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Thousand and one amino‐acid kinase 1 (TAOK1) is a MAP3K protein kinase, regulating different mitogen‐activated protein kinase pathways, thereby modulating a multitude of processes in the cell. Given the recent finding of TAOK1 involvement in neurodevelopmental disorders (NDDs), we investigated the role of TAOK1 in neuronal function and collected a cohort of 23 individuals with mostly de novo variants in TAOK1 to further define the associated NDD. Here, we provide evidence for an important role for TAOK1 in neuronal function, showing that altered TAOK1 expression levels in the embryonic mouse brain affect neural migration in vivo, as well as neuronal maturation in vitro. The molecular spectrum of the identified TAOK1 variants comprises largely truncating and nonsense variants, but also missense variants, for which we provide evidence that they can have a loss of function or dominant‐negative effect on TAOK1, expanding the potential underlying causative mechanisms resulting in NDD. Taken together, our data indicate that TAOK1 activity needs to be properly controlled for normal neuronal function and that TAOK1 dysregulation leads to a neurodevelopmental disorder mainly comprising similar facial features, developmental delay/intellectual disability and/or variable learning or behavioral problems, muscular hypotonia, infant feeding difficulties, and growth problems., In this study we expand the cohort of individuals with a neurodevelopmental disorder, carrying a de novo variant in TAOK1 (a), thereby further defining the neurodevelopmental disorder caused by TAOK1 malfunctioning. Using both in vivo (b) and in vitro (c) functional assays, we provide evidence that increased as well as decreased levels of TAOK1 cause disruption of neuronal development, showing that TAOK1 plays an important role in neuronal function. Additionally, our data suggests that both gain of function as well as loss of function mutations are potentially causative for the TAOK1‐related neurodevelopmental disorder.

The SET domain containing 2, histone lysine methyltransferase encoded by SETD2 is a dual-function methyltransferase for histones and microtubules and plays an important role for transcriptional regulation, genomic stability, and cytoskeletal functions. Specifically, SETD2 is associated with trimethylation of histone H3 at lysine 36 (H3K36me3) and methylation of α-tubulin at lysine 40. Heterozygous loss of function and missense variants have previously been described with Luscan-Lumish syndrome (LLS), which is characterized by overgrowth, neurodevelopmental features, and absence of overt congenital anomalies. We have identified 15 individuals with de novo variants in codon 1740 of SETD2 whose features differ from those with LLS. Group 1 consists of 12 individuals with heterozygous variant c.5218C>T p.(Arg1740Trp) and Group 2 consists of 3 individuals with heterozygous variant c.5219G>A p.(Arg1740Gln). The phenotype of Group 1 includes microcephaly, profound intellectual disability, congenital anomalies affecting several organ systems, and similar facial features. Individuals in Group 2 had moderate to severe intellectual disability, low normal head circumference, and absence of additional major congenital anomalies. While LLS is likely due to loss of function of SETD2, the clinical features seen in individuals with variants affecting codon 1740 are more severe suggesting an alternative mechanism, such as gain of function, effects on epigenetic regulation, or posttranslational modification of the cytoskeleton. Our report is a prime example of different mutations in the same gene causing diverging phenotypes and the features observed in Group 1 suggest a new clinically recognizable syndrome uniquely associated with the heterozygous variant c.5218C>T p.(Arg1740Trp) in SETD2.

Introduction: Chronic kidney disease (CKD) is a major public health issue in the USA. Identification of monogenic causes of CKD, which are present in ∼10% of adult cases, can impact prognosis and patient management. Broad gene panels can provide unbiased testing approaches, which are advantageous in phenotypically heterogeneous diseases. However, the use and yield of broad genetic panels by nephrologists in clinical practice is not yet well characterized. Methods: Renal genetic testing, ordered exclusively for clinical purposes, predominantly by general and transplant nephrologists within the USA, was performed on 1,007 consecutive unique patient samples. Testing was performed using a commercially available next-generation sequencing-based 382 gene kidney disease panel. Pathogenic (P) and likely pathogenic (LP) variants were reported. Positive findings included a monoallelic P/LP variant in an autosomal dominant or X-linked gene and biallelic P/LP variants in autosomal recessive genes. Results: Positive genetic findings were identified in 21.1% (212/1,007) of cases. A total of 220 positive results were identified across 48 genes. Positive results occurred most frequently in the PKD1 (34.1%), COL4A5 (10.9%), PKD2 (10.0%), COL4A4 (6.4%), COL4A3 (5.9%), and TTR (4.1%) genes. Variants identified in the remaining 42 genes comprised 28.6% of the total positive findings, including single positive results in 26 genes. Positive results in >1 gene were identified in 7.5% (16/212) of cases. Conclusions: Use of broad panel genetic testing by clinical nephrologists had a high success rate, similar to results obtained by academic centers specializing in genetics.

