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 (54.3%) had already introduced TMB analysis for research purposes and/or clinical applications. Fifty laboratories (72.5%) used targeted sequencing, although a number of different panels were employed. Most laboratories tested formalin-fixed paraffin-embedded material (94.2%), while 18/69 (26%) tested also cell-free DNA. Fifty-five laboratories used both single nucleotide variants and indels for TMB calculation; 20 centers included only non-synonymous variants. In conclusion, the data from this survey indicate that multiple global laboratories were capable of rapidly introducing routine clinical TMB testing. However, the variability of testing methods raises concerns about the reproducibility of results among centers.

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. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories. This study compared the results obtained from MPS testing of ten diagnostic AML bone marrow aspirate samples sent to eight participating laboratories across Australasia. A reassuringly high concordance of 94% was observed with regard to variant detection and characterisation of pathogenicity. The level of discordance observed, although low, demonstrates the need for ongoing assessment of concordance between diagnostic testing laboratories through quality assurance programs.

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. MATERIALS AND METHODS: A set of simulated ctDNA samples with known variants at known allelic frequencies and a corresponding case scenario were distributed to 66 genetic testing laboratories for ctDNA analysis. Written reports were collected to evaluate the detection accuracy, reporting integrity, and information sufficiency using 21 predefined criteria. RESULTS: Current reporting practices for NGS-based ctDNA analysis were found to be far from satisfactory, especially regarding testing interpretation and methodological details. Only 42.4% of laboratories reported the results in complete concordance with the expected results. Moreover, 74.2% of reports only listed aberrations with direct and well-known treatment consequences for the tumor type in question. Genetic aberrations for which experimental agents and/or drug access programs are available may thus be overlooked. Furthermore, methodological details for the interpretation of results were missing from the majority of reports (87.9%). CONCLUSION: This proof-of-principle study suggests that the capacity for accurate identification of variants, rational interpretation of genotypes, comprehensive recommendation of potential medications, and detailed description of methodologies need to be further improved before ctDNA analysis can be formally implemented in the clinic. IMPLICATIONS FOR PRACTICE: Accurate, comprehensive, and standardized clinical sequencing reports can help to translate complex genetic information into patient-centered clinical decisions, thereby shepherding precision oncology into daily practice. However, standards, guidelines, and quality requirements for clinical reports of next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) analysis are currently absent. By using a set of simulated clinical ctDNA samples and a corresponding case scenario, current practices were evaluated to identify deficiencies in clinical sequencing reports of ctDNA analysis. The recommendations provided here may serve as a roadmap for the improved implementation of NGS-based ctDNA analysis in the clinic.

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. This study therefore evaluated the performance of diagnostic laboratories currently performing cfDNA testing in China, Australia and New Zealand. A total of 89 laboratories participated in this EQA program. Reference testing material comprised of cfDNA manufactured to contain six different genotypes in four different genes (EGFR, KRAS, BRAF, NRAS). The predicted genotypic variant allelic frequencies ranged between 0.5% - 2.5%. Proficiency testing used a z-score on the laboratory consensus allelic frequency data to compare laboratory performance for the detection of the different genotypes. Allelic frequency genotyping data were received from 88 of the 89 laboratories. Next generation sequencing and digital PCR testing platforms were primarily used by participants in this pilot EQA. The average consensus data for each cfDNA genotype identified allelic frequencies ranging between 0.39% - 4.4%. Z-score proficiency testing found that >92% of clinical laboratories were concordant for detecting the cfDNA variants. The data from this pilot study suggest that current cfDNA testing platforms can detect cfDNA allelic frequency variants from 0.39% and above with high levels of confidence. In addition, these data highlight the importance of laboratories enrolling on EQA programs so that proficiency in cfDNA diagnostic testing can be determined and potential sources of error identified and addressed.

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. In addition, multiple proteomic testing pipelines have not been fully assessed through external quality assurance (EQA). This trial was therefore devised to evaluate the performance of a small number of mass spectrometry (MS) testing facilities to (i) evaluate the EQA material for potential usage in a proteomic quality assurance program, and to (ii) identify key problem areas associated with human peptide testing. Five laboratories were sent six peptide reference testing samples formulated to contain a total of 35 peptides in differing ratios of light (natural) to heavy (labelled) peptides. Proficiency assessment of laboratory data used a modified approach to similarity and dissimilarity testing that was based on Bray-Curtis and Sorensen indices. Proficiency EQA concordant consensus values could not be derived from the assessed data since none of the laboratories correctly identified all reference testing peptides in all samples. However, the produced data may be reflective of specific inter-laboratory differences for detecting multiple peptides since no two testing pipelines used were the same for any laboratory. In addition, laboratory feedback indicated that peptide filtering of the reference material was a common key problem area prior to analysis. These data highlight the importance of an EQA programme for identifying underlying testing issues so that improvements can be made and confidence for clinical diagnostic analysis can be attained.

