Making Sense of Intratumor Genetic Heterogeneity: Altered Frequency of Androgen Receptor CAG Repeat Length Variants in Breast Cancer Tissues

To examine the significance of intratumor genetic heterogeneity (ITGH) of the androgen receptor (AR) gene in breast cancer, patient‐matched samples of laser capture microdissected breast tumor cells, adjacent normal breast epithelia cells, and peripheral blood leukocytes were sequenced using a novel next generation sequencing protocol. This protocol measured the frequency of distribution of a variable AR CAG repeat length, a functional polymorphism associated with breast cancer risk. All samples exhibited some degree of ITGH with up to 30 CAG repeat length variants identified. Each type of tissue exhibited a different distribution profile of CAG repeat lengths with substantial differences in the frequencies of zero and 18–25 CAG AR variants. Tissue differences in the frequency of ARs with each of these CAG repeat lengths were significant as measured by paired, twin t‐tests. These results suggest that preferential selection of 18–25 CAG repeat length variants in breast tumors may be associated with breast cancer, and support the observation that shorter CAG repeats may protect against breast cancer. They also suggest that merely identifying variant genes will be insufficient to determine the critical mutational events of oncogenesis, which will require measuring the frequency of distribution of mutations within cancerous and matching normal tissues.     

High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets

Developing T cells face a series of cell fate choices in the thymus and in the periphery. The role of the individual T cell receptor (TCR) in determining decisions of cell fate remains unresolved. The stochastic/selection model postulates that the initial fate of the cell is independent of TCR specificity, with survival dependent on additional TCR/coreceptor “rescue” signals. The “instructive” model holds that cell fate is initiated by the interaction of the TCR with a cognate peptide-MHC complex. T cells are then segregated on the basis of TCR specificity with the aid of critical coreceptors and signal modulators [Chan S, Correia-Neves M, Benoist C, Mathis (1998) Immunol Rev 165: 195–207]. The former would predict a random representation of individual TCR across divergent T cell lineages whereas the latter would predict minimal overlap between divergent T cell subsets. To address this issue, we have used high-throughput sequencing to evaluate the TCR distribution among key T cell developmental and effector subsets from a single donor. We found numerous examples of individual subsets sharing identical TCR sequence, supporting a model of a stochastic process of cell fate determination coupled with dynamic patterns of clonal expansion of T cells bearing the same TCR sequence among both CD4⁺ and CD8+ populations.     

Lack of association of CCR2-64I and CCR5-Delta 32 with type 1 diabetes and latent autoimmune diabetes in adults

It is well known that type 1 diabetes mellitus (T1DM) is a complex genetic disease resulting from the autoimmune destruction of pancreatic beta cells. Several genes have been associated with susceptibility and/or protection for T1DM, but the disease risk is mostly influenced by genes located in the class II region of the major histocompatibility complex. The attraction of leukocytes to tissues is essential for inflammation and the beginning of autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytolines. Some studies have shown that CCR2-64I and CCR5-Delta 32 might be important for protection of susceptibility to some immunologically-mediated disorders. In the present study, we demonstrate the lack of association between CCR2-64I and CCR5-Delta 32 gene polymorphism and TIDM and we describe a new method for a simple and more precise genotyping of the CCR2 gene.     

Type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology

DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.     

Lack of association of human chemokine receptor gene polymorphisms CCR2‐64I and CCR5‐Δ32 with autoimmune Addison's disease

The attraction of leukocytes to tissues is essential for inflammation and the initiation of the autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytokines. We investigated whether human chemokine receptor gene polymorphisms, namely CCR5‐Δ32 and CCR2‐64I, are associated with susceptibility to autoimmune Addison's disease. Genotyping was performed in 56 patients and 127 healthy controls by a new method using pyrosequencing for CCR2‐64I and by polymerase chain reaction and detecting gel for CCR5‐Δ32. None of the CCR2 or CCR5 alleles was found to be associated, either positively or negatively, with disease risk. Our results indicate that the CCR2‐64I and CCR5‐Δ32 gene polymorphisms do not play a major role in conferring genetic risk for, and/or protection against, autoimmune Addison's disease.     

Influence of eotaxin 67G>A polymorphism on plasma eotaxin concentrations in myocardial infarction survivors and healthy controls

Eotaxin (CCL11) is a CC chemokine, whose systemic levels might be associated with coronary artery disease (CAD) and genetic variants predispose to the myocardial infarction (MI). However, the relationship between eotaxin genetic variants and plasma concentrations in CAD patients is still incompletely characterized. We genotyped 311 patients, who survived first MI and 338 controls for a 67G>A single nucleotide polymorphism in the eotaxin gene. By measuring plasma eotaxin concentrations in those subjects we related the former to the presence of 67G>A SNP. There were no differences in eotaxin genotype frequencies between patients and controls. Patient G/G carriers had higher circulating eotaxin levels compared both to G/A and A/A patients (P=0.046) and G/G controls (P=0.028), which might indicate the influence of additional factors (e.g. inflammatory mediators) on eotaxin secretion in those patients. At the same time, eotaxin levels did not differ between patients and controls irrespective of the 67G>A SNP variants they carried. There were no associations between plasma eotaxin levels, biochemical indicators of CAD and the degree of coronary artery stenosis in post-MI patients. Interestingly, some medications taken by the patients (e.g. diuretics and short-acting nitrates) might affect plasma eotaxin levels. In conclusion, our results show that there is no clear association between the presence of eotaxin 67G>A SNP, its plasma levels and CAD parameters in post-MI patients and that circulating eotaxin levels do not differ between subjects with clinical manifestations of coronary atherosclerosis and healthy controls.     

Assessment of physical functioning in the clinical care of the patient with advanced kidney disease

Maintenance of independent living is the top health priority among patients with advanced chronic kidney disease (CKD). Mobility limitation is often the first sign of functional limitation leading to loss of independence. Regular assessments of physical capacity can help provide kidney health providers identify patients at risk of frailty and other adverse health‐related outcomes that contribute to the loss of functional independence. These physical capacities can be measured with commonly used self‐reported measures of physical function or by objective physical performance testing. The current review describes commonly used assessments of self‐reported physical function and physical performance. First, we describe the disablement process and how these assessments can be performed with commonly used quality of life instruments measuring self‐reported physical function or objective physical performance tests. Second, we identify the determinants and correlates of self‐reported physical function and physical performance and their contribution to the frailty phenotype. Third, we describe the association of physical capacities with clinical outcomes. We conclude with on possible approach to identifying and intervening on persons with CKD at high risk of functional decline.     

Pyrosequencing for discovery and analysis of DNA sequence variations

Since the invention of pyrosequencing, more than 500 articles have been published describing different applications of this technology, most notably for DNA structure variation and microbial detection. Technological advances have been made to enhance the robustness and accuracy of this technique as well as to reduce the cost and increase the throughput. This review intends to cover recent advances in this technology and discuss its application for low and high-throughput DNA variation studies.