PURPOSE: We sought to delineate the genotypic and phenotypic spectrum of female and male individuals with X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). METHODS: Twenty-five individuals (15 males, 10 females) with causative variants in MSL3 were ascertained through exome or genome sequencing at ten different sequencing centers. RESULTS: We identified multiple variant types in MSL3 (ten nonsense, six frameshift, four splice site, three missense, one in-frame-deletion, one multi-exon deletion), most proven to be de novo, and clustering in the terminal eight exons suggesting that truncating variants in the first five exons might be compensated by an alternative MSL3 transcript. Three-dimensional modeling of missense and splice variants indicated that these have a deleterious effect. The main clinical findings comprised developmental delay and intellectual disability ranging from mild to severe. Autism spectrum disorder, muscle tone abnormalities, and macrocephaly were common as well as hearing impairment and gastrointestinal problems. Hypoplasia of the cerebellar vermis emerged as a consistent magnetic resonance image (MRI) finding. Females and males were equally affected. Using facial analysis technology, a recognizable facial gestalt was determined. CONCLUSION: Our aggregated data illustrate the genotypic and phenotypic spectrum of X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). Our cohort improves the understanding of disease related morbidity and allows us to propose detailed surveillance guidelines for affected individuals.

NCKAP1/NAP1 regulates neuronal cytoskeletal dynamics and is essential for neuronal differentiation in the developing brain. Deleterious variants in NCKAP1 have been identified in individuals with autism spectrum disorder (ASD) and intellectual disability; however, its clinical significance remains unclear. To determine its significance, we assemble genotype and phenotype data for 21 affected individuals from 20 unrelated families with predicted deleterious variants in NCKAP1. This includes 16 individuals with de novo (n = 8), transmitted (n = 6), or inheritance unknown (n = 2) truncating variants, two individuals with structural variants, and three with potentially disruptive de novo missense variants. We report a de novo and ultra-rare deleterious variant burden of NCKAP1 in individuals with neurodevelopmental disorders which needs further replication. ASD or autistic features, language and motor delay, and variable expression of intellectual or learning disability are common clinical features. Among inherited cases, there is evidence of deleterious variants segregating with neuropsychiatric disorders. Based on available human brain transcriptomic data, we show that NCKAP1 is broadly and highly expressed in both prenatal and postnatal periods and demostrate enriched expression in excitatory neurons and radial glias but depleted expression in inhibitory neurons. Mouse in utero electroporation experiments reveal that Nckap1 loss of function promotes neuronal migration during early cortical development. Combined, these data support a role for disruptive NCKAP1 variants in neurodevelopmental delay/autism, possibly by interfering with neuronal migration early in cortical development.

De novo pathogenic variants in the additional sex combs-like 3 (ASXL3) gene cause a rare multi-systemic neurodevelopmental disorder. There is growing evidence that germline and somatic mosaicism are more common and play a greater role in genetic disorders than previously acknowledged. There is one previous report of ASXL3-related syndrome caused by de novo pathogenic variants in two siblings suggesting gonadal mosaicism. In this report, we present five patients with ASXL3-related syndrome, describing two families comprising two non-twin siblings harbouring apparent de novo pathogenic variants in ASXL3. Parents were clinically unaffected and there was no evidence of mosaicism from genomic DNA on exome-trio data, suggesting germline mosaicism in one of the parents. We also describe clinical details of a patient with typical features of ASXL3-related syndrome and mosaic de novo pathogenic variant in ASXL3 in 30-35% of both blood and saliva sample on trio-exome sequencing. We expand the known genetic basis of ASXL3-related syndromes and discuss mosaicism as a disease mechanism in five patients from three unrelated families. The findings of this report highlight the importance of taking gonadal mosaicism into consideration when counselling families regarding recurrence risk. We also discuss postzygotic mosaicism as a cause of fully penetrant ASXL3-related syndrome.