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. To date, there has been little scrutiny on the accuracy and reporting of results by laboratories new to HLA typing. In response to clinician feedback that identified potentially clinically significant discrepancies in HLA typing results, the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) undertook a pilot study to assess laboratory performance in the detection of HLA-DQ2/DQ8 and their associated HLA-DQA1 and HLA-DQB1 alleles. METHODS: DNA was extracted from 5 patients and sent to 10 laboratories for external quality assurance (EQA) testing. Laboratories were assessed for reporting in genotyping, interpretation and methodology. RESULTS: Our findings showed that at least 80% of laboratories underperform with respect to recommended guidelines for HLA typing and reporting for CD, with 40% of laboratories failing to provide any clinical interpretation or full genotyping data. This suboptimal level of reporting may lead to ambiguities for downstream clinical interpretation that may compromise patient management. CONCLUSIONS: These findings highlight the importance of adherence to standardised guidelines for optimal performance and reporting of HLA results and substantiate the need for EQA and proficiency testing for laboratories providing this service.

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. Lymphocytes in liver, spleen, Peyers patches, mesenteric lymph nodes, and inguinal lymph nodes were evaluated using flow cytometry. Low protein intake reversed aging changes in splenic CD4 and CD8 T cells, CD4:CD8 T cell ratio, memory/effector CD4 T cells and naïve CD4 T cells. A similar influence of total caloric intake in these ad-libitum fed mice was not apparent. Protein intake also influenced hepatic NK cells and B cells, while protein to carbohydrate ratio influenced hepatic NKT cells. Hepatosteatosis was associated with increased energy and fat intake and changes in hepatic Tregs, effector/memory T, and NK cells. Hepatic NK cells were also associated with body fat, glucose tolerance, and leptin levels while hepatic Tregs were associated with hydrogen peroxide production by hepatic mitochondria. Dietary macronutrients, particularly protein, influence splanchnic lymphocytes in old age, with downstream associations with mitochondrial function, liver pathology, and obesity-related phenotype.

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. Mitochondrial-nuclear crosstalk is critical for the maintenance of cellular homeostasis, and some mitochondrial DNA mutations may perturb crosstalk signaling. However, ascertaining whether mitochondrial malfunction is a cause or a consequence of disease development will be key to determining whether or not impaired crosstalk signaling is of direct pathological and hence therapeutic relevance. Here, we review what is known about the nuclear adaptive compensatory mechanisms induced in response to mitochondrial malfunction. We discuss the role of mitochondrial DNA variants in modulating the penetrance of human inherited disease caused by mutations in the nuclear genome and explore the underlying mechanisms by which they influence the retrograde response. We conclude that mitochondrial DNA variants have the potential to induce molecular signals through the mitochondrial-nuclear crosstalk mechanism, thereby promoting nuclear compensation in response to mitochondrial malfunction. The implications for the development of genetic or pharmaceutical interventions for the treatment of mitochondrial malfunction in complex disease are also explored.

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. Impaired proteosomal/lysosomal degradation can promote the uptake and accumulation of α-syn in enteric neurons. Enteric neurons exposed to α-syn can also lead to impaired mitochondrial complex I activity, reduced mitochondrial function, and NAD(+) depletion culminating in cell death via energy restriction. These findings demonstrate neuron-to-neuron transmission of α-syn in enteric neurons, providing renewed evidence for Braak's hypothesis and the aetiology of PD.

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.

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. These metrics were combined and interpreted for potential functional impact. A notable strength of the 3-D model is that it can interpret and predict the structural consequences of amino-acid variation in all 13 protein subunits. Importantly, the influence of compensatory changes can also be modeled. We examine mutations listed in the human mutation database Mitomap, and in 100 older men, and compare the results from the 3-D model against the automated MutPred web application tool. In combination, these comparisons suggest that the 3-D model predicts more functionally significant mutations than does MutPred. We conclude that the model has useful functional prediction capability but may need modification as functional data on specific mutations becomes known.

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. We then consider the utility of each methodology in studying three biological systems: isolated mitochondria, cultured cells, and cell fibers and tissues. Finally, we discuss the application of each methodology in the study of mitochondrial dysfunction in Alzheimer's disease, type 2 diabetes mellitus, and aging-associated autophagy impairment and mitochondrial malfunction. We conclude that the methodologies are complementary, and researchers may need to examine multiple biological systems to unravel complex diseases of aging.

