Publications by authors named "Thomas Ohnesorg"

24 Publications

  • Page 1 of 1

ClinSV: clinical grade structural and copy number variant detection from whole genome sequencing data.

Genome Med 2021 02 25;13(1):32. Epub 2021 Feb 25.

Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Australia.

Whole genome sequencing (WGS) has the potential to outperform clinical microarrays for the detection of structural variants (SV) including copy number variants (CNVs), but has been challenged by high false positive rates. Here we present ClinSV, a WGS based SV integration, annotation, prioritization, and visualization framework, which identified 99.8% of simulated pathogenic ClinVar CNVs > 10 kb and 11/11 pathogenic variants from matched microarrays. The false positive rate was low (1.5-4.5%) and reproducibility high (95-99%). In clinical practice, ClinSV identified reportable variants in 22 of 485 patients (4.7%) of which 35-63% were not detectable by current clinical microarray designs. ClinSV is available at https://github.com/KCCG/ClinSV .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-021-00841-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908648PMC
February 2021

Genomic diagnostics in polycystic kidney disease: an assessment of real-world use of whole-genome sequencing.

Eur J Hum Genet 2021 05 12;29(5):760-770. Epub 2021 Jan 12.

Division of Genomics and Epigenetics, Garvan Institute of Medical Research, Sydney, NSW, Australia.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is common, with a prevalence of 1/1000 and predominantly caused by disease-causing variants in PKD1 or PKD2. Clinical diagnosis is usually by age-dependent imaging criteria, which is challenging in patients with atypical clinical features, without family history, or younger age. However, there is increasing need for definitive diagnosis of ADPKD with new treatments available. Sequencing is complicated by six pseudogenes that share 97% homology to PKD1 and by recently identified phenocopy genes. Whole-genome sequencing can definitively diagnose ADPKD, but requires validation for clinical use. We initially performed a validation study, in which 42 ADPKD patients underwent sequencing of PKD1 and PKD2 by both whole-genome and Sanger sequencing, using a blinded, cross-over method. Whole-genome sequencing identified all PKD1 and PKD2 germline pathogenic variants in the validation study (sensitivity and specificity 100%). Two mosaic variants outside pipeline thresholds were not detected. We then examined the first 144 samples referred to a clinically-accredited diagnostic laboratory for clinical whole-genome sequencing, with targeted-analysis to a polycystic kidney disease gene-panel. In this unselected, diagnostic cohort (71 males :73 females), the diagnostic rate was 70%, including a diagnostic rate of 81% in patients with typical ADPKD (98% with PKD1/PKD2 variants) and 60% in those with atypical features (56% PKD1/PKD2; 44% PKHD1/HNF1B/GANAB/ DNAJB11/PRKCSH/TSC2). Most patients with atypical disease did not have clinical features that predicted likelihood of a genetic diagnosis. These results suggest clinicians should consider diagnostic genomics as part of their assessment in polycystic kidney disease, particularly in atypical disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41431-020-00796-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110527PMC
May 2021

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9.

Nat Commun 2018 12 14;9(1):5319. Epub 2018 Dec 14.

Murdoch Children's Research Institute, Melbourne, 3052, VIC, Australia.

Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. Variants in two key genes, SRY and its target SOX9, are an established cause of 46,XY DSD, but the genetic basis of many DSDs remains unknown. SRY-mediated SOX9 upregulation in the early gonad is crucial for testis development, yet the regulatory elements underlying this have not been identified in humans. Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9. Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testis. This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07784-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293998PMC
December 2018

The Role of Copy Number Variants in Disorders of Sex Development.

Sex Dev 2018 17;12(1-3):19-29. Epub 2017 Nov 17.

Murdoch Children's Research Institute, Melbourne, VIC, Australia.

