The Role of Noncoding Variants in Heritable Disease.

The genetic basis of disease has largely focused on coding regions. However, it has become clear that a large proportion of the noncoding genome is functional and harbors genetic variants that contribute to disease etiology. Here, we review recent examples of inherited noncoding alterations that are responsible for Mendelian disorders or act to influence complex traits. We explore both rare and common genetic variants and discuss the wide range of mechanisms by which they affect gene regulation to promote disease. We also debate the challenges and progress associated with identifying and interpreting the functional and clinical significance of genetic variation in the context of the noncoding regulatory landscape.

A bruise-like patch in a 4-year-old girl.

Dermatofibrosarcoma protuberans (DFSP), a rare medium grade sarcoma, occasionally occurs in childhood and is even more rarely present at birth. In children, the clinical appearance may be mistaken for a vascular malformation and so delayed diagnosis is not uncommon. Dermatofibrosarcoma protuberans is locally invasive and notorious for its high recurrence rate even after attempted wide local excision owing to extensive subclinical and asymmetrical extensions. In adult DFSP, Mohs Micrographic Surgery (MMS) is the treatment of choice because it offers a higher clearance rate compared to wide local excision. However, MMS may result in extended operating times owing to tissue processing and multiple stages. In children, this means a prolonged period under general anesthetic, which may be undesirable. We describe an interesting case of a 4- year-old girl diagnosed with DFSP. She underwent a modified MMS procedure in which she had two short general anesthetics. The advantage of MMS technique in which the full peripheral and deep margin of the specimen was examined.

Molecular identification of Na(+)-H(+) exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus.

In the current study, reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR were used to clone full-length putative Na(+)-H(+) exchanger isoforms (NHE2a) cDNA from the gills of Fundulus heteroclitus. The 2480 bp cDNA includes a coding region for a protein that shows a 57% amino acid homology to rabbit NHE2. These sequences allowed data mining of available fish genome data, which revealed at least three NHE2 subtypes in some teleost species.

Composite mesh design for delivery of autologous mesenchymal stem cells influences mesh integration, exposure and biocompatibility in an ovine model of pelvic organ prolapse.

The widespread use of synthetic transvaginal polypropylene mesh for treating Pelvic Organ Prolapse (POP) has been curtailed due to serious adverse effects highlighted in 2008 and 2011 FDA warnings and subsequent legal action. We are developing new synthetic mesh to deliver endometrial mesenchymal stem cells (eMSC) to improve mesh biocompatibility and restore strength to prolapsed vaginal tissue. Here we evaluated knitted polyamide (PA) mesh in an ovine multiparous model using transvaginal implantation and matched for the degree of POP. Polyamide mesh dip-coated in gelatin and stabilised with 0.5% glutaraldehyde (PA/G) were used either alone or seeded with autologous ovine eMSC (eMSC/PA/G), which resulted in substantial mesh folding, poor tissue integration and 42% mesh exposure in the ovine model. In contrast, a two-step insertion protocol, whereby the uncoated PA mesh was inserted transvaginally followed by application of autologous eMSC in a gelatin hydrogel onto the mesh and crosslinked with blue light (PA + eMSC/G), integrated well with little folding and no mesh exposure. The autologous ovine eMSC survived 30 days in vivo but had no effect on mesh integration. The stiff PA/G constructs provoked greater myofibroblast and inflammatory responses in the vaginal wall, disrupted the muscularis layer and reduced elastin fibres compared to PA + eMSC/G constructs. This study identified the superiority of a two-step protocol for implanting synthetic mesh in cellular compatible composite constructs and simpler surgical application, providing additional translational value.

Laue diffraction study on the structure of cytochrome c peroxidase compound I.

BACKGROUND: Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group. RESULTS: Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures. CONCLUSIONS: The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

MicroRNA-206 is differentially expressed in Brca1-deficient mice and regulates epithelial and stromal cell compartments of the mouse mammary gland.

Depletion of Brca1 leads to defects in mouse mammary gland development and mammary tumors in humans and mice. To explore the role of microRNAs (miRNAs) in this process, we examined the mammary glands of MMTV-Cre Brca1(Co/Co) mice for differential miRNA expression using a candidate approach. Several miRNAs were differentially expressed in mammary tissue at day 1 of lactation and in mammary epithelial cell lines in which Brca1 messenger RNA (mRNA) levels have been reduced. Functional studies revealed that several of these miRNAs regulate mammary epithelial cell function in vitro, including miR-206. Creation and analysis of MMTV-miR-206 transgenic mice showed no effect on lactational mammary development and no tumors, but indicates a role in mammary tissue remodeling in mature mice, potentially involving Igf-1 and Sfrp1. These results indicate the potential of miRNAs to mediate the consequences of Brca1 loss and suggest a novel function for miR-206.

