Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration.

Late-onset retinal degeneration (L-ORD) is a rare autosomal dominant retinal dystrophy, characterised by extensive sub-retinal pigment epithelium (RPE) deposits, RPE atrophy, choroidal neovascularisation and photoreceptor cell death associated with severe visual loss. L-ORD shows striking phenotypic similarities to age-related macular degeneration (AMD), a common and genetically complex disorder, which can lead to misdiagnosis in the early stages. To date, a single missense mutation (S163R) in the C1QTNF5 gene, encoding C1q And Tumor Necrosis Factor Related Protein 5 (C1QTNF5) has been shown to cause L-ORD in a subset of affected families. Here, we describe the identification and characterisation of three novel pathogenic mutations in C1QTNF5 in order to elucidate disease mechanisms. In silico and in vitro characterisation show that these mutations perturb protein folding, assembly or polarity of secretion of C1QTNF5 and, importantly, all appear to destabilise the wildtype protein in co-transfection experiments in a human RPE cell line. This suggests that the heterozygous mutations in L-ORD show a dominant negative, rather than a haploinsufficient, disease mechanism. The function of C1QTNF5 remains unclear but this new insight into the pathogenetic basis of L-ORD has implications for future therapeutic strategies such as gene augmentation therapy.

Investigating the Expression of Oncogenic and Tumor Suppressive MicroRNA in DLBCL.

Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of lymphoma, accounting for 40 percent of newly diagnosed cases each year. DLBCL is an aggressive abnormal growth of tissue characterized by the accumulation of abnormal B-lymphocytes in the lymphatics of affected individuals. The goal of this study was to analyze microRNA (miRNA) as an alternative method of diagnosis and treatment for patients affected with the observed cancer. MiRNAs are small, non-coding, endogenous RNA that control gene expression at the post-transcriptional level. Emerging evidence suggests that miRNA-mediated gene regulation has a functional role in cancer and could prove to be crucial targets for therapeutic intervention. Here, we provide a quantitative study on the expression of a diverse class of oncogenic and tumor suppressive miRNA that have shown to regulate oncoproteins involved in differentiation, proliferation, and/or apoptosis.

BK virus as a potential co-factor for HPV in the development of cervical neoplasia.

Cervical cancer is the third most common type of cancer in women worldwide. A persistent infection with high risk (HR) human papillomavirus (HPV) is necessary for cervical cancer to occur. However, the great majority of women that are infected with HR-HPV will not develop cervical cancer, indicating that HR-HPV alone is not adequate to drive the development of cervical cancer, suggesting the involvement of cofactors. The BK polyomavirus (BKV) establishes latency near cervical tissue in the urogenital tract and is frequently detected in the urine, especially in immunosuppressed patients, and hence may coexist with HR-HPV. Current experimental evidence indicates that both HR-HPV and BKV are capable of altering cell-cycle control and inhibit apoptosis. Therefore, they may act additively or synergistically to promote malignant transformation. We hypothesize that BKV is a co-factor for HR-HPV in cervical cancer. In this study, we examined 249 cervical swabs that were submitted for routine HR-HPV screening test in the Molecular Diagnostics Laboratory at the University of Texas Medical Branch (UTMB). Our results showed that 107 samples contained HR-HPV at an overall rate of 43% (107/249); BKV was present in 4 (3.7%) of the 107 HR-HPV positive specimens and in 12 (8.5%) of the 142 HR-HPV negative samples with an overall positive rate of 6.4% (16/249). Although there was no statistical significance between HR-HPV and BKV co-infection (P=0.19, Fisher's exact test), our results support the hypothesis that BKV can co-exist with HR-HPV in cervical specimens.

Characterisation of a C1qtnf5 Ser163Arg knock-in mouse model of late-onset retinal macular degeneration.

A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD) in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse "knock-in" model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct.

Molecular identification of bacteria associated with canine periodontal disease.

