Genetic studies of osteoporosis: A rethink required

It has been 10 years since the seminal paper by Morrison and colleagues reporting the association of alleles of the vitamin D receptor and bone density [1], a paper which arguably kick-started the study of osteoporosis genetics. Since that report there have been literally thousands of osteoporosis genetic studies published, and large numbers of genes have been reported to be associated with the condition [2]. Although some of these reported associations are undoubtedly true, this snow-storm of papers and abstracts has clouded the field to such a great extent that it is very difficult to be certain of the veracity of most genetic associations reported hereto. The field needs to take stock and reconsider the best way forward, taking into account the biology of skeletal development and technological and statistical advances in human genetics, before more effort and money is wasted on continuing a process in which the primary achievement could be said to be a massive paper mountain. I propose in this review that the primary reasons for the paucity of success in osteoporosis genetics has been: •the absence of a major gene effect on bone mineral density (BMD), the most commonly studied bone phenotype; •failure to consider issues such as genetic heterogeneity, gene–environment interaction, and gene–gene interaction; •small sample sizes and over-optimistic data interpretation; and •incomplete assessment of the genetic variation in candidate genes studied.

Genetic factors in the pathogenesis of CPPD crystal deposition disease

Crystal deposition is a very complex process ruled by numerous factors. A small but important proportion of cases of chondrocalcinosis are monogenic, and many of the genes involved have been identified. These genetic findings strongly point to control of the level of extracellular inorganic pyrophosphate as the primary mechanism for their association with either calcium pyrophosphate dihydrate or hydroxyapatite deposition. However, effects on extracellular inorganic pyrophosphate levels do not explain the mechanism of association in all of these monogenic diseases. Further, there are likely to be several as yet unidentified genes that are important in this common condition. This review highlights what genetic studies have demonstrated about the processes involved in these diverse but related disorders.

Genome-wide association study to identify genetic determinants of severe asthma

Background: The genetic basis for developing asthma has been extensively studied. However, association studies to date have mostly focused on mild to moderate disease and genetic risk factors for severe asthma remain unclear. Objective: To identify common genetic variants affecting susceptibility to severe asthma. Methods: A genome-wide association study was undertaken in 933 European ancestry individuals with severe asthma based on Global Initiative for Asthma (GINA) criteria 3 or above and 3346 clean controls. After standard quality control measures, the association of 480 889 genotyped single nucleotide polymorphisms (SNPs) was tested. To improve the resolution of the association signals identified, non-genotyped SNPs were imputed in these regions using a dense reference panel of SNP genotypes from the 1000 Genomes Project. Then replication of SNPs of interest was undertaken in a further 231 cases and 1345 controls and a meta-analysis was performed to combine the results across studies. Results: An association was confirmed in subjects with severe asthma of loci previously identified for association with mild to moderate asthma. The strongest evidence was seen for the ORMDL3/GSDMB locus on chromosome 17q12-21 (rs4794820, p=1.03×10 (-8)following meta-analysis) meeting genome-wide significance. Strong evidence was also found for the IL1RL1/IL18R1 locus on 2q12 (rs9807989, p=5.59×10 (-8) following meta-analysis) just below this threshold. No novel loci for susceptibility to severe asthma met strict criteria for genome-wide significance. Conclusions: The largest genome-wide association study of severe asthma to date was carried out and strong evidence found for the association of two previously identified asthma susceptibility loci in patients with severe disease. A number of novel regions with suggestive evidence were also identified warranting further study.

Investigating ANKH and ENPP1 in Slovakian families with chondrocalcinosis

Familial articular chondrocalcinosis (CC) was Wrst reported in 1963. It is characterised by multiple calciWcations of hyaline and Wbrous cartilage in the joints and intervertebral discs. Mutations in ANKH have been identified in several pedigrees as a monogenic cause for this disorder. ANKH is a key protein in pyrophosphate metabolism and is involved in pyrophosphate transport across the cell membrane. The objective of this work was to screen ANKH and ENPP1, two key genes in pyrophosphate metabolism, in Slovakian kindreds with familial CC. DNA samples from 25 individuals (10 aVected, 15 unaVected) from 8 families were obtained. The promoter, coding regions and intron-exon boundaries of ANKH and ENPP1 were sequenced. Twelve DNA sequence variants, six in each gene, were identiWed. All the variants had been previously identified. None segregated with the disease. Our results suggest that neither ANKH nor ENPP1 mutations are the cause of CC in these families, indicating that possibly other major genes are involved in the aethiopathogenesis of this condition in these families.

Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21

We performed a genome-wide association study (GWAS) in 1705 Parkinson's disease (PD) UK patients and 5175 UK controls, the largest sample size so far for a PD GWAS. Replication was attempted in an additional cohort of 1039 French PD cases and 1984 controls for the 27 regions showing the strongest evidence of association (P < 10 4). We replicated published associations in the 4q22/SNCA and 17q21/MAPT chromosome regions (P < 10 10) and found evidence for an additional independent association in 4q22/SNCA.A detailed analysis of the haplotype structure at 17q21 showed that there are three separate risk groups within this region. We found weak but consistent evidence of association for common variants located in three previously published associated regions (4p15/BST1, 4p16/GAK and 1q32/PARK16). We found no support for the previously reported SNP association in 12q12/LRRK2. We also found an association of the two SNPs in 4q22/SNCA with the age of onset of the disease. © The Author 2010. Published by Oxford University Press.

The multiple sclerosis whole blood mRNA transcriptome and genetic associations indicate dysregulation of specific T cell pathways in pathogenesis

Multiple sclerosis (MS) is an autoimmune disease with a genetic component, caused at least in part by aberrant lymphocyte activity. The whole blood mRNA transcriptome was measured for 99 untreated MS patients: 43 primary progressive MS, 20 secondary progressive MS, 36 relapsing remitting MS and 45 age-matched healthy controls. The ANZgene Multiple Sclerosis Genetics Consortium genotyped more than 300 000 SNPs for 115 of these samples. Transcription from genes on translational regulation, oxidative phosphorylation, immune synapse and antigen presentation pathways was markedly increased in all forms of MS. Expression of genes tagging T cells was also upregulated (P < 10-12) in MS. A T cell gene signature predicts disease state with a concordance index of 0.79 with age and gender as co-variables, but the signature is not associated with clinical course or disability. The ANZgene genome wide association screen identified two novel regions with genome wide significance: one encoding the T cell co-stimulatory molecule, CD40; the other a region on chromosome 12q13-14. The CD40 haplotype associated with increased MS susceptibility has decreased gene expression in MS (P < 0.0007). The second MS susceptibility region includes 17 genes on 12q13-14 in tight linkage disequilibrium. Of these, only 13 are expressed in leukocytes, and of these the expression of one, FAM119B, is much lower in the susceptibility haplotype (P tdthomlt; 10-14). Overall, these data indicate dysregulation of T cells can be detected in the whole blood of untreated MS patients, and supports targeting of activated T cells in therapy for all forms of MS.

Genetic Options for Drought Management in Groundnut

Groundnut is one of the principal oilseeds in the world. It is cultivated on 24.8 million ha with a total production of 32.8 million t and an average productivity of 1.32 t ha-'. Developing countries account for 96.9% of the world groundnut area and 93.8% of total production. Production is concentrated in Asia (56.8% area and 66.5% production of the world) and Africa (38.0% area and 24.7% production). The groundnut productivity in Africa is only 0.86 t ha-' compared with 1.55 t hx1 of Asia. The world groundnut economy-facts, trends and outlook are desaibed in detail by Freeman et al., 1999. Briefly, in medium-term (i.e. up to 2010), 'groundnut production and consumption is likely to shift increasingly to developing countries; production will grow in all regions but most rapidly in Asia, slowly in sub-Saharan Africa and decline in Latin America; and utilizationwill continue to shift away from groundnut oil toward groundnut meal, specially confectionery products'.

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST–RFLP loci in the F1(NA6 × AU6) population. A comprehensive set of EST–SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA6 genetic map contains 88 EST–RFLP and 71 EST–SSR loci with a total map length of 963 cM, while the AU6 genetic map contains 67 EST–RFLP and 58 EST–SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium aphanidermatum populations infecting cucumber in Oman

Seventy three isolates of Pythium aphanidermatum obtained from cucumber from four different regions of Oman and 16 isolates of muskmelon from the Batinah region in Oman were characterized for aggressiveness, sensitivity to metalaxyl and genetic diversity using AFLP fingerprinting. Twenty isolates of P. aphanidermatum from diverse hosts from different countries were also included in the study. Most isolates from Oman were found to be aggressive on cucumber seedlings and all were highly sensitive to metalaxyl (EC50 < 0•80 µg mL−1). Isolates from cucumber and muskmelon were as aggressive as each other on both hosts (P > 0.05), which implies a lack of host specialization in P. aphanidermatum on these two hosts in Oman. AFLP analysis of all isolates using four primer-pair combinations resolved 152 bands, of which 61 (~40%) were polymorphic. Isolates of P. aphanidermatum from Oman and other countries exhibited high genetic similarity (mean = 94.1%) and produced 59 different AFLP profiles. Analysis of molecular variance indicated that most AFLP variation among populations of P. aphanidermatum in Oman was associated with geographical regions (FST = 0.118; P < 0.0001), not hosts (FST = -0.004; P = 0.4323). These data were supported by the high rate of recovery (24%) of identical phenotypes between cucumber and muskmelon fields in the same region as compared to the low recovery (10%) across regions in Oman, which suggests more frequent movement of Pythium inoculum among muskmelon and cucumber fields in the same region compared to movement across geographically separated regions. However, recovering clones among regions and different countries may imply circulation of Pythium inoculum via common sources in Oman and also intercontinental spread of isolates.

Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium spinosum infecting cucumber in Oman

A total of 24 isolates of Pythium spinosum from cucumber obtained from five regions in Oman were characterized for genetic diversity using amplified fragment length polymorphism (AFLP) fingerprinting and three isolates from the Netherlands, South Africa and Japan were included for comparison. Isolates from Oman were also characterized for aggressiveness on cucumber seedlings and sensitivity to metalaxyl. Identity of all isolates was confirmed using sequences of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), which showed more than 99% nucleotide similarity among all isolates. Using six primer-pair combinations, AFLP fingerprinting resolved 295 AFLP markers of which 193 were polymorphic among isolates from other countries and only six were polymorphic among isolates of P. spinosum from Oman. Seven different AFLP phenotypes of P. spinosum were recovered in Oman; two of them were found to contain over 79% of isolates and one was recovered from all regions in Oman. Phenotypes from Oman showed very high (?99%) levels of genetic similarity to each other compared to moderate (mean =53%) levels of genetic similarity with phenotypes from other countries. In addition, all isolates from Oman were found to be highly sensitive to metalaxyl and all were aggressive on cucumber seedlings at 25°C. The high genetic similarity among phenotypes of P. spinosum in Oman as well as recovering two major clones across regions may suggest that P. spinosum has been recently introduced in Oman via a common source.

Genetic Impacts of the Hull on Barley Grain Quality

Barley hull plays an important role in malt and feed quality and processing. In this study we measured the variation in hull con-tent along with other grain quality traits namely, kernel discolouration and degree of pre-harvest sprouting, in a single map-ping population. There were significant (p < 0.05) genetic as well as environment effects. In addition, heritability was calculated for hull content to be 29% and 47% for two years’ data. From the analysis, major QTL markers were identified in con-trolling the expression of hull content on chromosomes 2 (2H), and 6 (6H) with significant (P < 0.05) LOD scores of 5.4 and 3.46 respectively. Minor QTLs were identified on 1 (7H), 4 (4H), 5 (1H) and 7 (5H). The region at marker Bmac310 on 4(4H) could be associated with dormancy gene SD4. A number of the QTLs also coincided with regions for either kernel discolouration or preharvest sprouting resistance (dormancy). The results indicate that variation exists for hull content, which is influenced by both growing environment as well as genetically, although the genetic variance explained less than half of the total variance. Further, hull content also impacts on other grain quality attributes including dormancy, sprouting resistance and kernel appearance.

Measurement of genetic and environmental variation in barley (Hordeum vulgare) grain hardness

In this study, we assessed a broad range of barley breeding lines and commercial varieties by three hardness methods (two particle size methods and one crush resistance method (SKCS—Single-Kernel Characterization System), grown at multiple sites to see if there was variation in barley hardness and if that variation was genetic or environmentally controlled. We also developed near-infrared reflectance (NIR) calibrations for these three hardness methods to ascertain if NIR technology was suitable for rapid screening of breeding lines or specific populations. In addition, we used this data to identify genetic regions that may be associated with hardness. There were significant (p<0.05) genetic effects for the three hardness methods. There were also environmental effects, possibly linked to the effect of protein on hardness, i.e. increasing protein resulted in harder grain. Heritability values were calculated at >85% for all methods. The NIR calibrations, with R2 values of >90%, had Standard Error of Prediction values of 0.90, 72 and 4.0, respectively, for the three hardness methods. These equations were used to predict hardness values of a mapping population which resulted in genetic markers being identified on all chromosomes but chromosomes 2H, 3H, 5H, 6H and 7H had markers with significant LOD scores. The two regions on 5H were on the distal end of both the long and short arms. The region that showed significant LOD score was on the long arm. However, the region on the short arm associated with the hardness (hordoindoline) genes did not have significant LOD scores. The results indicate that barley hardness is influenced by both genotype and environment and that the trait is heritable, which would allow breeders to develop very hard or soft varieties if required. In addition, NIR was shown to be a reliable tool for screening for hardness. While the data set used in this study has a relatively low variation in hardness, the tools developed could be applied to breeding populations that have large variation in barley grain hardness.