Crystallization and purification of the enzyme anthranilate phosphoribosyl transferase.

Anthranilate phosphoribosyl transferase from the bacterium Hafnia alvei has been crystallized. This enzyme is one of a small number that constitute the biosynthetic pathway for tryptophan. Large cubic crystals were grown at 4 degrees C by dialyzing away the glycerol from a protein solution that included ammonium sulfate, polyethylene glycol and glycerol. The crystals were much more temperature stable and resistant to X-ray deterioration than a previous, similar crystal form that had included glycerol. The crystals belong to the space group I432, a = b = c = 189 A (1 A = 0.1 nm). The ratio of the monomer molecular weight, 37,000, to the volume of the unit cell suggests that there is one homodimer per asymmetric unit. The crystals diffracted to a resolution of 3.0 A at the Stanford Synchotron Radiation Laboratory X-ray source.

Temporal changes in the biomechanical properties of endometrial mesenchymal stem cell seeded scaffolds in a rat model.

Use of synthetic clinical meshes in pelvic organ prolapse (POP) repair can lead to poor mechanical compliance in vivo, as a result of a foreign body reaction leading to excessive scar tissue formation. Seeding mesh with mesenchymal stem cells (MSCs) prior to implantation may reduce the foreign body reaction and lead to improved biomechanical properties of the mesh-tissue complex. This study investigates the influence of seeding human endometrial mesenchymal stem cells (eMSCs) on novel gelatin-coated polyamide scaffolds, to identify differences in scaffold/tissue biomechanical properties and new tissue growth following up to 90 days' implantation, in a subcutaneous rat model of wound repair. Scaffolds were subcutaneously implanted, either with or without eMSCs, in immunocompromised rats and following 7, 30, 60 and 90 days were removed and assessed for their biomechanical properties using uniaxial tensile testing. Following 7, 30 and 90 days' implantation scaffolds were assessed for tissue ingrowth and organization using histological staining and scanning electron microscopy. The eMSCs were associated with altered collagen growth and organization around the mesh filaments of the scaffold, affecting the physiologically relevant tensile properties of the scaffold-tissue complex, in the toe region of the load-elongation curve. Scaffolds seeded with eMSCs were significantly less stiff on initial stretching than scaffolds implanted without eMSCs. Collagen growth and organization were enhanced in the long-term in eMSC-seeded scaffolds, with improved fascicle formation and crimp configuration. Results suggest that neo-tissue formation and remodelling may be enhanced through seeding scaffolds with eMSCs.

Chemically distinct preganglionic inputs to iris-projecting postganglionic neurons in the rat: A light and electron microscopic study.

Individual autonomic postganglionic neurons are surrounded by pericellular baskets of preganglionic terminals that are easily identifiable with the light microscope. It has been assumed that the target cell of a pericellular basket of preganglionic terminals is the neuron at the centre of the basket. This assumption has enabled the connectivity of preganglionic neurons to be determined at the light microscopic level. However, if the preganglionic terminals in a pericellular basket make synapses with the dendrites of nearby, but functionally different, postganglionic neurons, then the conclusions of light microscopic studies are far less certain. We have used a serial section ultrastructural study to determine the target of the preganglionic pericellular basket in a situation where the apparent target cell is surrounded by neurons of dissimilar function. In the rat superior cervical ganglion, postganglionic neurons projecting to the iris were identified, using retrograde tracers, as single neurons (i.e., not in clusters). We have used immunohistochemistry to show that iris-projecting neurons are surrounded by preganglionic nerve terminals containing calcitonin gene-related peptide (CGRP). We have demonstrated that the pericellular basket of CGRP-immunoreactive preganglionic terminals provides inputs only to the soma at the centre of the basket and not to the dendrites of surrounding neurons. This suggests that, in autonomic ganglia, light microscopic identification of the preganglionic terminal baskets is likely to be a reliable method for identifying the targets of subclasses of preganglionic neurons.

Dietary sugar and colon cancer.