Despite considerable research effort and significant advances in sequencing technologies, the majority of disorders of sex development (DSD) cases still lack a molecular genetic diagnosis. While coding variants have been discovered in known and candidate DSD genes, comparatively little is known about copy number variations (CNVs) affecting both coding and noncoding regions. Due to rapidly falling costs of whole genome sequencing, many more CNVs in individuals with DSD will be identified. These CNVs may explain a significant number of hitherto undiagnosed cases of DSD. In this review, we provide an overview of CNVs that are known to cause DSD and discuss approaches to identify and verify causative CNVs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000481896DOI Listing
November 2018

XX Disorder of Sex Development is associated with an insertion on chromosome 9 and downregulation of RSPO1 in dogs (Canis lupus familiaris).

PLoS One 2017 20;12(10):e0186331. Epub 2017 Oct 20.

Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia.

Remarkable progress has been achieved in understanding the mechanisms controlling sex determination, yet the cause for many Disorders of Sex Development (DSD) remains unknown. Of particular interest is a rare XX DSD subtype in which individuals are negative for SRY, the testis determining factor on the Y chromosome, yet develop testes or ovotestes, and both of these phenotypes occur in the same family. This is a naturally occurring disorder in humans (Homo sapiens) and dogs (C. familiaris). Phenotypes in the canine XX DSD model are strikingly similar to those of the human XX DSD subtype. The purposes of this study were to identify 1) a variant associated with XX DSD in the canine model and 2) gene expression alterations in canine embryonic gonads that could be informative to causation. Using a genome wide association study (GWAS) and whole genome sequencing (WGS), we identified a variant on C. familiaris autosome 9 (CFA9) that is associated with XX DSD in the canine model and in affected purebred dogs. This is the first marker identified for inherited canine XX DSD. It lies upstream of SOX9 within the canine ortholog for the human disorder, which resides on 17q24. Inheritance of this variant indicates that XX DSD is a complex trait in which breed genetic background affects penetrance. Furthermore, the homozygous variant genotype is associated with embryonic lethality in at least one breed. Our analysis of gene expression studies (RNA-seq and PRO-seq) in embryonic gonads at risk of XX DSD from the canine model identified significant RSPO1 downregulation in comparison to XX controls, without significant upregulation of SOX9 or other known testis pathway genes. Based on these data, a novel mechanism is proposed in which molecular lesions acting upstream of RSPO1 induce epigenomic gonadal mosaicism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186331PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650465PMC
November 2017

Functional characterization of novel NR5A1 variants reveals multiple complex roles in disorders of sex development.

Hum Mutat 2018 01 2;39(1):124-139. Epub 2017 Nov 2.

Murdoch Children's Research Institute, Melbourne, Australia.

Variants in the NR5A1 gene encoding SF1 have been described in a diverse spectrum of disorders of sex development (DSD). Recently, we reported the use of a targeted gene panel for DSD where we identified 15 individuals with a variant in NR5A1, nine of which are novel. Here, we examine the functional effect of these changes in relation to the patient phenotype. All novel variants tested had reduced trans-activational activity, while several had altered protein level, localization, or conformation. In addition, we found evidence of new roles for SF1 protein domains including a region within the ligand binding domain that appears to contribute to SF1 regulation of Müllerian development. There was little correlation between the severity of the phenotype and the nature of the NR5A1 variant. We report two familial cases of NR5A1 deficiency with evidence of variable expressivity; we also report on individuals with oligogenic inheritance. Finally, we found that the nature of the NR5A1 variant does not inform patient outcomes (including pubertal androgenization and malignancy risk). This study adds nine novel pathogenic NR5A1 variants to the pool of diagnostic variants. It highlights a greater need for understanding the complexity of SF1 function and the additional factors that contribute.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.23354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765430PMC
January 2018

Painful ovulation in a 46,XX SRY -ve adult male with duplication.

Endocrinol Diabetes Metab Case Rep 2017 7;2017. Epub 2017 Jun 7.

ANZAC Research Institute, University of Sydney, Sydney, New South WalesAustralia.

