Genetic control of bone density and turnover: Role of the collagen 1α1, estrogen receptor, and vitamin D receptor genes

Genetic factors are known to influence both the peak bone mass and probably the rate of change in bone density. A range of regulatory and structural genes has been proposed to be involved including collagen 1α1 (COL1A1), the estrogen receptor (ER), and the vitamin D receptor (VDR), but the actual genes involved are uncertain. We therefore studied the role of the COL1A1 and VDR loci in control of bone density by linkage in 45 dizygotic twin pairs and 29 nuclear families comprising 120 individuals. The influences on bone density of polymorphisms of COL1A1, VDR, and ER were studied by association both cross-sectionally and longitudinally in 193 elderly postmenopausal women (average age, 69 years) over a mean follow-up time of 6.3 years. Weak linkage of the COL1A1 locus with bone density was observed in both twins and families (p = 0.02 in both data sets), confirming previous observations of linkage of this locus with bone density. Association between the MscI polymorphism of COL1A1 and rate of lumbar spine bone loss was observed with significant gene-environment interaction related to dietary calcium intake (p = 0.0006). In the lowest tertile of dietary calcium intake, carriers of "s" alleles lost more bone than "SS" homozygotes (p = 0.01), whereas the opposite was observed in the highest dietary calcium intake (p = 0.003). Association also was observed between rate of bone loss at both the femoral neck and the lumbar spine and the TaqI VDR polymorphism (p = 0.03). This association was strongest in those in the lowest tertile of calcium intake, also suggesting the presence of gene-environment interaction involving dietary calcium and VDR, influencing bone turnover. No significant association was observed between the PvuII ER polymorphism alone or in combination with VDR or COL1A1 genotypes, with either bone density or its rate of change. These data support the involvement of COL1A1 in determination of bone density and the interaction of both COL1A1 and VDR with calcium intake in regulation of change of bone density over time.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

SSR analysis of cultivated groundnut (Arachis hypogaea L.) germplasm resistant to rust and late leaf spot diseases

Cultivated groundnut (Arachis hypogaea L.) is an agronomically and economically important oilseed crop grown extensively throughout the semi-arid tropics of Asia, Africa and Latin America. Rust (Puccinia arachidis) and late leaf spot (LLS, Phaseoisariopsis personata) are among the major diseases causing significant yield loss in groundnut. The development of varieties with high levels of resistance has been constrained by adaptation of disease isolates to resistance sources and incomplete resistance in resistant sources. Despite the wide range of morphological diversity observed in the cultivated groundnut gene pool, molecular marker analyses have thus far been unable to detect a parallel level of genetic diversity. However, the recent development of simple sequence repeat (SSR) markers presents new opportunities for molecular diversity analysis of cultivate groundnut. The current study was conducted to identify diverse disease resistant germplasm for the development of mapping populations and for their introduction into breeding programs. Twenty-three SSRs were screened across 22 groundnut genotypes with differing levels of resistance to rust and LLS. Overall, 135 alleles across 23 loci were observed in the 22 genotypes screened. Twelve of the 23 SSRs (52%) showed a high level of polymorphism, with PIC values ≥0.5. This is the first report detecting such high levels of genetic polymorphism in cultivated groundnut. Multi-dimensional scaling and cluster analyses revealed three well-separated groups of genotypes. Locus by locus AMOVA and Kruskal-Wallis one-way ANOVA identified candidate SSR loci that may be valuable for mapping rust and LLS resistance. The molecular diversity analysis presented here provides valuable information for groundnut breeders designing strategies for incorporating and pyramiding rust and late leaf spot resistances and for molecular biologists wishing to create recombinant inbred line populations to map these traits.

Achievements in forest tree genetic improvement in Australia and New Zealand 2: Development of Corymbia species and hybrids for plantations in eastern Australia

This paper describes the establishment of provenance seedling seed orchards of three spotted gums and cadaga (all species of Corymbia ex Eucalyptus). It also discusses the limitations of growing the spotted gums as pure species including: lack of mass flowering, susceptibility to a fungal shoot blight and low amenability to vegetative propagation. These limitations, together with observation of putative natural hybrids of the spotted gums with cadaga, and the early promise of manipulated hybrids, led to an intensive breeding and testing program. Many hybrid families have significant advantages in growth and tolerance to disease, insects and frost, and can be vegetatively propagated. They also exhibit broad environmental plasticity, allowing the best varieties to be planted across a wider range of sites than the spotted gums, resulting in more land being suitable for plantation development.

Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries.