It has been hypothesized that levels of triglycerides, glucose, and insulin are associated with risk of colon cancer and that diets high in simple sugars increase risk of colon cancer because of their impact on these factors. Limited epidemiological evidence supports the association between simple carbohydrates and risk of colon cancer. Using data from a population-based case-control study (n = 1993 cases and 2410 controls), we examined the associations between dietary sugars, foods containing high level of sugars, and dietary glycemic index (GI) and colon cancer. A dietary GI was developed to estimate metabolic response to a diet that may increase plasma glucose levels. Dietary data were obtained using a validated diet history questionnaire. High levels of sucrose intake were associated with increased risk of colon cancer among younger men [odds ratio (OR) for highest quintile relative to lowest, 1.59; 95% confidence interval (CI), 1.07-2.37]. There was also a trend of increasing colon cancer risk associated with a higher sucrose:dietary ratio for proximal tumors in both men and women. Individuals with proximal tumors who consumed a diet ranked as having a high GI were at increased risk (for men, comparing highest quintile to lowest quintile: OR, 1.58; 95% CI, 1.06-2.36; P trend 0.04; for women: OR, 1.72; 95% CI, 1.11-2.67; P trend 0.04). Those at greatest risk from a high dietary GI were those who were sedentary (for men, relative to those who were most active and had a low-GI diet: OR, 3.46; 95% CI, 1.78-6.70; for women: OR, 2.00; 95% CI, 0.98-4.07). We also observed that people who had a high sucrose: dietary fiber ration and who also were sedentary and had a large body mass index were at increased risk (OR, 4.58; 95% CI, 2.33-8.98) relative to those who had a low sucrose:dietary fiber ratio, were active, and had low body mass indices. These findings support previous reports that dietary sugars, especially diet high in simple carbohydrates relative to complex carbohydrates, increase risk of colon cancer, possibly through their impact on plasma glucose levels.

Nitric oxide synthase and chemical coding in cat sympathetic postganglionic neurons.

Nitric oxide synthase-like immunoreactivity was found in a subpopulation of sympathetic postganglionic neurons in the cat stellate and lower lumbar ganglia. In the ganglia of other segments such cells were rare. Double staining for tyrosine hydroxylase-like immunoreactivity and nitric oxide synthase-like immunoreactivity or the reduced nicotinamide adenine dinucleotide phosphate diaphorase reaction indicated that nitric oxide synthase-like immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase reactivity was always co-localized and was confined to tyrosine hydroxylase-negative (presumably cholinergic) ganglion cells, and was present in most of them. The occurrence of nitric oxide synthase in two subpopulations of cholinergic postganglionic neurons was investigated in triple staining experiments. Presumptive sudomotor neurons have been previously defined as scattered cells containing calcitonin gene-related peptide-like immunoreactivity, usually accompanied by vasoactive intestinal peptide-like immunoreactivity: 99% of these contained nitric oxide synthase. Presumptive muscle vasodilator neurons have been previously identified as clumped cells with strong vasoactive intestinal peptide-like immunoreactivity but no calcitonin gene-related peptide-like immunoreactivity: 70% of these contained nitric oxide synthase. Sweat glands were found in the paw pad skin surrounded by varicose fibres showing calcitonin gene-related peptide-like immunoreactivity and vasoactive intestinal peptide-like immunoreactivity, confirming previous work. Such fibres also stained for nitric oxide synthase-like immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase reactivity, although their staining was relatively weaker than in the corresponding cell bodies. Varicose fibres with the same chemical coding were also found around all large and most medium and small arteries in the paw skin as well as around arteriovenous anastomoses. Fibres with the muscle vasodilator coding (vasoactive intestinal peptide-like immunoreactivity without calcitonin gene-related peptide-like immunoreactivity) were not seen in paw skin. These results suggest that nitric oxide may act as a co-transmitter (with acetylcholine, substance P, vasoactive intestinal peptide and calcitonin gene-related peptide) in sudomotor neurons and (with acetylcholine and vasoactive intestinal peptide) in vasodilator neurons. Collateral branches of sudomotor neurons may innervate skin vessels, and release vasodilator transmitters including nitric oxide to cause the vasodilatation which provides the fluid supply for sweat formation. Alternatively, separate vasodilator neurons to skin may share the same chemical code as sudomotor neurons.

Distinct preganglionic neurons innervate noradrenaline and adrenaline cells in the cat adrenal medulla.

Calretinin immunoreactivity was present in a subset of preganglionic neurons retrogradely labelled from the cat adrenal gland. Overall, one-third of adrenal preganglionic neurons showed calretinin immunoreactivity, and their proportion increased in the more caudal spinal cord segments. Calretinin-immunoreactive nerve terminals were prominent within the adrenal gland, but were found only in areas of noradrenergic chromaffin cells (approximately one-third of the area of the adrenal medulla). Synaptophysin immunoreactivity was used to label terminals with and without calretinin immunoreactivity. Nerve terminals lacking calretinin immunoreactivity were present among the adrenergic chromaffin-cells and also comprised 20% of the nerve terminals innervating noradrenergic chromaffin cells. Calretinin immunoreactivity thus labels a subpopulation of cat adrenal preganglionic neurons that innervate the noradrenergic chromaffin cells.