46,XX disorders of sexual development (DSDs) occur rarely and result from disruptions of the genetic pathways underlying gonadal development and differentiation. We present a case of a young phenotypic male with 46,XX SRY-negative ovotesticular DSD resulting from a duplication upstream of presenting with a painful testicular mass resulting from ovulation into an ovotestis. We present a literature review of ovulation in phenotypic men and discuss the role of and in testicular development, including the role of upstream enhancer region duplication in female-to-male sex reversal.

Learning Points: In mammals, the early gonad is bipotent and can differentiate into either a testis or an ovary. SRY is the master switch in testis determination, responsible for differentiation of the bipotent gonad into testis.SRY activates gene, as a transcription factor is the second major gene involved in male sex determination. drives the proliferation of Sertoli cells and activates AMH/MIS repressing the ovary. is sufficient to induce testis formation and can substitute for SRY function.Assessing karyotype and then determination of the presence or absence of Mullerian structures are necessary serial investigations in any case of DSD, except for mixed gonadal dysgenesis identified by karyotype alone.Treatment is ideal in a multidisciplinary setting with considerations to genetic (implications to family and reproductive recurrence risk), psychological aspects (sensitive individualized counseling including patient gender identity and preference), endocrinological (hormone replacement), surgical (cosmetic, prophylactic gonadectomy) fertility preservation and reproductive opportunities and metabolic health (cardiovascular and bones).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/EDM-17-0045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467653PMC
June 2017

Review disorders of sex development: The evolving role of genomics in diagnosis and gene discovery.

Birth Defects Res C Embryo Today 2016 Dec;108(4):337-350

Murdoch Children's Research Institute, Melbourne, Victoria, Australia.

Disorders of Sex Development (DSDs) are a major paediatric concern and are estimated to occur in around 1.7% of all live births (Fausto-Sterling, Sexing the Body: Gender Politics and the Construction of Sexuality, Basic Books, New York, 2000). They are often caused by the breakdown in the complex genetic mechanisms that underlie gonadal development and differentiation. Having a genetic diagnosis can be important for patients with a DSD: it can increase acceptance of a disorder often surrounded by stigma, alter clinical management and it can assist in reproductive planning. While Massively Parallel Sequencing (MPS) is advancing the genetic diagnosis of rare Mendelian disorders, it is not yet clear which MPS assay is best suited for the clinical diagnosis of DSD patients and to what extent other established methods are still relevant. To complicate matters, DSDs represent a wide spectrum of disorders caused by an array of different genetic changes, many of which are yet unknown. Here we discuss the different genetic lesions that are known to contribute to different DSDs, and review the utility of a range of MPS approaches for diagnosing DSD patients. Birth Defects Research (Part C) 108:337-350, 2016. © 2016 Wiley Periodicals, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bdrc.21148DOI Listing
December 2016

Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort.

Genome Biol 2016 11 29;17(1):243. Epub 2016 Nov 29.

Department of Medical Genetics, Sydney Children's Hospital, Randwick, NSW, Australia.

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously.

Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management.

Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-016-1105-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126855PMC
November 2016

Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion.

PLoS Genet 2016 05 26;12(5):e1006067. Epub 2016 May 26.

Translational Research Unit, Canberra Hospital, Woden, Australian Capital Territory, Australia.

Most humans harbor both CD177neg and CD177pos neutrophils but 1-10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1), which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1006067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882059PMC
May 2016

Using ROADMAP Data to Identify Enhancers Associated with Disorders of Sex Development.

Sex Dev 2016 15;10(2):59-65. Epub 2016 Apr 15.

Murdoch Children's Research Institute, Melbourne, Vic., Australia.