Periodontal disease is one of the most common diseases of adult dogs, with up to 80% of animals affected. The aetiology of the disease is poorly studied, although bacteria are known to play a major role. The purpose of this study was to identify the bacteria associated with canine gingivitis and periodontitis and to compare this with the normal oral flora. Swabs were obtained from the gingival margin of three dogs with gingivitis and three orally healthy controls, and subgingival plaque was collected from three dogs with periodontitis. Samples were subjected to routine bacterial culture. The prevalent species identified in the normal, gingivitis and periodontitis groups were uncultured bacterium (12.5% of isolates), Bacteroides heparinolyticus/Pasteurella dagmatis (10.0%) and Actinomyces canis (19.4%), respectively. Bacteria were also identified using culture-independent methods (16S rRNA gene sequencing) and the predominant species identified were Pseudomonas sp. (30.9% of clones analysed), Porphyromonas cangingivalis (16.1%) and Desulfomicrobium orale (12.0%) in the normal, gingivitis and periodontitis groups, respectively. Uncultured species accounted for 13.2%, 2.0% and 10.5%, and potentially novel species for 38.2%, 38.3% and 35.3%, of clones in the normal, gingivitis and periodontitis groups, respectively. This is the first study to use utilise culture-independent methods for the identification of bacteria associated with this disease. It is concluded that the canine oral flora in health and disease is highly diverse and also contains a high proportion of uncultured and, in particular, potentially novel species.

Knockdown of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene results in retinal degeneration.

PURPOSE: The authors investigated the expression and function of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene. METHODS: Zebrafish RP2 (ZFRP2) cDNA was isolated from adult eye mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Gene expression was examined by RT-PCR. The deduced peptide sequence was aligned with RP2 orthologues from different species. Translational suppression (knockdown) of zebrafish RP2 was carried out by antisense morpholino-injection. The phenotype of ZFRP2 knockdown morphants was characterized by immunohistology and histology. Human wild-type and mutant RP2 mRNAs were coinjected with ZFRP2 morpholinos to test whether human RP2 mRNA could rescue ZFRP2 knockdown phenotypes. RESULTS: ZFRP2 encodes a protein of 376 amino acids containing an N-terminal tubulin folding cofactor C-like domain and a C-terminal nucleoside diphosphate kinase-like domain. It shares 63% to 65% amino acid identity with human, mouse and bovine RP2. RP2 is expressed at the earliest stages of zebrafish development and persists into adulthood. Knockdown of RP2 in zebrafish causes a curved body axis and small eye phenotype, associated with increased cell death throughout the retina. Human wild-type RP2 mRNA could rescue the body curvature phenotype of ZFRP2 morphants, and the eye size of the resultant morphants was significantly increased over that of morphants in which ZFRP2 had been depleted. CONCLUSIONS: Zebrafish RP2 is widely expressed throughout development. ZFRP2 knockdown caused retinal degeneration in zebrafish. Human RP2 could partially rescue the small eye phenotype of ZFRP2 morphants.

Identification of bacteria associated with feline chronic gingivostomatitis using culture-dependent and culture-independent methods.

Feline chronic gingivostomatitis (FCGS) is a chronic inflammatory disease of the oral cavity that causes severe pain and distress. There are currently no specific treatment methods available and little is known regarding its aetiology, although bacteria are thought to play a major role. The purpose of this study was to identify the oral bacterial flora in normal and diseased cats. Oral swabs were obtained from the palatoglossal folds of eight cats (three normal and five FCGS) and were subjected to microbiological culture. Pasteurella pneumotropica and Pasteurella multocida subsp. multocida were the most prevalent species identified by culture methods in the normal and FCGS samples, respectively. Bacteria were also identified using culture-independent methods (bacterial 16S rRNA gene sequencing). For the normal samples, 158 clones were analysed and 85 clones were sequenced. Capnocytophaga canimorsus (10.8% of clones analysed) was the predominant species. Uncultured species accounted for 8.2% of clones analysed, and 43.7% of clones analysed represented potentially novel species. For the FCGS samples, 253 clones were analysed and 91 clones were sequenced. The predominant species was P. multocida subsp. multocida (51.8% of clones analysed). Uncultured species accounted for 8.7% of clones analysed, and 4.7% of clones analysed represented potentially novel species. It is concluded that the oral flora in cats with FCGS appears to be less diverse than that found in normal cats. However, P. multocida subsp. multocida is found to be significantly more prevalent in FCGS than in normal cats and consequently may be of aetiological significance in this disease.