Measurement errors stemming from nonrespondents present at in-person interviews.

PURPOSE: Data are frequently collected from in-person interviews in epidemiologic studies. Despite the advantages of this mode of data collection, the presence of a third party during the interview can contribute to measurement error, especially if third-party presence is related to case status. METHODS: Using data obtained from a case-control study of colon cancer, we evaluated the frequency of third-party presence during in-person interviews, and how having someone else present during the interview influences reporting of exposure data. RESULTS: Interviews were conducted in the presence of a third party for 28% of cases and 22% of controls who lived in a household of two or more individuals. Men with a third party present reported significantly lower age-adjusted mean levels of alcohol consumption (P < 0.01). Associations, as indicated by odds ratios, between colon cancer and alcohol intake were not statistically different among those with a third party present and those without a third party present. Although not statistically significant, energy intake was more strongly associated with colon cancer among those without a third party present during the interview. CONCLUSIONS: These results emphasize the need to review questions to be asked and decide whether privacy should be emphasized before data collection begins. If privacy is required, interviewers need to be given better skills to ensure privacy during interview.

Physical activity and colon cancer: a public health perspective.

PURPOSE: It has been suggested that performing physical activity for at least 30 min on most days of the week will improve health. The purpose of this study was to assess the association between physical activity and colon cancer as it relates to this public health recommendation. METHODS: A large population-based case-control study of colon cancer was conducted. Study participants came from three areas of the United States: Northern California, Utah, and the Twin Cities Metropolitan Area in Minnesota. RESULTS: Long-term involvement in high levels of activity, equivalent to > or = 60 min of vigorous activity per session, was associated with decreased risk (odds ration [OR], 0.68; 95% confidence interval [CI] 0.52-0.87). The amount of time involved in the activity appeared to have a greater impact than the number of days per week that activities were performed. Those reporting the highest level of activity, as defined by both duration and vigorous intensity, were at the lowest risk (OR, 0.62; 95% CI, 0.52-0.75) relative to those who were sedentary; associations did not differ by age at diagnosis, site of the tumor within the colon, or sex. The inverse association between colon cancer and long-term vigorous leisure-time activity was slightly stronger among those without a family history of colorectal cancer than among those with a family history of colorectal cancer. From these data we estimate that 13% of colon cancer could be attributed to lack of vigorous leisure-time activity in the population; we estimate that 4.3 cases of colon cancer/100,000 population are prevented each year because people are involved in vigorous leisure-time physical activity. CONCLUSIONS: Data from this study suggest that a high level of vigorous leisure-time activity performed over the past 20 years was important in reducing colon cancer risk; the greatest inverse association was observed when activities were performed for longer periods of time per session for the past 20 years. These and other data indicate that it is important to identify ways to facilitate an increase in leisure-time physical activity within the population.

Response rates among control subjects in case-control studies.

Response rates are an important component of epidemiologic research. The purposes of this study are (a) to evaluate how response rates are defined and calculated for control subjects in epidemiologic case-control studies, and (b) to explore factors that may impact response in epidemiologic studies. Our results show that the method of control subject selection has an impact on study response. Gender of respondent does not appear to impact response rates. However, response rates are generally worse for individuals less than 45 years old. Methods used to calculate response have a great impact on "response rate"; therefore, it is important for researchers to define exactly what the reported response rates represent and how they are derived so that data can be interpreted appropriately.

Stage of colon cancer at diagnosis: implications for risk factor associations?

BACKGROUND: A potential source of bias in epidemiological studies comes from studying people at different stages of disease progression. This can result in biased selection of cases or in errors of measurement of exposures. METHODS: We use stage of disease at the time of diagnosis to evaluate how inclusion of people at different stages in the disease process can influence associations between environmental exposures and colon cancer. Data used were generated from a large case-control study of colon cancer. RESULTS: For most environmental exposures evaluated, including physical activity, body size, use of aspirin and of non-steroidal anti-inflammatory drugs, and dietary intake of folate and fibre, we did not observe differences in patterns of association by stage of disease at diagnosis. However, for total energy and red meat intake (men only), alcohol consumption, cigarette smoking, and family history of colorectal cancer among first degree relatives, patterns of associations were stronger when colon cancer was detected at an earlier stage of disease progression than when it was detected at a more advanced stage. CONCLUSIONS: Most exposures did not differ by stage of disease, thus selectively excluding cases at different disease stages should not influence associations between these exposures and colon cancer. Associations for other factors, such as alcohol consumption and cigarette smoking, may be biased from asking cases with advanced disease to recall a non-disease-free time period. Associations with family history may also be biased if those with a family history of colorectal cancer are detected at an earlier stage and therefore more likely to participate in epidemiological studies.

