Benjamín Rodríguez-Santiago

Mosaic Uniparental Disomies and Aneuploidies as Large Structural Variants of the Human Genome

Mosaicism is defined as the coexistence of cells with different genetic composition within an individual, caused by postzygotic somatic mutation. Although somatic mosaicism for chromosomal abnormalities is a well-established cause of developmental and somatic disorders and has also been detected in different tissues, its frequency and extent in the adult normal population are still unknown. We provide here a genome-wide survey of mosaic genomic variation obtained by analyzing Illumina 1M SNP array data from blood or buccal DNA samples of 1991 adult individuals from the Spanish Bladder Cancer/EPICURO genome-wide association study. We found mosaic abnormalities in autosomes in 1.7% of samples, including 23 segmental uniparental disomies, 8 complete trisomies, and 11 large (1.5-37 Mb) copy-number variants. Alterations were observed across the different autosomes with recurrent events in chromosomes 9 and 20. No case-control differences were found in the frequency of events or the percentage of cells affected, thus indicating that most rearrangements found are not central to the development of bladder cancer. However, five out of six events tested were detected in both blood and bladder tissue from the same individual, indicating an early developmental origin. The high cellular frequency of the anomalies detected and their presence in normal adult individuals suggest that this type of mosaicism is a widespread phenomenon in the human genome. Somatic mosaicism should be considered in the expanding repertoire of inter- and intraindividual genetic variation, some of which may cause somatic human diseases but also contribute to modifying inherited disorders and/or late-onset multifactorial traits.

Autism-specific copy number variants further implicate the phosphatidylinositol signaling pathway and the glutamatergic synapse in the etiology of the disorder

Autism spectrum disorders (ASDs) constitute a group of severe neurodevelopmental conditions with complex multifactorial etiology. In order to explore the hypothesis that submicroscopic genomic rearrangements underlie some ASD cases, we have analyzed 96 Spanish patients with idiopathic ASD after extensive clinical and laboratory screening, by array comparative genomic hybridization (aCGH) using a homemade bacterial artificial chromosome (BAC) array. Only 13 of the 238 detected copy number alterations, ranging in size from 89 kb to 2.4 Mb, were present specifically in the autistic population (12 out of 96 individuals, 12.5%). Following validation by additional molecular techniques, we have characterized these novel candidate regions containing 24 different genes including alterations in two previously reported regions of chromosome 7 associated with the ASD phenotype. Some of the genes located in ASD-specific copy number variants act in common pathways, most notably the phosphatidylinositol signaling and the glutamatergic synapse, both known to be affected in several genetic syndromes related with autism and previously associated with ASD. Our work supports the idea that the functional alteration of genes in related neuronal networks is involved in the etiology of the ASD phenotype and confirms a significant diagnostic yield for aCGH, which should probably be included in the diagnostic workup of idiopathic ASD.

Association of common copy number variants at the glutathione S -transferase genes and rare novel genomic changes with schizophrenia

Copy number variants (CNVs) are a substantial source of human genetic diversity, influencing the variable susceptibility to multifactorial disorders. Schizophrenia is a complex illness thought to be caused by a number of genetic and environmental effects, few of which have been clearly defined. Recent reports have found several low prevalent CNVs associated with the disease. We have used a multiplex ligation-dependent probe amplification-based (MLPA) method to target 140 previously reported and putatively relevant gene-containing CNV regions in 654 schizophrenic patients and 604 controls for association studies. Most genotyped CNVs (95%) showed very low (<1%) population frequency. A few novel rare variants were only present in patients suggesting a possible pathogenic involvement, including 1.39 Mb overlapping duplications at 22q11.23 found in two unrelated patients, and duplications of the somatostatin receptor 5 gene (SSTR5) at 16p13.3 in three unrelated patients. Furthermore, among the few relatively common CNVs observed in patients and controls, the combined analysis of gene copy number genotypes at two glutathione S-transferase (GST) genes, GSTM1 (glutathione S-transferase mu 1) (1p13.3) and GSTT2 (glutathione S-transferase theta 2) (22q11.23), showed a statistically significant association of non-null genotypes at both loci with an additive effect for increased vulnerability to schizophrenia (odds ratio of 1.92; P=0.0008). Our data provide complementary evidences for low prevalent, but highly penetrant chromosomal variants associated with schizophrenia, as well as for common CNVs that may act as susceptibility factors by disturbing glutathione metabolism.

Assessment of copy number variation using the Illumina Infinium 1M SNP-array: a comparison of methodological approaches in the Spanish Bladder Cancer/EPICURO study†

High‐throughput single nucleotide polymorphism (SNP)‐array technologies allow to investigate copy number variants (CNVs) in genome‐wide scans and specific calling algorithms have been developed to determine CNV location and copy number. We report the results of a reliability analysis comparing data from 96 pairs of samples processed with CNVpartition, PennCNV, and QuantiSNP for Infinium Illumina Human 1Million probe chip data. We also performed a validity assessment with multiplex ligation‐dependent probe amplification (MLPA) as a reference standard. The number of CNVs per individual varied according to the calling algorithm. Higher numbers of CNVs were detected in saliva than in blood DNA samples regardless of the algorithm used. All algorithms presented low agreement with mean Kappa Index (KI) <66. PennCNV was the most reliable algorithm (KIw=98.96) when assessing the number of copies. The agreement observed in detecting CNV was higher in blood than in saliva samples. When comparing to MLPA, all algorithms identified poorly known copy aberrations (sensitivity = 0.19–0.28). In contrast, specificity was very high (0.97–0.99). Once a CNV was detected, the number of copies was truly assessed (sensitivity >0.62). Our results indicate that the current calling algorithms should be improved for high performance CNV analysis in genome‐wide scans. Further refinement is required to assess CNVs as risk factors in complex diseases.Hum Mutat 32:1–10, 2011.

