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1.  Differential gene expression in femoral bone from red junglefowl and domestic chicken, differing for bone phenotypic traits 
BMC Genomics  2007;8:208.
Osteoporosis is frequently observed among aging hens from egg-producing strains (layers) of domestic chicken. White Leghorn (WL) has been intensively selected for egg production and it manifests striking phenotypic differences for a number of traits including several bone phenotypes in comparison with the wild ancestor of chicken, the red junglefowl (RJ). Previously, we have identified four Quantitative Trait Loci (QTL) affecting bone mineral density and bone strength in an intercross between RJ and WL. With the aim of further elucidating the genetic basis of bone traits in chicken, we have now utilized cDNA-microarray technology in order to compare global RNA-expression in femoral bone from adult RJ and WL (five of each sex and population).
When contrasting microarray data for all WL-individuals to that of all RJ-individuals we observed differential expression (False discovery rate adjusted p-values < 0.015) for 604 microarray probes. In corresponding male and female contrasts, differential expression was observed for 410 and 270 probes, respectively. Altogether, the three contrasts between WL and RJ revealed differential expression of 779 unique transcripts, 57 of which are located to previously identified QTL-regions for bone traits. Some differentially expressed genes have previously been attributed roles in bone metabolism and these were: WNT inhibitory factor 1 (WIF1), WD repeat-containing protein 5 (WDR5) and Syndecan 3 (SDC3). Among differentially expressed transcripts, those encoding structural ribosomal proteins were highly enriched and all 15 had lower expression in WL.
We report the identification of 779 differentially expressed transcripts, several residing within QTL-regions for bone traits. Among differentially expressed transcripts, those encoding structural ribosomal proteins were highly enriched and all had lower expression levels in WL. In addition, transcripts encoding four translation initiation and translation elongation factor proteins also had lower expression levels in WL, possibly indicating perturbation of protein biosynthesis pathways between the two populations. Information derived from this study could be relevant to the bone research field and may also aid in further inference of genetic changes accompanying animal domestication.
PMCID: PMC1934367  PMID: 17605776
2.  Dominant Red Coat Color in Holstein Cattle Is Associated with a Missense Mutation in the Coatomer Protein Complex, Subunit Alpha (COPA) Gene 
PLoS ONE  2015;10(6):e0128969.
Coat color in Holstein dairy cattle is primarily controlled by the melanocortin 1 receptor (MC1R) gene, a central determinant of black (eumelanin) vs. red/brown pheomelanin synthesis across animal species. The major MC1R alleles in Holsteins are Dominant Black (MC1RD) and Recessive Red (MC1Re). A novel form of dominant red coat color was first observed in an animal born in 1980. The mutation underlying this phenotype was named Dominant Red and is epistatic to the constitutively activated MC1RD. Here we show that a missense mutation in the coatomer protein complex, subunit alpha (COPA), a gene with previously no known role in pigmentation synthesis, is completely associated with Dominant Red in Holstein dairy cattle. The mutation results in an arginine to cysteine substitution at an amino acid residue completely conserved across eukaryotes. Despite this high level of conservation we show that both heterozygotes and homozygotes are healthy and viable. Analysis of hair pigment composition shows that the Dominant Red phenotype is similar to the MC1R Recessive Red phenotype, although less effective at reducing eumelanin synthesis. RNA-seq data similarly show that Dominant Red animals achieve predominantly pheomelanin synthesis by downregulating genes normally required for eumelanin synthesis. COPA is a component of the coat protein I seven subunit complex that is involved with retrograde and cis-Golgi intracellular coated vesicle transport of both protein and RNA cargo. This suggests that Dominant Red may be caused by aberrant MC1R protein or mRNA trafficking within the highly compartmentalized melanocyte, mimicking the effect of the Recessive Red loss of function MC1R allele.
