A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Life without a wall or division machine in Bacillus subtilis.

The cell wall is an essential structure for virtually all bacteria, forming a tough outer shell that protects the cell from damage and osmotic lysis. It is the target of our best antibiotics. L-form strains are wall-deficient derivatives of common bacteria that have been studied for decades. However, they are difficult to generate and typically require growth for many generations on osmotically protective media with antibiotics or enzymes that kill walled forms. Despite their potential importance for understanding antibiotic resistance and pathogenesis, little is known about their basic cell biology or their means of propagation. We have developed a controllable system for generating L-forms in the highly tractable model bacterium Bacillus subtilis. Here, using genome sequencing, we identify a single point mutation that predisposes cells to grow without a wall. We show that propagation of L-forms does not require the normal FtsZ-dependent division machine but occurs by a remarkable extrusion-resolution mechanism. This novel form of propagation provides insights into how early forms of cellular life may have proliferated.

Cell kinetics and gene expression changes in colorectal cancer patients given resistant starch: a randomised controlled trial.

OBJECTIVE: This study investigated the effects of oral supplementation of resistant starch (RS) on tumour cell and colonic mucosal cell kinetics and on gene expression in patients with colorectal cancer (CRC), and its potential role in colon cancer prevention. METHODS: 65 patients with CRC were randomised to treatment with RS or ordinary starch (OS) and were given starch treatment for up to 4 weeks. Pretreatment and post-treatment biopsies were obtained from the tumour and colonic mucosa, and the effects of the starch treatment on cell proliferation and expression of the cell cycle regulatory genes CDK4 (cyclin-dependent kinase 4) and GADD45A (growth arrest and DNA damage-inducible, alpha) were investigated. RESULTS: The proportion of mitotic cells in the top half of the colonic crypt was significantly lower following RS treatment (3.1 (1.5), mean (SEM)) as compared with OS treatment (13.7 (3.2)) (p = 0.028). However, there was no effect of RS treatment on crypt dimensions and tumour cell proliferation index. There was significant upregulation in expression of CDK4 (p<0.01) and downregulation in expression of GADD45A (p<0.001) in the tumour tissue when compared with macroscopically normal mucosa. Following RS treatment, CDK4 expression in tumours (0.88 (0.15)) was twofold higher than that in the OS group (0.37 (0.16)) (p = 0.02). The expression of GADD45A, which was downregulated in the presence of cancer, was significantly upregulated (p = 0.048) following RS treatment (1.41 (0.26)) as compared with OS treatment (0.56 (0.3)). However, there were no significant differences in the expression of these genes in the normal mucosa following starch treatment. CONCLUSIONS: Cell proliferation in the upper part of colonic crypts is a premalignant marker and its reduction by RS supplementation is consistent with an antineoplastic action of this food component. Differential expression of the key cell cycle regulatory genes may contribute to the molecular mechanisms underlying these antineoplastic effects of RS.

Nutrition and DNA repair--potential molecular mechanisms of action.

At its most fundamental, cancer is a genetic disease in the sense that the primary events in tumorigenesis involve damage to the genome. The genome is subject to damage continuously from both exogenous agents and endogenous processes but this becomes functionally important only if the damage is not detected and resolved in a timely and effective manner. In mammals there are 5 DNA repair pathways, encoded by approximately 150 genes, which appear to have arisen early in evolution and which are highly conserved. Given the substantial epidemiological and experimental evidence that variation in dietary intake accounts for a significant proportion of the variance in cancer prevalence, an a priori case can be made that dietary factors may influence the effectiveness of DNA repair. A review of the literature has identified 4 observational and 8 intervention studies in human subjects where DNA repair (or a component thereof) has been measured in relation to nutrition. This rather limited evidence base precludes drawing definitive conclusions, but the fact that there were significant effects of dietary supplements in 5 out of the 8 intervention studies suggests that food components and/or nutritional status may influence DNA repair. This review considers possible molecular mechanisms through which such factors could modulate repair.

DNA mismatch repair status may influence anti-neoplastic effects of butyrate.