Integrated analysis of whole-exome sequencing and transcriptome profiling in males with autism spectrum disorders

BackgroundAutism spectrum disorders (ASD) are a group of neurodevelopmental disorders with high heritability. Recent findings support a highly heterogeneous and complex genetic etiology including rare de novo and inherited mutations or chromosomal rearrangements as well as double or multiple hits.MethodsWe performed whole-exome sequencing (WES) and blood cell transcriptome by RNAseq in a subset of male patients with idiopathic ASD (n = 36) in order to identify causative genes, transcriptomic alterations, and susceptibility variants.ResultsWe detected likely monogenic causes in seven cases: five de novo (SCN2A, MED13L, KCNV1, CUL3, and PTEN) and two inherited X-linked variants (MAOA and CDKL5). Transcriptomic analyses allowed the identification of intronic causative mutations missed by the usual filtering of WES and revealed functional consequences of some rare mutations. These included aberrant transcripts (PTEN, POLR3C), deregulated expression in 1.7% of mutated genes (that is, SEMA6B, MECP2, ANK3, CREBBP), allele-specific expression (FUS, MTOR, TAF1C), and non-sense-mediated decay (RIT1, ALG9). The analysis of rare inherited variants showed enrichment in relevant pathways such as the PI3K-Akt signaling and the axon guidance.ConclusionsIntegrative analysis of WES and blood RNAseq data has proven to be an efficient strategy to identify likely monogenic forms of ASD (19% in our cohort), as well as additional rare inherited mutations that can contribute to ASD risk in a multifactorial manner. Blood transcriptomic data, besides validating 88% of expressed variants, allowed the identification of missed intronic mutations and revealed functional correlations of genetic variants, including changes in splicing, expression levels, and allelic expression.

A fast and accurate method to detect allelic genomic imbalances underlying mosaic rearrangements using SNP array data

BackgroundMosaicism for copy number and copy neutral chromosomal rearrangements has been recently identified as a relatively common source of genetic variation in the normal population. However its prevalence is poorly defined since it has been only studied systematically in one large-scale study and by using non optimal ad-hoc SNP array data analysis tools, uncovering rather large alterations (> 1 Mb) and affecting a high proportion of cells. Here we propose a novel methodology, Mosaic Alteration Detection-MAD, by providing a software tool that is effective for capturing previously described alterations as wells as new variants that are smaller in size and/or affecting a low percentage of cells.ResultsThe developed method identified all previously known mosaic abnormalities reported in SNP array data obtained from controls, bladder cancer and HapMap individuals. In addition MAD tool was able to detect new mosaic variants not reported before that were smaller in size and with lower percentage of cells affected. The performance of the tool was analysed by studying simulated data for different scenarios. Our method showed high sensitivity and specificity for all assessed scenarios.ConclusionsThe tool presented here has the ability to identify mosaic abnormalities with high sensitivity and specificity. Our results confirm the lack of sensitivity of former methods by identifying new mosaic variants not reported in previously utilised datasets. Our work suggests that the prevalence of mosaic alterations could be higher than initially thought. The use of appropriate SNP array data analysis methods would help in defining the human genome mosaic map.

Is mitochondrial DNA depletion involved in Alzheimer's disease?

Several studies have suggested that mitochondrial metabolism disturbances and mitochondrial DNA (mtDNA) abnormalities may contribute to the progression of the pathology of Alzheimers disease (AD).In this study we have investigated whether the amount of mtDNA is modified in different brain regions (cerebellum, hippocampus and frontal cortex) of confirmed AD necropsies and in blood of living AD patients. We used a real-time PCR method to analyse the mtDNA relative abundance in brain regions from 12 ADand seven controls and from a group of blood samples (17 living AD patients and 11 controls). MtDNA from blood samples together with hippocampus and cerebellum brain areas did not show differences between controls and AD. However, AD patients showed a 28% decrease in the amount of mtDNA in the frontalcortex when compared to controls for this specific area. Since frontal cortex is a severely affected regionin AD, our results support the hypothesis that mitochondrial defects may play a role in the pathogenesis of AD.

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

BackgroundGermline 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.ResultsThis 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.ConclusionThis 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.

Mosaic loss of chromosome Y is associated with common variation near TCL1A

Mosaic loss of chromosome Y (mLOY) leading to gonosomal XY/XO commonly occurs during aging, particularly in smokers. We investigated whether mLOY was associated with non-hematological cancer in three prospective cohorts (8,679 cancer cases and 5,110 cancer-free controls) and genetic susceptibility to mLOY. Overall, mLOY was observed in 7% of men, and its prevalence increased with age (per-year odds ratio (OR) = 1.13, 95% confidence interval (CI) = 1.12–1.15; P < 2 × 10−16), reaching 18.7% among men over 80 years old. mLOY was associated with current smoking (OR = 2.35, 95% CI = 1.82–3.03; P = 5.55 × 10−11), but the association weakened with years after cessation. mLOY was not consistently associated with overall or specific cancer risk (for example, bladder, lung or prostate cancer) nor with cancer survival after diagnosis (multivariate-adjusted hazard ratio = 0.87, 95% CI = 0.73–1.04; P = 0.12). In a genome-wide association study, we observed the first example of a common susceptibility locus for genetic mosaicism, specifically mLOY, which maps to TCL1A at 14q32.13, marked by rs2887399 (OR = 1.55, 95% CI = 1.36–1.78; P = 1.37 × 10−10).

The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies.

BackgroundUrothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression.ResultsBased on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events.ConclusionsOverall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.