13q deletion syndrome and retinoblastoma in identical dichorionic diamniotic monozygotic twins.

PURPOSE: To report the case of identical dichorionic diamniotic female twins with unilateral retinoblastoma in 13q deletion syndrome. METHODS: Clinical and ophthalmoscopic evaluation, combination of multiple ligation-dependent probe amplification, array-comparative genomic hybridization analyses, and magnetic resonance imaging were performed. RESULTS: Peculiar facial features, marked hypotonia, gastroesophageal reflux, interatrial septal defect with left to right shunt and light dilatation of right chambers, 5th finger hypoplasia, 3rd-5th toes clinodactyly, 2nd toe overlapped to 3rd toe, and cutis marmorata were found. Ophthalmoscopic evaluation revealed unilateral retinoblastoma in both girls. Magnetic resonance imaging detected corpus callosum hypoplasia in both twins. A 34.4-Mb deletion involving bands 13q13.2-q21.33 and including the RB1 gene was identified in both twins. The deletion was not present in the DNA of their parents and older brother. CONCLUSIONS: Dysmorphic features in children must be always suspicious of 13q deletion syndrome and a short ophthalmoscopic follow-up is necessary to detect the presence of a retinoblastoma.

A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

Transcriptome analysis of the mobile genome ICEclc in Pseudomonas knackmussii B13.

Background: Integrative and conjugative elements (ICE) form a diverse group of DNA elements that are integrated in the chromosome of the bacterial host, but can occasionally excise and horizontally transfer to a new host cell. ICE come in different families, typically with a conserved core for functions controlling the element's behavior and a variable region providing auxiliary functions to the host. The ICEclc element of Pseudomonas knackmussii strain B13 is representative for a large family of chromosomal islands detected by genome sequencing approaches. It provides the host with the capacity to degrade chloroaromatics and 2-aminophenol. Results: Here we study the transcriptional organization of the ICEclc core region. By northern hybridizations, reverse-transcriptase polymerase chain reaction (RT-PCR) and Rapid Amplification of cDNA Ends (5'-RACE) fifteen transcripts were mapped in the core region. The occurrence and location of those transcripts were further confirmed by hybridizing labeled cDNA to a semi-tiling micro-array probing both strands of the ICEclc core region. Dot blot and semi-tiling array hybridizations demonstrated most of the core transcripts to be upregulated during stationary phase on 3-chlorobenzoate, but not on succinate or glucose. Conclusions: The transcription analysis of the ICEclc core region provides detailed insights in the mode of regulatory organization and will help to further understand the complex mode of behavior of this class of mobile elements. We conclude that ICEclc core transcription is concerted at a global level, more reminiscent of a phage program than of plasmid conjugation.

Comparison of three commercially available radio frequency coils for human brain imaging at 3 Tesla.

OBJECTIVE: To evaluate a transverse electromagnetic (TEM), a circularly polarized (CP) (birdcage), and a 12-channel phased array head coil at the clinical field strength of B0 = 3T in terms of signal-to-noise ratio (SNR), signal homogeneity, and maps of the effective flip angle alpha. MATERIALS AND METHODS: SNR measurements were performed on low flip angle gradient echo images. In addition, flip angle maps were generated for alpha(nominal) = 30 degrees using the double angle method. These evaluation steps were performed on phantom and human brain data acquired with each coil. Moreover, the signal intensity variation was computed for phantom data using five different regions of interest. RESULTS: In terms of SNR, the TEM coil performs slightly better than the CP coil, but is second to the smaller 12-channel coil for human data. As expected, both the TEM and the CP coils show superior image intensity homogeneity than the 12-channel coil, and achieve larger mean effective flip angles than the combination of body and 12-channel coil with reduced radio frequency power deposition. CONCLUSION: At 3T the benefits of TEM coil design over conventional lumped element(s) coil design start to emerge, though the phased array coil retains an advantage with respect to SNR performance.

The effect of homozygous deletion of the BBOX1 and Fibin genes on carnitine level and acyl carnitine profile.

