Tumor mutation burden testing: a survey of the International Quality Network for Pathology (IQN Path).

While tumour mutation burden (TMB) is emerging as a possible biomarker for immune-checkpoint inhibitors (ICI), methods for testing have not been standardised as yet. In April 2019, the International Quality Network for Pathology (IQN Path) launched a survey to assess the current practice of TMB testing. Of the 127 laboratories that replied, 69 (54.

Laboratory quality assessment of candidate gene panel testing for acute myeloid leukaemia: a joint ALLG / RCPAQAP initiative.

Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches.

From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis.

Background: With the accelerated development of next-generation sequencing (NGS), identified variants, and targeted therapies, clinicians who confront the complicated and multifarious genetic information may not effectively incorporate NGS-based circulating tumor DNA (ctDNA) analysis into routine patient care. Consequently, standardized ctDNA testing reports are of vital importance. In an effort to guarantee high-quality reporting performance, we conducted an investigation of the current detection and reporting practices for NGS-based ctDNA analysis.

External Quality Assurance of Current Technology for the Testing of Cancer-Associated Circulating Free DNA Variants.

Liquid biopsy testing is rapidly emerging as a diagnostic means of identifying circulating free DNA (cfDNA) disease-associated variants. However, the reporting of cfDNA variants remains inconsistent due in part to the application of multiple testing pipelines which raise uncertainty about current cfDNA detection efficiency. External quality assurance (EQA) programs are required to monitor, evaluate and help improve laboratory performance for cfDNA variant detection and in clinical interpretation.

An external quality assurance trial to assess mass spectrometry protein testing facilities for identifying multiple human peptides.

The application of proteomic liquid chromatography mass spectrometry (LC-MS) for identifying proteins and peptides associated with human disease is rapidly growing in clinical diagnostics. However, the ability to accurately and consistently detect disease-associated peptides remains clinically uncertain. Variability in diagnostic testing occurs in part due to the absence of appropriate reference testing materials and standardised clinical guidelines for proteomic testing.

High rates of variation in HLA-DQ2/DQ8 testing for coeliac disease: results from an RCPAQAP pilot program.

Aim: Coeliac disease(CD) is a highly prevalent, gluten-dependent, autoimmune enteropathy. While the diagnosis is based on serological and histological criteria, genotyping of the human leucocyte antigens (HLA) DQ2 and DQ8 has been shown to have substantial clinical utility, especially in excluding the diagnosis in patients who do not carry either antigen. As a result, HLA genotyping is now being performed by more laboratories and has recently become one of the most frequently requested genetic tests in Australia.

The Influence of Macronutrients on Splanchnic and Hepatic Lymphocytes in Aging Mice.

There is a strong association between aging, diet, and immunity. The effects of macronutrients and energy intake on splanchnic and hepatic lymphocytes were studied in 15 month old mice. The mice were ad-libitum fed 1 of 25 diets varying in the ratios and amounts of protein, carbohydrate, and fat over their lifetime.

The emergence of the mitochondrial genome as a partial regulator of nuclear function is providing new insights into the genetic mechanisms underlying age-related complex disease.

Mitochondrial malfunction appears to be intimately associated with age and age-related complex disorders but the precise pathological relevance of such malfunction remains unclear. Mitochondrial, and more specifically bioenergetic, malfunction is commonly encountered in cancer, degenerative disorders and aging. The identification of a mitochondrial-nuclear retrograde signaling pathway in yeast has facilitated the study of the corresponding retrograde signaling mechanisms induced in response to mitochondrial malfunction in mammals including human.

Uptake and mitochondrial dysfunction of alpha-synuclein in human astrocytes, cortical neurons and fibroblasts.

The accumulation and aggregation of alpha-synuclein (α-syn) in several tissue including the brain is a major pathological hallmark in Parkinson's disease (PD). In this study, we show that α-syn can be taken up by primary human cortical neurons, astrocytes and skin-derived fibroblasts in vitro. Our findings that brain and peripheral cells exposed to α-syn can lead to impaired mitochondrial function, leading to cellular degeneration and cell death, provides additional evidence for the involvement of mitochondrial dysfunction as a mechanism of toxicity of α-syn in human cells.