Key Clinical Message A research study utilizing whole‐genome sequence analysis for preconception carrier screening provided a genome‐first detection of a severe de novo Factor VIII mutation in a woman with implications for pregnancy management and life‐saving interventions of her newborn son, and a challenge to the existing paradigm regarding carrier testing.

Activation of the aryl hydrocarbon receptor (AhR) by immunosuppressive ligands promotes the development of regulatory T (Treg) cells. Although AhR-induced Foxp3+ Treg cells have been well studied, much less is known about the development and fate of AhR-induced Type 1 Treg (AhR-Tr1) cells. In the current study, we identified the unique transcriptional and functional changes in murine CD4+ T cells that accompany the differentiation of AhR-Tr1 cells during the CD4+ T-cell-dependent phase of an allospecific cytotoxic T lymphocyte (allo-CTL) response. AhR activation increased the expression of genes involved in T-cell activation, immune regulation and chemotaxis, as well as a global downregulation of genes involved in cell cycling. Increased IL-2 production was responsible for the early AhR-Tr1 activation phenotype previously characterized as CD25+CTLA4+GITR+ on day 2. The AhR-Tr1 phenotype was further defined by the coexpression of the immunoregulatory receptors Lag3 and Tim3 and non-overlapping expression of CCR4 and CCR9. Consistent with the increased expression of CCR9, real-time imaging showed enhanced migration of AhR-Tr1 cells to the lamina propria of the small intestine and colon. The discovery of mucosal imprinting of AhR-Tr1 cells provides an additional mechanism by which therapeutic AhR ligands can control immunopathology. This article is protected by copyright. All rights reserved

While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization. Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free-text responses that were later clarified with phone interviews. Twenty-one laboratories participated. Practices highly concordant across all groups included consent documentation, multiperson case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing, or patient access to data. This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas of development in best practice guidelines that may be helpful.Genet Med advance online publication 03 Novemeber 2016.

Advances in sequencing technologies permit the analysis of a larger selection of genes for preconception carrier screening. The study was designed as a sequential carrier screen using genome sequencing to analyze 728 gene-disorder pairs for carrier and medically actionable conditions in 131 women and their partners (n = 71) who were planning a pregnancy. We report here on the clinical laboratory results from this expanded carrier screening program. Variants were filtered and classified using the latest American College of Medical Genetics and Genomics (ACMG) guideline; only pathogenic and likely pathogenic variants were confirmed by orthologous methods before being reported. Novel missense variants were classified as variants of uncertain significance. We reported 304 variants in 202 participants. Twelve carrier couples (12/71 couples tested) were identified for common conditions; eight were carriers for hereditary hemochromatosis. Although both known and novel variants were reported, 48% of all reported variants were missense. For novel splice-site variants, RNA-splicing assays were performed to aid in classification. We reported ten copy-number variants and five variants in non-coding regions. One novel variant was reported in F8, associated with hemophilia A; prenatal testing showed that the male fetus harbored this variant and the neonate suffered a life-threatening hemorrhage which was anticipated and appropriately managed. Moreover, 3% of participants had variants that were medically actionable. Compared with targeted mutation screening, genome sequencing improves the sensitivity of detecting clinically significant variants. While certain novel variant interpretation remains challenging, the ACMG guidelines are useful to classify variants in a healthy population.

Genomics-based carrier screening is one of many opportunities to use genomic information to inform medical decision making, but clinicians, health care delivery systems, and payers need to determine whether to offer screening and how to do so in an efficient, ethical way. To shed light on this issue, we conducted a study in the period 2014-17 to inform the design of clinical screening programs and guide further health services research. Many of our results have been published elsewhere; this article summarizes the lessons we learned from that study and offers policy insights. Our experience can inform understanding of the potential impact of expanded carrier screening services on health system workflows and workforces-impacts that depend on the details of the screening approach. We found limited patient or health system harms from expanded screening. We also found that some patients valued the information they learned from the process. Future policy discussions should consider the value of offering such expanded carrier screening in health delivery systems with limited resources.