Despite recent advances in our understanding, most cases of disorders of sex development (DSD) cannot be explained by mutations in known genes. In genome-wide screens of DSD patients, we and others detected duplications or deletions of potential regulatory regions of known or suspected DSD genes. It is therefore likely that a significant proportion of DSD cases may be explained by disrupted transcriptional regulation of gonad genes. Despite many recent technological advances, limited availability of relevant tissues - especially human embryonic material - can make the identification of long-range regulatory elements extremely difficult. In an attempt to overcome this limitation, we evaluated the usefulness of publicly available DNaseI hypersensitivity data from the Roadmap Epigenomics Project. For this feasibility study we used the 'gene desert' around the SOX9 gene and a genomic locus downstream of GATA4. Over 60% of our selected candidate regions had significant enhancer activity in luciferase assays. We show that this approach facilitates the detection of strong enhancer candidates worthy of further analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000445398DOI Listing
November 2017

Genetic regulation of mammalian gonad development.

Nat Rev Endocrinol 2014 Nov 23;10(11):673-83. Epub 2014 Sep 23.

Murdoch Childrens Research Institute, Department of Paediatrics, The University of Melbourne, The Royal Children's Hospital, 50 Flemington Road, Melbourne, VIC 3052, Australia.

Sex-specific gonadal development starts with formation of the bipotential gonad, which then differentiates into either a mature testis or an ovary. This process is dependent on activation of either the testis-specific or the ovary-specific pathway while the opposite pathway is continuously repressed. A network of transcription factors tightly regulates initiation and maintenance of these distinct pathways; disruption of these networks can lead to disorders of sex development in humans and male-to-female or female-to-male sex reversal in mice. Sry is the Y-linked master switch that is both required and sufficient to drive the testis-determining pathway. Another key component of the testis pathway is Sox9, which acts immediately downstream of Sry. In contrast to the testis pathway, no single sex-determining factor has been identified in the ovary pathway; however, multiple genes, such as Foxl2, Rspo1, Ctnnb1, and Wnt4, seem to work synergistically and in parallel to ensure proper ovary development. Our understanding of the regulatory networks that underpin testis and ovary development has grown substantially over the past two decades.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nrendo.2014.163DOI Listing
November 2014

Development of retroviral vectors for tissue-restricted expression in chicken embryonic gonads.

PLoS One 2014 8;9(7):e101811. Epub 2014 Jul 8.

Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia; Department of Paediatrics, The University of Melbourne, Melbourne, VIC, Australia; Poultry Cooperative Research Centre, Armidale, NSW, Australia.

The chicken embryo has long been a useful model organism for studying development, including sex determination and gonadal differentiation. However, manipulating gene expression specifically in the embryonic avian gonad has been difficult. The viral vector RCASBP can be readily used for embryo-wide transgene expression; however global mis-expression using this method can cause deleterious off-target effects and embryo-lethality. In an attempt to develop vectors for the over-expression of sequences in chicken embryonic urogenital tissues, the viral vector RCANBP was engineered to contain predicted promoter sequences of gonadal-expressed genes. Several promoters were analysed and it was found that although the SF1 promoter produced a tissue-restricted expression pattern that was highest in the mesonephros and liver, it was also higher in the gonads compared to the rest of the body. The location of EGFP expression from the SF1 promoter overlapped with several key gonad-expressed sex development genes; however expression was generally low-level and was not seen in all gonadal cells. To further validate this sequence the key testis determinant DMRT1 was over-expressed in female embryos, which due to insufficient levels had no effect on gonad development. The female gene aromatase was then over-expressed in male embryos, which disrupted the testis pathway as demonstrated by a reduction in AMH protein. Taken together, although these data showed that the SF1 promoter can be used for functional studies in ovo, a stronger promoter sequence would likely be required for the functional analysis of gonad genes that require high-level expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101811PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4086957PMC
March 2015

The genetics of disorders of sex development in humans.

Sex Dev 2014 31;8(5):262-72. Epub 2014 Jan 31.

Murdoch Children's Research Institute and Department of Paediatrics, The University of Melbourne, Royal Children's Hospital, Melbourne, Vic., Australia.

