Blind study evaluation illustrates utility of the Ion PGM? system for use in human identity DNA typing

Aim

To perform a blind study to assess the capability of the Ion Personal Genome Machine™ (PGM) system to sequence forensically relevant genetic marker panels and to characterize unknown individuals for ancestry and possible relatedness.

Methods

Twelve genomic samples were provided by a third party for blinded genetic analysis. For these 12 samples, the mitochondrial genome and three PGM™ panels containing human identity single nucleotide polymorphisms (SNPs), ancestry informative SNPs, and short tandem repeats (STRs) were sequenced on the PGM™ system and analyzed.

Results

All four genetic systems were run and analyzed on the PGM™ system in a reasonably quick time frame. Completeness of genetic profiles, depth of coverage, strand balance, and allele balance were informative metrics that illustrated the quality and reliability of the data produced. SNP genotypes allowed for identification of sex, paternal lineage, and population ancestry. STR genotypes were shown to be in complete concordance with genotypes generated by standard capillary electrophoresis-based technologies. Variants in the mitochondrial genome data provided information on population background and maternal relationships.

Conclusion

All results from analysis of the 12 genomic samples were consistent with sample information provided by the sample providers at the end of the blinded study. The relatively easy identification of intra-STR allele SNPs offered the potential for increased discrimination power. The promising nature of these results warrants full validation studies of this massively parallel sequencing technology and its further development for forensic data analysis.The advent of massively parallel sequencing (MPS) technologies offers an alternative to current DNA typing methods. Comprehensive coverage of multiple forensically relevant genetic markers made possible by MPS technologies can provide a wealth of data for use in criminal investigations (1-6). While short tandem repeats (STRs) have been the primary marker system for human identity typing due to their polymorphic nature and high discrimination power, MPS allows for the examination of repeat and sequence variants in these STRs and for the inclusion of single nucleotide polymorphisms (SNPs) and mitochondrial DNA into the analysis pipeline. Using MPS to analyze STRs allows for the exact sequence of each allele to be obtained and for SNPs to be identified within the STR repeat structure. These intra-STR SNPs offer greater resolving power when analyzing mixtures and performing kinship analyses (1,4,7). Alternative marker types can facilitate analysis of degraded or low template samples. SNPs and mitochondrial DNA can aid in the analysis of degraded and low quantity samples. SNPs reflect a single base change, thus short amplicons can be used in their analysis (2,8). Sequencing the entire mitochondrial genome allows a greater discrimination power to be obtained and more accurate haplogroup assignments to be generated (3,5). This capability allows for better population background predictions and identification of maternal lineage relationships. The multitude of SNPs provides information on identity, ancestry, and lineage, which can help produce investigative leads that were not previously possible (6,8-11).The Ion Torrent Personal Genome Machine® (PGM™) (Thermo Fisher Scientific, Waltham, MA USA) is one available MPS benchtop platform. The PGM™ is a high-throughput sequencer that employs semiconductor-sequencing technology (12). This sequence-by-synthesis chemistry measures the release of hydrogen ions as nucleotides are incorporated into the growing DNA strand. The PGM™ measures the associated, real-time pH change of the surrounding solution on a semiconductor chip thereby allowing for direct translation of chemically-encoded information into digital information (12). This process uses customized chemistries in a laboratory workflow that enables high-throughput and fast run times at a reasonable cost. In fact, the PGM™’s read length, sequencing time, running costs, and scalability lend itself to effective incorporation into diagnostic workflow of the forensic laboratory.Twelve genomic samples were provided by a sample exchange group through the Green Mountain DNA Conference for a blinded genetic study. These samples were used to evaluate the potential of MPS on the PGM™ system to reliably analyze unknown samples, based on the self-declared information of the donors of these samples. The mitochondrial genome, identity SNPs, ancestry informative SNPs, and STRs (the latter three enabled by using PGM™ panels) were sequenced on the PGM™ system and resultant data were analyzed for these 12 samples.     

Responding to the global human resources crisis

The global community is in the midst of a growing response to health crises in developing countries, which is focused on mobilising financial resources and increasing access to essential medicines. However, the response has yet to tackle the most important aspect of health-care systems--the people that make them work. Human resources for health--the personnel that deliver public-health, clinical, and environmental services--are in disarray and decline in much of the developing world, particularly in sub-Saharan Africa. The reasons behind this disorder are complex. For decades, efforts have focused on building training institutions. What is becoming increasingly clear, however, is that issues of supply, demand, and mobility (transnational, regional, and local) are central to the human-resource problem. Without substantial improvements in workforces, newly mobilised funds and commodities will not deliver on their promise. The global community needs to engage in four core strategies: raise the profile of the issue of human resources; improve the conceptual base and statistical evidence available to decision makers; collect, share, and learn from country experiences; and begin to formulate and enact policies at the country level that affect all aspects of the crisis.     

