163 patients evaluated for the CV substudy were randomized in NETT (53 were ineligible), 54 to medical treatment and 56 to LVRS. Fifty-five of these patients had both baseline and repeat right heart catheterization 6 months postrandomization. Baseline demographics and lung function data revealed CV substudy patients to be similar to the remaining 1,163 randomized NETT patients in terms of age, sex, FEV(1), residual volume, diffusion capacity of carbon monoxide, Pa(O(2)), Pa(CO(2)), and six-minute-walk distance. CV substudy patients had moderate pulmonary hypertension at rest (Ppa, 24.8 +/- 4.9 mm Hg); baseline hemodynamic measurements were similar across groups. Changes from baseline pressures to 6 months post-treatment were similar across treatment groups, except for a smaller change in pulmonary capillary wedge pressure at end-expiration post-LVRS compared with medical treatment (-1.8 vs. 3.5 mm Hg, p = 0.04). CONCLUSIONS: In comparison to medical therapy, LVRS was not associated with an increase in pulmonary artery pressures.