Yeast XRS2 and human NBN gene: Experimental evidence for homology using codon optimized cDNA

The genes, XRS2 in Saccharomyces cerevisiae and NBN in mammals, have little sequence identity at the amino acid level. Nevertheless, they are both found together with MRE11 and RAD50 in a highly conserved protein complex which functions in the repair of DNA double-strand breaks. Here, we have examined the evolutionary and functional relationship of these two genes by cross-complementation experiments. These experiments necessitated sequence correction for specific codon usage before they could be successfully conducted. We present evidence that despite extreme sequence divergence nibrin can, at least partially, replace Xrs2 in the cellular DNA damage response, and Xrs2 is able to promote nuclear localization of MRE11 in NBS cells. We discuss that the extreme sequence divergence reflects a unique adaptive pressure during evolution related to the specific eukaryotic role for both Xrs2 and nibrin in the subcellular localisation of the DNA repair complex. This, we suggest, is of particular relevance when cells are infected by viruses. The conflict hypothesis of co-evolution of DNA repair genes and DNA viruses may thus explain the very low sequence identity of these two homologous genes

Selfish or altruistic? An analysis of alarm call function in wild capuchin monkeys, Cebus apella nigritus

Alarm calls facilitate some antipredatory benefits of group living but may endanger the caller by attracting the predator's attention. A number of hypotheses invoking kin selection and individual selection have been proposed to explain how such behaviour could evolve. This study tests eight hypotheses for alarm call evolution by examining the responses of tufted capuchin monkeys to models of felids, perched raptors and vipers. Specifically, this study examines: (1) differences between individuals in their propensity to call in response to different threat types, (2) whether there is an audience effect for alarm calling and (3) the response of conspecifics to alarms. Results indicate that the benefits likely to be afforded to the caller vary with stimulus type. Alarm calling in response to felids is most likely selfish, with calls apparently directed towards both the predator and potential conspecific mobbers. Alarm calling in response to vipers attracts additional mobbers as well, but also appears to be driven by kin selection in the case of males and parental care benefits in the case of females. Alarm responses to perched raptors are rare, but seem to be selfish, with callers benefiting by recruiting additional mobbers

Nijmegen breakage syndrome (NBS)

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive syndrome of chromosomal instability mainly characterized by microcephaly at birth, combined immunodeficiency and predisposition to malignancies. Due to a founder mutation in the underlying NBN gene (c.657_661del5) the disease is encountered most frequently among Slavic populations. The principal clinical manifestations of the syndrome are: microcephaly, present at birth and progressive with age, dysmorphic facial features, mild growth retardation, mild-to-moderate intellectual disability, and, in females, hypergonadotropic hypogonadism. Combined cellular and humoral immunodeficiency with recurrent sinopulmonary infections, a strong predisposition to develop malignancies (predominantly of lymphoid origin) and radiosensitivity are other integral manifestations of the syndrome. The NBN gene codes for nibrin which, as part of a DNA repair complex, plays a critical nuclear role wherever double-stranded DNA ends occur, either physiologically or as a result of mutagenic exposure. Laboratory findings include: (1) spontaneous chromosomal breakage in peripheral T lymphocytes with rearrangements preferentially involving chromosomes 7 and 14, (2) sensitivity to ionizing radiation or radiomimetics as demonstrated in vitro by cytogenetic methods or by colony survival assay, (3) radioresistant DNA synthesis, (4) biallelic hypomorphic mutations in the NBN gene, and (5) absence of full-length nibrin protein. Microcephaly and immunodeficiency are common to DNA ligase IV deficiency (LIG4 syndrome) and severe combined immunodeficiency with microcephaly, growth retardation, and sensitivity to ionizing radiation due to NHEJ1 deficiency (NHEJ1 syndrome). In fact, NBS was most commonly confused with Fanconi anaemia and LIG4 syndrome. Genetic counselling should inform parents of an affected child of the 25% risk for further children to be affected. Prenatal molecular genetic diagnosis is possible if disease-causing mutations in both alleles of the NBN gene are known. No specific therapy is available for NBS, however, hematopoietic stem cell transplantation may be one option for some patients. Prognosis is generally poor due to the extremely high rate of malignancies

