Francesco Maria Calabrese

HmtDB, a genomic resource for mitochondrion-based human variability studies

HmtDB (http://www.hmtdb.uniba.it:8080/hmdb) is a open resource created to support population genetics and mitochondrial disease studies. The database hosts human mitochondrial genome sequences annotated with population and variability data, the latter being estimated through the application of the SiteVar software based on site-specific nucleotide and amino acid variability calculations. The annotations are manually curated thus adding value to the quality of the information provided to the end-user. Classifier tools implemented in HmtDB allow the prediction of the haplogroup for any human mitochondrial genome currently stored in HmtDB or externally submitted de novo by an end-user. Haplogroup definition is based on the Phylotree system. End-users accessing HmtDB are hence allowed to (i) browse the database through the use of a multi-criterion ‘query’ system; (ii) analyze their own human mitochondrial sequences via the ‘classify’ tool (for complete genomes) or by downloading the ‘fragment-classifier’ tool (for partial sequences); (iii) download multi-alignments with reference genomes as well as variability data.

Extraction and annotation of human mitochondrial genomes from 1000 Genomes Whole Exome Sequencing data

BackgroundWhole Exome Sequencing (WES) is one of the most used and cost-effective next generation technologies that allows sequencing of all nuclear exons. Off-target regions may be captured if they present high sequence similarity with baits. Bioinformatics tools have been optimized to retrieve a large amount of WES off-target mitochondrial DNA (mtDNA), by exploiting the aspecificity of probes, partially overlapping to Nuclear mitochondrial Sequences (NumtS). The 1000 Genomes project represents one of the widest resources to extract mtDNA sequences from WES data, considering the large effort the scientific community is undertaking to reconstruct human population history using mtDNA as marker, and the involvement of mtDNA in pathology.ResultsA previously published pipeline aimed at assembling mitochondrial genomes from off-target WES reads and further improved to detect insertions and deletions (indels) and heteroplasmy in a dataset of 1242 samples from the 1000 Genomes project, enabled to obtain a nearly complete mitochondrial genome from 943 samples (76% analyzed exomes). The robustness of our computational strategy was highlighted by the reduction of reads amount recognized as mitochondrial in the original annotation produced by the Consortium, due to NumtS filtering.An accurate survey was carried out on 1242 individuals. 215 indels, mostly heteroplasmic, and 3407 single base variants were mapped. A homogeneous mismatches distribution was observed along the whole mitochondrial genome, while a lower frequency of indels was found within protein-coding regions, where frameshift mutations may be deleterious. The majority of indels and mismatches found were not previously annotated in mitochondrial databases since conventional sequencing methods were limited to homoplasmy or quasi-homoplasmy detection. Intriguingly, upon filtering out non haplogroup-defining variants, we detected a widespread population occurrence of rare events predicted to be damaging. Eventually, samples were stratified into blood- and lymphoblastoid-derived to detect possibly different trends of mutability in the two datasets, an analysis which did not yield significant discordances.ConclusionsTo the best of our knowledge, this is likely the most extended population-scale mitochondrial genotyping in humans enriched with the estimation of heteroplasmies.

The reference human nuclear mitochondrial sequences compilation validated and implemented on the UCSC genome browser

BackgroundEukaryotic nuclear genomes contain fragments of mitochondrial DNA called NumtS (Nuclear mitochondrial Sequences), whose mode and time of insertion, as well as their functional/structural role within the genome are debated issues. Insertion sites match with chromosomal breaks, revealing that micro-deletions usually occurring at non-homologous end joining loci become reduced in presence of NumtS. Some NumtS are involved in recombination events leading to fragment duplication. Moreover, NumtS are polymorphic, a feature that renders them candidates as population markers. Finally, they are a cause of contamination during human mtDNA sequencing, leading to the generation of false heteroplasmies.ResultsHere we present RHNumtS.2, the most exhaustive human NumtSome catalogue annotating 585 NumtS, 97% of which were here validated in a European individual and in HapMap samples. The NumtS complete dataset and related features have been made available at the UCSC Genome Browser. The produced sequences have been submitted to INSDC databases. The implementation of the RHNumtS.2 tracks within the UCSC Genome Browser has been carried out with the aim to facilitate browsing of the NumtS tracks to be exploited in a wide range of research applications.ConclusionsWe aimed at providing the scientific community with the most exhaustive overview on the human NumtSome, a resource whose aim is to support several research applications, such as studies concerning human structural variation, diversity, and disease, as well as the detection of false heteroplasmic mtDNA variants. Upon implementation of the NumtS tracks, the application of the BLAT program on the UCSC Genome Browser has now become an additional tool to check for heteroplasmic artefacts, supported by data available through the NumtS tracks.

