When it comes to needles and drawing blood, most patients agree that bigger is not better. But in the first study of its kind, Rice University bioengineers have found results from a single drop of blood are highly variable, and as many as six to nine drops must be combined to achieve consistent results.

The study examines the variation between blood drops drawn from a single finger-prick. The results suggest that health care professionals must take care to avoid skewed results as they design new protocols and technologies that rely on finger-prick blood.

“We began looking at this after we got some surprising results from our controls in an earlier study,” said lead investigator Rebecca Richards-Kortum, Rice’s Malcolm Gillis University Professor and director of Rice 360°: Institute for Global Health Technologies. “Students in my lab are developing novel, low-cost platforms for anaemia, platelet and white blood cell testing in low-resource settings, and one of my students, Meaghan Bond, noticed there was wide variation in some of the benchmark tests that she was performing on hospital-grade blood analysers.”

The benchmark controls are used to gauge the accuracy of test results from the new technology under study, so the variation among the control data was a sign that something was amiss. What wasn’t immediately clear was whether the readings resulted from a problem with the current experiments or actual variations in the amount of haemoglobin, platelets and white blood cells (WBC) in the different drops of blood.

Richards-Kortum and Bond designed a simple protocol to test whether there was actual variation, and if so, how much. They drew six successive 20-microliter droplets of blood from 11 donors. As an additional test to determine whether minimum droplet size might also affect the results, they drew 10 successive 10-microliter droplets from seven additional donors.

All droplets were drawn from the same finger-prick, and the researchers followed best practices in obtaining the droplets; the first drop was wiped away to remove contamination from disinfectants, and the finger was not squeezed or “milked,” which can lead to inaccurate results. For experimental controls, they use venipuncture, the standard of care in most hospitals, to draw tubes of blood from an arm vein.

Each 20-microliter droplet was analysed with a hospital-grade blood analyser for haemoglobin concentration, total WBC count, platelet count and three-part WBC differential, a test that measures the ratio of different types of white blood cells, including lymphocytes and granulocytes. Each 10-microliter droplet was tested for haemoglobin concentration with a popular point-of-care blood analyser used in many clinics and blood centres.

“A growing number of clinically important tests are performed using finger-prick blood, and this is especially true in low-resource settings,” Bond said. “It is important to understand how variations in finger-prick blood collection protocols can affect point-of-care test accuracy as well as how results might vary between different kinds of point-of-care tests that use finger-prick blood from the same patient.”

Bond and Richards-Kortum found that haemoglobin content, platelet count and WBC count each varied significantly from drop to drop.

“Some of the differences were surprising,” Bond said. “For example, in some donors, the haemoglobin concentration changed by more than two grams per deciliter in the span of two successive drops of blood.”

Bond and Richards-Kortum found that averaging the results of the droplet tests could produce results that were on par with venous blood tests, but tests on six to nine drops blood were needed to achieve consistent results.

“Finger-prick blood tests can be accurate and they are an important tool for health care providers, particularly in point-of-care and low-resource settings,” Bond said. “Our results show that people need to take care to administer finger-prick tests in a way that produces accurate results because accuracy in these tests is increasingly important for diagnosing conditions like anaemia, infections and sickle-cell anemia, malaria, HIV and other diseases.” Rice University

Researchers from the University of Miami Miller School of Medicine’s John P. Hussman Institute for Human Genomics and Bascom Palmer Eye Institute are part of a consortium that has significantly expanded the number of genetic factors known to play a role in age-related macular degeneration (AMD), a leading cause of vision loss among people age 50 and older. Supported by the National Eye Institute, part of the National Institutes of Health, the findings may help improve our understanding of the biological processes that lead to AMD and identify new therapeutic targets for potential drug development.
AMD is a progressive disease that causes the death of the retinal photoreceptors, the light-sensitive cells at the back of the eye. The most severe damage occurs in the macula, a small area of the retina that is needed for sharp, central vision necessary for reading, driving and other daily tasks. There are currently no FDA -approved treatments for the more common form of advanced AMD, called geographic atrophy or “dry” AMD. While therapies for the other advanced form, neovascular or “wet” AMD, can successfully halt the growth of abnormal, leaky blood vessels in the eye, the therapies do not cure the condition, nor do they work for everyone.