Identifying species boundaries within morphologically indistinguishable cryptic species complexes is often contentious. For the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae), the lack of a clear understanding about the genetic limits of the numerous genetic groups and biotypes so far identified has resulted in a lack of consistency in the application of the terms, the approaches use to apply them and in our understanding of what genetic structure within B. tabaci means. Our response has been to use mitochondrial gene cytochrome oxidase one to consider how to clearly and consistently define genetic separation. Using Bayesian phylogenetic analysis and analysis of sequence pairwise divergence we found a considerably higher to number of genetic groups than had been previously determined with two breaks in the distribution, one at 11% and another at 3.5%. At >11% divergence, 11 distinct groups were resolved, whereas at >3.5% divergence 24 groups were identified. Consensus sequences for each of these groups were determined and were shown to be useful in the correct assignment of sequences of unknown origin. The 3.5% divergence bound is consistent with species level separations in other insect taxa and Suggests that B. tabaci is it cryptic species composed of at least 24 distinct species. We further show that the placement of Bemesia atriplex (Froggatt) within the B. tabaci in, group adds further weight to the argument for species level separation within B. tabaci. This new analysis, which constructs consensus sequences and uses these its a standard against which unknown sequences call be compared, provides for the first time it consistent means of identifying the genetic hounds of each species with it high degree of certainty.

Cholesterol Disturbances in Inherited Neurodegenerative Diseases : Studies on Npc1, Ppt1 and Ctsd deficient mice

The central nervous system (CNS) is the most cholesterol-rich organ in the body. Cholesterol is essential to CNS functions such as synaptogenesis and formation of myelin. Significant differences exist in cholesterol metabolism between the CNS and the peripheral organs. However, the regulation of cholesterol metabolism in the CNS is poorly understood compared to our knowledge of the regulation of cholesterol homeostasis in organs reached by cholesterol-carrying lipoprotein particles in the circulation. Defects in CNS cholesterol homeostasis have been linked to a variety of neurodegenerative diseases, including common diseases with complex pathogenetic mechanisms such as Alzheimer s disease. In spite of intense effort, the mechanisms which link disturbed cholesterol homeostasis to these diseases remain elusive. We used three inherited recessive neurodegenerative disorders as models in the studies included in this thesis: Niemann-Pick type C (NPC), infantile neuronal ceroid lipofuscinosis and cathepsin D deficiency. Of these three, NPC has previously been linked to disturbed intracellular cholesterol metabolism. Elucidating the mechanisms with which disturbances of cholesterol homeostasis link to neurodegeneration in recessive inherited disorders with known genetic lesions should shed light on how cholesterol is handled in the healthy CNS and help to understand how these and more complex diseases develop. In the first study we analyzed the synthesis of sterols and the assembly and secretion of lipoprotein particles in Npc1 deficient primary astrocytes. We found that both wild type and Npc1 deficient astrocytes retain significant amounts of desmosterol and other cholesterol precursor sterols as membrane constituents. No difference was observed in the synthesis of sterols and the secretion of newly synthesized sterols between Npc1 wild type, heterozygote or knockout astrocytes. We found that the incorporation of newly synthesized sterols into secreted lipoprotein particles was not inhibited by Npc1 mutation, and the lipoprotein particles were similar to those excreted by wild type astrocytes in shape and size. The bulk of cholesterol was found to be secreted independently of secreted NPC2. These observations demonstrate the ability of Npc1 deficient astrocytes to handle de novo sterols, and highlight the unique sterol composition in the developing brain. Infantile neuronal ceroid lipofuscinosis is caused by the deficiency of a functional Ppt1 enzyme in the cells. In the second study, global gene expression studies of approximately 14000 mouse genes showed significant changes in the expression of 135 genes in Ppt1 deficient neurons compared to wild type. Several genes encoding for enzymes of the mevalonate pathway of cholesterol biosynthesis showed increased expression. As predicted by the expression data, sterol biosynthesis was found to be upregulated in the knockout neurons. These data link Ppt1 deficiency to disturbed cholesterol metabolism in CNS neurons. In the third study we investigated the effect of cathepsin D deficiency on the structure of myelin and lipid homeostasis in the brain. Our proteomics data, immunohistochemistry and western blotting data showed altered levels of the myelin protein components myelin basic protein, proteolipid protein and 2 , 3 -cyclic nucleotide 3 phosphodiesterase in the brains of cathepsin D deficient mice. Electron microscopy revealed altered myelin structure in cathepsin D deficient brains. Additionally, plasmalogen-derived alkenyl chains and 20- and 24-carbon saturated and monounsaturated fatty acids typical for glycosphingolipids were found to be significantly reduced, but polyunsaturated species were significantly increased in the knockout brains, pointing to a decrease in white matter. The levels of ApoE and ABCA1 proteins linked to cholesterol efflux in the CNS were found to be altered in the brains of cathepsin D deficient mice, along with an accumulation of cholesteryl esters and a decrease in triglycerols. Together these data demonstrate altered myelin architecture in cathepsin D deficient mice and link cathepsin D deficiency to aberrant cholesterol metabolism and trafficking. Basic research into rare monogenic diseases sheds light on the underlying biological processes which are perturbed in these conditions and contributes to our understanding of the physiological function of healthy cells. Eventually, understanding gained from the study of disease models may contribute towards establishing treatment for these disorders and further our understanding of the pathogenesis of other, more complex and common diseases.