Clinical molecular testing: subspecialty, entry-level or specialist certification?

Some clinical laboratories require workers who have basic knowledge in molecular techniques (such as fluorescent in situ hybridization and polymerase chain reaction). Exclusively molecular diagnostic laboratories need workers to be competent in a variety of cutting edge molecular technologies, such as DNA sequencing, array-based comparative genomic hybridization, quantitative polymerase chain reaction, and many other techniques. Having only one certification for molecular biology at the entry level, as newly prescribed by the Board of Certification, doesn't accurately define the two very differently trained types of people these differing types of laboratories require. Creating a second molecular certification, at the specialist level, would address this issue positively.

Zebrafish Rpgr is required for normal retinal development and plays a role in dynein-based retrograde transport processes.

Mutations in the human RPGR gene cause one of the most common and severe forms of inherited retinal dystrophy, but the function of its protein product remains unclear. We have identified two genes resembling human RPGR (ZFRPGR1, ZFRPGR2) in zebrafish (Danio rerio), both of which are expressed within the nascent and adult eye as well as more widely during development. ZFRPGR2 appears to be functionally orthologous to human RPGR, because it encodes similar protein isoforms (ZFRPGR2(ORF15), ZFRPGR2(ex1-17)) and causes developmental defects similar to other ciliary proteins, affecting gastrulation, tail and head development after morpholino-induced knockdown (translation suppression). These defects are consistent with a ciliary function and were rescued by human RPGR but not by RPGR mutants causing retinal dystrophy. Unlike mammals, RPGR knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. Developmental abnormalities in the eye included lamination defects, failure to develop photoreceptor outer segments and a small eye phenotype, associated with increased cell death throughout the retina. These defects could be rescued by expression of wild-type but not mutant forms of human RPGR. ZFRPGR2 knockdown also resulted in an intracellular transport defect affecting retrograde but not anterograde transport of organelles. ZFRPGR2 is therefore necessary both for the normal differentiation and lamination of the retina and to prevent apoptotic retinal cell death, which may relate to its proposed role in dynein-based retrograde transport processes.

Development of a novel PCR assay for detection of Prevotella oris in clinical specimens.

Prevotella oris is a nonpigmented, Gram-negative, anaerobic bacterium that has been associated with several serious oral and systemic infections. Prevotella oris has been identified in clinical specimens by bacterial culture and biochemical tests, which are generally unreliable. The aim of this study was to develop a PCR assay for the direct detection of P. oris in clinical specimens. PCR primers specific for P. oris were identified by alignment of bacterial 16S rRNA genes from closely related species and selection of PCR primers specific for P. oris at their 3' ends. Amplification of a 1110-bp product indicated PCR positivity for P. oris. The primers were shown to be specific for P. oris DNA, because no PCR products were obtained when DNA from other oral bacteria, including closely related Prevotella species, were used as test species, and this was confirmed by digestion of PCR products with RsaI and MnlI. Prevotella oris DNA was detected in 17 (36.2%) of 47 pus samples from subjects with dentoalveolar abscesses and in all three pus samples from subjects with spreading odontogenic infections. This PCR assay provides a sensitive, specific and reliable method for identifying P. oris in clinical specimens.

Identification of bacteria on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties by 16S rRNA gene sequencing and by microbiological culture.

