Breakpoint characterization of a rare alpha0 -thalassemia deletion using targeted locus amplification on genomic DNA.

INTRODUCTION: The high-sequence homology of the α-globin-gene cluster is responsible for microhomology-mediated recombination events during meiosis, resulting in a high density of deletion breakpoints within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation-dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long-range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site. METHODS: Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment-specific inverse primers, creating a library that is suitable for Illumina sequencing. RESULTS: Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results. CONCLUSIONS: The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha-globin cluster. The deletion was described only once in an earlier study as the --gb , but as it was not registered correctly in the available databases, it was not initially recognized as such.

Rare variants in non-coding regulatory regions of the genome that affect gene expression in systemic lupus erythematosus.

Personalized medicine approaches are increasingly sought for diseases with a heritable component. Systemic lupus erythematosus (SLE) is the prototypic autoimmune disease resulting from loss of immunologic tolerance, but the genetic basis of SLE remains incompletely understood. Genome wide association studies (GWAS) identify regions associated with disease, based on common single nucleotide polymorphisms (SNPs) within them, but these SNPs may simply be markers in linkage disequilibrium with other, causative mutations. Here we use an hierarchical screening approach for prediction and testing of true functional variants within regions identified in GWAS; this involved bioinformatic identification of putative regulatory elements within close proximity to SLE SNPs, screening those regions for potentially causative mutations by high resolution melt analysis, and functional validation using reporter assays. Using this approach, we screened 15 SLE associated loci in 143 SLE patients, identifying 7 new variants including 5 SNPs and 2 insertions. Reporter assays revealed that the 5 SNPs were functional, altering enhancer activity. One novel variant was linked to the relatively well characterized rs9888739 SNP at the ITGAM locus, and may explain some of the SLE heritability at this site. Our study demonstrates that non-coding regulatory elements can contain private sequence variants affecting gene expression, which may explain part of the heritability of SLE.

Skewed X-inactivation is common in the general female population.

X-inactivation is a well-established dosage compensation mechanism ensuring that X-chromosomal genes are expressed at comparable levels in males and females. Skewed X-inactivation is often explained by negative selection of one of the alleles. We demonstrate that imbalanced expression of the paternal and maternal X-chromosomes is common in the general population and that the random nature of the X-inactivation mechanism can be sufficient to explain the imbalance. To this end, we analyzed blood-derived RNA and whole-genome sequencing data from 79 female children and their parents from the Genome of the Netherlands project. We calculated the median ratio of the paternal over total counts at all X-chromosomal heterozygous single-nucleotide variants with coverage ≥10. We identified two individuals where the same X-chromosome was inactivated in all cells. Imbalanced expression of the two X-chromosomes (ratios ≤0.35 or ≥0.65) was observed in nearly 50% of the population. The empirically observed skewing is explained by a theoretical model where X-inactivation takes place in an embryonic stage in which eight cells give rise to the hematopoietic compartment. Genes escaping X-inactivation are expressed from both alleles and therefore demonstrate less skewing than inactivated genes. Using this characteristic, we identified three novel escapee genes (SSR4, REPS2, and SEPT6), but did not find support for many previously reported escapee genes in blood. Our collective data suggest that skewed X-inactivation is common in the general population. This may contribute to manifestation of symptoms in carriers of recessive X-linked disorders. We recommend that X-inactivation results should not be used lightly in the interpretation of X-linked variants.

Reduced PRC2 function alters male germline epigenetic programming and paternal inheritance.