The genetic diversity of ampeloviruses in Australian pineapples and their association with mealybug wilt disease

Pineapple mealybug wilt-associated virus 1 (PMWaV-1), 2 (PMWaV-2) and -3 (PMWaV-3) have been detected in Australian commercial pineapple crops, along with a previously undescribed ampelovirus, for which the name Pineapple mealybug wilt-associated virus 5 (PMWaV-5) is proposed. Partial sequences extending from open reading frame 1b through to the heat shock protein homologue were obtained for PMWaV-1, -3 and -5. Phylogenetic analyses of selected regions of these sequences indicated that PMWaV-5 is a distinct species and most closely related to PMWaV-1. The amino acid sequence variation observed in the RNA-dependent RNA polymerase region of PMWaV-1 isolates was 95.8–98.4% and of PMWaV-3 isolates was 92.2–99.5%. In surveys of mealybug wilt disease (MWD) affected crops, none of the four viruses was clearly associated with the disease at all survey sites. A statistically significant association (P < 0.001) between the presence of PMWaV-2 and symptoms was observed at one survey site (site 3), but the virus was at a low incidence at the remaining three survey sites. By contrast, although PMWaV-1 and -3 were equally distributed between symptomless and MWD-affected plants at site 3, there was a statistically significant (P < 0.001) association between each of these two viruses and MWD at sites 1 and 4. At site 2, there was a statistically significant (P < 0.001) association only between PMWaV-3 and MWD. PMWaV-1 was the most commonly found of the four viruses and conversely PMWaV-5 was only occasionally found. Australian isolates of PMWaV-1, -2 and -3 were transmitted by the mealybug species Dysmicoccus brevipes.

DNA amplification fingerprinting analysis of genetic variation within Fusarium oxysporum f.sp zingiberi

Genetic variation among 29 isolates of Fusarium oxysporum f.sp. zingiberi (Foz) collected from diseased ginger rhizome in production regions throughout Queensland was analysed using DNA amplification fingerprinting (DAF). Eight isolates of other Fusarium species and/or formae speciales were included for comparative analysis. Within the Foz isolates, three haplotypes were identified based on 17 polymorphic bands generated with five primers. Two groups showed very little genetic variation (98.6% similarity), whereas the third single isolate was quite distinct in terms of its molecular profile (77.2% similarity). Genetic similarity among the Fusarium solani, F. oxysporum f.sp. lycopersici and F. oxysporum f.sp. cubense races 1, 3 and 4 isolates compared well with the published literature.

A consensus genetic map of sorghum that integrates multiple component maps and high-throughput Diversity Array Technology (DArT) markers

Background: Sorghum genome mapping based on DNA markers began in the early 1990s and numerous genetic linkage maps of sorghum have been published in the last decade, based initially on RFLP markers with more recent maps including AFLPs and SSRs and very recently, Diversity Array Technology (DArT) markers. It is essential to integrate the rapidly growing body of genetic linkage data produced through DArT with the multiple genetic linkage maps for sorghum generated through other marker technologies. Here, we report on the colinearity of six independent sorghum component maps and on the integration of these component maps into a single reference resource that contains commonly utilized SSRs, AFLPs, and high-throughput DArT markers. Results: The six component maps were constructed using the MultiPoint software. The lengths of the resulting maps varied between 910 and 1528 cM. The order of the 498 markers that segregated in more than one population was highly consistent between the six individual mapping data sets. The framework consensus map was constructed using a "Neighbours" approach and contained 251 integrated bridge markers on the 10 sorghum chromosomes spanning 1355.4 cM with an average density of one marker every 5.4 cM, and were used for the projection of the remaining markers. In total, the sorghum consensus map consisted of a total of 1997 markers mapped to 2029 unique loci ( 1190 DArT loci and 839 other loci) spanning 1603.5 cM and with an average marker density of 1 marker/0.79 cM. In addition, 35 multicopy markers were identified. On average, each chromosome on the consensus map contained 203 markers of which 58.6% were DArT markers. Non-random patterns of DNA marker distribution were observed, with some clear marker-dense regions and some marker-rare regions. Conclusion: The final consensus map has allowed us to map a larger number of markers than possible in any individual map, to obtain a more complete coverage of the sorghum genome and to fill a number of gaps on individual maps. In addition to overall general consistency of marker order across individual component maps, good agreement in overall distances between common marker pairs across the component maps used in this study was determined, using a difference ratio calculation. The obtained consensus map can be used as a reference resource for genetic studies in different genetic backgrounds, in addition to providing a framework for transferring genetic information between different marker technologies and for integrating DArT markers with other genomic resources. DArT markers represent an affordable, high throughput marker system with great utility in molecular breeding programs, especially in crops such as sorghum where SNP arrays are not publicly available.