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. Although a potential alternative splice site spans the location of the +1169 SNP, polymerase chain reaction-based assays failed to yield any evidence for alternative splicing associated with either allele. To determine whether the +1169 SNP, in different allelic combinations with SNPs at -278 (G/T), -57 (T/G) and +2103 (C/T), exerts a direct effect on gene expression and/or GH secretion, we performed a series of transfections of various GH1 haplotype-expressing constructs into rat GC (somatotroph) cells. The results obtained provided evidence to support the contention that the +1169A allele contributes directly to the observed reduction in both GH1 gene expression and GH secretion. Part of the apparent influence of the +1169A-bearing allele on GH1 gene expression and GH secretion may still, however, be attributable to alleles of additional SNPs in cis to +1169A and located within either the promoter or the 3'-flanking region.

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). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55alpha, B56alpha, B56gamma, and B56delta). Overexpression of PP2A-Aalpha in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT(+) cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT(+) cancers.

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. The technique has been successful in providing pathological gene expression profiles in a number of common genetic disorders including diabetes, cardiovascular disease, Parkinson disease and Down syndrome. When combined with next generation sequencing platforms, SAGE has the potential to become a more powerful and sensitive technique making it more amenable for diagnostic use. This review will therefore discuss the application of SAGE to several common genetic disorders and will further evaluate its potential use in diagnosing human genetic disease.

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). The average gene conversion tract length was estimated to be 84 bp. A gene conversion hotspot was identified, spanning the GH1 transcriptional initiation site (positions -6 to +25). Although these findings serve to highlight the importance of gene conversion for the recent evolution of the human GH1 promoter, its relative frequency does not appear to be related simply to the presence of specific DNA sequence motifs or secondary structures, the degree of homology between GH paralogs, the distance between them, or their transcriptional orientation. The GH1 promoter was also found to be highly polymorphic in chimpanzee but not in macaque. This may reflect the lower degree of pair-wise similarity between the GH1 promoter and its paralogs in macaque (mean, 92.0%) as compared to chimpanzee (93.5%) and human (94.0%), and hence provides further support for the idea of a threshold (perhaps around 92%) below which gene conversion is reduced or abolished.

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%. Although four of these variants were novel missense substitutions (Ala13Val, Arg19His, Phe25Tyr and Ser95Arg), none of these had any measurable effect on either GH function or secretion in vitro. Some 37 different GH1 promoter haplotypes were identified, 23 of which are as yet unreported in Europeans. The mean in vitro expression level of the GH1 promoter haplotypes observed in the African population was significantly higher than that previously measured in Britons (p<0.001). A gene conversion in the GH1 promoter, previously reported in a single individual of British origin, was found to occur at polymorphic frequency (5%) in the West-African population and was associated with a 1.7-fold increase in promoter activity relative to the wild-type. The d3 allele of the GHR exon 3 deletion polymorphism, known to be associated with increased GH responsiveness, was also found to occur at an elevated frequency in these individuals from Benin. We speculate that both elevated GH1 gene expression and increased GHR-mediated GH responsiveness may constitute adaptive responses to the effects of scarce food supply in this West-African population since increased circulating GH appears to form part of a physiological response to nutritional deprivation.

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. GH1 promoter haplotypes were found to differ significantly not only between hypertensive patients (n = 111) and controls (n = 121) but also between stroke patients (n = 155) and controls (n = 158). Intriguingly, the association between GH1 promoter haplotype and risk of hypertension was much greater in females than in males. An inverse correlation between height and central systolic blood pressure was apparent in both hypertensive patients and normal controls but was much stronger in individuals carrying at least one GH1 promoter risk haplotype. The GH1 genotype therefore constitutes a risk factor for hypertension that interacts with stature. A strong association was found between the presence of at least one GH1 risk haplotype and a family history of stroke at an early age (odds ratio: 9.07, 95% confidence interval: 1.14-72.22). Three novel GH variants (Arg16His, Phe176Cys, Cys189Arg) were identified during the course of this study. Although two exhibited markedly reduced biological activity in vitro, their clinical significance remains unclear. No association was found between GHR genotype and either hypertension or stroke, nor was any interaction noted between GHR and GH1 genotypes in terms of a disease association. However, an association between GHRd3 genotype and hypertension was observed among stroke patients, particularly females. Elevated HDL was found to be a risk factor for hypertension in individuals lacking a copy of the GHRd3 allele. Weak associations with GHR genotype were also noted for peripheral systolic and diastolic blood pressure in hypertensive patients. Although the underlying mechanisms are still unclear, our findings are consistent with a complex relationship between height, hypertension, GH1 promoter haplotype, GHR polymorphism and the risk of 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. The latter is likely to be of pathological significance since it is predicted to lead to the synthesis of a truncated protein. The functional significance of three of the other variants (14 bp del, C + 261G, G + 462C) was explored by luciferase reporter gene expression and electrophoretic mobility shift assays. The del14 variant demonstrated allele-specific protein binding without altered reporter gene expression and the G + 462C allele showed slightly decreased reporter gene expression.