HNPCC (hereditary non-polyposis colon cancer) is an autosomal-dominant disorder characterized by early-onset CRC (colorectal cancer). HNPCC is most often associated with mutations in the MMR (mismatch repair) genes hMLH1, hMSH2, hMSH6 or hPMS2. The mutator phenotype of a defective MMR system is MSI (microsatellite instability), which also occurs in approx. 15-25% of sporadic CRC cases, where it is associated with the hypermethylation of the promoter region of hMLH1. Dietary factors, including excessive alcohol consumption, ingestion of red meat and low folate intake, may increase the risk of MSI high tumour development. In contrast, aspirin may suppress MSI in MMR-deficient CRC cell lines. Butyrate, a short-chain-fatty-acid end product of carbohydrate fermentation in the colon, shares a number of anti-neoplastic properties with aspirin, including inhibiting proliferation and inducing apoptosis of CRC cells. Recent in vitro studies suggest that physiological concentrations of butyrate (0.5-2 mM) may have more potent anti-neoplastic effects in CRC cell lines deficient in MMR, but mechanisms for such a differential response remain to be established.

Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene.

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20microg of 14C PhIP (182kBq, specific activity 2.05GBq/mmol) or 5microg of 14C B[a]P (36kBq, specific activity 1.81GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both 14C PhIP and 14C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22-477.35 and 6.61-208. 38 adducts/10(12) nucleotides following administration of 14C PhIP and 14C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.

Comparative biotransformation studies of MeIQx and PhIP in animal models and humans.

MeIQx and PhIP are putative carcinogenic heterocyclic amines formed during the cooking of meat and fish. Using accelerator mass spectrometry, we have investigated the metabolism and macromolecule binding of 14C-labelled MeIQx and PhIP in human cancer patients compared to the rat. Following oral administration of MeIQx and PhIP, more DNA adducts were formed in human colon tissue compared with rats. Differences were also observed between rats and humans in the metabolite profile and urine excretion for these compounds. These results suggest humans metabolise heterocyclic amines differently to laboratory rodents and question their use as models of human risk.

Detectable levels of serum aflatoxin B1-albumin adducts in the United Kingdom population: implications for aflatoxin-B1 exposure in the United Kingdom.

This study aimed to estimate aflatoxin B1 (AFB1) exposure in the United Kingdom population by measuring levels of serum AFB1-albumin (alb), using immunoassay and high-performance liquid chromatography (HPLC) with fluorescence detection. A self-questionnaire on dietary habits from 104 volunteers (47 men and 57 women) in York was completed, and blood samples were collected. Serum alb was extracted, and AFB1-lysine (lys), the digest product of AFB1-alb, was isolated and measured. A sensitive ELISA (detection limit, approximately 1.4 pg of AFB1-lys) was developed. A good correlation was found between calibration of ELISA results and scintillation counting, for rats dosed with [3H]AFB1 (r = 0.972; P < 0.001). This ELISA was subsequently used to analyze human serum alb. For United Kingdom human sera, the mean adduct levels were 29.3 +/- 14.8 pg AFB1-lys equivalents (eq) mg albumin (males) and 26.9 +/- 14.4 pg AFB1-lys eq/mg alb (females). Confirmation of the ELISA data was sought using reversed-phase HPLC with fluorescence detection. HPLC chromatograms of digested York serum alb were compared to digested serum alb for humans from Qidong County, People's Republic of China, and from AFB1-dosed rats. These all gave similar HPLC profiles. Each sample contained fluorescent material that coeluted with and just before the AFB1-lys standard. Fluorescent fractions were found to be inhibitory in a separate anti-AFB1-lys ELISA, indicating that these earlier fluorescent peaks contained AFB1 residues. Our results suggest that measurable internal AFB1 exposure may be occurring in some United Kingdom individuals, albeit at lower levels than those seen for areas with high AFB1 exposure. The source of this exposure may reflect the known difficulties in accurately monitoring regulated imported foodstuffs and/or the lack of regulations on other potentially contaminated imports. However, no positive correlations were found between our AFB1-lys measurements and any dietary questionnaire information. Animal studies, as well as human studies, have been important in developing exposure and internal adduct relationships in humans. Based on this literature, our AFB1-alb data indicate a mean daily exposure of 3 microg of AFB1 and a mean internal dose in liver DNA of 5.9 adducts/10(7) nucleotides. We believe this may be an overestimate of the AFB1 exposure level in the United Kingdom, and further studies are needed to accurately relate external dose and internal AFB1 biomarkers in humans.