Activation of the aryl hydrocarbon receptor (AhR) by immunosuppressive ligands promotes the development of regulatory T (Treg) cells. Although AhR-induced Foxp3+ Treg cells have been well studied, much less is known about the development and fate of AhR-induced Type 1 Treg (AhR-Tr1) cells. In the current study, we identified the unique transcriptional and functional changes in murine CD4+ T cells that accompany the differentiation of AhR-Tr1 cells during the CD4+ T-cell-dependent phase of an allospecific cytotoxic T lymphocyte (allo-CTL) response. AhR activation increased the expression of genes involved in T-cell activation, immune regulation and chemotaxis, as well as a global downregulation of genes involved in cell cycling. Increased IL-2 production was responsible for the early AhR-Tr1 activation phenotype previously characterized as CD25+CTLA4+GITR+ on day 2. The AhR-Tr1 phenotype was further defined by the coexpression of the immunoregulatory receptors Lag3 and Tim3 and non-overlapping expression of CCR4 and CCR9. Consistent with the increased expression of CCR9, real-time imaging showed enhanced migration of AhR-Tr1 cells to the lamina propria of the small intestine and colon. The discovery of mucosal imprinting of AhR-Tr1 cells provides an additional mechanism by which therapeutic AhR ligands can control immunopathology.

Activation of the aryl hydrocarbon receptor (AhR) by its prototypic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces potent suppression of an acute graft-versus-host (GVH) response and prevents GVH disease (GVHD). Suppression is associated with development of a regulatory population of donor CD4(+) CD25(+)T-cells that express high levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, a direct link between these AhR-induced Tregs (AhR-Tregs) and suppression of GVHD remains to be shown. CTLA-4 is a negative regulator of T-cell responses and is associated with the induction of tolerogenic dendritic cells (DCs) that produce indoleamine 2,3-dioxygenase (IDO). We hypothesized that AhR-Tregs mediate suppression via their enhanced expression of CTLA-4, which, in turn, induces IFN-γ and IDO in host DCs. Subsequent depletion of tryptophan by IDO leads to termination of the donor T-cell response prior to development of effector CTL. Here, we show that despite increased expression of Ifng, Irf3, Irf7, Ido1, and Ido2 in the lymph nodes of TCDD-treated host mice, inhibition of IDO enzyme activity by 1-methyl-tryptophan was unable to relieve TCDD-mediated suppression of the GVH response. Furthermore, treatment with an anti-CTLA-4 antibody that blocks CTLA-4 signaling was also unable to alleviate TCDD-mediated suppression. Alternatively, we investigated the possibility that donor-derived AhR-Tregs produce IFN-γ to suppress effector CTL development. However, suppression of GVHD by TCDD was not affected by the use of Ifng-deficient donor cells. Together, these results indicate that neither overexpression of CTLA-4 nor production of IFN-γ by AhR-Tregs plays a major role in the manifestation of their immunosuppressive function in vivo.

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine.

Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.