BACKGROUND: Defining the mechanisms that establish and regulate the transmission of epigenetic information from parent to offspring is critical for understanding disease heredity. Currently, the molecular pathways that regulate epigenetic information in the germline and its transmission to offspring are poorly understood. RESULTS: Here we provide evidence that Polycomb Repressive Complex 2 (PRC2) regulates paternal inheritance. Reduced PRC2 function in mice resulted in male sub-fertility and altered epigenetic and transcriptional control of retrotransposed elements in foetal male germ cells. Males with reduced PRC2 function produced offspring that over-expressed retrotransposed pseudogenes and had altered preimplantation embryo cleavage rates and cell cycle control. CONCLUSION: This study reveals a novel role for the histone-modifying complex, PRC2, in paternal intergenerational transmission of epigenetic effects on offspring, with important implications for understanding disease inheritance.

Loss of maternal EED results in postnatal overgrowth.

BACKGROUND: Investigating how epigenetic information is transmitted through the mammalian germline is the key to understanding how this information impacts on health and disease susceptibility in offspring. EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes. RESULTS: In this study, we used oocyte-specific Zp3-Cre recombinase (Zp3Cre) to delete Eed specifically in mouse growing oocytes, permitting the study of EED function in oocytes and the impact of depleting EED in oocytes on outcomes in offspring. As EED deletion occurred only in growing oocytes and females were mated to normal wild type males, this model allowed the study of oocyte programming without confounding factors such as altered in utero environment. Loss of EED from growing oocytes resulted in a significant overgrowth phenotype that persisted into adult life. Significantly, this involved increased adiposity (total fat) and bone mineral density in offspring. Similar overgrowth occurs in humans with Cohen-Gibson (OMIM 617561) and Weaver (OMIM 277590) syndromes, that result from de novo germline mutations in EED or its co-factor EZH2, respectively. Consistent with a role for EZH2 in human oocytes, we demonstrate that de novo germline mutations in EZH2 occurred in the maternal germline in some cases of Weaver syndrome. However, deletion of Ezh2 in mouse oocytes resulted in a distinct phenotype compared to that resulting from oocyte-specific deletion of Eed. CONCLUSIONS: This study provides novel evidence that altering EED-dependent oocyte programming leads to compromised offspring growth and development in the next generation.

Critical points for an accurate human genome analysis.

Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.

Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing.

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.

Genotyping Multiallelic Copy Number Variation with Multiplex Ligation-Dependent Probe Amplification (MLPA).

Multiallelic copy number variants are genomic loci that can be present in a range of different copy numbers between individuals. High or low copy numbers of specific genes have been associated with different diseases. Precise genotyping of these loci can be complicated, and relies on accurate assays. Multiplex ligation-dependent probe amplification (MLPA) is a PCR-based approach that allows copy number determination of up to 50 genomic loci in a single reaction. In this chapter, we outline the basic protocol, with a particular emphasis on the appropriate approach to accurately genotype multiallelic copy numbers.

Quantitative DNA Analysis Using Droplet Digital PCR.

Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries.

Targeted Locus Amplification and Next-Generation Sequencing.

Despite developments in targeted and whole-genome sequencing, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Targeted locus amplification (TLA) is a cross-linking-based technique that generates complex DNA libraries covering >100 kb of contiguous sequence surrounding one primer pair complementary to a short locus-specific sequence. In combination with next-generation sequencing, TLA enables the complete sequencing and haplotyping of targeted regions of interest. Here we outline the basis of TLA, together with a detailed protocol of the technique.

Multiallelic copy number variation in the complement component 4A (C4A) gene is associated with late-stage age-related macular degeneration (AMD).

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of vision loss in Western societies with a strong genetic component. Candidate gene studies as well as genome-wide association studies strongly implicated genetic variations in complement genes to be involved in disease risk. So far, no association of AMD with complement component 4 (C4) was reported probably due to the complex nature of the C4 locus on chromosome 6. METHODS: We used multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of the C4 gene as well as of both relevant isoforms, C4A and C4B, and assessed their association with AMD using logistic regression models. RESULTS: Here, we report on the analysis of 2645 individuals (1536 probands and 1109 unaffected controls), across three different centers, for multiallelic copy number variation (CNV) at the C4 locus. We find strong statistical significance for association of increased copy number of C4A (OR 0.81 (0.73; 0.89);P = 4.4 × 10(-5)), with the effect most pronounced in individuals over 78 years (OR 0.67 (0.55; 0.81)) and females (OR 0.77 (0.68; 0.87)). Furthermore, this association is independent of known AMD-associated risk variants in the nearby CFB/C2 locus, particularly in females and in individuals over 78 years. CONCLUSIONS: Our data strengthen the notion that complement dysregulation plays a crucial role in AMD etiology, an important finding for early intervention strategies and future therapeutics. In addition, for the first time, we provide evidence that multiallelic CNVs are associated with AMD pathology.

