Systematic analysis and functional annotation of variations in the genome of an Indian individual

Whole genome sequencing of personal genomes has revealed a large repertoire of genomic variations and has provided a rich template for identification of common and rare variants in genomes in addition to understanding the genetic basis of diseases. The widespread application of personal genome sequencing in clinical settings for predictive and preventive medicine has been limited due to the lack of comprehensive computational analysis pipelines. We have used next-generation sequencing technology to sequence the whole genome of a self-declared healthy male of Indian origin. We have generated around 28X of the reference human genome with over 99% coverage. Analysis revealed over 3 million single nucleotide variations and about 490,000 small insertion-deletion events including several novel variants. Using this dataset as a template, we designed a comprehensive computational analysis pipeline for the systematic analysis and annotation of functionally relevant variants in the genome. This study follows a systematic and intuitive data analysis workflow to annotate genome variations and its potential functional effects. Moreover, we integrate predictive analysis of pharmacogenomic traits with emphasis on drugs for which pharmacogenomic testing has been recommended. This study thus provides the template for genome-scale analysis of personal genomes for personalized medicine.     

Low Prepregnancy Adiponectin Concentrations Are Associated With a Marked Increase in Risk for Development of Gestational Diabetes Mellitus

OBJECTIVE

To examine whether circulating total and high–molecular weight (HMW) adiponectin concentrations, measured before pregnancy, are associated with subsequent risk of gestational diabetes mellitus (GDM).

RESEARCH DESIGN AND METHODS

This was a nested case-control study among women who participated in the Kaiser Permanente Northern California Multiphasic Health Check-up exam (1984–1996) with a serum sample obtained and who had a subsequent pregnancy (1984–2009). Eligible women were free of recognized diabetes. Case subjects were the 256 women who developed GDM. Two control subjects were selected for each case and matched for year of blood draw, age at exam, age at pregnancy, and number of intervening pregnancies.

RESULTS

Compared with the highest quartile of adiponectin, the risk of GDM increased with decreasing quartile (odds ratio [OR] 1.5 [95% CI 0.7–2.9], 3.7 [1.9–7.2], and 5.2 [2.6–10.1]; P_trend <0.001) after adjustment for family history of diabetes, BMI, parity, race/ethnicity, cigarette smoking, and glucose and insulin concentrations. Similar estimates were observed for HMW (P_trend <0.001). The combined effects of having total adiponectin levels below the median (<10.29 mg/mL) and being overweight or obese (BMI ≥25.0 kg/m²) were associated with a sevenfold increased risk of GDM compared with normal-weight women with adiponectin levels above the median (OR 6.7 [95% CI 3.6–12.5]).

CONCLUSIONS

Prepregnancy low adiponectin concentrations, a marker of decreased insulin sensitivity and altered adipocyte endocrine function, is associated with reduced glucose tolerance during pregnancy and may identify women at high risk for GDM to target for early intervention.Gestational diabetes mellitus (GDM), defined as glucose intolerance with onset or first diagnosis during pregnancy, is a common complication of pregnancy. Women with a history of GDM have a sevenfold increased risk of developing type 2 diabetes after delivery (1), and the children of women with GDM are more likely to be obese and develop diabetes (2,3). The underlying etiology of GDM appears to be similar to the physiological abnormalities that characterize diabetes outside of pregnancy and is thought to be due to an inability of the pancreatic β-cells to compensate for the increased insulin resistance induced by pregnancy (4,5). The extent to which insulin resistance or reduced insulin sensitivity leading to GDM occurs even years before pregnancy has not been determined in population-based studies. There is increasing interest in identifying prepregnancy risk factors and biomarkers for GDM to inform future preconception prevention strategies, given the proven success of specific prevention strategies for type 2 diabetes in high-risk populations (6).Adiponectin is an abundant adipocyte-derived hormone demonstrated to have actions consistent with protection against insulin resistance, inflammation, and atherosclerosis (7). Total adiponectin circulates in the bloodstream as three discrete complexes: a lower–molecular weight trimer, a mid–molecular weight hexamer, and a high–molecular weight (HMW) complex (8). Some evidence suggests that HMW adiponectin is the isoform that mediates the insulin-sensitizing and antiatherogenic effects (9,10). Prospective studies examining adiponectin and incident type 2 diabetes reported that lower circulating total adiponectin concentrations were associated with a higher risk of type 2 diabetes in a dose-response relationship (11). Both total adiponectin (12) and HMW adiponectin (13) are known to decrease significantly in normal pregnancies in response to decreased insulin sensitivity; therefore, it is important to determine whether prepregnancy levels of adiponectin are related to subsequent risk of GDM in order to clarify the temporal sequence of the association. The aim of this study is to examine the association between prepregnancy total and HMW adiponectin concentrations and the risk of developing GDM and to determine whether these associations are independent of known metabolic risk factors for GDM.     