PMCID: PMC4456281  PMID: 26042826
3.  A Genomic Duplication is Associated with Ectopic Eomesodermin Expression in the Embryonic Chicken Comb and Two Duplex-comb Phenotypes 
PLoS Genetics  2015;11(3):e1004947.
Duplex-comb (D) is one of three major loci affecting comb morphology in the domestic chicken. Here we show that the two Duplex-comb alleles, V-shaped (D*V) and Buttercup (D*C), are both associated with a 20 Kb tandem duplication containing several conserved putative regulatory elements located 200 Kb upstream of the eomesodermin gene (EOMES). EOMES is a T-box transcription factor that is involved in mesoderm specification during gastrulation. In D*V and D*C chicken embryos we find that EOMES is ectopically expressed in the ectoderm of the comb-developing region as compared to wild-type embryos. The confinement of the ectopic expression of EOMES to the ectoderm is in stark contrast to the causal mechanisms underlying the two other major comb loci in the chicken (Rose-comb and Pea-comb) in which the transcription factors MNR2 and SOX5 are ectopically expressed strictly in the mesenchyme. Interestingly, the causal mutations of all three major comb loci in the chicken are now known to be composed of large-scale structural genomic variants that each result in ectopic expression of transcription factors. The Duplex-comb locus also illustrates the evolution of alleles in domestic animals, which means that alleles evolve by the accumulation of two or more consecutive mutations affecting the phenotype. We do not yet know whether the V-shaped or Buttercup allele correspond to the second mutation that occurred on the haplotype of the original duplication event.
Author Summary
There are three major variant comb types found in the domestic chicken; Rose-comb, Pea-comb and Duplex-comb. Within the Duplex-comb there are two distinct types, V-shaped and Buttercup. Previous experiments have shown that these two Duplex-comb types represent different alleles at a single locus. We have mapped the location of the Duplex-comb locus and identified a 20 Kb duplication that is present only in chickens that have a Duplex-comb phenotype. The 20 Kb duplication is located 200 Kb upstream of EOMES, a gene that was found to be abnormally expressed in the comb-developing region of V-shaped and Buttercup comb chicken embryos. This suggests that the 20 Kb duplication contains regulatory elements affecting EOMES expression. These findings complete our characterization of the genetic basis of the three major comb loci in the chicken, all of which are caused by large-scale structural genomic variants that drive ectopic expression of transcription factors in the comb region during chicken embryo development.
PMCID: PMC4366209  PMID: 25789773
4.  A cis-Regulatory Mutation of PDSS2 Causes Silky-Feather in Chickens 
PLoS Genetics  2014;10(8):e1004576.
Silky-feather has been selected and fixed in some breeds due to its unique appearance. This phenotype is caused by a single recessive gene (hookless, h). Here we map the silky-feather locus to chromosome 3 by linkage analysis and subsequently fine-map it to an 18.9 kb interval using the identical by descent (IBD) method. Further analysis reveals that a C to G transversion located upstream of the prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2) gene is causing silky-feather. All silky-feather birds are homozygous for the G allele. The silky-feather mutation significantly decreases the expression of PDSS2 during feather development in vivo. Consistent with the regulatory effect, the C to G transversion is shown to remarkably reduce PDSS2 promoter activity in vitro. We report a new example of feather structure variation associated with a spontaneous mutation and provide new insight into the PDSS2 function.
Author Summary
The feather is an excellent model for evolution and development due to its complex structure and vast diversity. Some chickens have silky-feather because of a loss of hooklets in pennaceous feathers, while most chickens have the wild-type normal feather. Hooklets are formed in the last differentiation stage of the life cycle of a pennaceous feather. Chickens with silky-feather are homozygous for a recessive allele (hookless, h). Silkie chicken from China is one of the breeds showing the fascinating silky-feather phenotype and the breed has been known for hundreds of years. In this study, we mapped the silky-feather locus to an 18.9 kb interval and identified a single nucleotide polymorphism (SNP) completely associated with silky-feather. The causative mutation is located 103 base pairs upstream of the coding sequence of prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2). The expression of the PDSS2 gene is decreased in silky-feather skin during feather development in vivo. The silky-feather allele also reduces the PDSS2 promoter activity in vitro. This is the first report of feather structure variation associated with PDSS2 and provides new insight into molecular signaling in the late development stage of feather morphogenesis.