Up to this point, researchers had identified 21 loci that influence the risk of AMD. The new research raises the number of loci to 34. The Miller School’s Margaret A. Pericak-Vance, Ph.D., the Dr. John T. Macdonald Foundation Professor of Human Genomics and Director of the John P. Hussman Institute for Human Genomics, and William K. Scott, Ph.D., professor and Vice Chair for Education and Training at the Dr. John T. Macdonald Foundation Department of Human Genetics and the John P. Hussman Institute for Human Genomics, and professor of public health sciences, were two of the senior authors on the study.

The International AMD Genomics Consortium, which includes 26 centres worldwide, collected and analysed the genetic data from 43,566 people of predominantly European ancestry to systematically identify common and rare variations in genetic coding — called variants — associated with AMD. Pericak-Vance is the co-Principal Investigator of the National Eye Institute-funded consortium. Common variants generally have an indirect association with a disease. Rare variants, by contrast, are more likely to alter protein expression or function and therefore have a direct or causal association with a disease. Rare variants were defined as those found in less than 1 percent of the study population.

The study included about 23,000 participants with AMD and 20,000 without it. Researchers analysed DNA samples from both groups, surveying most of the genome, but also focusing on distinct loci already known or suspected to be associated with AMD. Next, they compared the participants’ DNA to a reference dataset called the 1,000 Genomes project, yielding more than 12 million genetic variants of potential interest. Finally, they went back to the participants’ DNA samples, looking at all 12 million variants, to see if any were found more or less often in people with AMD than those without it.

The study findings also bolster associations between AMD and two genes, CFH and TIMP3, which had each previously been linked to AMD. CFH was the very first disease-linked gene to be found through a genome-wide association study. TIMP3 had earlier been linked to Sorsby’s fundus dystrophy, a rare disease that is similar to AMD clinically, but which tends to affect people before the age of 45.
For the first time the researchers also identified a variant specific to the neovascular form of AMD, which may point to reasons why therapy for this form of AMD is effective for some people but not everyone. Miller School of Medicine

The research suggests genetic inheritance is much more important in testicular cancer than in most other cancer types, where genetics typically accounts for less than 20% of risk.

The findings suggest testing for a range of genetic variants linked to testicular cancer could be effective in picking out patients who are at substantially increased risk – potentially opening up ways of preventing the disease.

Scientists at The Institute of Cancer Research, London, along with colleagues in Germany, Sweden and the US, used two independent approaches to analyse the risk of testicular germ cell tumours – easily the most common type of testicular cancer. Their research is the largest study ever to explore testicular germ cell tumours in detail.

Researchers first used statistical analysis to examine patterns of ancestral testicular cancer in family groups across 15.7 million people from the Swedish Population Registry cancer family database, including 9,324 cases of testicular cancer.

They then looked in detail at the genetic code of 6,000 UK men from two previous testicular cancer studies, 986 of whom had been diagnosed with the disease.

The combined analysis revealed that 49% of all the possible factors contributing to testicular cancer risk are inherited.

It found that the inherited risk comes from a large number of minor variations in DNA code, rather than one faulty gene with a big effect.

Although substantial inroads have been made over the last five years at the ICR into identifying mutations associated with risk of testicular cancer, the study also showed that these known mutations only account for 9.1% of the risk of developing the disease. Therefore the majority of the genetic variants that raise testicular cancer risk have yet to be identified.

Identifying more of these ‘hidden’ mutations could allow doctors to screen men for testicular cancer risk, increasing the chance of preventing the disease or catching it early.