Hormonal evaluation in relation to phenotype and genotype in 286 patients with a disorder of sex development from Indonesia.

OBJECTIVE: The objective of this study was to determine the aetiological spectrum of disorders of sex development (DSD) in a large cohort of underprivileged and undiagnosed patients from Indonesia. METHODS: A total of 286 patients with atypical external and/or internal genitalia were evaluated using clinical, hormonal, molecular genetic and histological parameters. RESULTS: The age (years) at presentation was 0-0·5 in 41 (14·3%), >0·5-12 in 181 (63·3%) and >12 in 64 cases (22·4%). 46,XY DSD was most common (68·2%, n = 195), 46,XX DSD was found in 23·4% (n = 67) and sex chromosomal DSD in 8·4% (n = 24). In 61·2% of 46,XX DSD patients, 17·9% of 46,XY DSD patients and all sex chromosome DSD patients (29·4% in total), a final diagnosis was reached based on genetic or histological gonadal tissue evaluation. 17-hydroxyprogesterone and androstenedione levels were the most distinctive parameters in 46,XX DSD patients. In 46,XY DSD, diagnostic groups were identified based on the external masculinization score: androgen action disorder (AAD), unknown male undermasculinization (UMU), and gonadal dysgenesis (GD). LH, FSH and testosterone levels were most informative especially in the older age group. HCG tests were of no additional value as no patients with androgen synthesis disorders were found. Hormonal profiles of patients with sex chromosome DSD and a Y-chromosome sequence containing karyotype showed high levels of LH and FSH, and low levels of AMH, inhibin B and testosterone compared with the normal male range. Gene mutations were found in all patients with CAH, but in only 24·5% and 1·8% of patients with AAD and UMU. In 32% of 46,XY GD patients, copy number variants of different genes were found. CONCLUSION: A stepwise diagnostic approach led to a molecularly or histologically proven final diagnosis in 29·4% of the patients. The most informative parameters were serum levels of 17-hydroxyprogesterone and androstenedione in 46,XX DSD patients, and serum LH, FSH and testosterone levels in 46,XY DSD patients.

Glucocorticoid-induced leucine zipper (GILZ) inhibits B cell activation in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids. METHODS: We conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice. RESULTS: Reduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli. CONCLUSIONS: Our findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

Deletion of the Complex I Subunit NDUFS4 Adversely Modulates Cellular Differentiation.

The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth. Symptoms include motor and intellectual retardation, often accompanied by dystonia, ataxia, and growth retardation, and most patients die by 3 years of age. To understand the origins of this disease, we have generated a series of mouse embryonic stem cell lines from blastocysts that were wild type, heterozygous, and homozygous for the deletion of the Ndufs4 gene. We have demonstrated their pluripotency and potential to differentiate into all cell types of the body. Although the loss of Ndufs4 did not affect the stability of the mitochondrial and nuclear genomes, there were significant differences in patterns of chromosomal gene expression following both spontaneous differentiation and directed neural differentiation into astrocytes. The defect also affected the potential of the cells to generate beating embryoid bodies. These outcomes demonstrate that defects associated with Complex I deficiency affect early gene expression patterns, which escalate during early and later stages of differentiation and are mediated by the defect and not other chromosomal or mitochondrial DNA defects.