The economic consequences of prostate and bladder cancer in the UK

OBJECTIVE: To compare the costs of managing prostate and bladder cancer and relate them to current expenditure on research, as the increasing prevalence of both necessitates the adequate direction of resources. METHODS: All new prostate and bladder cancers diagnosed in 2001-2002 were identified from British Association of Urological Surgeons Section of Oncology database (national and local). The total cost of diagnosing, treating and following patients for 5 years was estimated as the sum of direct costs (National Health Service) and indirect costs (loss of earnings). Annual research fund allocation (RFA) for each cancer were obtained from the National Cancer Research Institute UK. RESULTS: There were 15 099 and 7703 patients with newly diagnosed prostate (mean age 72.3 years) and bladder cancers (mean age 71.3 years). The total cost for prostate cancer was estimated at 92.74 million UK pounds, with hormonal therapy alone costing 63.1 million UK pounds. The total cost for bladder cancer was 55.39 million UK pounds, of which superficial disease cost 35.25 million. The mean cost per patient was more for bladder than for prostate cancer (8349 UK pounds vs. 7294). The RFA allocation during this period was 20.56 million UK pounds and 4.62 million UK pounds for prostate and bladder cancer, respectively, and the respective RFA allotment per pound spent on the mean cost of disease management per patient was 2818 UK pounds and 553 UK pounds. CONCLUSION: Individual patient management is more costly for bladder cancer but less is invested in research than for prostate cancer. This study suggests a need to re-evaluate future strategies.     

Prefrontal cortex, thalamus, and cerebellar volumes in adolescents and young adults with adolescent-onset alcohol use disorders and comorbid mental disorders

BACKGROUND: In adults, prefrontal, thalamic, and cerebellar brain injury is associated with excessive ethanol intake. As these brain structures are actively maturing during adolescence, we hypothesized that subjects with adolescent-onset alcohol use disorders, compared with control subjects, would have smaller brain volumes in these areas. Thus, we compared prefrontal-thalamic-cerebellar measures of adolescents and young adults with adolescent-onset alcohol use disorders (AUD, defined as DSM-IV alcohol dependence or abuse) with those of sociodemographically similar control subjects. METHODS: Magnetic resonance imaging was used to measure prefrontal cortex, thalamic, and cerebellar volumes in 14 subjects (eight males, six females) with an AUD (mean age, 17.0+/-2.1 years) and 28 control subjects (16 males, 12 females; 16.9+/-2.3 years). All AUD subjects were recruited from substance abuse treatment programs and had comorbid mental disorders. RESULTS: Subjects with alcohol use disorders had smaller prefrontal cortex and prefrontal cortex white matter volumes compared with control subjects. Right, left, and total thalamic, pons/brainstem, right and left cerebellar hemispheric, total cerebellar, and cerebellar vermis volumes did not differ between groups. There was a significant sex-by-group effect, indicating that males with an adolescent-onset AUD compared with control males had smaller cerebellar volumes, whereas the two female groups did not differ in cerebellar volumes. Prefrontal cortex volume variables significantly correlated with measures of alcohol consumption. CONCLUSIONS: These findings suggest that a smaller prefrontal cortex is associated with early-onset drinking in individuals with comorbid mental disorders. Further studies are warranted to examine if a smaller prefrontal cortex represents a vulnerability to, or a consequence of, early-onset drinking.     

Sex differences in the prevalence of peripheral artery disease in patients undergoing coronary catheterization

To determine whether there are sex differences in the prevalence of peripheral artery disease, we performed an observational study of 1014 men and 547 women, aged ≥ 40 years, referred for elective coronary angiography. Women were slightly older, more obese, had higher low-density lipoprotein cholesterol (LDL-C) levels and systolic blood pressure (BP), and were more likely to be African American. Women had higher high-density lipoprotein cholesterol (HDL-C) levels, lower diastolic BP, and were less likely to smoke or to have a history of cardiovascular disease. Women had less prevalent (62% vs 81%) and less severe coronary artery disease (CAD) (p < 0.001 for both) by coronary angiography, but more prevalent peripheral artery disease (PAD) as determined by the ankle-brachial index (ABI) than men (23.6% versus 17.2%). Independent predictors of lower ABI were female sex, black race, older age, tobacco use, CAD, diabetes, and triglyceride level. In a full multivariable logistic regression model, women had a risk-adjusted odds ratio for PAD of 1.78 (95% CI 1.25-2.54) relative to men. Among patients referred for coronary angiography, women have less prevalent and less severe CAD, but more prevalent PAD, a sex difference that is not explained by traditional cardiovascular disease risk factors or CAD severity. Clinical Trial Registration-URL: http://clinicaltrials.gov. Unique identifier: NCT00380185.     