Alpha-synuclein transmission and mitochondrial toxicity in primary human foetal enteric neurons in vitro.

Parkinson's disease (PD) is a multicentred neurodegenerative disorder characterised by the accumulation and aggregation of alpha-synuclein (α-syn) in several parts of the central nervous system. However, it is well established that PD can generate symptoms of constipation and other gastrointestinal problems and α-syn containing lesions have been identified in intestinal nerve cells. In this study, we show that α-syn can be taken up and accumulate in primary human foetal enteric neurons from the gastrointestinal tract and can be transferred between foetal enteric neurons.

Quaternary protein modeling to predict the function of DNA variation found in human mitochondrial cytochrome c oxidase.

Cytochrome c oxidase (COX) of the electron transport system is thought to be the rate-limiting step in cellular respiration and is found mutated in numerous human pathologies. Here, we employ quaternary three-dimensional (3-D) modeling to construct a model for human COX. The model was used to predict the functional consequences of amino-acid mutations based on phylogenetic conservation of amino acids together with volume and/or steric perturbations, participation in subunit-subunit interfaces and non-covalent energy loss or incompatibilities.

Review: quantifying mitochondrial dysfunction in complex diseases of aging.

There is accumulating evidence that mitochondrial respiratory malfunction is associated with aging-associated complex diseases. However, progress in our understanding of these diseases has been hampered by the sensitivity and throughput of systems employed to quantify dysfunction and inherent limitations of the biological systems studied. In this review, we describe and contrast two methodologies that have been developed for measuring mitochondrial function to address the need for improved sensitivity and increased throughput.

Characterisation of a functional intronic polymorphism in the human growth hormone (GH1) gene.

The +1169A allele of the A/T single nucleotide polymorphism (SNP; rs2665802), located within intron 4 of the human growth hormone 1 ( GH1 ) gene, has been associated with reduced levels of circulating GH and insulin-like growth factor 1, a reduced risk of colorectal cancer and a predisposition to osteoporosis. Whether this intronic SNP is itself the functional polymorphism responsible for exerting a direct effect on GH1 gene expression, however, or whether it is instead in linkage disequilibrium with the functional SNP, has been an open question. The evolutionary conservation of the +1169T allele (and the surrounding intronic sequence) in the bovine genome, as well as in primate genomes, is, however, suggestive of its functionality.

Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers.

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A).

Application of serial analysis of gene expression to the study of human genetic disease.

Sequence tag analysis using serial analysis of gene expression (SAGE) is a powerful strategy for the quantitative analysis of gene expression in human genetic disorders. SAGE facilitates the measurement of mRNA transcripts and generates a non-biased gene expression profile of normal and pathological disease tissue. In addition, the SAGE technique has the capacity of detecting the expression of novel transcripts allowing for the identification of previously uncharacterised genes, thus providing a unique advantage over the traditional microarray-based approach for expression profiling.

Growth hormone (GH1) gene variation and the growth hormone receptor (GHR) exon 3 deletion polymorphism in a West-African population.

Among Europeans, functionally significant GH1 gene variants occur not only in individuals with idiopathic growth hormone (GH) deficiency and/or short stature but also fairly frequently in the general population. To assess the generality of these findings, 163 individuals from Benin, West Africa were screened for mutations and polymorphisms in their GH1 genes. A total of 37 different sequence variants were identified in the GH1 gene region, 24 of which occurred with a frequency of >1%.

A gene conversion hotspot in the human growth hormone (GH1) gene promoter.

