The azo dye Disperse Orange 1 induces DNA damage and cytotoxic effects but does not cause ecotoxic effects in Daphnia similis and Vibrio fischeri

Azo dyes constitute the largest group of colorants used in industry and can pass through municipal waste water plants nearly unchanged due to their resistance to aerobic treatment, which potentially exposes humans and local biota to adverse effects. Unfortunately, little is known about their environmental fate. Under anaerobic conditions, some azo dyes are cleaved by microorganisms forming potentially carcinogenic aromatic amines. In the present study, the azo dye Disperse Orange 1, widely used in textile dyeing, was tested using the comet, Salmonella/microsome mutagenicity, cell viability, Daphnia similis and Microtox (R) assays. The human hepatoma cell line (HepG2) was used in the comet assay and for cell viability. In the mutagenicity assay. Salmonella typhimurium strains with different levels of nitroreductase and o-acetyltransferase were used. The dye showed genotoxic effects with respect to HepG2 cells at concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0 mu g/mL. In the mutagenicity assay, greater responses were obtained with the strains TA98 and YG1041, suggesting that this compound mainly induces frameshift mutations. Moreover, the mutagenicity was greatly enhanced with the strains overproducing nitroreductase and o-acetyltransferase, showing the importance of these enzymes in the mutagenicity of this dye. In addition, the compound induced apoptosis after 72 h in contact with the HepG2 cells. No toxic effects were observed for either D. similis or Vibrio fischeri. (C) 2011 Elsevier B.V. All rights reserved.

Identification of two novel mutations in the hydroxymethylbilane synthase gene in three patients from two unrelated families with acute intermittent porphyria

We have screened the hydroxymethylbilane synthase cDNAs of 3 patients from 2 families suffering from acute intermittent porphyria (AIP) from Scotland and South Africa using heteroduplex and chemical cleavage of mismatch analyses, Direct sequencing was used to characterise the mutations, The two novel mutations identified were a missense mutation at nucleotide position 64 in exon 3 (R22C) and a single base-pair deletion in exon 15, These mutations are predicted to affect the normal function of the enzyme and, therefore, are expected to be the primary cause of disease in these patients.

Mutational analysis of the cystathionine beta-synthase gene: A splicing mutation, two missense mutations and an insertion in patients with homocystinuria

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G-->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G-->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA. (C) 1998 Wiley-Liss, Inc.

Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype-phenotype correlations in phenylketonuria

Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories:'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes; domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.

Sodium channel alpha 1-subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms

Background: Mutations in SCN1A, the gene encoding the alpha1 subunit of the sodium channel, have been found in severe myoclonic epilepsy of infancy (SMEI) and generalized epilepsy with febrile seizures plus (GEFS(+)). Mutations in SMEI include missense, nonsense, and frameshift mutations more commonly arising de novo in affected patients. This finding is difficult to reconcile with the family history of GEFS(+) in a significant proportion of patients with SMEI Infantile spasms (IS), or West syndrome, is a severe epileptic encephalopathy that is usually symptomatic. In some cases, no etiology is found and there is a family history of epilepsy. Method: The authors screened SCN1A in 24 patients with SMEI and 23 with IS. Results: Mutations were found in 8 of 24 (33%) SMEI patients, a frequency much lower than initial reports from Europe and Japan. One mutation near the carboxy terminus was identified in an IS patient. A family history of seizures was found in 17 of 24 patients with SMEI. Conclusions: The rate of SCN1A mutations in this cohort of SMEI patients suggests that other factors may be important in SMEI. Less severe mutations associated with GEFS(+) could interact with other loci to cause SMEI in cases with a family history of GEFS(+). This study extends the phenotypic heterogeneity of mutations in SCN1A to include IS.