One of the defining events during human embryonic development with the most far-reaching effects for the individual is whether the embryo develops as male or female. The crucial step in this process is the differentiation of the bipotential embryonic gonads into either testes or ovaries. If the embryo inherits X and Y sex chromosomes, the Y-linked SRY (sex determining region in Y) gene initiates a network of genes that results in a functional testis and ultimately a male phenotype. By contrast, in an embryo with 2 X chromosomes, the undifferentiated gonad develops as an ovary resulting in a female phenotype. Perturbation of any of the genes in either the testicular or ovarian developmental pathway can result in individuals with disorders of sex development. In this review, we provide a summary of known components of testicular or ovarian pathways and their antagonistic actions and give a brief overview of new technologies currently used to identify the missing pieces of the sex development network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000357956DOI Listing
July 2015

Detecting DNaseI-hypersensitivity sites with MLPA.

Methods Mol Biol 2012 ;786:201-10

Molecular Development Laboratory, Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia.

DNaseI-hypersensitive sites within chromatin are indicative of genomic loci with regulatory function. Several techniques have been described for analyzing these regions, but are either laborious, offer low-throughput possibilities, or are expensive. We have developed a new approach based on a modified version of multiplex ligation-dependent probe amplification (MLPA). Using this method, it is possible to analyse up to 50 defined genomic regions for DNaseI-hypersensitivity in a single PCR-based reaction. This chapter outlines the approach and discusses the critical features of each step of the procedure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-61779-292-2_12DOI Listing
February 2012

Copy number variation in patients with disorders of sex development due to 46,XY gonadal dysgenesis.

PLoS One 2011 Mar 7;6(3):e17793. Epub 2011 Mar 7.

Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017793PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049794PMC
March 2011

The many faces of MLPA.

Methods Mol Biol 2011 ;687:193-205

Molecular Development, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.

Multiplex Ligation-dependent Probe Amplification (MLPA) is a PCR-based technique that was developed for identifying deletions and duplications in genomic DNA. The simplicity and sensitivity of this approach has led to it being implemented in many laboratories around the world. Since the original publication, there have been several variants of MLPA described, allowing the quantitative analysis of mRNA transcript levels, CpG methylation, complex genomic regions, and DNaseI hypersensitive sites. This chapter outlines the basic MLPA protocol, describes the different modifications and applications that have been published, and discusses the critical points during each of the steps.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-60761-944-4_13DOI Listing
February 2011

Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach.

BMC Genomics 2009 Sep 4;10:412. Epub 2009 Sep 4.

Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, VIC, Australia.

Background: Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.

Results: We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.

Conclusion: This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-10-412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2748097PMC
September 2009

The avian Z-linked gene DMRT1 is required for male sex determination in the chicken.

Nature 2009 Sep 26;461(7261):267-71. Epub 2009 Aug 26.

Murdoch Children's Research Institute and Department of Paediatrics, The University of Melbourne, Royal Children's Hospital, Melbourne, Victoria 3052, Australia.

Sex in birds is chromosomally based, as in mammals, but the sex chromosomes are different and the mechanism of avian sex determination has been a long-standing mystery. In the chicken and all other birds, the homogametic sex is male (ZZ) and the heterogametic sex is female (ZW). Two hypotheses have been proposed for the mechanism of avian sex determination. The W (female) chromosome may carry a dominant-acting ovary determinant. Alternatively, the dosage of a Z-linked gene may mediate sex determination, two doses being required for male development (ZZ). A strong candidate avian sex-determinant under the dosage hypothesis is the conserved Z-linked gene, DMRT1 (doublesex and mab-3-related transcription factor 1). Here we used RNA interference (RNAi) to knock down DMRT1 in early chicken embryos. Reduction of DMRT1 protein expression in ovo leads to feminization of the embryonic gonads in genetically male (ZZ) embryos. Affected males show partial sex reversal, characterized by feminization of the gonads. The feminized left gonad shows female-like histology, disorganized testis cords and a decline in the testicular marker, SOX9. The ovarian marker, aromatase, is ectopically activated. The feminized right gonad shows a more variable loss of DMRT1 and ectopic aromatase activation, suggesting differential sensitivity to DMRT1 between left and right gonads. Germ cells also show a female pattern of distribution in the feminized male gonads. These results indicate that DMRT1 is required for testis determination in the chicken. Our data support the Z dosage hypothesis for avian sex determination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature08298DOI Listing
September 2009

Transcriptional regulation of human and murine 17beta-hydroxysteroid dehydrogenase type-7 confers its participation in cholesterol biosynthesis.