Aryl hydrocarbon receptor (AhR) activation by high-affinity ligands mediates immunosuppression in association with increased regulatory T cells (Tregs), making this transcription factor an attractive therapeutic target for autoimmune diseases. We recently discovered 10-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (10-Cl-BBQ), a nanomolar affinity AhR ligand with immunosuppressive activity and favorable pharmacologic properties. In this study, we tested the consequences of AhR activation in the NOD model. Oral 10-Cl-BBQ treatment prevented islet infiltration without clinical toxicity, whereas AhR-deficient NOD mice were not protected. Suppression of insulitis was associated with an increased frequency, but not total number, of Foxp3+ Tregs in the pancreas and pancreatic lymph nodes. The requirement for Foxp3+ cells in AhR-induced suppression of insulitis was tested using NOD.Foxp3DTR mice, which show extensive islet infiltration upon treatment with diphtheria toxin. AhR activation prevented the development of insulitis caused by the depletion of Foxp3+ cells, demonstrating that Foxp3+ cells are not required for AhR-mediated suppression and furthermore that the AhR pathway is able to compensate for the absence of Foxp3+ Tregs, countering current dogma. Concurrently, the development of disease-associated CD4+Nrp1+Foxp3−RORγt+ cells was inhibited by AhR activation. Taken together, 10-Cl-BBQ is an effective, nontoxic AhR ligand for the intervention of immune-mediated diseases that functions independently of Foxp3+ Tregs to suppress pathogenic T cell development.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and a member of the basic helix-loop-helix PER/ARNT/SIM family of chemosensors and developmental regulators. The AhR is widely known as a mediator of dioxin toxicity; however, it also suppresses cancer cell proliferation and recent findings have implicated its role as a tumor suppressor. We conducted a chemical library screen to identify nontoxic AhR ligands with anti-cancer effects and discovered flutamide (Eulexin) as a putative AhR ligand. Flutamide is an androgen receptor (AR) antagonist approved by the United States Food and Drug Administration for the treatment of prostate cancer. We found that flutamide inhibited the growth of several cancer cell lines independent of AR status, and that suppression of AhR expression reversed the anti-proliferative effects of flutamide. We investigated the AhR-dependent mechanism of action of flutamide in human hepatocellular carcinoma cells and identified that transforming growth factor-β1 (TGF-β1) is induced by flutamide in an AhR-dependent manner. In contrast, the potent AhR agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin had no effect on TGF-β1 expression, indicating the ligand specificity of AhR activation. We also determined that TGF-β1 induction is required for the AhR-dependent growth inhibitory effects of flutamide. Therefore, flutamide may be effective in AhR-positive cancers that are sensitive to TGF-β1 signaling, such as hepatocellular carcinoma.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays multiple roles in regulation of immune and inflammatory responses. The ability of certain AhR ligands to induce regulatory T cells (Tregs) has generated interest in developing AhR ligands for therapeutic treatment of immune-mediated diseases. To this end, we designed a screen for novel Treg-inducing compounds based on our understanding of the mechanisms of Treg induction by the well-characterized immunosuppressive AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We screened a ChemBridge small molecule library and identified 10-chloro-7H-benzimidazo[2,1-a]benzo[de]Iso-quinolin-7-one (10-Cl-BBQ) as a potent AhR ligand that was rapidly metabolized and not cytotoxic to proliferating T cells. Like TCDD,10-Cl-BBQ altered donor CD4(+) T cell differentiation during the early stages of a graft versus host (GVH) response resulting in expression of high levels of CD25, CTLA-4 and ICOS, as well as several genes associated with Treg function. The Treg phenotype required AhR expression in the donor CD4(+) T cells. Foxp3 was not expressed in the AhR-induced Tregs implicating AhR as an independent transcription factor for Treg induction. Structure-activity studies showed that unsubstituted BBQ as well as 4, 11-dichloro-BBQ were capable of inducing AhR-Tregs. Other substitutions reduced activation of AhR. Daily treatment with 10-Cl-BBQ during the GVH response prevented development of GVH disease in an AhR-dependent manner with no overt toxicity. Together, our data provide strong support for development of select BBQs that activate the AhR to induce Tregs for treatment of immune-mediated diseases.

Azo dyes are commonly used in many commercial industries. Some of the azo dyes can produce carcinogenic compounds after being metabolized by azoreductase. Several human intestinal microbiota possess azoreductase activity which plays an important role in the toxicity and mutagenicity of these azo dye compounds. The acpD gene product (AzoEf1) responsible for the azoreductase activity of Enterococcus faecium, an intestinal bacterium, was heterologously expressed, purified and characterized. The protein sequence shares 67% identity with the azoreductase from Enterococcus faecalis, AzoA. Although AzoEf1 possesses many commonalities with AzoA, there are differences in coenzyme preference, residues associated with FMN binding, substrate specificity, and specific activity. AzoEf1 utilized both NADH and NADPH for the reduction of azo dyes, and it contains a leucyl residue at position 104 and threonyl residue at position 19 which differ from AzoA at the active site. Its specific activity was 5095 M/min/mg and its catalytic efficiency for Methyl red reduction was lower than AzoA.

Azo dyes are commonly used in many commercial industries. Some of the azo dyes can produce carcinogenic compounds after being metabolized by azoreductase. Several human intestinal microbiota possess azoreductase activity which plays an important role in the toxicity and mutagenicity of these azo dye compounds. The acpD gene product (AzoEf1) responsible for the azoreductase activity of Enterococcus faecium, an intestinal bacterium, was heterologously expressed, purified and characterized. The protein sequence shares 67% identity with the azoreductase from Enterococcus faecalis, AzoA. Although AzoEf1 possesses many commonalities with AzoA, there are differences in coenzyme preference, residues associated with FMN binding, substrate specificity, and specific activity. AzoEf1 utilized both NADH and NADPH for the reduction of azo dyes, and it contains a leucyl residue at position 104 and threonyl residue at position 19 which differ from AzoA at the active site. Its specific activity was 5095 &mu, M/min/mg and its catalytic efficiency for Methyl red reduction was lower than AzoA.