Dr Clare Turnbull, Senior Researcher in Genetics and Epidemiology at the ICR, said: “Our study has shown that testicular cancer is a strongly heritable disease. Around half of a man’s risk of developing testicular cancer comes from the genes he inherits from his parents – with environmental and behavioural factors contributing to the other half.

“Our findings have important implications in that they show that if we can discover these genetic causes, screening of men with a family history of testicular cancer could help to diagnose those at greatest risk, and help them to manage that risk. Institute of Cancer Research

A particular location in DNA, called the Dlk1-Gtl2 locus, plays a critical role in protecting hematopoietic, or blood-forming, stem cells–a discovery revealing a critical role of metabolic control in adult stem cells, and providing insight for potentially diagnosing and treating cancer, according to researchers from the Stowers Institute for Medical Research.

In their study, Stowers Investigator Linheng Li, Ph.D., and first author Pengxu Qian, Ph.D., along with other collaborators, reveal how the mammalian imprinted Gtl2, located on mouse chromosome 12qF1, protects adult hematopoietic stem cells by restricting metabolic activity in the cells’ mitochondria.

The research focused on imprinted genes–genes ‘stamped’ according to whether they are inherited from the mother or father. With imprinted genes, one working copy, or allele, is inherited instead of two. Either the copy from the mother or father is inactivated or ‘silenced.’ Typically, the paternally inherited allele’s expression promotes growth, while the maternally inherited allele’s expression suppresses it.

The researchers found that when the Gtl2 locus is expressed from the maternally inherited allele, it produces non-coding RNAs to curb metabolic activity. Mechanistically, Gtl2’s ‘megacluster’ of microRNA, the largest cluster of microRNA in the mammalian genome, suppresses the mTOR signaling pathway and downstream mitochondrial biogenesis and metabolism, thus blocking mitochondrial-associated byproducts called reactive oxygen species (ROS) that can damage adult stem cells.

‘Reactive oxygen species are like the potentially harmful by-products that come from industrial manufacturing,’ says Li. ‘ROS are unavoidable derivatives of the mitochondrial metabolic process and need to be managed by the cell,’ he explains.

Hematopoietic stems cells renew themselves and differentiate into other cells, including white blood cells, red blood cells, and platelets, constantly renewing the body’s blood supply in a process called hematopoiesis. Because of their extraordinary transformative qualities, the transplantation or transfusion of isolated human hematopoietic stem cells has been used in the treatment of anemia, immune deficiencies, and other diseases, including cancer.

While hematopoietic stem cells have gained attention in research, it remains largely unknown how cell metabolic states are controlled. The new findings shed light on the delicate metabolic control required to balance hematopoietic stem cell maintenance and action and the associated healthy hematopoiesis.

An upset in that balance can cause cells to grow abnormally and lead to disease. Abnormalities in the Gtl2 locus on human chromosome 14q32.2 are associated with uniparental disomy in which an individual receives two copies of a chromosome from one parent and no copy from the other parent. Uniparental disomy may cause delayed development, mental retardation, or other medical problems. Differences in gene expression at the Gtl2 locus have also been linked to fetal alcohol exposure disorder.

But when working properly, the Gtl2 locus acts as a great protector of cells.

‘Most of the non-coding RNAs at the Gtl2 locus have been documented to function as tumor suppressors to maintain normal cell function,’ Qian says.

Li’s team zeroed in on Gtl2 by studying hematopoietic stem cells in mice with support from Stowers core centers including cytometry, bioinformatics, histology and electron microscopy, molecular biology, and tissue culture. Other collaborators included researchers from the University of Kansas; the University of Kansas Medical Center; Tianjin Medical University, China; Christian Medical College, Vellore, India; Tokyo University of Agriculture, Japan; and University of Cambridge, United Kingdom.

Over the three-year study, investigators used transcriptome profiling to analyze 17 hematopoietic cell types and found that non-coding RNAs expressed from the Gtl2 locus are predominantly located in a subset of the cell types, including adult ‘long-term’ hematopoietic stem cells which have long-term self-renewal capacity. In subsequent experiments, deleting the locus from the maternally inherited allele in hematopoietic stem cells increased mitochondrial biogenesis and subsequent metabolic activity as well as increased ROS levels, with the latter inducing cell death.