Hematopoietic stem cell and progenitor cell mechanisms in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of disorders characterized by variable cytopenias and ineffective hematopoiesis. Hematopoietic stem cells (HSCs) and myeloid progenitors in MDS have not been extensively characterized. We transplanted purified human HSCs from MDS samples into immunodeficient mice and show that HSCs are the disease-initiating cells in MDS. We identify a recurrent loss of granulocyte-macrophage progenitors (GMPs) in the bone marrow of low risk MDS patients that can distinguish low risk MDS from clinical mimics, thus providing a simple diagnostic tool. The loss of GMPs is likely due to increased apoptosis and increased phagocytosis, the latter due to the up-regulation of cell surface calreticulin, a prophagocytic marker. Blocking calreticulin on low risk MDS myeloid progenitors rescues them from phagocytosis in vitro. However, in the high-risk refractory anemia with excess blasts (RAEB) stages of MDS, the GMP population is increased in frequency compared with normal, and myeloid progenitors evade phagocytosis due to up-regulation of CD47, an antiphagocytic marker. Blocking CD47 leads to the selective phagocytosis of this population. We propose that MDS HSCs compete with normal HSCs in the patients by increasing their frequency at the expense of normal hematopoiesis, that the loss of MDS myeloid progenitors by programmed cell death and programmed cell removal are, in part, responsible for the cytopenias, and that up-regulation of the “don’t eat me” signal CD47 on MDS myeloid progenitors is an important transition step leading from low risk MDS to high risk MDS and, possibly, to acute myeloid leukemia.     

Glucosamine-Linked Near-Infrared Fluorescent Probes for Imaging of Solid Tumor Xenografts

Purpose

Near-infrared fluorescence (NIRF) imaging is an attractive technique for studying diseases at the molecular level in vivo. Glucose transporters are often used as targets for in vivo imaging of tumors. The efficiency of a tumor-seeking fluorescent probe can be enhanced by attaching one or more glucosamine (GlcN) moieties. This study was designed to evaluate the use of previously developed GlcN-linked NIRF probes for in vitro and in vivo optical imaging of cancer.

Procedures

Cellular uptake of the probes (1???M) was investigated in monolayer cultures of luciferase-expressing PC3 (PC3-luc) cells. The prostate tumors were established as subcutaneous xenografts using PC3-luc cells in nude mice. The biodistributions and tumor-targeting specificities of cypate (cyp), cypate-d-(+)-glucosamine (cyp-GlcN), and d-(+)-gluosamine-cypate-d-(+)-gluosamine (cyp-2GlcN) were studied. The tumor, muscle, and major organs were collected for ex vivo optical imaging.

Results

The tumor cell uptake of the probe containing two glucosamine residues, cyp-2GlcN, was significantly higher than the uptake of both the probe with one glucosamine residue, cyp-GlcN, and the probe without glucosamine, cyp only. Similarly, in in vivo experiments, cyp-2GlcN demonstrated higher maximum fluorescence intensity and longer residence lifetime in tumors than cyp-GlcN or cyp. The ex vivo biodistribution analysis revealed that tumor uptake of cyp-2GlcN and cyp-GlcN was four- and twofold higher than that of cyp at 24?h post-injection, respectively.

Conclusion

Both cyp-GlcN and cyp-2GlcN NIRF probes exhibited good tumor-targeting properties in prostate cancer cell cultures and live mice. The cyp-2GlcN probe showed the highest uptake with good retention characteristics in vivo. The uptake of cyp-2GlcN and cyp-GlcN is likely mediated by glucosamine-recognizing transporters. The uptake mechanism is being explored further for developing cypate-glucosamine-based probes for in vivo imaging.     

Primary cutaneous marginal zone lymphoma (immunocytoma like) with lymphoepithelioid or Lennert's lymphoma like involvement of nodes

Primary cutaneous marginal zone lymphomas (PCMZL) have a wide range of morphology from tumors with monocytoid B cells to those composed entirely of plasma cells and the T-cell rich variants. We report a 60-year-old male with a PCMZL rich in plasma cells of the foot with a lymphoepithelioid-like pattern of dissemination to the lymph nodes posing problems in the diagnosis. The patient had a lesion on the dorsum of the foot which histologically revealed dense perivascular collections of lymphoid cells and plasma cells amidst fibrous tissue. Though the plasma cells did show light chain restriction, CD20 and CD3 did not reveal an overwhelming B/T-cell population and hence a diagnosis of a reactive process was offered. Subsequently the patient developed inguinal nodes with diffuse loss of architecture and replacement by epithelioid histiocytes and reactive T cells with few large B cells (lymphoepithelioid-like pattern). On pathology review it was realized that the two lesions may be related and clonality studies were asked for. The skin lesion showed clonally rearranged IgH receptor while the T-cell receptor rearrangement was negative. The patient developed disseminated disease and received six cycles of chemotherapy with partial response and 6 years after the initial presentation was alive with nonprogressive disease. Thus, the polymorphous background in PCMZL is evolving and an immunocytoma-like tumor can show a T-cell rich or Lennert's like growth pattern of spread and early recognition these odd patterns may aid in appropriate management of patients.