Peptide hormone analysis in diagnosis and treatment of Differences of Sex Development: joint position paper of EU COST Action 'DSDnet' and European Reference Network on Rare Endocrine Conditions.

Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.

Steroid hormone analysis in diagnosis and treatment of DSD: position paper of EU COST Action BM 1303 'DSDnet'.

Disorders or differences in sex development (DSD) comprise a heterogeneous group of conditions with an atypical sex development. For optimal diagnosis, highly specialised laboratory analyses are required across European countries. Working group 3 of EU COST (European Cooperation in Science and Technology) Action BM 1303 'DSDnet' 'Harmonisation of Laboratory Assessment' has developed recommendations on laboratory assessment for DSD regarding the use of technologies and analytes to be investigated. This position paper on steroid hormone analysis in diagnosis and treatment of DSD was compiled by a group of specialists in DSD and/or hormonal analysis, either from participating European countries or international partner countries. The topics discussed comprised analytical methods (immunoassay/mass spectrometry-based methods), matrices (urine/serum/saliva) and harmonisation of laboratory tests. The following positions were agreed upon: support of the appropriate use of immunoassay- and mass spectrometry-based methods for diagnosis and monitoring of DSD. Serum/plasma and urine are established matrices for analysis. Laboratories performing analyses for DSD need to operate within a quality framework and actively engage in harmonisation processes so that results and their interpretation are the same irrespective of the laboratory they are performed in. Participation in activities of peer comparison such as sample exchange or when available subscribing to a relevant external quality assurance program should be achieved. The ultimate aim of the guidelines is the implementation of clinical standards for diagnosis and appropriate treatment of DSD to achieve the best outcome for patients, no matter where patients are investigated or managed.

Construction of recombinant human cytomegalovirus.

The use of reverse genetics to generate recombinant viruses allows the researcher to investigate the exact functional significance of particular viral genes during the virus life cycle, by means of their deletion or modification in the viral genome. These studies can extend to the introduction of viral or foreign genetic material into ectopic sites in the viral genome, to investigate viral cis-control sequences or to phenotypically modify or tag the recombinant virus. Genetically modified viruses may also have applications in biotechnology and gene therapy. Such studies are well advanced for herpes simplex virus (HSV), a relative of human cytomegalovirus (HCMV), but have been relatively limited for HCMV itself.

Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant.

To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely-impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfiel.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the ie1-ie2 locus, revealing that this intron was dispensable for growth in cell culture.

A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation.

Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IE1(491aa) and IE2(579aa), produced from differentially spliced transcripts under control of the ie1/ie2 promoter-enhancer. A deletion mutation removing all 406 IE1(491aa)-specific amino acids was engineered into the viral genome and this mutant (RC303 delta Acc) was propagated on an IE1(491aa)-expressing human fibroblast cell line (ihfie1.3). RC303 delta Acc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois > 3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC303 delta Acc correlated with the ability to express IE2(579aa), consistent with a role for IE1(491aa) in positive autoregulation of the ie1/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IE1(491aa) under high moi conditions. ie1-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.

Selectable insertion and deletion mutagenesis of the human cytomegalovirus genome using the Escherichia coli guanosine phosphoribosyl transferase (gpt) gene.

We describe the mutagenesis of the IRSI-US5 region of the human cytomegalovirus genome, demonstrating the potential of the E. coli guanosine phosphoribosyl transferase (gpt) gene as a selectable marker for insertion and deletion mutagenesis of high passage (AD169, Towne) as well as low passage (Toledo) strains of virus. Despite evidence suggesting that the US3 gene product may play a regulatory role, disruption of this gene with a gpt insert had no effect on growth of any of these strains of virus in resting or dividing human fibroblasts, or in human thymus plus liver implants in SCID-hu mice. Transcripts of the gpt gene, under control of the herpes simplex virus thymidine kinase promoter adjacent to the US3 enhancer in the viral genome, accumulated with delayed early (beta) kinetics. Mutants with deletions in the IRS1 and US3-US5 regions were isolated by back-selection against gpt with the drug 6-thioguanine by growing virus in human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyl transferase deficient) skin fibroblasts immortalized with human papillomavirus oncogenes. Thus, we demonstrate a dependable method for insertion and deletion mutagenesis that can be applied to any region of the viral genome.

Sequence, function, and regulation of the Vmw65 gene of herpes simplex virus type 2.

We determined the sequence of the gene for the virion transactivator protein Vmw65 of herpes simplex virus type 2 (HSV-2), strain 333. An analysis of the coding sequence revealed an overall high degree of primary sequence conservation (86%) relative to the HSV-1 protein, although the carboxy-terminal region which encompasses the powerful acidic transactivation domain of the HSV-1 protein was slightly less well conserved (70%). One important change in this region was the presence of a proline residue in a region of the HSV-2 protein which is thought to form an amphipathic alpha-helix in the HSV-1 homolog. Despite the occurrence of this helix-disrupting residue, the HSV-2 protein exhibited powerful transactivation properties for immediate-early target promoters. We also demonstrated that the HSV-2 protein forms a transcriptional complex (TRF.C) with the cellular Oct-1 protein and target TAATGARAT elements from immediate-early promoters. A comparison of upstream sequences from the two Vmw65 genes revealed good conservation of proximal promoter elements but considerable divergence elsewhere. Specifically, the HSV-2 promoter alone carries 9.5 copies of a 9-bp direct repeat (GGGGCGGGA) ending 85 bp upstream of the conserved TTAAAT element. An analysis of transcription factor binding sites in vitro revealed that cellular factor Sp1 bound to the direct repeat sequence of the HSV-2 promoter and that cellular factor USF bound to a proximal element present in both HSV-1 and HSV-2 promoters. Mutational analysis of the HSV-2 promoter demonstrated that the integrity of both of these binding sites was important for the full activity of the promoter.

Structural requirements in the herpes simplex virus type 1 transactivator Vmw65 for interaction with the cellular octamer-binding protein and target TAATGARAT sequences.

Herpes simplex virus type 1 virion protein Vmw65 forms a complex (TRF.C) with TAATGARAT sequences and the cellular transcription factor oct-1, which has been implicated as an intermediate in the activation of gene expression by Vmw65. To examine structural requirements within Vmw65 for this interaction, we analyzed extracts of transfected cells that express mutant Vmw65 proteins by gel retardation assay and identified two regions in the primary sequence of Vmw65 which are necessary for in vitro assembly of TRF.C. The amino-terminal boundary for complex assembly and trans activation mapped between residues 49 and 75. At the carboxyl terminus, deletion as far as residue 388 did not affect in vitro TRF.C assembly, although trans-activating activity was abolished. Deletion beyond residue 388 rapidly impaired the ability of the protein to participate in the TRF.C complex, such that a truncated mutant of 380 residues was completely inactive. These requirements towards the carboxyl terminus overlap a region of strong local sequence similarity between Vmw65 and terminal protein p3 of bacteriophage phi 29. Although substitution of corresponding p3 residues into Vmw65 failed to produce a functional chimera, site-directed mutagenesis within the region of similarity identified a number of single-point mutant proteins which were completely deficient for TRF.C formation. These mutant proteins were also unable to trans activate expression from immediate-early promoters, despite the integrity of the acidic carboxyl terminus. The extreme sensitivity of both TRF.C formation and trans activation to single-residue substitutions within this region of Vmw65 suggests that it is directly involved in the protein-protein or protein-DNA interactions required for assembly of a transcriptional complex containing oct-1.