PMCID: PMC4148213  PMID: 25166907
5.  ZBED6 Modulates the Transcription of Myogenic Genes in Mouse Myoblast Cells 
PLoS ONE  2014;9(4):e94187.
ZBED6 is a recently discovered transcription factor, unique to placental mammals, that has evolved from a domesticated DNA transposon. It acts as a repressor at the IGF2 locus. Here we show that ZBED6 acts as a transcriptional modulator in mouse myoblast cells, where more than 700 genes were differentially expressed after Zbed6-silencing. The most significantly enriched GO term was muscle protein and contractile fiber, which was consistent with increased myotube formation. Twenty small nucleolar RNAs all showed increased expression after Zbed6-silencing. The co-localization of histone marks and ZBED6 binding sites and the effect of Zbed6-silencing on distribution of histone marks was evaluated by ChIP-seq analysis. There was a strong association between ZBED6 binding sites and the H3K4me3, H3K4me2 and H3K27ac modifications, which are usually found at active promoters, but no association with the repressive mark H3K27me3. Zbed6-silencing led to increased enrichment of active marks at myogenic genes, in agreement with the RNA-seq findings. We propose that ZBED6 preferentially binds to active promoters and modulates transcriptional activity without recruiting repressive histone modifications.
PMCID: PMC3979763  PMID: 24714595
6.  Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels - a Novel Therapeutic Approach in Osteogenesis Imperfecta 
Osteogenesis imperfecta, also known as “brittle bone disease”, is a heterogeneous disorder of connective tissue generally caused by dominant mutations in the genes COL1A1 and COL1A2, encoding the α1 and α2 chains of type I (pro)collagen. Symptomatic patients are usually prescribed bisphosphonates, but this treatment is neither curative nor sufficient. A promising field is gene silencing through RNA interference. In this study small interfering RNAs (siRNAs) were designed to target each allele of 3'UTR insertion/deletion polymorphisms (indels) in COL1A1 (rs3840870) and COL1A2 (rs3917). For both indels, the frequency of heterozygous individuals was determined to be approximately 50% in Swedish cohorts of healthy controls as well as in patients with osteogenesis imperfecta. Cultures of primary human bone derived cells were transfected with siRNAs through magnet-assisted transfection. cDNA from transfected cells was sequenced in order to measure targeted allele/non-targeted allele ratios and the overall degree of silencing was assessed by quantitative PCR. Successful allele dependent silencing was observed, with promising results for siRNAs complementary to both the insertion and non-insertion harboring alleles. In COL1A1 cDNA the indel allele ratios were shifted from 1 to 0.09 and 0.19 for the insertion and non-insertion allele respectively while the equivalent resulting ratios for COL1A2 were 0.05 and 0.01. Reductions in mRNA abundance were also demonstrated; in cells treated with siRNAs targeting the COL1A1 alleles the average COL1A1 mRNA levels were reduced 65% and 78% compared to negative control levels and in cells treated with COL1A2 siRNAs the average COL1A2 mRNA levels were decreased 26% and 49% of those observed in the corresponding negative controls. In conclusion, allele dependent silencing of collagen type I utilizing 3'UTR indels common in the general population constitutes a promising mutation independent therapeutic approach for osteogenesis imperfecta.
PMCID: PMC3752721  PMID: 23983594
osteogenesis imperfecta; collagen type I; siRNA
7.  Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice 
Nature  2012;488(7413):642-646.
Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement1. These networks produce left–right alternation of limbs as well as coordinated activation of flexor and extensor muscles2. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.
