Evidence for time dependency of molecular rate estimates

Long-term changes in the genetic composition of a population occur by the fixation of new mutations, a process known as substitution. The rate at which mutations arise in a population and the rate at which they are fixed are expected to be equal under neutral conditions (Kimura, 1968). Between the appearance of a new mutation and its eventual fate of fixation or loss, there will be a period in which it exists as a transient polymorphism in the population (Kimura and Ohta, 1971). If the majority of mutations are deleterious (and nonlethal), the fixation probabilities of these transient polymorphisms are reduced and the mutation rate will exceed the substitution rate (Kimura, 1983). Consequently, different apparent rates may be observed on different time scales of the molecular evolutionary process (Penny, 2005; Penny and Holmes, 2001). The substitution rate of the mitochondrial protein-coding genes of birds and mammals has been traditionally recognized to be about 0.01 substitutions/site/million years (Myr) (Brown et al., 1979; Ho, 2007; Irwin et al., 1991; Shields and Wilson, 1987), with the noncoding D-loop evolving several times more quickly (e.g., Pesole et al., 1992; Quinn, 1992). Over the past decade, there has been mounting evidence that instantaneous mutation rates substantially exceed substitution rates, in a range of organisms (e.g., Denver et al., 2000; Howell et al., 2003; Lambert et al., 2002; Mao et al., 2006; Mumm et al., 1997; Parsons et al., 1997; Santos et al., 2005). The immediate reaction to the first of these findings was that the polymorphisms generated by the elevated mutation rate are short-lived, perhaps extending back only a few hundred years (Gibbons, 1998; Macaulay et al., 1997). That is, purifying selection was thought to remove these polymorphisms very rapidly.

Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism

We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.

Haplotype analysis of two recurrent CDKN2A mutations in 10 melanoma families : evidence for common founders and independent mutations

Germ-line mutations in CDKN2A have been shown to predispose to cutaneous malignant melanoma. We have identified 2 new melanoma kindreds which carry a duplication of a 24bp repeat present in the 5' region of CDKN2A previously identified in melanoma families from Australia and the United States. This mutation has now been reported in 5 melanoma families from 3 continents: Europe, North America, and Australasia. The M53I mutation in exon 2 of CDKN2A has also been documented in 5 melanoma families from Australia and North America. The aim of this study was to determine whether the occurrence of the mutations in these families from geographically diverse populations represented mutation hotspots within CDKN2A or were due to common ancestors. Haplotypes of 11 microsatellite markers flanking CDKN2A were constructed in 5 families carrying the M53I mutation and 5 families carrying the 24bp duplication. There were some differences in the segregating haplotypes due primarily to recombinations and mutations within the short tandem-repeat markers; however, the data provide evidence to indicate that there were at least 3 independent 24bp duplication events and possibly only 1 original M53I mutation. This is the first study to date which indicates common founders in melanoma families from different continents.

Characterization of ENU-induced mutations in red blood cell structural proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

Cytochrome p450 1b1, a new keystone in gene-environment interactions related to human head and neck cancer?

Alcohol consumption and tobacco smoking are major causes of head and neck cancers, and regional differences point to the importance of research into gene-environment interactions. Much interest has been focused on polymorphisms of CYP1A1 and of GSTM1 and GSTT1, but a number of studies have not demonstrated significant effects. This has mostly been ascribed to small sample sizes. In general, the impact of polymorphisms of metabolic enzymes appears inconsistent, with some reports of weak-to-moderate associations, and with others of no elevation of risks. The classical cytochrome P450 isoenzyme considered for metabolic activation of polycyclic aromatic hydrocarbons (PAH) is CYP1A1. A new member of the CYP1 family, CYP1B1, was cloned in 1994, currently representing the only member of the CYP1B subfamily. A number of single nucleotide polymorphisms of the CYP1B1 gene have been reported. The amino acid substitutions Val432Leu (CYP1B1*3) and Asn453Ser (CYP1B1*4), located in the heme binding domain of CYP1B1, appear as likely candidates to be linked with biological effects. CYP1B1 activates a wide range of PAH, aromatic and heterocyclic amines. Very recently, the CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squamous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has pointed to interactive effects. This provides new molecular evidence that tobacco smoke-specific compounds relevant to head and neck carcinogenesis are metabolically activated through CYP1B1 and is consistent with a major pathogenetic relevance of PAH as ingredients of tobacco smoke.

Super DNAging - New insights into DNA integrity, genome stability and telomeres in the oldest old

Reductions in DNA integrity, genome stability, and telomere length are strongly associated with the aging process, age-related diseases as well as the age-related loss of muscle mass. However, in people reaching an age far beyond their statistical life expectancy the prevalence of diseases, such as cancer, cardiovascular disease, diabetes or dementia, is much lower compared to “averagely” aged humans. These inverse observations in nonagenarians (90–99 years), centenarians (100–109 years) and super-centenarians (110 years and older) require a closer look into dynamics underlying DNA damage within the oldest old of our society. Available data indicate improved DNA repair and antioxidant defense mechanisms in “super old” humans, which are comparable with much younger cohorts. Partly as a result of these enhanced endogenous repair and protective mechanisms, the oldest old humans appear to cope better with risk factors for DNA damage over their lifetime compared to subjects whose lifespan coincides with the statistical life expectancy. This model is supported by study results demonstrating superior chromosomal stability, telomere dynamics and DNA integrity in “successful agers”. There is also compelling evidence suggesting that life-style related factors including regular physical activity, a well-balanced diet and minimized psycho-social stress can reduce DNA damage and improve chromosomal stability. The most conclusive picture that emerges from reviewing the literature is that reaching “super old” age appears to be primarily determined by hereditary/genetic factors, while a healthy lifestyle additionally contributes to achieving the individual maximum lifespan in humans. More research is required in this rapidly growing population of super old people. In particular, there is need for more comprehensive investigations including short- and long-term lifestyle interventions as well as investigations focusing on the mechanisms causing DNA damage, mutations, and telomere shortening.