Genetic studies of body mass index yield new insights for obesity biology

Obesity is heritable and predisposes to many diseases. To understand the genetic basis of obesity better, here we conduct a genome-wide association study and Metabochip meta-analysis of body mass index (BMI), a measure commonly used to define obesity and assess adiposity, in up to 339,224 individuals. This analysis identifies 97 BMI-associated loci (P < 5 × 10−8), 56 of which are novel. Five loci demonstrate clear evidence of several independent association signals, and many loci have significant effects on other metabolic phenotypes. The 97 loci account for ~2.7% of BMI variation, and genome-wide estimates suggest that common variation accounts for >20% of BMI variation. Pathway analyses provide strong support for a role of the central nervous system in obesity susceptibility and implicate new genes and pathways, including those related to synaptic function, glutamate signalling, insulin secretion/action, energy metabolism, lipid biology and adipogenesis.

Six novel susceptibility Loci for early-onset androgenetic alopecia and their unexpected association with common diseases

Androgenetic alopecia (AGA) is a highly heritable condition and the most common form of hair loss in humans. Susceptibility loci have been described on the X chromosome and chromosome 20, but these loci explain a minority of its heritable variance. We conducted a large-scale meta-analysis of seven genome-wide association studies for early-onset AGA in 12,806 individuals of European ancestry. While replicating the two AGA loci on the X chromosome and chromosome 20, six novel susceptibility loci reached genome-wide significance (p = 2.62x10(-)(9)-1.01x10(-)(1)(2)). Unexpectedly, we identified a risk allele at 17q21.31 that was recently associated with Parkinson's disease (PD) at a genome-wide significant level. We then tested the association between early-onset AGA and the risk of PD in a cross-sectional analysis of 568 PD cases and 7,664 controls. Early-onset AGA cases had significantly increased odds of subsequent PD (OR = 1.28, 95% confidence interval: 1.06-1.55, p = 8.9x10(-)(3)). Further, the AGA susceptibility alleles at the 17q21.31 locus are on the H1 haplotype, which is under negative selection in Europeans and has been linked to decreased fertility. Combining the risk alleles of six novel and two established susceptibility loci, we created a genotype risk score and tested its association with AGA in an additional sample. Individuals in the highest risk quartile of a genotype score had an approximately six-fold increased risk of early-onset AGA [odds ratio (OR) = 5.78, p = 1.4x10(-)(8)(8)]. Our results highlight unexpected associations between early-onset AGA, Parkinson's disease, and decreased fertility, providing important insights into the pathophysiology of these conditions.

Genetic Options for nematode control

Root-lesion nematodes (Pratylenchus thornei and P. neglectus) cause severe economic loss in wheat in Australia. This project aims to develop adaptaed wheat lines with resistance and tolerance to both species. These lines will be made available to Australian wheat breeding companies for further crossing and development of resistant and tolerant wheat varieties. Sources of resistance will be synthetic hexaploid and landrace wheats from the Middle East. Suitable double haploid populations will be phenotyped for the development of molecular markers to resistance and tolerance genes. The value of resistance and tolerance will be extended to growers through collaboration in demonstration trials with NGA and ORANA and presentations at GRDC Updates.