PMCID: PMC3523687  PMID: 22932389
8.  A Sexual Ornament in Chickens Is Affected by Pleiotropic Alleles at HAO1 and BMP2, Selected during Domestication 
PLoS Genetics  2012;8(8):e1002914.
Domestication is one of the strongest forms of short-term, directional selection. Although selection is typically only exerted on one or a few target traits, domestication can lead to numerous changes in many seemingly unrelated phenotypes. It is unknown whether such correlated responses are due to pleiotropy or linkage between separate genetic architectures. Using three separate intercrosses between wild and domestic chickens, a locus affecting comb mass (a sexual ornament in the chicken) and several fitness traits (primarily medullary bone allocation and fecundity) was identified. This locus contains two tightly-linked genes, BMP2 and HAO1, which together produce the range of pleiotropic effects seen. This study demonstrates the importance of pleiotropy (or extremely close linkage) in domestication. The nature of this pleiotropy also provides insights into how this sexual ornament could be maintained in wild populations.
Author Summary
The genetic analysis of phenotypes and the identification of the causative underlying genes remain central to molecular and evolutionary biology. By utilizing the domestication process, it is possible to exploit the large differences between domesticated animals and their wild counterparts to study both this and the mechanism of domestication itself. Domestication has been central to the advent of modern civilization; and yet, despite domesticated animals displaying similar adaptations in morphology, physiology, and behaviour, the genetic basis of these changes are unknown. In addition, though sexual selection theory has been the subject of a vast amount of study, very little is known about which genes are underpinning such traits. We have generated multiple intercrosses and advanced intercrosses based on wild-derived and domestic chickens to fine-map genomic regions affecting a sexual ornament. These regions have been over-laid with putative selective sweeps identified in domestic chickens and found to be significantly associated with them. By using expression QTL analysis, we show that two genes in one region, HAO1 and BMP2, are controlling multiple aspects of the domestication phenotype, from a sexual ornament to multiple life history traits. This demonstrates the importance of pleiotropy (or extremely close linkage) in controlling these genetic changes.
PMCID: PMC3431302  PMID: 22956912
9.  The Rose-comb Mutation in Chickens Constitutes a Structural Rearrangement Causing Both Altered Comb Morphology and Defective Sperm Motility 
PLoS Genetics  2012;8(6):e1002775.
Rose-comb, a classical monogenic trait of chickens, is characterized by a drastically altered comb morphology compared to the single-combed wild-type. Here we show that Rose-comb is caused by a 7.4 Mb inversion on chromosome 7 and that a second Rose-comb allele arose by unequal crossing over between a Rose-comb and wild-type chromosome. The comb phenotype is caused by the relocalization of the MNR2 homeodomain protein gene leading to transient ectopic expression of MNR2 during comb development. We also provide a molecular explanation for the first example of epistatic interaction reported by Bateson and Punnett 104 years ago, namely that walnut-comb is caused by the combined effects of the Rose-comb and Pea-comb alleles. Transient ectopic expression of MNR2 and SOX5 (causing the Pea-comb phenotype) occurs in the same population of mesenchymal cells and with at least partially overlapping expression in individual cells in the comb primordium. Rose-comb has pleiotropic effects, as homozygosity in males has been associated with poor sperm motility. We postulate that this is caused by the disruption of the CCDC108 gene located at one of the inversion breakpoints. CCDC108 is a poorly characterized protein, but it contains a MSP (major sperm protein) domain and is expressed in testis. The study illustrates several characteristic features of the genetic diversity present in domestic animals, including the evolution of alleles by two or more consecutive mutations and the fact that structural changes have contributed to fast phenotypic evolution.
