High accuracy of HLA-B*27 genotyping by allele-specific real-time polymerase chain reaction in a heterogeneous population compared to flow cytometry and single nucleotide polymorphism detection assays.

HLA-B27 is a risk marker for ankylosing spondylitis and other associated seronegative spondyloarthropathies. We compared three methods of HLA-B*27 typing in a New South Wales (NSW) population: flow cytometry, rs4349859 single nucleotide polymorphism (SNP) detection assay, and allele-specific real-time polymerase chain reaction (RT-PCR) analysis of exons 2 and 3. Over a 5-month period, 543 samples underwent flow cytometric testing and RT-PCR high-resolution melt analysis of rs4349859 SNP and of exon 2 (5' fragment) and exon 3. In the third method, positive samples were further analysed with fluorescent resonance emission transfer (FRET) RT-PCR of exon 2 fragments, 2a and 2b. HLA-B*27 and other genotypes were confirmed by Sanger sequencing of a 600 base pair fragment of exons 2 and 3. In our cohort, the rs4349859 SNP method had 78.6% sensitivity and 98.7% specificity. Screening with exon 2 (5' fragment) and exon 3 RT-PCR provided 100% sensitivity. Further testing with exon 2a and 2b FRET RT-PCR produced 100% specificity. This cascade approach with allele-specific RT-PCR assays was able to differentiate all samples into HLA-B*27 subtypes. HLA-B*27 genotyping with allele-specific RT-PCR assays, to screen for and confirm HLA-B27 positive samples, was more sensitive and specific than flow cytometry and rs4349859 SNP assays. It is a potentially cost-effective method for differentiating HLA-B27 subtypes. Our cascade genetic testing approach is suitable for replacing the current flow cytometric HLA-B27 assay for the heterogeneous NSW population.

SDH-deficient renal cell carcinoma associated with biallelic mutation in succinate dehydrogenase A: comprehensive genetic profiling and its relation to therapy response.

Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is a rare RCC subtype that is caused by biallelic mutation of one of the four subunits of the SDH complex (SDHA, B, C, and D) and results in inactivation of the SDH enzyme. Here we describe a case of genetically characterized SDH-deficient RCC caused by biallelic (germline plus somatic) SDHA mutations. SDHA pathogenic variants were detected using comprehensive genomic profiling and SDH absence was subsequently confirmed by immunohistochemistry. Very little is known regarding the genomic context of SDH-deficient RCC. Interestingly we found genomic amplifications commonly observed in RCC but there was an absence of additional variants in common cancer driver genes. Prior to genetic testing a PD-1 inhibitor treatment was administered. However, following the genetic results a succession of tyrosine kinase inhibitors were administered as targeted treatment options and we highlight how the genetic results provide a rationale for their effectiveness. We also describe how the genetic results benefited the patient by empowering him to adopt dietary and lifestyle changes in accordance with knowledge of the mechanisms of SDH-related tumorigenesis.

Effect of NT-proBNP testing on diagnostic certainty in patients admitted to the emergency department with possible heart failure.

BACKGROUND: Difficulty in distinguishing congestive heart failure (HF) from other causes of dyspnoea in the emergency department (ED) may result in delay in appropriate treatment and referral. Although the diagnostic value of serum amino-terminal pro-B-type natriuretic peptide (NT-proBNP) is well documented, the impact on diagnostic certainty of providing these results to ED physicians is not well studied. We sought to determine the effect of providing NT-proBNP results on diagnostic certainty of physicians managing patients presenting to the ED with suspected HF. METHODS: A randomized controlled study was conducted in 68 patients presenting to the ED with dyspnoea. ED clinicians initially rated the probability of HF as the cause of dyspnoea without the knowledge of the result. A scale of 1-7 was used, with 1 representing a high degree of certainty of a diagnosis other than HF and 7 representing a high degree of certainty of HF being the cause of dyspnoea. In 38 patients, the ED physician then reassessed the probability of HF as the cause of dyspnoea after receiving the NT-proBNP result. A cardiologist blinded to the NT-proBNP result determined the final diagnosis after review of medical records and investigations. RESULTS: Providing the NT-proBNP result reduced diagnostic uncertainty, defined as a test score of 3-5, from 66% of cases to 18% of cases (P < 0.0001) and improved diagnostic accuracy from 53% to 71% (P = 0.016). CONCLUSION: Measurement of NT-proBNP concentrations reduces diagnostic uncertainty and improves diagnostic accuracy in patients presenting to the ED with dyspnoea and possible HF.