BTB-ZF transcriptional regulator PLZF modifies chromatin to restrain inflammatory signaling programs.

Inflammation is critical for host defense, but without appropriate control, it can cause chronic disease or even provoke fatal responses. Here we identify a mechanism that limits the inflammatory response. Probing the responses of macrophages to the key sensory Toll-like receptors, we identify that the Broad-complex, Tramtrack and Bric-a-brac/poxvirus and zinc finger (BTB/POZ), transcriptional regulator promyelocytic leukemia zinc finger (PLZF) limits the expression of inflammatory gene products. In accord with this finding, PLZF-deficient animals express higher levels of potent inflammatory cytokines and mount exaggerated inflammatory responses to infectious stimuli. Temporal quantitation of inflammatory gene transcripts shows increased gene induction in the absence of PLZF. Genome-wide analysis of histone modifications distinguish that PLZF establishes basal activity states of early response genes to maintain immune homeostasis and limit damaging inflammation. We show that PLZF stabilizes a corepressor complex that encompasses histone deacetylase activity to control chromatin. Together with our previous demonstration that PLZF promotes the antiviral response, these results suggest a strategy that could realize one of the major goals of immune therapy to retain immune resistance to pathogens while curbing damaging inflammation.

Copy number variation associated with meiotic arrest in idiopathic male infertility.

OBJECTIVE: To assess the association between copy number variations (CNVs) and meiotic arrest and azoospermic men. DESIGN: Genetic association study. SETTING: University. PATIENT(S): Australian men: 19 with histologically confirmed meiotic arrest, 110 men with azoospermia in the absence of histologic data, and 97 fertile men (controls). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The identification of CNV by microarray and/or multiplex ligation-dependent probe amplification (MLPA), and the localization of unique CNV encoded proteins to the human testis. RESULT(S): Microarray identified two CNVs unique to meiosis arrest patients. One containing the MYRIP gene and a second containing LRRC4C and the long noncoding RNA LOC100507205. All three genes are transcribed in the human testis, and MYRIP and LRRC4C localize to meiotic cells. The reverse genetic screen for CNVs in meiosis genes identified in mouse models further identified CNVs including HSPA2 as being associated with azoospermia. CONCLUSION(S): These data raise the possibility that, while relatively rare, CNVs may contribute to human male infertility and that CNV screening should be incorporated into long-term plans for genome profiling as a diagnostic tool.

Seminoma and embryonal carcinoma footprints identified by analysis of integrated genome-wide epigenetic and expression profiles of germ cell cancer cell lines.

BACKGROUND: Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments. RESULTS: This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape. CONCLUSION: The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines.

Technical considerations for genotyping multi-allelic copy number variation (CNV), in regions of segmental duplication.

BACKGROUND: Intrachromosomal segmental duplications provide the substrate for non-allelic homologous recombination, facilitating extensive copy number variation in the human genome. Many multi-copy gene families are embedded within genomic regions with high levels of sequence identity (>95%) and therefore pose considerable analytical challenges. In some cases, the complexity involved in analyzing such regions is largely underestimated. Rapid, cost effective analysis of multi-copy gene regions have typically implemented quantitative approaches, however quantitative data are not an absolute means of certainty. Therefore any technique prone to degrees of measurement error can produce ambiguous results that may lead to spurious associations with complex disease. RESULTS: In this study we have focused on testing the accuracy and reproducibility of quantitative analysis techniques. With reference to the C-C Chemokine Ligand-3-like-1 (CCL3L1) gene, we performed analysis using real-time Quantitative PCR (QPCR), Multiplex Ligation-dependent Probe Amplification (MLPA) and Paralogue Ratio Test (PRT). After controlling for potential outside variables on assay performance, including DNA concentration, quality, preparation and storage conditions, we find that real-time QPCR produces data that does not cluster tightly around copy number integer values, with variation substantially greater than that of the MLPA or PRT systems. We find that the method of rounding real-time QPCR measurements can potentially lead to mis-scoring of copy number genotypes and suggest caution should be exercised in interpreting QPCR data. CONCLUSIONS: We conclude that real-time QPCR is inherently prone to measurement error, even under conditions that would seem favorable for association studies. Our results indicate that potential variability in the physicochemical properties of the DNA samples cannot solely explain the poor performance exhibited by the real-time QPCR systems. We recommend that more robust approaches such as PRT or MLPA should be used to genotype multi-allelic copy number variation in disease association studies and suggest several approaches which can be implemented to ensure the quality of the copy number typing using quantitative methods.