Author Summary
Comb morphology is a trait that shows considerable variability among domestic chickens. The Rose-comb mutation causes a drastically altered shape of the comb, whereas the Pea-comb mutation leads to a considerable reduction in the size of the comb. The combined effect of Rose-comb and Pea-comb is a comb shaped like a walnut, and the phenotype is consequently named walnut-comb. Both Pea-comb and Rose-comb are caused by structural changes in the genome leading to altered expression of important transcription factors. In a previous study we showed that Pea-comb is caused by misexpression of SOX5 during the development of the comb. In this study we report that Rose-comb is caused by a large inversion on chicken chromosome 7. The inversion moves the MNR2 gene to a new genomic location. This leads to misexpression of MNR2 during comb development, similar to the defect causing Pea-comb. Roosters that are homozygous for the Rose-comb inversion show poor sperm motility, and our results suggest that this is caused by the disruption of the CCDC108 gene that is located at one of the inversion breakpoints. CCDC108 is well conserved between chickens and humans, and this study establishes CCDC108 as a candidate gene for sperm motility disorders in humans.
PMCID: PMC3386170  PMID: 22761584
10.  COL1 C-propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta 
Human mutation  2011;32(6):598-609.
Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.
PMCID: PMC3103631  PMID: 21344539
Osteogenesis imperfecta; C-propeptide; collagen; C-proteinase; mineralization; high bone mass
11.  Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens 
BMC Genomics  2012;13:59.
Variations in gene expression, mediated by epigenetic mechanisms, may cause broad phenotypic effects in animals. However, it has been debated to what extent expression variation and epigenetic modifications, such as patterns of DNA methylation, are transferred across generations, and therefore it is uncertain what role epigenetic variation may play in adaptation.
In Red Junglefowl, ancestor of domestic chickens, gene expression and methylation profiles in thalamus/hypothalamus differed substantially from that of a domesticated egg laying breed. Expression as well as methylation differences were largely maintained in the offspring, demonstrating reliable inheritance of epigenetic variation. Some of the inherited methylation differences were tissue-specific, and the differential methylation at specific loci were little changed after eight generations of intercrossing between Red Junglefowl and domesticated laying hens. There was an over-representation of differentially expressed and methylated genes in selective sweep regions associated with chicken domestication.
Our results show that epigenetic variation is inherited in chickens, and we suggest that selection of favourable epigenomes, either by selection of genotypes affecting epigenetic states, or by selection of methylation states which are inherited independently of sequence differences, may have been an important aspect of chicken domestication.
PMCID: PMC3297523  PMID: 22305654
Domestication; gene expression; tiling array; behaviour; methylation
12.  A Complex Genomic Rearrangement Involving the Endothelin 3 Locus Causes Dermal Hyperpigmentation in the Chicken 
PLoS Genetics  2011;7(12):e1002412.
Dermal hyperpigmentation or Fibromelanosis (FM) is one of the few examples of skin pigmentation phenotypes in the chicken, where most other pigmentation variants influence feather color and patterning. The Silkie chicken is the most widespread and well-studied breed displaying this phenotype. The presence of the dominant FM allele results in extensive pigmentation of the dermal layer of skin and the majority of internal connective tissue. Here we identify the causal mutation of FM as an inverted duplication and junction of two genomic regions separated by more than 400 kb in wild-type individuals. One of these duplicated regions contains endothelin 3 (EDN3), a gene with a known role in promoting melanoblast proliferation. We show that EDN3 expression is increased in the developing Silkie embryo during the time in which melanoblasts are migrating, and elevated levels of expression are maintained in the adult skin tissue. We have examined four different chicken breeds from both Asia and Europe displaying dermal hyperpigmentation and conclude that the same structural variant underlies this phenotype in all chicken breeds. This complex genomic rearrangement causing a specific monogenic trait in the chicken illustrates how novel mutations with major phenotypic effects have been reused during breed formation in domestic animals.