Azoreductases reduce the azo bond (N=N) in azo dyes to produce colorless amine products. Crude cell extracts from Enterococcus faecalis have been shown to utilize both NADH and NADPH as electron donors for azo dye reduction. An azoreductase was purified from E. faecalis by hydrophobic, anion exchange and affinity chromatography. The azoreductase activity of the purified preparation was tested on a polyacrylamide gel after electrophoresis under native conditions and the protein that decolorized the azo dye (Methyl Red) with both NADH and NADPH was identified by mass spectrometry to be AzoA. Previously, the heterologously expressed and purified AzoA was shown to utilize NADH only for the reduction of Methyl Red. However, AzoA purified from the wild-type organism was shown to utilize both coenzymes but with more than 180-fold preference for NADH over NADPH as an electron donor to reduce Methyl Red. Also, its specific activity was more than 150-fold higher than the previous study on AzoAwhen NADH was used as the electron donor. The catalytic efficiency for Methyl Red reduction by AzoA from E. faecalis was several orders of magnitude higher than other azoreductases that were purified from a heterologous source.

Azoreductases reduce the azo bond (N=N) in azo dyes to produce colorless amine products. Crude cell extracts from Enterococcus faecalis have been shown to utilize both NADH and NADPH as electron donors for azo dye reduction. An azoreductase was purified from E. faecalis by hydrophobic, anion exchange and affinity chromatography. The azoreductase activity of the purified preparation was tested on a polyacrylamide gel after electrophoresis under native conditions and the protein that decolorized the azo dye (Methyl Red) with both NADH and NADPH was identified by mass spectrometry to be AzoA. Previously, the heterologously expressed and purified AzoA was shown to utilize NADH only for the reduction of Methyl Red. However, AzoA purified from the wild-type organism was shown to utilize both coenzymes but with more than 180-fold preference for NADH over NADPH as an electron donor to reduce Methyl Red. Also, its specific activity was more than 150-fold higher than the previous study on AzoA when NADH was used as the electron donor. The catalytic efficiency for Methyl Red reduction by AzoA from E. faecalis was several orders of magnitude higher than other azoreductases that were purified from a heterologous source.

Azo dyes are widely used in the food, pharmaceutical, paper, and textile industries. Some azo dyes are known to produce carcinogenic compounds upon reductive cleavage of the azo bond (N=N) by intestinal flora. There is not much information available on the effect of these dyes on the physiology of the gut microflora as well as their kinetics of reduction in different environments. The azoreductase activity of Enterococcus faecalis , an important opportunistic intestinal pathogen, was tested using the azo dyes Methyl Red, Direct Blue 15 and tartrazine under the following conditions: a complex supplemented brain heart infusion (BHIS) medium under aerobic and anaerobic conditions, a simple phosphate-buffered saline glucose (PBSG) medium under anaerobic conditions, and cell extracts using NADH and NADPH as electron donors, under aerobic conditions. The kinetics of reduction in all these conditions was determined. The effect of high and low concentrations of each dye on the growth of E. faecalis was also tested. Our results show that the sulfonated azo dye tartrazine was completely reduced extracellularly in BHIS under aerobic and anaerobic conditions while the other sulfonated azo dye, Direct Blue 15 was completely reduced only under anaerobic conditions. The non-polar azo dye, Methyl Red was also completely reduced, with the highest rate of reduction in both conditions. The generation time of E. faecalis in the presence of high concentration of tartrazine was significantly lower than the control, while the generation time was unaffected in the presence of low and high concentrations of Methyl Red, Direct Blue 15 and 20 uM tartrazine. Our results suggest that the metabolism of different azo dyes by E. faecalis depends on the physiochemical properties of the azo dyes and the conditions of the surrounding environment in the presence of actively dividing cells. Key words: azo dyes, Methyl Red, Direct Blue 15, tartrazine, Enterococcus faecalis, growth phase, azoreductase