The identification of mitochondrial DNA variants in glioblastoma multiforme.

BACKGROUND: Mitochondrial DNA (mtDNA) encodes key proteins of the electron transfer chain (ETC), which produces ATP through oxidative phosphorylation (OXPHOS) and is essential for cells to perform specialised functions. Tumor-initiating cells use aerobic glycolysis, a combination of glycolysis and low levels of OXPHOS, to promote rapid cell proliferation and tumor growth. Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor and mitochondria have been proposed to play a vital role in GBM tumorigenesis. RESULTS: Using next generation sequencing and high resolution melt analysis, we identified a large number of mtDNA variants within coding and non-coding regions of GBM cell lines and predicted their disease-causing potential through in silico modeling. The frequency of variants was greatest in the D-loop and origin of light strand replication in non-coding regions. ND6 was the most susceptible coding gene to mutation whilst ND4 had the highest frequency of mutation. Both genes encode subunits of complex I of the ETC. These variants were not detected in unaffected brain samples and many have not been previously reported. Depletion of HSR-GBM1 cells to varying degrees of their mtDNA followed by transplantation into immunedeficient mice resulted in the repopulation of the same variants during tumorigenesis. Likewise, de novo variants identified in other GBM cell lines were also incorporated. Nevertheless, ND4 and ND6 were still the most affected genes. We confirmed the presence of these variants in high grade gliomas. CONCLUSIONS: These novel variants contribute to GBM by rendering the ETC. partially dysfunctional. This restricts metabolism to anaerobic glycolysis and promotes cell proliferation.

No TAP63 promoter mutation is detected in bladder exstrophy-epispadias complex patients.

BACKGROUND/PURPOSE: Bladder exstrophy-epispadias complex (BEEC) is thought to have a genetic component in its pathogenesis. Previously we found that p63(-/-) mice show increased ventral apoptosis and develop a BEEC phenotype. Down-regulation of the anti-apoptotic ΔNP63 and an up-regulation of pro-apoptotic TAP63 isoforms have been demonstrated in BEEC patient bladder tissues. We have previously shown that insertion/deletion polymorphisms of the ΔNp63 promoter are associated with an increased risk of BEEC. In this study, we specifically examined the TAP63 promoter to see if any sequence changes might lead to up-regulation of TAP63 and exaggerated apoptosis in BEEC patients. METHODS: i) Bioinformatic analysis of the TAP63 promoter was performed to identify putative regulatory regions. ii) High-resolution Melt and Sanger sequencing was used to screen targeted regions in 112 BEEC patient DNA samples for potential sequence variants. iii) Sequence variation was analysed for significance against normal population frequency data. RESULTS: i) We identified multiple epigenetic markers of transcriptional regulation within highly conserved areas of the TAP63 promoter sequence. ii) Of the 112 buccal swab DNA samples, adequate and successful screening ranged between 48 and 67 for each region. iii) No novel sequence variation or mutation was uncovered. iv) Two known SNPs were identified. However, allele frequency analysis was not statistically significant. CONCLUSION: Our data do not associate genetic variation within the TAP63 promoter region with an increased risk of BEEC. Our data so far suggests that only ΔNP63 promoter aberration is involved in BEEC pathogenesis.