DNA Damage in Nijmegen Breakage Syndrome Cells Leads to PARP Hyperactivation and Increased Oxidative Stress

Nijmegen Breakage Syndrome (NBS), an autosomal recessive genetic instability syndrome, is caused by hypomorphic mutation of the NBN gene, which codes for the protein nibrin. Nibrin is an integral member of the MRE11/RAD50/NBN (MRN) complex essential for processing DNA double-strand breaks. Cardinal features of NBS are immunodeficiency and an extremely high incidence of hematological malignancies. Recent studies in conditional null mutant mice have indicated disturbances in redox homeostasis due to impaired DSB processing. Clearly this could contribute to DNA damage, chromosomal instability, and cancer occurrence. Here we show, in the complete absence of nibrin in null mutant mouse cells, high levels of reactive oxygen species several hours after exposure to a mutagen. We show further that NBS patient cells, which unlike mouse null mutant cells have a truncated nibrin protein, also have high levels of reactive oxygen after DNA damage and that this increased oxidative stress is caused by depletion of NAD+ due to hyperactivation of the strand-break sensor, Poly(ADP-ribose) polymerase. Both hyperactivation of Poly(ADP-ribose) polymerase and increased ROS levels were reversed by use of a specific Poly(ADP-ribose) polymerase inhibitor. The extremely high incidence of malignancy among NBS patients is the result of the combination of a primary DSB repair deficiency with secondary oxidative DNA damage

Somatic mutations and progressive monosomy modify SAMD9-related phenotypes in humans

It is well established that somatic genomic changes can influence phenotypes in cancer, but the role of adaptive changes in developmental disorders is less well understood. Here we have used next-generation sequencing approaches to identify de novo heterozygous mutations in sterile α motif domain–containing protein 9 (SAMD9, located on chromosome 7q21.2) in 8 children with a multisystem disorder termed MIRAGE syndrome that is characterized by intrauterine growth restriction (IUGR) with gonadal, adrenal, and bone marrow failure, predisposition to infections, and high mortality. These mutations result in gain of function of the growth repressor product SAMD9. Progressive loss of mutated SAMD9 through the development of monosomy 7 (–7), deletions of 7q (7q–), and secondary somatic loss-of-function (nonsense and frameshift) mutations in SAMD9 rescued the growth-restricting effects of mutant SAMD9 proteins in bone marrow and was associated with increased length of survival. However, 2 patients with –7 and 7q– developed myelodysplastic syndrome, most likely due to haploinsufficiency of related 7q21.2 genes. Taken together, these findings provide strong evidence that progressive somatic changes can occur in specific tissues and can subsequently modify disease phenotype and influence survival. Such tissue-specific adaptability may be a more common mechanism modifying the expression of human genetic conditions than is currently recognized

The NBN founder mutation—Evidence for a country specific difference in age at cancer manifestation

Background: Nijmegen breakage syndrome (NBS) is an autosomal-recessive chromosome instability disorder characterized by, among others, hypersensitivity to X-irradiation and an exceptionally high risk for lymphoid malignancy. The vast majority of NBS patients is homozygous for a common Slavic founder mutation, c.657del5, of the NBN gene, which is involved in the repair of DNA double-strand breaks (DSBs). The founder mutation also predisposes heterozygous carriers to cancer, apparently however, with a higher risk in the Czech Republic/Slovakia (CS) than in Poland. Aim: To examine whether the age of cancer manifestation and cancer death of NBN homozygotes is different between probands from CS and Poland. Methods: The study is restricted to probands born until 1989, before replacement of the communist regime by a democratic system in CS and Poland, and a substantial transition of the health care systems. Moreover, all patients were recruited without knowledge of their genetic status since the NBN gene was not identified until 1998. Results: Here, we show that cancer manifestation of NBN homozygotes is at a significantly earlier age in probands from CS than from Poland. This is explained by the difference in natural and medical radiation exposure, though within the permissible dosage. Conclusion: It is reasonable to assume that this finding also sheds light on the higher cancer risk of NBN heterozygotes in CS than in Poland. This has implications for genetic counseling and individualized medicine also of probands with other DNA repair defects