The MYH7 p.R787H mutation causes hypertrophic cardiomyopathy in two unrelated families

BACKGROUND:

Familial hypertrophic cardiomyopathy (FHC) is a Mendelian disorder usually caused by mutations in any one of more than 12 genes, most of which encode sarcomere proteins. The disease exhibits extensive genetic heterogeneity, and it is important to identify mutations that result in adverse symptoms and/or lethality in affected individuals. An analysis of disease-causing mutations has been initiated in the Indian population to determine prevalent mutations.

METHODS:

FHC was detected using echocardiography and by analysis of clinical symptoms and family history. The disease-causing mutation was identified using polymerase chain reaction DNA sequencing.

RESULTS:

The p.R787H mutation was identified in the MYH7 gene in two FHC families. Sequence and structure analysis suggested impaired binding of the mutant protein to the myosin essential light chain.

CONCLUSIONS:

Although the mutation results in variable clinical symptoms in the affected individuals, probably owing to the effect of modifier genes and/or environmental factors, it does not appear to be a lethal mutation.     

Thoracic ultrasonography for the pulmonary specialist

Thoracic ultrasonography is a noninvasive and readily available imaging modality that has important applications in pulmonary medicine outside of the ICU. It allows the clinician to diagnose a variety of thoracic disorders at the point of care. Ultrasonography is useful in imaging lung consolidation, pleural-based masses and effusions, pneumothorax, and diaphragmatic dysfunction. It can identify complex or loculated effusions and be useful in planning treatment. Identifying intrathoracic mass lesions can guide sampling by aspiration and biopsy. This article summarizes thoracic ultrasonography applications for the pulmonary specialist, related procedural codes, and reimbursement. The major concepts are illustrated with cases. These case summaries are enhanced with online supplemental videos and chest radiograph, chest CT scan, and ultrasound correlation.     

Renal sympathetic denervation for treatment of resistant hypertension – Indigenous technique

BackgroundThe endovascular approach of ablation of renal sympathetic nerves is found to be effective in the treatment of uncontrolled hypertension. We report here our experience with the procedure in eight patients with drug resistant hypertension.MethodsWe included patients in whom the blood pressure remained above 150/90 mmHg despite being on minimum three antihypertensive drugs. Radiofrequency ablation of the sympathetic nerves of both the renal arteries was done using conventional ablation catheters. The patients were followed at 1-month, 3 months and 6 months post procedure and blood pressure recorded.ResultsAll patients underwent successful renal sympathetic denervation. The mean blood pressure of the patients was 181/102.5 mmHg before the procedure and the average requirement of antihypertensive drugs per day was 4. A significant reduction in both systolic and diastolic blood pressure was observed post procedure which sustained over the follow up period of six months. The mean blood pressure observed at 1-month, 3 and 6 months were 137.5/80 mmHg, 136/81 mmHg and 137.5/81 mmHg, respectively. The average requirement of the number of antihypertensives also was reduced to 2.5 at the end of 6 months. There were no procedural complications.ConclusionCatheter based renal denervation causes substantial and sustained blood pressure reduction without serious adverse events in patients with resistant hypertension.     

Focal para-hisian atrial tachycardia with dual exits

Focal atrial tachycardias (AT) in the right atrium (RA) tend to cluster around the crista terminalis, coronary sinus (CS) region, tricuspid annulus, and para-hisian region. In most cases, the AT focus can be identified by careful activation mapping, and completely eliminated by radiofrequency (RF) catheter ablation. However, RF ablation near the His bundle (HB) carries a risk of inadvertent damage to the atrioventricular (AV) conduction system. Here we describe a patient with an AT originating in the vicinity of the AV node, which was successfully ablated earlier from non-coronary aortic cusp (NCC), and recurred with an exit from para-hisian location. Respiratory excursions of the catheter were associated with migration to the area of HIs. This was successfully ablated during controlled apnoea, using 3D electroanatomic mapping.