COL1A1 C-propeptide cleavage site mutation causes high bone mass, bone fragility and jaw lesions: A new cause of gnathodiaphyseal dysplasia?

Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant condition characterized by bone fragility, irregular bone mineral density (BMD) and fibro-osseous lesions in the skull and jaw. Mutations in Anoctamin-5 (ANO5) have been identified in some cases. We aimed to identify the causative mutation in a family with features of GDD but no mutation in ANO5, using whole exome capture and massive parallel sequencing (WES). WES of two affected individuals (a mother and son) and the mother's unaffected parents identified a mutation in the C-propeptide cleavage site of COL1A1. Similar mutations have been reported in individuals with osteogenesis imperfecta (OI) and paradoxically increased BMD. C-propeptide cleavage site mutations in COL1A1 may not only cause 'high bone mass OI', but also the clinical features of GDD, specifically irregular sclerotic BMD and fibro-osseous lesions in the skull and jaw. GDD patients negative for ANO5 mutations should be assessed for mutations in type I collagen C-propeptide cleavage sites.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10-5), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10-4) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10-9). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Genome-wide study of familial juvenile hyperuricaemic (gouty) nephropathy (FJHN) indicates a new locus, FJHN3, linked to chromosome 2p22.1-p21

Familial juvenile hyperuricaemic (gouty) nephropathy (FJHN), is an autosomal dominant disease associated with a reduced fractional excretion of urate, and progressive renal failure. FJHN is genetically heterogeneous and due to mutations of three genes: uromodulin (UMOD), renin (REN) and hepatocyte nuclear factor-1beta (HNF-1β) on chromosomes 16p12, 1q32.1, and 17q12, respectively. However, UMOD, REN or HNF-1β mutations are found in only ~45% of FJHN probands, indicating the involvement of other genetic loci in ~55% of probands. To identify other FJHN loci, we performed a single nucleotide polymorphism (SNP)-based genome-wide linkage analysis, in six FJHN families in whom UMOD, HNF-1β and REN mutations had been excluded. Parametric linkage analysis using a 'rare dominant' model established linkage in five of the six FJHN families, with a LOD score >+3, at 0% recombination, between FJHN and SNPs at chromosome 2p22.1-p21. Analysis of individual recombinants in two unrelated affected individuals defined a ~5.5 Mbp interval, flanked telomerically by SNP RS372139 and centromerically by RS896986 that contained the locus, designated FJHN3. The interval contains 28 genes, and DNA sequence analysis of the most likely candidate, solute carrier family 8 member 1 (SLC8A1), did not identify any abnormalities in the FJHN3 probands. FJHN3 is likely located within a ~5.5 Mbp interval on chromosome 2p22.1-p21, and identifying the genetic abnormality will help to further elucidate mechanisms predisposing to gout and renal failure.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition. © The Author 2014.

New dimeric supramolecular structure of mixed (phthalocyaninato)(porphyrinato)-europium(III) sandwiches : preparation and spectroscopic characteristics

The mixed double-decker Eu\[Pc(15C5)4](TPP) (1) was obtained by base-catalysed tetramerisation of 4,5-dicyanobenzo-15-crown-5 using the half-sandwich complex Eu(TPP)(acac) (acac = acetylacetonate), generated in situ, as the template. For comparative studies, the mixed triple-decker complexes Eu2\[Pc(15C5)4](TPP)2 (2) and Eu2\[Pc(15C5)4]2(TPP) (3) were also synthesised by the raise-by-one-story method. These mixed ring sandwich complexes were characterised by various spectroscopic methods. Up to four one-electron oxidations and two one-electron reductions were revealed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). As shown by electronic absorption and infrared spectroscopy, supramolecular dimers (SM1 and SM3) were formed from the corresponding double-decker 1 and triple-decker 3 in the presence of potassium ions in MeOH/CHCl3.

Disorderly categories in picture postcards from colonial Papua and New Guinea

At the turn of the century in Melbourne, a notice typed on the verso of a postcard stated that the South Yarra Baptist Young Men's class was meeting on the following Sunday at 2.45 p.m. The card, published in the United Kingdom, was numbered 51828 in the Valentine series of Papuan postcards.1 The image, a photograph of Hanuabada village taken in the early 1880s, and the text, written early in 1900, are contradictory and constitute separate realms of evidence that invite a renegotiation of meaning, analysis, and interpretation of the relationships between images, tourism, colonial rule, and ethnographic knowing. The visual evidence suggests the postcard may have played an ethnographic, educative role in the public understanding of Papua, which had just become an Australian Territory and was not yet well known. It is also suggestive of educative roles related to mission endeavours, subimperialist ambitions and the new tourist traffic through the ports of Port Moresby, Samarai, and Rabaul.