Author Summary
The process of animal domestication has been a long and ongoing effort of the human race to cultivate beneficial traits in agriculturally productive or otherwise beneficial species. We are just now beginning to understand the effect this type of selection pressure has had on genetic variation and overall genome architecture using quickly advancing modern genetic and genomic technologies. Here we show how along the path of animal domestication a single large rearrangement involving a duplication and inversion of two distinct regions of the chicken genome occurred, likely disrupting long-range cis-regulatory elements of endothelin 3 (EDN3) and resulting in a very extreme skin pigmentation phenotype. Dermal hyperpigmentation, or Fibromelanosis (FM), is a defining characteristic of the Silkie chicken breed, which originates in China. Chickens very similar to the Silkie have been described in ancient Chinese texts on traditional medicine, illustrating how unique phenotypes in domesticated animals are incorporated into human culture and tradition that persists to this day. The presence of the same rearrangement in other FM chicken breeds found around the world highlights both the causality of this mutation as well as how humans serve to spread genetic variation linked to novel traits in domestic animals.
PMCID: PMC3245302  PMID: 22216010
13.  ZBED6 
Transcription  2010;1(3):144-148.
A DNA transposon integrated into -the genome of a primitive mammal some 200 million years ago and, millions of years later, it evolved an essential function in the common ancestor of all placental mammals. This protein, now named ZBED6, was recently discovered because a mutation disrupting one of its binding sites, in an intron of the IGF2 gene, makes pigs grow more muscle. These findings have revealed a new mechanism for regulating muscle growth as well as a novel transcription factor that appears to be of major importance for transcriptional regulation in placental mammals.
PMCID: PMC3023575  PMID: 21326889
ZBED6; IGF2; transcriptional regulation; evolution; ChIP-seq; muscle growth
14.  Copy Number Variation in Intron 1 of SOX5 Causes the Pea-comb Phenotype in Chickens 
PLoS Genetics  2009;5(6):e1000512.
Pea-comb is a dominant mutation in chickens that drastically reduces the size of the comb and wattles. It is an adaptive trait in cold climates as it reduces heat loss and makes the chicken less susceptible to frost lesions. Here we report that Pea-comb is caused by a massive amplification of a duplicated sequence located near evolutionary conserved non-coding sequences in intron 1 of the gene encoding the SOX5 transcription factor. This must be the causative mutation since all other polymorphisms associated with the Pea-comb allele were excluded by genetic analysis. SOX5 controls cell fate and differentiation and is essential for skeletal development, chondrocyte differentiation, and extracellular matrix production. Immunostaining in early embryos demonstrated that Pea-comb is associated with ectopic expression of SOX5 in mesenchymal cells located just beneath the surface ectoderm where the comb and wattles will subsequently develop. The results imply that the duplication expansion interferes with the regulation of SOX5 expression during the differentiation of cells crucial for the development of comb and wattles. The study provides novel insight into the nature of mutations that contribute to phenotypic evolution and is the first description of a spontaneous and fully viable mutation in this developmentally important gene.
Author Summary
The featherless comb and wattles are defining features of the chicken. Whilst the Pea-comb allele was known to show a dominant inheritance and drastically reduce the size of both comb and wattles, the genetics underlying the mutation remained elusive. Chicken comb is primarily composed of collagen and hyaluronan, which are produced by chondrocytes. These cells are formed through the condensation and differentiation of mesenchyme cells during the chondrogenesis pathway, the early stages of which are regulated by SOX transcription factors. Here we pinpoint a massive amplification of a duplicated sequence in the first intron of SOX5 as causing the Pea-comb phenotype. By studying early embryos, we show that SOX5 is ectopically expressed during a restricted stage of development in the cells which underlie the comb and wattles of Pea-comb animals. We hypothesise that the sequence duplication alters the regulation of SOX5 expression when the differentiation of cells essential for comb and wattle development is taking place. Pea-comb adds to the growing list of phenotypic variation which is explained by regulatory mutations and so demonstrates the evolutionary significance of such events.
