Genotypic classification of patients with Wolfram syndrome: insights into the natural history of the disease and correlation with phenotype.

PURPOSE: Wolfram syndrome is a degenerative, recessive rare disease with an onset in childhood. It is caused by mutations in WFS1 or CISD2 genes. More than 200 different variations in WFS1 have been described in patients with Wolfram syndrome, which complicates the establishment of clear genotype-phenotype correlation. The purpose of this study was to elucidate the role of WFS1 mutations and update the natural history of the disease. METHODS: This study analyzed clinical and genetic data of 412 patients with Wolfram syndrome published in the last 15 years. RESULTS: (i) 15% of published patients do not fulfill the current -inclusion criterion; (ii) genotypic prevalence differences may exist among countries; (iii) diabetes mellitus and optic atrophy might not be the first two clinical features in some patients; (iv) mutations are nonuniformly distributed in WFS1; (v) age at onset of diabetes mellitus, hearing defects, and diabetes insipidus may depend on the patient's genotypic class; and (vi) disease progression rate might depend on genotypic class. CONCLUSION: New genotype-phenotype correlations were established, disease progression rate for the general population and for the genotypic classes has been calculated, and new diagnostic criteria have been proposed. The conclusions raised could be important for patient management and counseling as well as for the development of treatments for Wolfram syndrome.

EURO-WABB: an EU rare diseases registry for Wolfram syndrome, Alström syndrome and Bardet-Biedl syndrome.

BACKGROUND: Wolfram, Alström and Bardet-Biedl (WABB) syndromes are rare diseases with overlapping features of multiple sensory and metabolic impairments, including diabetes mellitus, which have caused diagnostic confusion. There are as yet no specific treatments available, little or no access to well characterized cohorts of patients, and limited information on the natural history of the diseases. We aim to establish a Europe-wide registry for these diseases to inform patient care and research. METHODS: EURO-WABB is an international multicenter large-scale observational study capturing longitudinal clinical and outcome data for patients with WABB diagnoses. Three hundred participants will be recruited over 3 years from different sites throughout Europe. Comprehensive clinical, genetic and patient experience data will be collated into an anonymized disease registry. Data collection will be web-based, and forms part of the project's Virtual Research and Information Environment (VRIE). Participants who haven't undergone genetic diagnostic testing for their condition will be able to do so via the project. CONCLUSIONS: The registry data will be used to increase the understanding of the natural history of WABB diseases, to serve as an evidence base for clinical management, and to aid the identification of opportunities for intervention to stop or delay the progress of the disease. The detailed clinical characterisation will allow inclusion of patients into studies of novel treatment interventions, including targeted interventions in small scale open label studies; and enrolment into multi-national clinical trials. The registry will also support wider access to genetic testing, and encourage international collaborations for patient benefit.

Genotype and Clinical Characteristics of Patients with Wolfram Syndrome and WFS1-related Disorders.

Objective: Wolfram syndrome (WFS) is an autosomal recessive disorder associated with juvenile-onset diabetes mellitus, optic atrophy, diabetes insipidus, and sensorineural hearing loss. We sought to elucidate the relationship between genotypic and phenotypic presentations of Wolfram syndrome which would assist clinicians in classifying the severity and prognosis of Wolfram syndrome more accurately. Approach: Patient data from the Washington University International Registry and Clinical Study for Wolfram Syndrome and patient case reports were analyzed to select for patients with two recessive mutations in the WFS1 gene. Mutations were classified as being either nonsense/frameshift variants or missense/in-frame insertion/deletion variants and statistical analysis was performed using unpaired and paired t-tests and one- and two-way ANOVA with Tukey's or Dunnett's tests. Results: A greater number of genotype variants correlated with earlier onset and a more severe presentation of Wolfram syndrome. Secondly, non-sense and frameshift variants had more severe phenotypic presentations than missense variants, as evidenced by optic atrophy emerging significantly earlier in patients with 2 nonsense/frameshift alleles compared with 0 missense transmembrane variants. In addition, the number of transmembrane in-frame variants demonstrated a statistically significant dose-effect on age of onset of diabetes mellitus and optic atrophy. Summary / Conclusions: The results contribute to our current understanding of the genotype-phenotype relationship of Wolfram syndrome, suggesting that alterations in coding sequences result in significant changes in the presentation and severity of Wolfram. The impact of these findings is significant, as the results will aid clinicians in predicting more accurate prognoses and pave the way for personalized treatments for Wolfram syndrome.

Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction.