Mutations of the KISS1 Gene in Disorders of Puberty

Context: Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP). Objective: Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH. Patients: Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development. Methods: The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells. Results: Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group. Conclusion: Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype. (J Clin Endocrinol Metab 95: 2276-2280, 2010)

Absence of GH-Releasing Hormone (GHRH) Mutations in Selected Patients with Isolated GH Deficiency

Context: Although numerous reports of mutations in GH1 and GHRHR (GHRH receptor) causing isolated GH deficiency (IGHD) have been published, mutations in GHRH itself have not been hitherto reported but are obvious candidates for GH deficiency. Objective: The aim of this study was to identify mutations in GHRH in a large cohort of patients with IGHD. Patients and Methods: DNA was isolated from 151 patients diagnosed with IGHD at national and international centers. Seventy-two patients fulfilled all the following criteria: severe short stature (height SD score <= -2.5), low peakGHafter stimulation (peak <= 5 ng/ml), eutopic posterior pituitary lobe, and absence of mutations in GH1 and GHRHR and therefore were strong candidates for GHRH mutations. The coding sequence and splice sites of GHRH were amplified by PCR with intronic primers and sequenced. Results: In five of 151 patients (four of 42 from Brazil), the GHRH c. 223 C>T, p. L75F change was identified in heterozygosity. This variant has been previously reported as a polymorphism and is more frequent in African than European and Asian populations. Six allelic variants (five novel) that do not predict change of amino acids or splice sites were identified in five patients: c. 147 C>T, p.S49S, IVS1 -70 G>A, IVS1 -74 T>C, IVS3 -47 del1, and IVS3 +7 G>A/IVS3 + 41 G>A. No functional mutations were found in this cohort. Conclusions: GHRH mutations were not identified in a selected cohort of patients with IGHD, suggesting that, if they exist, they may be an extremely rare cause of IGHD. Other, as-yet-unidentified genetic factors may be implicated in the genetic etiology of IGHD in our cohort. (J Clin Endocrinol Metab 96: E1457-E1460, 2011)

Germline mutations in TMEM127 confer susceptibility to pheochromocytoma

Pheochromocytomas, which are catecholamine-secreting tumors of neural crest origin, are frequently hereditary(1). However, the molecular basis of the majority of these tumors is unknown(2). We identified the transmembrane-encoding gene TMEM127 on chromosome 2q11 as a new pheochromocytoma susceptibility gene. In a cohort of 103 samples, we detected truncating germline TMEM127 mutations in approximately 30% of familial tumors and about 3% of sporadic-appearing pheochromocytomas without a known genetic cause. The wild-type allele was consistently deleted in tumor DNA, suggesting a classic mechanism of tumor suppressor gene inactivation. Pheochromocytomas with mutations in TMEM127 are transcriptionally related to tumors bearing NF1 mutations and, similarly, show hyperphosphorylation of mammalian target of rapamycin (mTOR) effector proteins. Accordingly, in vitro gain-of-function and loss-of-function analyses indicate that TMEM127 is a negative regulator of mTOR. TMEM127 dynamically associates with the endomembrane system and colocalizes with perinuclear (activated) mTOR, suggesting a subcompartmental-specific effect. Our studies identify TMEM127 as a tumor suppressor gene and validate the power of hereditary tumors to elucidate cancer pathogenesis.

NKX2.5 mutations in patients with non-syndromic congenital heart disease

Background: Cardiac development is a complex and multifactorial biological process. Heterozygous mutations in the transcription factor NKX2.5 are between the first evidence of a genetic cause for congenital heart defects in human beings. In this study, we evaluated the presence and frequency of mutations in the NKX2.5 gene on 159 unrelated patients with a diverse range of non-syndromic congenital heart defects (conotruncal anomalies, septal defects, left-sided lesions, right-sided lesions, patent ductus arteriosus and Ebstein`s anomaly). Methods: The coding region of the NKX2.5 locus was amplified by polymerase chain reaction and mutational analysis was performed using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. Results: We identified two distinct mutations in the NKX2.5 coding region among the 159 (1.26%) individuals evaluated. An Arg25Cys mutation was identified in a patient with Tetralogy of Fallot. The second mutation found was an Ala42Pro in a patient with Ebstein`s anomaly. Conclusions: The association of NKX2.5 mutations is present in a small percentage of patients with non-syndromic congenital heart defects and may explain only a few cases of the disease. Screening strategies considering the identification of germ-line molecular defects in congenital heart disease are still unwarranted and should consider other genes besides NKX2.5. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

De novo mutations of the Patched gene in nevoid basal cell carcinoma syndrome help to define the clinical phenotype

The demonstration that mutations in the Patched (PTCH) gene cause nevoid basal cell carcinoma syndrome (NBCCS) has led to the identification of the exact molecular lesion in a percentage of individuals with the syndrome, In addition, it has been possible to determine, through molecular analysis of parents and other relatives of these individuals, if the mutation is inherited or has arisen de novo, We have previously reported 28 mutations in individuals with NBCCS, and here we present an additional 4 novel mutations, We have also analyzed relatives of a number of the individuals in whom we have found mutations, In total we have identified 8 individuals who carry a de novo mutation in the PTCH gene, In 5 of these cases, clinical and radiological examination had not unequivocally ruled out a diagnosis in one of the parents, This helps to define the clinical phenotype and suggests that diagnostic criteria in this complex syndrome may require review. (C) 1997 Wiley-Liss, Inc.