Problematic pigmented lesions: approach to diagnosis.

A number of pigmented lesions are difficult to classify and raise the possibility of a melanoma diagnosis. Care should be exercised to exclude non-melanocytic lesions, and benign melanocytic entities, both of which can mimic melanoma histologically. In addition, the possibility of the lesion being a melanoma variant or epidermotropic metastasis should be considered. There will still be some cases that are difficult to resolve. These usually fall into one of three categories: atypical junctional melanocytic lesion versus early melanoma; naevus versus naevoid melanoma; and atypical Spitz, cellular blue, and deep penetrating naevi versus thick melanoma. These will pose problems even for experts. The atypical Spitz lesions are perhaps the most important category because they tend to be from younger individuals, the differential diagnosis is thick melanoma, and there is no single discriminating histological feature.

Preoperative histological classification of primary lung cancer: accuracy of diagnosis and use of the non-small cell category.

AIMS: To compare the preoperative classification of lung carcinoma made on cytological and histological specimens with the postoperative classification made on the resected specimen. In addition, to find out how often the term "non-small cell lung cancer, not otherwise specified" (NSCLC) was used, and in such cases to note the final diagnosis. METHODS: Between 1991 and 1995, 303 patients had a lung resection in Aberdeen for primary carcinoma. For each patient, the departmental records were examined for preoperative specimens (cytological and histological). A note was made of whether each specimen was positive or negative for malignancy and, if positive, what the cell type was. Where patients had more than one sample submitted, the most specific result was taken. RESULTS: Fifty four per cent of patients had a correct specific preoperative diagnosis of malignancy, whereas 34% were labelled as NSCLC. Patients with squamous carcinoma were more likely to have a diagnosis of malignancy (88%) that was specifically correct (75%). Patients who had adenocarcinoma were less likely to have a preoperative diagnosis of malignancy (64%) that was specifically correct (35%). For those in whom a diagnosis of NSCLC was made, 55% turned out to have adenocarcinoma whereas 24% had squamous carcinoma. CONCLUSIONS: By adhering strictly to criteria, a high accuracy of diagnosis can be achieved for squamous carcinoma, but the diagnosis of adenocarcinoma seems to be more of a challenge. NSCLC is a useful and appropriate classification, the use of which reduces the rate of inaccurate specific diagnosis. There are occasions when pathologists can provide a more accurate diagnosis by being less precise.

Intraperitoneal injections of Fluorogold reliably labels all sympathetic preganglionic neurons in the rat.

The ability of intraperitoneal injections of a retrograde neuronal tracer, Fluorogold, to label the entire population of sympathetic preganglionic neurones was tested with a double-labelling strategy. Animals were injected intraperitoneally (i.p.) with Fluorogold, while Fast Blue or subunit B of cholera toxin were injected into a peripheral autonomic ganglion or into the adrenal gland. Sympathetic preganglionic neurones were then examined for retrogradely transported tracers. In all cases, preganglionic neurones labelled with Fast Blue or cholera toxin also contained Fluorogold, indicating that i.p. injections of Fluorogold do reliably label the entire population of sympathetic preganglionic neurones.

The distribution of nitric oxide synthase-containing autonomic preganglionic terminals in the rat.

Nitric oxide synthase (NOS)-immunoreactivity was co-localised with NADPH diaphorase activity in preganglionic sympathetic neurons and in their terminals in pre- and paravertebral sympathetic ganglia. The density of NOS-containing terminals varied between ganglia. Reactive terminals were densest in the superior cervical, stellate and inferior mesenteric ganglia, where the majority of the neurons were surrounded by reactive fibres, and the coeliac and superior mesenteric ganglia, where about half the postganglionic somata were surrounded by reactive terminals. Fibres were least abundant in the pelvic ganglia and thoracic and lumbar sympathetic chain ganglia. NOS reactivity did not coincide with the distribution of calcitonin gene related peptide immunoreactivity, a marker for the terminals of NOS-containing sensory neurons in the rat. The distribution of nerve cells and terminals suggests that NOS is present in more than one functional subpopulation of sympathetic preganglionic neurons.