BACKGROUND: Carnitine is a key molecule in energy metabolism that helps transport activated fatty acids into the mitochondria. Its homeostasis is achieved through oral intake, renal reabsorption and de novo biosynthesis. Unlike dietary intake and renal reabsorption, the importance of de novo biosynthesis pathway in carnitine homeostasis remains unclear, due to lack of animal models and description of a single patient defective in this pathway. CASE PRESENTATION: We identified by array comparative genomic hybridization a 42 months-old girl homozygote for a 221 Kb interstitial deletions at 11p14.2, that overlaps the genes encoding Fibin and butyrobetaine-gamma 2-oxoglutarate dioxygenase 1 (BBOX1), an enzyme essential for the biosynthesis of carnitine de novo. She presented microcephaly, speech delay, growth retardation and minor facial anomalies. The levels of almost all evaluated metabolites were normal. Her serum level of free carnitine was at the lower limit of the reference range, while her acylcarnitine to free carnitine ratio was normal. CONCLUSIONS: We present an individual with a completely defective carnitine de novo biosynthesis. This condition results in mildly decreased free carnitine level, but not in clinical manifestations characteristic of carnitine deficiency disorders, suggesting that dietary carnitine intake and renal reabsorption are sufficient to carnitine homeostasis. Our results also demonstrate that haploinsufficiency of BBOX1 and/or Fibin is not associated with Primrose syndrome as previously suggested.

Genome-wide mRNA expression correlates of viral control in CD4+ T-cells from HIV-1-infected individuals.

There is great interindividual variability in HIV-1 viral setpoint after seroconversion, some of which is known to be due to genetic differences among infected individuals. Here, our focus is on determining, genome-wide, the contribution of variable gene expression to viral control, and to relate it to genomic DNA polymorphism. RNA was extracted from purified CD4+ T-cells from 137 HIV-1 seroconverters, 16 elite controllers, and 3 healthy blood donors. Expression levels of more than 48,000 mRNA transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina). Genome-wide SNP data was generated from genomic DNA using the HumanHap550 Genotyping BeadChip (Illumina). We observed two distinct profiles with 260 genes differentially expressed depending on HIV-1 viral load. There was significant upregulation of expression of interferon stimulated genes with increasing viral load, including genes of the intrinsic antiretroviral defense. Upon successful antiretroviral treatment, the transcriptome profile of previously viremic individuals reverted to a pattern comparable to that of elite controllers and of uninfected individuals. Genome-wide evaluation of cis-acting SNPs identified genetic variants modulating expression of 190 genes. Those were compared to the genes whose expression was found associated with viral load: expression of one interferon stimulated gene, OAS1, was found to be regulated by a SNP (rs3177979, p = 4.9E-12); however, we could not detect an independent association of the SNP with viral setpoint. Thus, this study represents an attempt to integrate genome-wide SNP signals with genome-wide expression profiles in the search for biological correlates of HIV-1 control. It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. It also shows that elite controllers do not have a fully distinctive mRNA expression pattern in CD4+ T cells. Overall, changes in global RNA expression reflect responses to viral replication rather than a mechanism that might explain viral control.

A genome-wide association search for type 2 diabetes genes in African Americans.

African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P<0.0071), were directionally consistent in the Replication cohort and were associated with T2DM in subjects without nephropathy (P<0.05). Meta-analysis in all cases and controls revealed a single SNP reaching genome-wide significance (P<2.5×10(-8)). SNP rs7560163 (P = 7.0×10(-9), OR (95% CI) = 0.75 (0.67-0.84)) is located intergenically between RND3 and RBM43. Four additional loci (rs7542900, rs4659485, rs2722769 and rs7107217) were associated with T2DM (P<0.05) and reached more nominal levels of significance (P<2.5×10(-5)) in the overall analysis and may represent novel loci that contribute to T2DM. We have identified novel T2DM-susceptibility variants in the African-American population. Notably, T2DM risk was associated with the major allele and implies an interesting genetic architecture in this population. These results suggest that multiple loci underlie T2DM susceptibility in the African-American population and that these loci are distinct from those identified in other ethnic populations.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.

Glutathione Precursor, N-Acetyl-Cysteine, Modulates EEG Synchronization in Schizophrenia Patients

Background: Glutathione (GSH) dysregulation at the gene, protein and functional levels observed in schizophrenia patients, and schizophrenia-like anomalies in GSH deficit experimental models, suggest that genetic glutathione synthesis impairments represent one major risk factor for the disease (Do et al., 2009). In a randomized, double blind, placebo controlled, add-on clinical trial of 140 patients, the GSH precursor N-Acetyl-Cysteine (NAC, 2g/day, 6 months) significantly improved the negative symptoms and reduced sideeffects due to antipsychotics (Berk et al., 2008). In a subset of patients (n=7), NAC (2g/day, 2 months, cross-over design) also improved auditory evoked potentials, the NMDA-dependent mismatch negativity (Lavoie et al, 2008). Methods: To determine whether increased GSH levels would modulate the topography of functional brain connectivity, we applied a multivariate phase synchronization (MPS) estimator (Knyazeva et al, 2008) to dense-array EEGs recorded during rest with eyes closed at the protocol onset, the point of crossover, and at its end. Results: The whole-head imaging revealed a specific synchronization landscape in NAC compared to placebo condition. In particular, NAC increased MPS over frontal and left temporal regions in a frequency-specific manner. The topography and direction of MPS changes were similar and robust in all 7 patients. Moreover, these changes correlated with the changes in the Liddle's score of disorganization, thus linking EEG synchronization to the improvement of the clinical picture. Conclusions: The data suggest an important pathway towards new therapeutic strategies that target GSH dysregulation in schizophrenia. They also show the utility of MPS mapping as a marker of treatment efficacy.

Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis.

BACKGROUND: Histologic grade in breast cancer provides clinically important prognostic information. However, 30%-60% of tumors are classified as histologic grade 2. This grade is associated with an intermediate risk of recurrence and is thus not informative for clinical decision making. We examined whether histologic grade was associated with gene expression profiles of breast cancers and whether such profiles could be used to improve histologic grading. METHODS: We analyzed microarray data from 189 invasive breast carcinomas and from three published gene expression datasets from breast carcinomas. We identified differentially expressed genes in a training set of 64 estrogen receptor (ER)-positive tumor samples by comparing expression profiles between histologic grade 3 tumors and histologic grade 1 tumors and used the expression of these genes to define the gene expression grade index. Data from 597 independent tumors were used to evaluate the association between relapse-free survival and the gene expression grade index in a Kaplan-Meier analysis. All statistical tests were two-sided. RESULTS: We identified 97 genes in our training set that were associated with histologic grade; most of these genes were involved in cell cycle regulation and proliferation. In validation datasets, the gene expression grade index was strongly associated with histologic grade 1 and 3 status; however, among histologic grade 2 tumors, the index spanned the values for histologic grade 1-3 tumors. Among patients with histologic grade 2 tumors, a high gene expression grade index was associated with a higher risk of recurrence than a low gene expression grade index (hazard ratio = 3.61, 95% confidence interval = 2.25 to 5.78; P < .001, log-rank test). CONCLUSIONS: Gene expression grade index appeared to reclassify patients with histologic grade 2 tumors into two groups with high versus low risks of recurrence. This approach may improve the accuracy of tumor grading and thus its prognostic value.

The future is genome-wide.

A report of the annual meeting of the European Society of Human Genetics, Amsterdam, 6-9 May 2006.

Identification of structural variation in mouse genomes.

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Infrequent promoter methylation of the MGMT gene in liver metastases from uveal melanoma.

Uveal melanoma is associated with a high mortality rate once metastases occur, with over >90% of metastatic patients dying within less than 1 year from metastases to the liver. The intraarterial hepatic (iah) administration of the alkylating agent fotemustine holds some promise with response rates of 36% and median survival of 15 months. Here, we investigated whether the DNA-repair-protein MGMT may be involved in the variability of response to fotemustine and temozolomide in uveal melanoma. Epigenetic inactivation of MGMT has been demonstrated to be a predictive marker for benefit from alkylating agent therapy in glioblastoma. We found a methylated MGMT promoter in 6% of liver metastases from 34 uveal melanoma patients. The mean MGMT activity measured in liver metastases with negligible liver tissue content was significantly lower than in liver tissue (146 versus 523 fmol/mg protein, p = 0.002). Expression of the MGMT protein was detectable in 50% of 88 metastases by immunohistochemistry on a tissue microarray. Expression was heterogeneous, and in accordance with MGMT activity data, usually lower than in the surrounding liver. Differential MGMT activity/expression between metastasis and liver tissue and more efficient depletion of MGMT with higher doses of alkylating agent therapy using iah delivery may provide the pharmacologic window for the higher response rate. However, these results do not support MGMT methylation status or protein expression as predictive markers for treatment outcome to iah chemotherapy with alkylating agents.

MDM2 and CDK4 immunohistochemistry is a valuable tool in the differential diagnosis of low-grade osteosarcomas and other primary fibro-osseous lesions of the bone.

Low-grade osteosarcoma is a rare malignancy that may be subdivided into two main subgroups on the basis of location in relation to the bone cortex, that is, parosteal osteosarcoma and low-grade central osteosarcoma. Their histological appearance is quite similar and characterized by spindle cell stroma with low-to-moderate cellularity and well-differentiated anastomosing bone trabeculae. Low-grade osteosarcomas have a simple genetic profile with supernumerary ring chromosomes comprising amplification of chromosome 12q13-15, including the cyclin-dependent kinase 4 (CDK4) and murine double-minute type 2 (MDM2) gene region. Low-grade osteosarcoma can be confused with fibrous and fibro-osseous lesions such as fibromatosis and fibrous dysplasia on radiological and histological findings. We investigated MDM2-CDK4 immunohistochemical expression in a series of 72 low-grade osteosarcomas and 107 fibrous or fibro-osseous lesions of the bone or paraosseous soft tissue. The MDM2-CDK4 amplification status of low-grade osteosarcoma was also evaluated by comparative genomic hybridization array in 18 cases, and the MDM2 amplification status was evaluated by fluorescence in situ hybridization or quantitative real-time polymerase chain reaction in 31 cases of benign fibrous and fibro-osseous lesions. MDM2-CDK4 immunostaining and MDM2 amplification by fluorescence in situ hybridization or quantitative real-time polymerase chain reaction were investigated in a control group of 23 cases of primary high-grade bone sarcoma, including 20 conventional high-grade osteosarcomas, two pleomorphic spindle cell sarcomas/malignant fibrous histiocytomas and one leiomyosarcoma. The results showed that MDM2 and/or CDK4 immunoreactivity was present in 89% of low-grade osteosarcoma specimens. All benign fibrous and fibro-osseous lesions and the tumors of the control group were negative for MDM2 and CDK4. These results were consistent with the MDM2 and CDK4 amplification results. In conclusion, immunohistochemical expression of MDM2 and CDK4 is specific and provides sensitive markers for the diagnosis of low-grade osteosarcomas, helping to differentiate them from benign fibrous and fibro-osseous lesions, particularly in cases with atypical radio-clinical presentation and/or limited biopsy samples.

The role of astroglia in neuroprotection.

Astrocytes are the main neural cell type responsible for the maintenance of brain homeostasis. They form highly organized anatomical domains that are interconnected into extensive networks. These features, along with the expression of a wide array of receptors, transporters, and ion channels, ideally position them to sense and dynamically modulate neuronal activity. Astrocytes cooperate with neurons on several levels, including neurotransmitter trafficking and recycling, ion homeostasis, energy metabolism, and defense against oxidative stress. The critical dependence of neurons upon their constant support confers astrocytes with intrinsic neuroprotective properties which are discussed here. Conversely, pathogenic stimuli may disturb astrocytic function, thus compromising neuronal functionality and viability. Using neuroinflammation, Alzheimer's disease, and hepatic encephalopathy as examples, we discuss how astrocytic defense mechanisms may be overwhelmed in pathological conditions, contributing to disease progression.