Methicillin Resistance In Staphylococci : Horizontal Transfer of Mobile Genetic Element (SCCmec) Between Staphylococcal Species

Regardless of the existence of antibiotics, infectious diseases are the leading causes of death in the world. Staphylococci cause many infections of varying severity, although they can also exist peacefully in many parts of the human body. Most often Staphylococcus aureus colonises the nose, and that colonisation is considered to be a risk factor for spread of this bacterium. S. aureus is considered to be the most important Staphylococcus species. It poses a challenge to the field of medicine, and one of the most problematic aspects is the drastic increase of the methicillin-resistant S. aureus (MRSA) strains in hospitals and community world-wide, including Finland. In addition, most of the clinical coagulase-negative staphylococcus (CNS) isolates express resistance to methicillin. Methicillin-resistance in S. aureus is caused by the mecA gene that encodes an extra penicillin-binding protein (PBP) 2a. The mecA gene is found in a mobile genomic island called staphylococcal chromosome cassette mec (SCCmec). The SCCmec consists of the mec gene and cassette chromosome recombinase (ccr)gene complexes. The areas of the SCCmec element outside the ccr and mec complex are known as the junkyard J regions. So far, eight types of SCCmec(SCCmec I- SCCmec VIII) and a number of variants have been described. The SCCmec island is an acquired element in S. aureus. Lately, it appears that CNS might be the storage place of the SCCmec that aid the S. aureus by providing it with the resistant elements. The SCCmec is known to exist only in the staphylococci. The aim of the present study was to investigate the horizontal transfer of SCCmec between the S. aureus and CNS. One specific aim was to study whether or not some methicillin-sensitive S. aureus (MSSA) strains are more inclined to receive the SCCmec than others. This was done by comparing the genetic background of clinical MSSA isolates in the health care facilities of the Helsinki and Uusimaa Hospital District in 2001 to the representatives of the epidemic MRSA (EMRSA) genotypes, which have been encountered in Finland during 1992-2004. Majority of the clinical MSSA strains were related to the EMRSA strains. This finding suggests that horizontal transfer of SCCmec from unknown donor(s) to several MSSA background genotypes has occurred in Finland. The molecular characteristics of representative clinical methicillin-resistant S. epidermidis (MRSE) isolates recovered in Finnish hospitals between 1990 and 1998 were also studied, examining their genetic relation to each other and to the internationally recognised MRSE clones as well, so as to ascertain the common traits between the SCCmec elements in MRSE and MRSA. The clinical MRSE strains were genetically related to each other; eleven PFGE types were associated with sequence type ST2 that has been identified world-wide. A single MRSE strain may possess two SCCmec types III and IV, which were recognised among the MRSA strains. Moreover, six months after the onset of an outbreak of MRSA possessing a SCCmec type V in a long-term care facility in Northern Finland (LTCF) in 2003, the SCCmec element of nasally carried methicillin-resistant staphylococci was studied. Among the residents of a LTCF, nasal carriage of MR-CNS was common with extreme diversity of SCCmec types. MRSE was the most prevalent CNS species. Horizontal transfer of SCCmec elements is speculated to be based on the sharing of SCCmec type V between MRSA and MRSE in the same person. Additionally, the SCCmec element of the clinical human S. sciuri isolates was studied. Some of the SCCmec regions were present in S. sciuri and the pls gene was common in it. This finding supports the hypothesis of genetic exchange happening between staphylococcal species. Evaluation of the epidemiology of methicillin-resistant staphylococcal colonisation is necessary in order to understand the apparent emergence of these strains and to develop appropriate control strategies. SCCmec typing is essential for understanding the emergence of MRSA strains from CNS, considering that the MR-CNS may represent the gene pool for the continuous creation of new SCCmec types from which MRSA might originate.