IRF8 Mutations and Human Dendritic-Cell Immunodeficiency

BACKGROUND The genetic analysis of human primary immunodeficiencies has defined the contribution of specific cell populations and molecular pathways in the host defense against infection. Disseminated infection caused by bacille Calmette-Guerin (BCG) vaccines is an early manifestation of primary immunodeficiencies, such as severe combined immunodeficiency. In many affected persons, the cause of disseminated BCG disease is unexplained. METHODS We evaluated an infant presenting with features of severe immunodeficiency, including early-onset disseminated BCG disease, who required hematopoietic stem-cell transplantation. We also studied two otherwise healthy subjects with a history of disseminated but curable BCG disease in childhood. We characterized the monocyte and dendritic-cell compartments in these three subjects and sequenced candidate genes in which mutations could plausibly confer susceptibility to BCG disease. RESULTS We detected two distinct disease-causing mutations affecting interferon regulatory factor 8 (IRF8). Both K108E and T80A mutations impair IRF8 transcriptional activity by disrupting the interaction between IRF8 and DNA. The K108E variant was associated with an autosomal recessive severe immunodeficiency with a complete lack of circulating monocytes and dendritic cells. The T80A variant was associated with an autosomal dominant, milder immunodeficiency and a selective depletion of CD11c+CD1c+ circulating dendritic cells. CONCLUSIONS These findings define a class of human primary immunodeficiencies that affect the differentiation of mononuclear phagocytes. They also show that human IRF8 is critical for the development of monocytes and dendritic cells and for antimycobacterial immunity. (Funded by the Medical Research Council and others.)

Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P<0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.

A single nucleotide deletion at the C1 inhibitor gene as the cause of hereditary angioedema: insights from a Brazilian family

Background: Hereditary angioedema is an autosomal dominant disease characterized by episodes of subcutaneous and submucosal edema. It is caused by deficiency of the C1 inhibitor protein, leading to elevated levels of bradykinin. More than 200 mutations in C1 inhibitor gene have been reported. The aim of this study was to analyze clinical features of a large family with an index case of hereditary angioedema and to determine the disease-causing mutation in this family. Methods: Family pedigree was constructed with 275 individuals distributed in five generations. One hundred and sixty-five subjects were interviewed and investigated for mutation at the C1 inhibitor gene. Subjects reporting a history of recurrent episodes of angioedema and/or abdominal pain attacks underwent evaluation for hereditary angioedema. Results: We have identified a novel mutation at the C1 inhibitor gene, c.351delC, which is a single-nucleotide deletion of a cytosine on exon 3, resulting in frameshift with premature stop codon. Sequencing analysis of the hypothetical truncated C1 inhibitor protein allowed us to conclude that, if transcription occurs, this protein has no biological activity. Twenty-eight members of the family fulfilled diagnostic criteria for hereditary angioedema and all of them presented the c.351delC mutation. Variation in clinical presentation and severity of disease was observed among these patients. One hundred and thirty-seven subjects without hereditary angioedema did not have the c.351delC mutation. Conclusion: The present study provides definitive evidence to link a novel genetic mutation to the development of hereditary angioedema in patients from a Brazilian family.

Multi-system neurological disease is common in patients with OPA1 mutations

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal `dominant optic atrophy plus` variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

Mutations involved in Aicardi-Goutieres syndrome implicate SAMHD1 as regulator of the innate immune response

Aicardi-Goutieres syndrome is a mendelian mimic of congenital infection and also shows overlap with systemic lupus erythematosus at both a clinical and biochemical level. The recent identification of mutations in TREX1 and genes encoding the RNASEH2 complex and studies of the function of TREX1 in DNA metabolism have defined a previously unknown mechanism for the initiation of autoimmunity by interferon-stimulatory nucleic acid. Here we describe mutations in SAMHD1 as the cause of AGS at the AGS5 locus and present data to show that SAMHD1 may act as a negative regulator of the cell-intrinsic antiviral response.