The finding opens the possibility for Gtl2 to be used as a biomarker because it could help label dormant (or reserve) stem cells in normal or potentially cancerous stem cell populations, Li says. The addition of a fluorescent tag to the Gtl2 locus could allow researchers to mark other adult stem cells in the gut, hair follicle, muscle, and neural systems. EurekAlert

Scientists who analysed the genes involved in 10 autoimmune diseases that begin in childhood have discovered 22 genome-wide signals shared by two or more diseases. These shared gene sites may reveal potential new targets for treating many of these diseases, in some cases with existing drugs already available for non-autoimmune disorders.

Autoimmune diseases, such as type 1 diabetes, Crohn’s disease and juvenile idiopathic arthritis, collectively affect 7 to 10 percent of the population in the Western Hemisphere.

Dr. Hakonarson“Our approach did more than finding genetic associations among a group of diseases,” said study leader, Hakon Hakonarson, MD, PhD, director of the Center for Applied Genomics at The Children’s Hospital of Philadelphia (CHOP). “We identified genes with a biological relevance to these diseases, acting along gene networks and pathways that may offer very useful targets for therapy.”

The international study team performed a meta-analysis, including a case-control study of 6,035 subjects with automimmune disease and 10,700 controls, all of European ancestry. The study’s lead analyst, Yun (Rose) Li, an MD/PhD graduate student at the University of Pennsylvania and the Center for Applied Genomics, mentored by Hakonarson and his research team, applied highly innovative and integrative approaches in supporting the study of pathogenic roles of the genes uncovered across multiple diseases.

The research encompassed 10 clinically distinct autoimmune diseases with onset during childhood: type 1 diabetes, celiac disease, juvenile idiopathic arthritis, common variable immunodeficiency disease, systemic lupus erythematosus, Crohn’s disease, ulcerative colitis, psoriasis, autoimmune thyroiditis and ankylosing spondylitis.

Because many of these diseases run in families and because individual patients often have more than one autoimmune condition, clinicians have long suspected these conditions have shared genetic predispositions. Previous genome-wide association studies have identified hundreds of susceptibility genes among autoimmune diseases, largely affecting adults.

The current research was a systematic analysis of multiple paediatric-onset diseases simultaneously. The study team found 27 genome-wide loci, including five novel loci, among the diseases examined. Of those 27 signals, 22 were shared by at least two of the autoimmune diseases, and 19 of them were shared by at least three of them.

Many gene signals found on biological pathways linked to cell activation, proliferation and signalling

Many of the gene signals the investigators discovered were on biological pathways functionally linked to cell activation, cell proliferation and signalling systems important in immune processes. One of the five novel signals, near the CD40LG gene, was especially compelling, said Hakonarson, who added, “That gene encodes the ligand for the CD40 receptor, which is associated with Crohn’s disease, ulcerative colitis and celiac disease. This ligand may represent another promising drug target in treating these diseases.”

Gene signals have biological relevance to autoimmune disease processes, opportunities to better target gene networks and pathways

Many of the 27 gene signals the investigators uncovered have a biological relevance to autoimmune disease processes, Hakonarson said. “Rather than looking at overall gene expression in all cells, we focused on how these genes upregulated gene expression in specific cell types and tissues, and found patterns that were directly relevant to specific diseases. For instance, among several of the diseases, we saw genes with stronger expression in B cells. Looking at diseases such as lupus or juvenile idiopathic arthritis, which feature dysfunctions in B cells, we can start to design therapies to dial down over-expression in those cells.”

He added that “the level of granularity the study team uncovered offers opportunities for researchers to better target gene networks and pathways in specific autoimmune diseases, and perhaps to fine tune and expedite drug development by repurposing existing drugs, based on our findings.” The Children’s Hospital of Philadelphia