To assess the evolutionary importance of nonallelic (or interlocus) gene conversion for the highly polymorphic human growth hormone (GH1) gene promoter, sequence variation in this region was studied in four different ethnic groups. For 14 SNPs in the proximal GH1 promoter (535 bp), 60 different haplotypes were observed in 577 individuals (156 Britons, 116 Spaniards, 163 West-Africans, 142 Asians). Using a novel coalescence-based statistical test, significant evidence was found in the British, Spanish, and African groups for GH1 having acted as an acceptor of gene conversion, with at least one of the four paralogous GH gene promoters serving as the donor (and specifically GH2 in the Britons and Spaniards).

Genetic variation at the growth hormone (GH1) and growth hormone receptor (GHR) loci as a risk factor for hypertension and stroke.

An increased prevalence of both hypertension and cerebrovascular stroke is apparent in growth hormone (GH) deficiency whilst hypertension is a frequent complication in acromegaly. This has suggested a possible link between GH, stature and arterial function. Since the risk of both hypertension and stroke also appears to be inversely correlated with adult height, we have instigated an exploratory study to assess whether inter-individual variation in the genes encoding human growth hormone (GH1) and the GH receptor (GHR) might be associated with an increased risk of hypertension and stroke.

Functional analysis of polymorphic variation within the promoter and 5' untranslated region of the neurofibromatosis type 1 (NF1) gene.

The regulatory regions of the neurofibromatosis type 1 (NF1) gene have scarcely been screened either for mutations of potential pathological importance or for functional polymorphisms. To address this question, a 987 bp sequence spanning the promoter and 5' flanking sequence of the human NF1 gene was screened for sequence variants in 570 unrelated NF1 patients and 105 controls. Five novel sequence variants were identified, comprising a 14 bp deletion at -142 within the promoter region, three single nucleotide substitutions in the 5'UTR (C + 247T, C + 261G, G + 462C), and a substitution (C + 514T) at the 5' end of the coding region that served to generate a Stop codon.

A novel dysfunctional growth hormone variant (Ile179Met) exhibits a decreased ability to activate the extracellular signal-regulated kinase pathway.

The pituitary-expressed GH1 gene was screened for mutation in a group of 74 children with familial short stature. Two novel mutations were identified: an Ile179Met substitution and a -360A-->G promoter variant. The Ile179Met variant was shown to exhibit a similar degree of resistance to proteolysis as wild-type GH, indicating that the introduction of Met does not cause significant misfolding.

Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors.

One of the main features of neurofibromatosis type 1 (NF1) is benign neurofibromas, 10-20% of which become transformed into malignant peripheral nerve sheath tumors (MPNSTs). The molecular basis of NF1 tumorigenesis is, however, still unclear. Ninety-one tumors from 31 NF1 patients were screened for gross changes in the NF1 gene using microsatellite/restriction fragment length polymorphism (RFLP) markers; loss of heterozygosity (LOH) was found in 17 out of 91 (19%) tumors (including two out of seven MPNSTs).

Novel mutations of the growth hormone 1 (GH1) gene disclosed by modulation of the clinical selection criteria for individuals with short stature.

Subtle mutations in the growth hormone 1 (GH1) gene have been regarded as a comparatively rare cause of short stature. Such lesions were sought in a group of 41 individuals selected for short stature, reduced height velocity, and bone age delay; a group of 11 individuals with short stature and idiopathic growth hormone deficiency (IGHD); and a group of 154 controls. Heterozygous mutations were identified in all three groups but disproportionately in the individuals with short stature, both with (odds ratio 25.

Human growth hormone 1 (GH1) gene expression: complex haplotype-dependent influence of polymorphic variation in the proximal promoter and locus control region.

The proximal promoter region of the human pituitary expressed growth hormone (GH1) gene is highly polymorphic, containing at least 15 single nucleotide polymorphisms (SNPs). This variation is manifest in 40 different haplotypes, the high diversity being explicable in terms of gene conversion, recurrent mutation, and selection. Functional analysis showed that 12 haplotypes were associated with a significantly reduced level of reporter gene expression whereas 10 haplotypes were associated with a significantly increased level.