It has been postulated that bacteria attached to the surface of prosthetic hip joints can cause localised inflammation, resulting in failure of the replacement joint. However, diagnosis of infection is difficult with traditional microbiological culture methods, and evidence exists that highly fastidious or non-cultivable organisms have a role in implant infections. The purpose of this study was to use culture and culture-independent methods to detect the bacteria present on the surface of prosthetic hip joints removed during revision arthroplasties. Ten consecutive revisions were performed by two surgeons, which were all clinically and radiologically loose. Five of the hip replacement revision surgeries were performed because of clinical infections and five because of aseptic loosening. Preoperative and perioperative specimens were obtained from each patient and subjected to routine microbiological culture. The prostheses removed from each patient were subjected to mild ultrasonication to dislodge adherent bacteria, followed by aerobic and anaerobic microbiological culture. Bacterial DNA was extracted from each sonicate and the 16S rRNA gene was amplified with the universal primer pair 27f/1387r. All 10 specimens were positive for the presence of bacteria by both culture and PCR. PCR products were then cloned, organised into groups by RFLP analysis and one clone from each group was sequenced. Bacteria were identified by comparison of the 16S rRNA gene sequences obtained with those deposited in public access sequence databases. A total of 512 clones were analysed by RFLP analysis, of which 118 were sequenced. Culture methods identified species from the genera Leifsonia (54.3%), Staphylococcus (21.7%), Proteus (8.7%), Brevundimonas (6.5%), Salibacillus (4.3%), Methylobacterium (2.2%) and Zimmermannella (2.2%). Molecular detection methods identified a more diverse microflora. The predominant genus detected was Lysobacter, representing 312 (60.9%) of 512 clones analysed. In all, 28 phylotypes were identified: Lysobacter enzymogenes was the most abundant phylotype (31.4%), followed by Lysobacter sp. C3 (28.3%), gamma proteobacterium N4-7 (6.6%), Methylobacterium SM4 (4.7%) and Staphylococcus epidermidis (4.7%); 36 clones (7.0%) represented uncultivable phylotypes. We conclude that a diverse range of bacterial species are found within biofilms on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties.

Identification of bacteria associated with spreading odontogenic infections by 16S rRNA gene sequencing.

OBJECTIVE: To determine the bacterial species associated with spreading odontogenic infections (SOIs). STUDY DESIGN: Pus samples from 4 cases of SOI were analyzed by microbiological culture methods for the presence of bacteria, and by polymerase chain reaction (PCR) amplification, cloning, and sequencing of bacterial 16S rRNA genes. RESULTS: Culture methods identified species from the genera Prevotella, Streptococcus, and Fusobacterium, as well as anaerobic streptococci. Molecular detection methods identified a far more diverse microflora. The predominant genus detected was Prevotella, representing 102 (50.2%) of 203 clones analyzed. Prevotella oris was the most abundant species identified, representing 45 (22.2%) of 203 clones analyzed. Twelve clones (5.9%) represented uncultivable species, namely Prevotella PUS9.180, an uncultured Peptostreptococcus species, and an uncultured bacterium belonging to the Bacteroidetes phylum. CONCLUSIONS: Prevotella species may play an important role in SOIs, and further work to examine in more detail the pathogenicity determinants of these organisms and associated host responses is warranted.

Disease mechanisms in late-onset retinal macular degeneration associated with mutation in C1QTNF5.

Late-onset retinal macular degeneration (L-ORMD) is an autosomal dominant condition resembling age-related macular degeneration (AMD) in which a key pathological feature is a thick extracellular sub-retinal pigment epithelial (RPE) deposit. L-ORMD is caused by mutation in the C1QTNF5 (CTRP5) short-chain collagen gene, but the disease mechanism is unknown. Here, we first show that wild-type C1QTNF5 is secreted, whereas mutant C1QTNF5 is misfolded and retained within the endoplasmic reticulum (ER). Secondly, the ER retained mutant protein has a shorter half-life than wild-type C1QTNF5 and is preferentially degraded by proteasomes. Thirdly, C1QTNF5 is shown to interact with the membrane-type frizzled related protein (MFRP), on the basis of yeast two-hybrid, protein pull-down and co-immunoprecipitation assays and RPE co-localization. These data suggest that L-ORMD is due to insufficient levels of secreted C1QTNF5, compromised RPE cell function resulting from ER retention of the mutant protein or both mechanisms.

Mutation analysis of NR2E3 and NRL genes in Enhanced S Cone Syndrome.