PMCID: PMC2685452  PMID: 19521496
15.  Allele dependent silencing of COL1A2 using small interfering RNAs 
Osteogenesis imperfecta (OI) is generally caused by a dominant mutation in Collagen I, encoded by the genes COL1A1 and COL1A2. To date there is no satisfactory therapy for OI, but inactivation of the mutant allele through small interfering RNAs (siRNA) is a promising approach, as siRNAs targeting each allele of a polymorphism could be used for allele-specific silencing irrespective of the location of the actual mutations. In this study we examined the allele dependent effects of several tiled siRNAs targeting a region surrounding an exonic COL1A2 T/C polymorphism (rs1800222) in heterozygous primary human bone cells. Relative abundances of COL1A2 alleles were determined by cDNA sequencing and overall COL1A2 abundance was analyzed by quantitative PCR. One of the siRNAs decreased overall COL1A2 abundance by 71% of which 75% was due to silencing of the targeted T-allele. In conclusion, allele-preferential silencing of Collagen type I genes may be a future therapeutic approach for OI.
PMCID: PMC2583335  PMID: 19015742
COL1A2; allele-preferential silencing; Osteogenesis imperfecta
17.  Multiplex Real-Time PCR Targeting the RNase P RNA Gene for Detection and Identification of Candida Species in Blood▿  
Journal of Clinical Microbiology  2007;45(3):874-880.
We have developed a single-tube multiplex real-time PCR method for the detection of the eight most common Candida species causing septicemia: Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis. The method developed targets the RNase P RNA gene RPR1. Sequences of this gene were determined for seven of the Candida species and showed surprisingly large sequence variation. C. glabrata was found to have a gene that was five times longer gene than those of the other species, and the nucleotide sequence similarity between C. krusei and C. albicans was as low as 55%. The multiplex PCR contained three probes that enabled the specific detection of C. albicans, C. glabrata, and C. krusei and a fourth probe that allowed the general detection of the remaining species. The method was able to detect 1 to 10 genome copies when the detection limit was tested repeatedly for the four species C. albicans, C. glabrata, C. krusei, and C. guilliermondii. No significant difference in the detection limit was seen when the multiplex format was compared with single-species PCR, i.e., two primers and one probe. The method detected eight clinically relevant Candida species and did not react with other tested non-Candida species or human DNA. The assay was applied to 20 blood samples from nine patients and showed a sensitivity similar to that of culture.
PMCID: PMC1829127  PMID: 17215340
18.  Comparison of a Duplex Quantitative Real-Time PCR Assay and the COBAS Amplicor CMV Monitor Test for Detection of Cytomegalovirus 
Journal of Clinical Microbiology  2004;42(5):1909-1914.
A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 103 to 108 copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with ≥105 copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.
PMCID: PMC404600  PMID: 15131148
19.  Characterization of ompA Genotypes by Sequence Analysis of DNA from All Detected Cases of Chlamydia trachomatis Infections during 1 Year of Contact Tracing in a Swedish County 
Journal of Clinical Microbiology  2004;42(4):1641-1647.
In this study we aimed to characterize the ompA gene by sequencing DNA from all detected cases of Chlamydia trachomatis infection in a Swedish county during 2001, in order to improve the efficiency of contact tracing. Approximately 990 bp of the ompA gene was amplified, and sequence analysis was achieved in 678 (94%) of 725 C. trachomatis-positive cases in this unselected population. The most prevalent genotype was serotype E (39%), followed by F (21%), G (11%), D (9%), K (9%), J (7%), H (2%), B (1%), and Ia (1%). Serotype E was found in five genotype variants, with the reference sequence comprising 96% of all E cases. Serotype D was the most variable, and of seven sequence variants, three were identified as recombinants with serotype E. Altogether 29 genetic variants were detected, and mutations and recombination events are discussed. Clinical manifestations were not associated with genotypes. Sequence variation was linked to sexual networks identified by contact tracing and improved epidemiological knowledge but was of limited benefit.
PMCID: PMC387550  PMID: 15071019

Results 1-19 (19)