Genotyping for genetic association studies: Methods and applications

In this thesis, two separate single nucleotide polymorphism (SNP) genotyping techniques were set up at the Finnish Genome Center, pooled genotyping was evaluated as a screening method for large-scale association studies, and finally, the former approaches were used to identify genetic factors predisposing to two distinct complex diseases by utilizing large epidemiological cohorts and also taking environmental factors into account. The first genotyping platform was based on traditional but improved restriction-fragment-length-polymorphism (RFLP) utilizing 384-microtiter well plates, multiplexing, small reaction volumes (5 µl), and automated genotype calling. We participated in the development of the second genotyping method, based on single nucleotide primer extension (SNuPeTM by Amersham Biosciences), by carrying out the alpha- and beta tests for the chemistry and the allele-calling software. Both techniques proved to be accurate, reliable, and suitable for projects with thousands of samples and tens of markers. Pooled genotyping (genotyping of pooled instead of individual DNA samples) was evaluated with Sequenom s MassArray MALDI-TOF, in addition to SNuPeTM and PCR-RFLP techniques. We used MassArray mainly as a point of comparison, because it is known to be well suited for pooled genotyping. All three methods were shown to be accurate, the standard deviations between measurements being 0.017 for the MassArray, 0.022 for the PCR-RFLP, and 0.026 for the SNuPeTM. The largest source of error in the process of pooled genotyping was shown to be the volumetric error, i.e., the preparation of pools. We also demonstrated that it would have been possible to narrow down the genetic locus underlying congenital chloride diarrhea (CLD), an autosomal recessive disorder, by using the pooling technique instead of genotyping individual samples. Although the approach seems to be well suited for traditional case-control studies, it is difficult to apply if any kind of stratification based on environmental factors is needed. Therefore we chose to continue with individual genotyping in the following association studies. Samples in the two separate large epidemiological cohorts were genotyped with the PCR-RFLP and SNuPeTM techniques. The first of these association studies concerned various pregnancy complications among 100,000 consecutive pregnancies in Finland, of which we genotyped 2292 patients and controls, in addition to a population sample of 644 blood donors, with 7 polymorphisms in the potentially thrombotic genes. In this thesis, the analysis of a sub-study of pregnancy-related venous thromboses was included. We showed that the impact of factor V Leiden polymorphism on pregnancy-related venous thrombosis, but not the other tested polymorphisms, was fairly large (odds ratio 11.6; 95% CI 3.6-33.6), and increased multiplicatively when combined with other risk factors such as obesity or advanced age. Owing to our study design, we were also able to estimate the risks at the population level. The second epidemiological cohort was the Helsinki Birth Cohort of men and women who were born during 1924-1933 in Helsinki. The aim was to identify genetic factors that might modify the well known link between small birth size and adult metabolic diseases, such as type 2 diabetes and impaired glucose tolerance. Among ~500 individuals with detailed birth measurements and current metabolic profile, we found that an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene was associated with the duration of gestation, and weight and length at birth. Interestingly, the ACE insertion allele was also associated with higher indices of insulin secretion (p=0.0004) in adult life, but only among individuals who were born small (those among the lowest third of birth weight). Likewise, low birth weight was associated with higher indices of insulin secretion (p=0.003), but only among carriers of the ACE insertion allele. The association with birth measurements was also found with a common haplotype of the glucocorticoid receptor (GR) gene. Furthermore, the association between short length at birth and adult impaired glucose tolerance was confined to carriers of this haplotype (p=0.007). These associations exemplify the interaction between environmental factors and genotype, which, possibly due to altered gene expression, predisposes to complex metabolic diseases. Indeed, we showed that the common GR gene haplotype associated with reduced mRNA expression in thymus of three individuals (p=0.0002).

DAQ566 Genetic approaches to resist to Fusarium and Bipolaris in wheat/barley

Genetic approaches to resistance of bread wheat to crown rot and common root rot.

GM solutions to root diseases of cereals - A situation analysis and business case

Root disease causes about $503 million in losses annually to Australia's wheat and barley industries. Because of these large losses and in many cases the difficulty in reducing these losses through breeding or management, root diseases are candidates for solutions through genetic modification (GM). Through an extensive review of the scientific literature and patents, a range of approaches to GM solutions to root diseases are critically discussed. Given the high cost of regulatory approval for GM crops and a complex intellectual property (IP) landscape, it is likely that research in this area will be done in collaboration with international partners.

Genetic Structure of Cochliobolus sativus Populations Sampled from Roots and Leaves of Barley and Wheat in North Dakota

Common root rot (CRR) and spot blotch, caused by Cochliobolus sativus (Ito and Kurib.) Drechsl. ex Dast., are important diseases of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) worldwide. However, the population biology of C. sativus is still poorly understood. In this study, the genetic structure of three C. sativus populations, consisting of isolates sampled respectively from barley leaves (BL), barley roots (BR) and wheat roots (WR) in North Dakota, was analysed with amplified fragment length polymorphism (AFLP) markers. A total of 127 AFLP loci were generated among 208 C. sativus isolates analysed with three primer combinations. Gene diversity (H = 0.277-0.335) were high in all three populations. Genetic variation among C. sativus individuals within population accounted for 74%, whereas 26% of the genetic variation was explained among populations. Genetic differentiation was high (empty set PT = 0.261, corrected G ''(st)= 0.39), whereas gene flow (Nm) ranged from 1.27 to 1.56 among the three populations analysed. The multilocus linkage disequilibrium (LD) ((r) over bard = 0.0760.117) was moderate in C. sativus populations. Cluster analyses indicate that C. sativus populations differentiated according to the hosts (barley and wheat) and tissues (root and leaf) although generalists also exist in North Dakota. Crop breeding may benefit from combining genes for resistance against both specialists and generalists of C. sativus.