Ten new and seventeen previously reported Enhanced S Cone Syndrome (ESCS) subjects were used to search for genetic heterogeneity. All subjects were diagnosed with ESCS on the basis of clinical, psychophysical and/or electroretinography testing using published criteria. Mutation analysis was performed on the NR2E3 nuclear receptor gene by single strand conformation analysis and direct sequencing, which revealed either homozygous (N=13) or compound heterozygous (N=11) mutations in 24 subjects (89%), heterozygous mutations in 2 subjects (7%) and no mutations in 1 subject (4%). Fifteen different mutations were identified, including six not previously reported. The subject (Patient A) with no detected NR2E3 mutation had features not usually associated with ESCS, in particular moderate rod photoreceptor function in peripheral retina and an abnormally thick retinal nerve fibre layer. Mutation analysis of the NRL, CRX, NR1D1 and THRB genes in this individual revealed a heterozygous one base-pair insertion in exon 2 of the NRL gene, which results in a predicted truncation of the NRL protein. Loss-of-function NRL alleles have not been described previously in humans, but since the same mutation was present in unaffected family members, it raises the possibility that the abnormal ESCS phenotype in Patient A may result from a digenic mechanism, with a heterozygous NRL mutation and a mutation in another unknown gene.

Determination of secondary chromosomal aberrations of chronic myelocytic leukemia.

Chronic myeloctyic leukemia (CML) is a stem cell disorder characterized by the cytogenetic abnormality of t(9;22)(q34;q11.2), which progresses from a chronic phase to an accelerated phase (AP), and/or a blast phase (BP) of myelocytic or lymphoid phenotype. This progression is frequently preceded or accompanied by recurring secondary chromosomal abnormalities (SCA) that are believed to play a role in the transformation and may also serve as valuable prognostic indicators. Failure to note such abnormalities may lead to an inappropriate clinical evaluation. We observed CML patients with AP or BP who did not show SCA by routine cytogenetic analysis. To determine the presence or absence of specific SCA in those cases, we applied fluorescence in situ hybridization (FISH) to four CML cases with pseudodiploid cytogenetics [t(9;22)(q34;11.2) as the sole abnormality] by conventional karyotyping. Bone marrow biopsies from two AP and two BP of CML patients with pseudodiploid karyotypes by conventional cytogenetics were examined by FISH for trisomy 8 and i(17q). These SCA are major secondary chromosomal changes seen in BP of CML patients. Results were considered positive if more than 2.4% of cells had +8 and >6.25% for i(17q) by FISH. Four out of four patients were positive for +8. These results indicate that FISH techniques are valuable in the determination of SCA in CML, which were t(9;22)(q34;q11.2) positive as the sole cytogenetic abnormality with standard G-banding karyotyping and can be helpful for the early diagnosis of CML progression.

Identification of oral peptostreptococcus isolates by PCR-restriction fragment length polymorphism analysis of 16S rRNA genes.

Oral Peptostreptococcus isolates tentatively identified by conventional microbiological culture methods were identified to the species level by a combination of PCR amplification of 16S rRNA genes and restriction enzyme analysis of the amplified products. This method is a reliable and rapid alternative to conventional methods for identification of these bacterial species.

Mutation in a short-chain collagen gene, CTRP5, results in extracellular deposit formation in late-onset retinal degeneration: a genetic model for age-related macular degeneration.

A primary feature of age-related macular degeneration (AMD) is the presence of extracellular deposits between the retinal pigment epithelium (RPE) and underlying Bruch's membrane, leading to RPE dysfunction, photoreceptor death and severe visual loss. AMD accounts for about 50% of blind registrations in Western countries and is a common, genetically complex disorder. Very little is known regarding its molecular basis. Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder with striking clinical and pathological similarity to AMD. Here we show that L-ORD is genetically heterogeneous and that a proposed founder mutation in the CTRP5 (C1QTNF5) gene, which encodes a novel short-chain collagen, changes a highly conserved serine to arginine (Ser163Arg) in 7/14 L-ORD families and 0/1000 control individuals. The mutation occurs in the gC1q domain of CTRP5 and results in abnormal high molecular weight aggregate formation which may alter its higher-order structure and interactions. These results indicate a novel disease mechanism involving abnormal adhesion between RPE and Bruch's membrane.