J Mol Endocrinol 2006 Aug;37(1):185-97

School of Paediatrics and Reproductive Health, Discipline of Obstetrics and Gynaecology, Adelaide University, Adelaide 5005, South Australia, Australia.

In both humans and mice, 17beta-hydroxysteroid dehydrogenase type-7 (HSD17B7) was described as possessing dual enzymatic functionality. The enzyme was first shown to be able to convert estrone to estradiol in vitro. Later involvement of this enzyme in postsqualene cholesterol biosynthesis was postulated (conversion of zymosterone to zymosterol) and could be proven in vitro. In this work, we performed a detailed analysis of the transcriptional regulation of both the human and murine genes. Despite relatively low sequence similarity, both promoters contain similar contexts of transcription factor-binding sites. The participation of these sites in transcriptional regulation of HSD17B7 was proven by electro-mobility shift assay and site-directed mutagenesis of the corresponding binding sites. We describe novel involvement of vitamin D receptor/retinoid X receptor and provide new information on the regulation of HSD17B7 expression by sterol regulatory element-binding protein and hepatocyte nuclear factor 4, the latter known from other genes of cholesterogenic enzymes. The results of our study provide unequivocal evidence for a role of HSD17B7 in cholesterol biosynthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1677/jme.1.02043DOI Listing
August 2006

Analysis of the 5' flanking regions of human and murine HSD17B7: identification of a cholesterol dependent enhancer region.

Mol Cell Endocrinol 2006 Mar 13;248(1-2):164-7. Epub 2005 Dec 13.

GSF-National Research Center for Environment and Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.

17Beta hydroxysteroid dehydrogenase type 7 (HSD17B7) was described to possess dual functionality in steroidogenesis as well as in postsqualene cholesterol biosynthesis in vitro. In order to gain insight into the transcriptional regulation, and thereby into in vivo functionality of HSD17B7, we analyzed and compared the 5' flanking regions of the corresponding human and murine genes. For this task we used bioinformatic and experimental approaches. The identified proximal promoter regions of both human and murine HSD17B7 genes contain multiple transcription factor binding sites and show strong similarity to cholesterogenic genes, especially to other postsqualene genes, but not to other steroidogenic genes. In liver cell lines, the transcriptional activity is dependent on the level of cholesterol, but not estradiol. The results of our study lead us to the conclusion that HSD17B7 is involved in postsqualene cholesterol biosynthesis in both human and mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mce.2005.10.016DOI Listing
March 2006

Promoter analyses of human and mouse 17beta-hydroxysteroid dehydrogenase type 7.

J Steroid Biochem Mol Biol 2005 Feb 24;94(1-3):259-61. Epub 2005 Feb 24.

GSF-National Research Center for Environment and Health, Institute of Experimental Genetics, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.

In this study we examined and compared the 5' flanking regions (promoter regions) of human and murine 17beta-hydroxysteroid dehydrogenase type 7 genes (HSD17B7). In 1998, this enzyme was described to convert estrone to biologically more active estradiol. However, in 2001, it was predicted to be involved in cholesterol biosynthesis. To elucidate the mechanism governing HSD17B7 expression and its function, we analyzed the promoter regions by bioinformatic and experimental approaches. Our study provides new insights to the transcriptional regulation of HSD17B7 and further hints to its involvement in cholesterol biosynthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsbmb.2005.01.012DOI Listing
February 2005
-->