BACKGROUND: Genome-wide studies have begun to link subtle variations in both allelic DNA methylation and parent-of-origin genetic effects with early development. Numerous reports have highlighted that the placenta plays a critical role in coordinating fetal growth, with many key functions regulated by genomic imprinting. With the recent description of wide-spread polymorphic placenta-specific imprinting, the molecular mechanisms leading to this curious polymorphic epigenetic phenomenon is unknown, as is their involvement in pregnancies complications. RESULTS: Profiling of 35 ubiquitous and 112 placenta-specific imprinted differentially methylated regions (DMRs) using high-density methylation arrays and pyrosequencing revealed isolated aberrant methylation at ubiquitous DMRs as well as abundant hypomethylation at placenta-specific DMRs. Analysis of the underlying chromatin state revealed that the polymorphic nature is not only evident at the level of allelic methylation, but DMRs can also adopt an unusual epigenetic signature where the underlying histones are biallelically enrichment of H3K4 methylation, a modification normally mutually exclusive with DNA methylation. Quantitative expression analysis in placenta identified two genes, GPR1-AS1 and ZDBF2, that were differentially expressed between IUGRs and control samples after adjusting for clinical factors, revealing coordinated deregulation at the chromosome 2q33 imprinted locus. CONCLUSIONS: DNA methylation is less stable at placenta-specific imprinted DMRs compared to ubiquitous DMRs and contributes to privileged state of the placenta epigenome. IUGR-associated expression differences were identified for several imprinted transcripts independent of allelic methylation. Further work is required to determine if these differences are the cause IUGR or reflect unique adaption by the placenta to developmental stresses.

Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility.

BACKGROUND: Germline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally. RESULTS: This study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC. CONCLUSION: This study proposes that variation at putative 8q24 cis-regulator(s) of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene.

The bioenergetic signature of cancer: a marker of tumor progression.

Mitochondrial H+-ATP synthase is required for cellular energy provision and for efficient execution of apoptosis. Almost one century ago, Otto Warburg proposed the hypothesis that mitochondrial function might be impaired in cancer cells. However, his hypothesis was never demonstrated in human carcinomas. In this study, we have analyzed the expression of the beta-catalytic subunit of the H+-ATP synthase (beta-F1-ATPase) of mitochondria in carcinomas of the human liver, kidney, and colon. We show that carcinogenesis in the liver involves a depletion of the cellular mitochondrial content, as revealed by reduced content of mitochondrial markers, whereas in kidney and colon carcinomas, it involves a selective repression of the expression of the beta-F1-ATPase concurrent with an increase in the expression of the glycolytic glyceraldehyde-3-phosphate dehydrogenase. Both mechanisms limit mitochondrial cellular activity in cancer, strongly supporting Warburg's hypothesis, and suggest a mechanism for the resistance and compromised apoptotic potential of tumor cells. Furthermore, we show that the metabolic state of the cell, as defined by a bioenergetic mitochondrial index relative to the cellular glycolytic potential, provides a signature of carcinogenesis of prognostic value in assessing the progression of colorectal carcinomas.

Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction.

Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

BootstRatio: A web-based statistical analysis of fold-change in qPCR and RT-qPCR data using resampling methods.

Real-time quantitative polymerase chain reaction (qPCR) is widely used in biomedical sciences quantifying its results through the relative expression (RE) of a target gene versus a reference one. Obtaining significance levels for RE assuming an underlying probability distribution of the data may be difficult to assess. We have developed the web-based application BootstRatio, which tackles the statistical significance of the RE and the probability that RE>1 through resampling methods without any assumption on the underlying probability distribution for the data analyzed. BootstRatio perform these statistical analyses of gene expression ratios in two settings: (1) when data have been already normalized against a control sample and (2) when the data control samples are provided. Since the estimation of the probability that RE>1 is an important feature for this type of analysis, as it is used to assign statistical significance and it can be also computed under the Bayesian framework, a simulation study has been carried out comparing the performance of BootstRatio versus a Bayesian approach in the estimation of that probability. In addition, two analyses, one for each setting, carried out with data from real experiments are presented showing the performance of BootstRatio. Our simulation study suggests that Bootstratio approach performs better than the Bayesian one excepting in certain situations of very small sample size (N≤12). The web application BootstRatio is accessible through http://regstattools.net/br and developed for the purpose of these intensive computation statistical analyses.

Altered expression of 12S/MT-RNR1, MT-CO2/COX2, and MT-ATP6 mitochondrial genes in prostate cancer.

BACKGROUND: Prostate cancer is one of the commonest cancers worldwide and is responsible for nearly 6% of all male cancer deaths. Despite this relevance, the mechanisms involved in the development and progression of this malignancy remain unknown. The involvement of polypeptides of the mitochondrial respiratory chain, the Krebs cycle and the glutathione antioxidant system in this type of cancer has been previously described, although no publication has focused on the expression of mitochondrial genes in the prostate of PCa patients. METHODS: We have determined by reverse transcription-quantitative PCR (RT-qPCR) the relative amount of the transcripts of eight mitochondrial genes (MT-ND2, MT-ND4, MT-ND6, MT-CYB, 12S/MT-RNR1, 16S/MT-RNR2, MT-CO2/COX2, MT-ATP6), and four nuclear genes (COX11, GSR, CS, ACO2), all of them key players in the normal metabolism of mitochondria. Additionally we analyzed the expression of Cyclophilin A (PPIA). RESULTS: We observed differential expression of mitochondrial 12S/MT-RNR1, MT-CO2/COX2, and MT-ATP6 transcripts in tumor samples when compared to their paired normal samples. CONCLUSIONS: The amount of mitochondrial 12S/MT-RNR1, MT-CO2/COX2, and MT-ATP6 transcripts is significantly decreased in tumor samples when compared to their paired normal sample, suggesting that mitochondrial gene expression is altered in PCa.