Production and perception of situationally variable alarm calls in wild tufted capuchin monkeys (Cebus apella nigritus)

Many mammalian and avian species produce conspicuous vocalizations upon encountering a predator, but vary their calling based on risk urgency and/or predator type. Calls falling into the latter category are termed “functionally referential” if they also elicit predator-appropriate reactions in listeners. Functionally referential alarm calling has been well documented in a number of Old World monkeys and lemurs, but evidence among Neotropical primates is limited. This study investigates the alarm call system of tufted capuchin monkeys (Cebus apella nigritus) by examining responses to predator and snake decoys encountered at various distances (reflecting differences in risk urgency). Observations in natural situations were conducted to determine if predator-associated calls were given in additional contexts. Results indicate the use of three call types. “Barks” are elicited exclusively by aerial threats, but the call most commonly given to terrestrial threats (the “hiccup”) is given in nonpredatory contexts. The rate in which this latter call is produced reflects risk urgency. Playbacks of these two call types indicate that each elicits appropriate antipredator behaviors. The third call type, the “peep,” seems to be specific to terrestrial threats, but it is unknown if the call elicits predator-specific responses. “Barks” are thus functionally referential aerial predator calls, while “hiccups” are better seen as generalized disturbance calls which reflect risk urgency. Further evidence is needed to draw conclusions regarding the “peep.” These results add to the evidence that functionally referential aerial predator alarm calls are ubiquitous in primates, but that noncatarrhine primates use generalized disturbance calls in response to terrestrial threats

Glucocorticoids regulate AKR1D1 activity in human liver in vitro and in vivo

Steroid 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates endogenous glucocorticoids and catalyses an important step in bile acid synthesis. Endogenous and synthetic glucocorticoids are potent regulators of metabolic phenotype and play a crucial role in hepatic glucose metabolism. However, the potential of synthetic glucocorticoids to be metabolised by AKR1D1 as well as to regulate its expression and activity has not been investigated. The impact of glucocorticoids on AKR1D1 activity was assessed in human liver HepG2 and Huh7 cells; AKR1D1 expression was assessed by qPCR and Western blotting. Genetic manipulation of AKR1D1 expression was conducted in HepG2 and Huh7 cells and metabolic assessments were made using qPCR. Urinary steroid metabolite profiling in healthy volunteers was performed pre- and post-dexamethasone treatment, using gas chromatography-mass spectrometry. AKR1D1 metabolised endogenous cortisol, but cleared prednisolone and dexamethasone less efficiently. In vitro and in vivo, dexamethasone decreased AKR1D1 expression and activity, further limiting glucocorticoid clearance and augmenting action. Dexamethasone enhanced gluconeogenic and glycogen synthesis gene expression in liver cell models and these changes were mirrored by genetic knockdown of AKR1D1 expression. The effects of AKR1D1 knockdown were mediated through multiple nuclear hormone receptors, including the glucocorticoid, pregnane X and farnesoid X receptors. Glucocorticoids down-regulate AKR1D1 expression and activity and thereby reduce glucocorticoid clearance. In addition, AKR1D1 down-regulation alters the activation of multiple nuclear hormone receptors to drive changes in gluconeogenic and glycogen synthesis gene expression profiles, which may exacerbate the adverse impact of exogenous glucocorticoids

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

BACKGROUND: The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst approximately 8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs. CONCLUSIONS/SIGNIFICANCE: In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress