Characterization of the human SLC2A11 (GLUT11) gene: alternative promoter usage,function, expression,and subcellular distribution of three isoforms,and lack of mouse orthologue

GLUT11 (SLC2A11) is a class II sugar transport facilitator which exhibits highest similarity with the fructose transporter GLUT5 (about 42%). Here we demonstrate that separate exons 1 (exon 1A, exon 1B, and exon 1C) of the SLC2A11 gene generate mRNAs of three GLUT11 variants (GLUT11-A, GLUT11-B, and GLUT11-C) that differ in the amino acid sequence of their N-termini. All three 5′-flanking regions of exon 1A, exon 1B and exon 1C exhibited promoter activity when expressed as luciferase fusion constructs in COS-7 cells. 5′-RACE-PCR, quantitative real-time PCR, and Northern blot analysis performed with specific probes for exon 1A, 1B and 1C demonstrated that GLUT11-A is expressed in heart, skeletal muscle, and kidney, GLUT11-B in kidney, adipose tissue, and placenta, and GLUT11-C in adipose tissue, heart, skeletal muscle, and pancreas. Surprisingly, mice and rats lack the SLC2A11 gene. When expressed in Xenopus oocytes, all three GLUT11 isoforms transport glucose and fructose but not galactose. There was no apparent difference in the subcellular distribution of the three isoforms expressed in COS-7 cells. Our data indicate that different promoters and splicing of the human SLC2A11 gene generate three GLUT11 isoforms which are expressed in a tissue specific manner but do not appear to differ in their functional characteristics.     

A classification model for distinguishing copy number variants from cancer-related alterations

Background  

Both somatic copy number alterations (CNAs) and germline copy number variants (CNVs) that are prevalent in healthy individuals can appear as recurrent changes in comparative genomic hybridization (CGH) analyses of tumors. In order to identify important cancer genes CNAs and CNVs must be distinguished. Although the Database of Genomic Variants (DGV) contains a list of all known CNVs, there is no standard methodology to use the database effectively.     

Genome-wide detection of copy number variants in European autochthonous and commercial pig breeds by whole-genome sequencing of DNA pools identified breed-characterising copy number states

In this study, we identified copy number variants (CNVs) in 19 European autochthonous pig breeds and in two commercial breeds (Italian Large White and Italian Duroc) that represent important genetic resources for this species. The genome of 725 pigs was sequenced using a breed-specific DNA pooling approach (30–35 animals per pool) obtaining an average depth per pool of 42×. This approach maximised CNV discovery as well as the related copy number states characterising, on average, the analysed breeds. By mining more than 17.5 billion reads, we identified a total of 9592 CNVs (~683 CNVs per breed) and 3710 CNV regions (CNVRs; 1.15% of the reference pig genome), with an average of 77 CNVRs per breed that were considered as private. A few CNVRs were analysed in more detail, together with other information derived from sequencing data. For example, the CNVR encompassing the KIT gene was associated with coat colour phenotypes in the analysed breeds, confirming the role of the multiple copies in determining breed-specific coat colours. The CNVR covering the MSRB3 gene was associated with ear size in most breeds. The CNVRs affecting the ELOVL6 and ZNF622 genes were private features observed in the Lithuanian Indigenous Wattle and in the Turopolje pig breeds respectively. Overall, the genome variability unravelled here can explain part of the genetic diversity among breeds and might contribute to explain their origin, history and adaptation to a variety of production systems.     

Complex Analysis of Urate Transporters SLC2A9, SLC22A12 and Functional Characterization of Non-Synonymous Allelic Variants of GLUT9 in the Czech Population: No Evidence of Effect on Hyperuricemia and Gout

Objective

Using European descent Czech populations, we performed a study of SLC2A9 and SLC22A12 genes previously identified as being associated with serum uric acid concentrations and gout. This is the first study of the impact of non-synonymous allelic variants on the function of GLUT9 except for patients suffering from renal hypouricemia type 2.

Methods

The cohort consisted of 250 individuals (150 controls, 54 nonspecific hyperuricemics and 46 primary gout and/or hyperuricemia subjects). We analyzed 13 exons of SLC2A9 (GLUT9 variant 1 and GLUT9 variant 2) and 10 exons of SLC22A12 by PCR amplification and sequenced directly. Allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. The functional studies were analyzed using the non-parametric Wilcoxon and Kruskall-Wallis tests; the association study used the Fisher exact test and linear regression approach.

Results

We identified a total of 52 sequence variants (12 unpublished). Eight non-synonymous allelic variants were found only in SLC2A9: rs6820230, rs2276961, rs144196049, rs112404957, rs73225891, rs16890979, rs3733591 and rs2280205. None of these variants showed any significant difference in the expression of GLUT9 and in urate transport. In the association study, eight variants showed a possible association with hyperuricemia. However, seven of these were in introns and the one exon located variant, rs7932775, did not show a statistically significant association with serum uric acid concentration.

Conclusion

Our results did not confirm any effect of SLC22A12 and SLC2A9 variants on serum uric acid concentration. Our complex approach using association analysis together with functional and immunohistochemical characterization of non-synonymous allelic variants did not show any influence on expression, subcellular localization and urate uptake of GLUT9.     

Synergistic Interactions between Drosophila Orthologues of Genes Spanned by De Novo Human CNVs Support Multiple-Hit Models of Autism

Autism spectrum disorders (ASDs) are highly heritable and characterised by deficits in social interaction and communication, as well as restricted and repetitive behaviours. Although a number of highly penetrant ASD gene variants have been identified, there is growing evidence to support a causal role for combinatorial effects arising from the contributions of multiple loci. By examining synaptic and circadian neurological phenotypes resulting from the dosage variants of unique human:fly orthologues in Drosophila, we observe numerous synergistic interactions between pairs of informatically-identified candidate genes whose orthologues are jointly affected by large de novo copy number variants (CNVs). These CNVs were found in the genomes of individuals with autism, including a patient carrying a 22q11.2 deletion. We first demonstrate that dosage alterations of the unique Drosophila orthologues of candidate genes from de novo CNVs that harbour only a single candidate gene display neurological defects similar to those previously reported in Drosophila models of ASD-associated variants. We then considered pairwise dosage changes within the set of orthologues of candidate genes that were affected by the same single human de novo CNV. For three of four CNVs with complete orthologous relationships, we observed significant synergistic effects following the simultaneous dosage change of gene pairs drawn from a single CNV. The phenotypic variation observed at the Drosophila synapse that results from these interacting genetic variants supports a concordant phenotypic outcome across all interacting gene pairs following the direction of human gene copy number change. We observe both specificity and transitivity between interactors, both within and between CNV candidate gene sets, supporting shared and distinct genetic aetiologies. We then show that different interactions affect divergent synaptic processes, demonstrating distinct molecular aetiologies. Our study illustrates mechanisms through which synergistic effects resulting from large structural variation can contribute to human disease.     

Allele-specific disparity in breast cancer

Background

Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.

Methods

We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.

Results

DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.

Conclusions

This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.     

Integrative Analysis of Genome, 3D Genome,and Transcriptome Alterations of Clinical Lung Cancer Samples

Genomic studies of cancer cell alterations, such as mutations, copy number variations (CNVs), and translocations, greatly promote our understanding of the genesis and development of cancers. However, the 3D genome architecture of cancers remains less studied due to the complexity of cancer genomes and technical difficulties. To explore the 3D genome structure in clinical lung cancer, we performed Hi-C experiments using paired normal and tumor cells harvested from patients with lung cancer, combining with RNA sequenceing analysis. We demonstrated the feasibility of studying 3D genome of clinical lung cancer samples with a small number of cells (1 × 10⁴), compared the genome architecture between clinical samples and cell lines of lung cancer, and identified conserved and changed spatial chromatin structures between normal and cancer samples. We also showed that Hi-C data can be used to infer CNVs and point mutations in cancer. By integrating those different types of cancer alterations, we showed significant associations between CNVs, 3D genome, and gene expression. We propose that 3D genome mediates the effects of cancer genomic alterations on gene expression through altering regulatory chromatin structures. Our study highlights the importance of analyzing 3D genomes of clinical cancer samples in addition to cancer cell lines and provides an integrative genomic analysis pipeline for future larger-scale studies in lung cancer and other cancers.     

Rare De Novo Copy Number Variants in Patients with Congenital Pulmonary Atresia

Background

Ongoing studies using genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly ignored in the past. The aim of this study was to identify rare copy number variants in individuals with congenital pulmonary atresia (PA).

Methods and Results

Based on the hypothesis that rare structural variants encompassing key genes play an important role in heart development in PA patients, we performed high-resolution genome-wide microarrays for copy number variations (CNVs) in 82 PA patient-parent trios and 189 controls with an Illumina SNP array platform. CNVs were identified in 17/82 patients (20.7%), and eight of these CNVs (9.8%) are considered potentially pathogenic. Five de novo CNVs occurred at two known congenital heart disease (CHD) loci (16p13.1 and 22q11.2). Two de novo CNVs that may affect folate and vitamin B₁₂ metabolism were identified for the first time. A de novo 1-Mb deletion at 17p13.2 may represent a rare genomic disorder that involves mild intellectual disability and associated facial features.

Conclusions

Rare CNVs contribute to the pathogenesis of PA (9.8%), suggesting that the causes of PA are heterogeneous and pleiotropic. Together with previous data from animal models, our results might help identify a link between CHD and folate-mediated one-carbon metabolism (FOCM). With the accumulation of high-resolution SNP array data, these previously undescribed rare CNVs may help reveal critical gene(s) in CHD and may provide novel insights about CHD pathogenesis.     

ATAQS: A computational software tool for high throughput transition optimization and validation for selected reaction monitoring mass spectrometry

Background

Copy number variants (CNVs), including deletions, amplifications, and other rearrangements, are common in human and cancer genomes. Copy number data from array comparative genome hybridization (aCGH) and next-generation DNA sequencing is widely used to measure copy number variants. Comparison of copy number data from multiple individuals reveals recurrent variants. Typically, the interior of a recurrent CNV is examined for genes or other loci associated with a phenotype. However, in some cases, such as gene truncations and fusion genes, the target of variant lies at the boundary of the variant.

Results

We introduce Neighborhood Breakpoint Conservation (NBC), an algorithm for identifying rearrangement breakpoints that are highly conserved at the same locus in multiple individuals. NBC detects recurrent breakpoints at varying levels of resolution, including breakpoints whose location is exactly conserved and breakpoints whose location varies within a gene. NBC also identifies pairs of recurrent breakpoints such as those that result from fusion genes. We apply NBC to aCGH data from 36 primary prostate tumors and identify 12 novel rearrangements, one of which is the well-known TMPRSS2-ERG fusion gene. We also apply NBC to 227 glioblastoma tumors and predict 93 novel rearrangements which we further classify as gene truncations, germline structural variants, and fusion genes. A number of these variants involve the protein phosphatase PTPN12 suggesting that deregulation of PTPN12, via a variety of rearrangements, is common in glioblastoma.

Conclusions

We demonstrate that NBC is useful for detection of recurrent breakpoints resulting from copy number variants or other structural variants, and in particular identifies recurrent breakpoints that result in gene truncations or fusion genes. Software is available at http://http.//cs.brown.edu/people/braphael/software.html.     

Characterization of the human SLC2A11 (GLUT11) gene: alternative promoter usage, function, expression, and subcellular distribution of three isoforms, and lack of mouse orthologue

GLUT11 (SLC2A11) is a class II sugar transport facilitator which exhibits highest similarity with the fructose transporter GLUT5 (about 42%). Here we demonstrate that separate exons 1 (exon 1A, exon 1B, and exon 1C) of the SLC2A11 gene generate mRNAs of three GLUT11 variants (GLUT11-A, GLUT11-B, and GLUT11-C) that differ in the amino acid sequence of their N-termini. All three 5'-flanking regions of exon 1A, exon 1B and exon 1C exhibited promoter activity when expressed as luciferase fusion constructs in COS-7 cells. 5'-RACE-PCR, quantitative real-time PCR, and Northern blot analysis performed with specific probes for exon 1A, 1B and 1C demonstrated that GLUT11-A is expressed in heart, skeletal muscle, and kidney, GLUT11-B in kidney, adipose tissue, and placenta, and GLUT11-C in adipose tissue, heart, skeletal muscle, and pancreas. Surprisingly, mice and rats lack the SLC2A11 gene. When expressed in Xenopus oocytes, all three GLUT11 isoforms transport glucose and fructose but not galactose. There was no apparent difference in the subcellular distribution of the three isoforms expressed in COS-7 cells. Our data indicate that different promoters and splicing of the human SLC2A11 gene generate three GLUT11 isoforms which are expressed in a tissue specific manner but do not appear to differ in their functional characteristics.     

SLC2A12 of SLC2 Gene Family in Bird Provides Functional Compensation for the Loss of SLC2A4 Gene in Other Vertebrates

Avian genomes are small and lack some genes that are conserved in the genomes of most other vertebrates including nonavian sauropsids. One hypothesis stated that paralogs may provide biochemical or physiological compensation for certain gene losses; however, no functional evidence has been reported to date. By integrating evolutionary analysis, physiological genomics, and experimental gene interference, we clearly demonstrate functional compensation for gene loss. A large-scale phylogenetic analysis of over 1,400 SLC2 gene sequences identifies six new SLC2 genes from nonmammalian vertebrates and divides the SLC2 gene family into four classes. Vertebrates retain class III SLC2 genes but partially lack the more recent duplicates of classes I and II. Birds appear to have completely lost the SLC2A4 gene that encodes an important insulin-sensitive GLUT in mammals. We found strong evidence for positive selection, indicating that the N-termini of SLC2A4 and SLC2A12 have undergone diversifying selection in birds and mammals, and there is a significant correlation between SLC2A12 functionality and basal metabolic rates in endotherms. Physiological genomics have uncovered that SLC2A12 expression and allelic variants are associated with insulin sensitivity and blood glucose levels in wild birds. Functional tests have indicated that SLC2A12 abrogation causes hyperglycemia, insulin resistance, and high relative activity, thus increasing energy expenditures that resemble a diabetic phenotype. These analyses suggest that the SLC2A12 gene not only functionally compensates insulin response for SLC2A4 loss but also affects daily physical behavior and basal metabolic rate during bird evolution, highlighting that older genes retain a higher level of functional diversification.     

Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT''s limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.     

The hyper-IgE syndrome is not caused by a microdeletion syndrome

The hyper-immunoglobulin E syndrome (HIES) is a rare primary immunodeficiency characterized by recurrent infections, elevated serum IgE-levels, and involvement of the soft- and bony tissues. We speculated that this complex disease may be caused by a microdeletion syndrome. We therefore analyzed 30 sporadic HIES patients for the presence of chromosomal imbalances using Affymetrix 50k XbaI and 23 of the 30 patients with the higher-resolution 250k StyI SNP mapping arrays. We detected only eight different copy number alterations in six patients with the 50k approach, and seven of these presented known polymorphic regions not associated with disease. However, one patient showed a unique gain on chromosome 20p. 250k array analysis identified this gain as a rare polymorphism segregating in the patient’s family, but not associated with the HIES phenotype. In addition, 265 known and novel copy number variants (CNVs) were identified with the 250k arrays, but no recurrent imbalances reminescent of a microdeletion syndrome were found. We aligned the identified CNVs with loci that have been associated with HIES or phenotypically overlapping syndromes. Doing so, a 2-Mb deletion spanning the PEPD gene on 19q13.11 was identified on one allele of one patient. Homozygous mutations in PEPD are responsible for the autosomal-recessive prolidase deficiency which resembles HIES in some aspects. Sequencing of the healthy allele, however, revealed a wild-type sequence. In summary, our results suggest that HIES is not likely to be a microdeletion syndrome. Dietmar Pfeifer and Cristina Woellner contributed equally to the work and are considered aequo loco.     

Elusive Copy Number Variation in the Mouse Genome

Background

Array comparative genomic hybridization (aCGH) to detect copy number variants (CNVs) in mammalian genomes has led to a growing awareness of the potential importance of this category of sequence variation as a cause of phenotypic variation. Yet there are large discrepancies between studies, so that the extent of the genome affected by CNVs is unknown. We combined molecular and aCGH analyses of CNVs in inbred mouse strains to investigate this question.

Principal Findings

Using a 2.1 million probe array we identified 1,477 deletions and 499 gains in 7 inbred mouse strains. Molecular characterization indicated that approximately one third of the CNVs detected by the array were false positives and we estimate the false negative rate to be more than 50%. We show that low concordance between studies is largely due to the molecular nature of CNVs, many of which consist of a series of smaller deletions and gains interspersed by regions where the DNA copy number is normal.

Conclusions

Our results indicate that CNVs detected by arrays may be the coincidental co-localization of smaller CNVs, whose presence is more likely to perturb an aCGH hybridization profile than the effect of an isolated, small, copy number alteration. Our findings help explain the hitherto unexplored discrepancies between array-based studies of copy number variation in the mouse genome.