Somatic complex I disruptive mitochondrial DNA mutations are modifiers of tumorigenesis that correlate with low genomic instability in pituitary adenomas

Mitochondrial DNA (mtDNA) mutations leading to the disruption of respiratory complex I (CI) have been shown to exhibit anti-tumorigenic effects, at variance with those impairing only the function but not the assembly of the complex, which appear to contribute positively to cancer development. Owing to the challenges in the analysis of the multi-copy mitochondrial genome, it is yet to be determined whether tumour-associated mtDNA lesions occur as somatic modifying factors or as germ-line predisposing elements. Here we investigated the whole mitochondrial genome sequence of 20 pituitary adenomas with oncocytic phenotype and identified pathogenic and/or novel mtDNA mutations in 60% of the cases. Using highly sensitive techniques, namely fluorescent PCR and allele-specific locked nucleic acid quantitative PCR, we identified the most likely somatic nature of these mutations in our sample set, since none of the mutations was detected in the corresponding blood tissue of the patients analysed. Furthermore, we have subjected a series of 48 pituitary adenomas to a high-resolution array comparative genomic hybridization analysis, which revealed that CI disruptive mutations, and the oncocytic phenotype, significantly correlate with low number of chromosomal aberrations in the nuclear genome. We conclude that CI disruptive mutations in pituitary adenomas are somatic modifiers of tumorigenesis most likely contributing not only to the development of oncocytic change, but also to a less aggressive tumour phenotype, as indicated by a stable karyotype.

Primates and mouse NumtS in the UCSC Genome Browser

BackgroundNumtS (N uclear M iT ochondrial S equences) are mitochondrial DNA sequences that, after stress events involving the mitochondrion, colonized the nuclear genome. Accurate mapping of NumtS avoids contamination during mtDNA PCR amplification, thus supplying reliable bases for detecting false heteroplasmies. In addition, since they commonly populate mammalian genomes (especially primates) and are polymorphic, in terms of presence/absence and content of SNPs, they may be used as evolutionary markers in intra- and inter-species population analyses.ResultsThe need for an exhaustive NumtS annotation led us to produce the Reference Human NumtS compilation, followed, as reported in this paper, by those for chimpanzee, rhesus macaque and mouse ones. Identification of NumtS inside the UCSC Genome Browser and their inter-species comparison required the design and the implementation of NumtS tracks, starting from the compilation data. NumtS retrieval through the UCSC Genome Browser, in the species examined, is now feasible at a glance.ConclusionsAnalyses involving NumtS tracks, together with other genome element tracks publicly available at the UCSC Genome Browser, can provide deep insight into genome evolution and comparative genomics, thus improving studies dealing with the mechanisms that drove the generation of NumtS. In addition, the NumtS tracks constitute a useful tool in the design of mitochondrial DNA primers.

Polymorphic NumtS trace human population relationships

The human genome is constantly subjected to evolutionary forces which shape its architecture. Insertions of mitochondrial DNA sequences into nuclear genome (NumtS) have been described in several eukaryotic species, including Homo sapiens and other primates. The ongoing process of the generation of NumtS has made them valuable markers in primate phylogenetic studies, as well as potentially informative loci for reconstructing the genetic history of modern humans. Here, we report the identification of 53 human-specific NumtS by inspection of the UCSC genome browser, showing that they may be direct insertions of mitochondrial DNA into the human nuclear DNA after the human-chimpanzee split. In silico analyses allowed us to identify 14 NumtS which are polymorphic in terms of their presence/absence within the human genome in individuals of different ancestry. The allele frequencies of these polymorphic NumtS were calculated for 1000 Genomes Project sequence data from 13 populations worldwide, and principal components analysis and hierarchical clustering methods allowed the detection of strong signals of geographical structure related to the genetic diversity of these loci. All identified polymorphic human-specific NumtS together with a tandemly duplicated NumtS have also been validated by PCR amplification on a panel of 60 samples belonging to five native populations worldwide, confirming the expected NumtS variability. On the basis of these findings, we have succeeded in depicting the landscape of variation of a series of NumtS in several ethnic groups, making an advance in their identification as useful markers in the study on human population genetics.

Genome Sequence of the Polychlorinated-Biphenyl Degrader Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 is a soil polychlorinated biphenyl (PCB) degrader, able to grow both planktonically and as a biofilm in the presence of various toxic metals and metalloids. Here we report the genome sequence (5,957,359 bp) of P. pseudoalcaligenes KF707, which provides insights into metabolic degradation pathways, flagellar motility, and chemotaxis.

Exome and deep sequencing of clinically aggressive neuroblastoma reveal somatic mutations that affect key pathways involved in cancer progression

The spectrum of somatic mutation of the most aggressive forms of neuroblastoma is not completely determined. We sought to identify potential cancer drivers in clinically aggressive neuroblastoma. Whole exome sequencing was conducted on 17 germline and tumor DNA samples from high-risk patients with adverse events within 36 months from diagnosis (HR-Event3) to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in additional 48 germline and tumor pairs (62.5% HR-Event3 and high-risk patients), 17 HR-Event3 tumors and 17 human-derived neuroblastoma cell lines. We revealed 22 significantly mutated genes, many of which implicated in cancer progression. Fifteen genes (68.2%) were highly expressed in neuroblastoma supporting their involvement in the disease. CHD9, a cancer driver gene, was the most significantly altered (4.0% of cases) after ALK. Other genes (PTK2, NAV3, NAV1, FZD1 and ATRX), expressed in neuroblastoma and involved in cell invasion and migration were mutated at frequency ranged from 4% to 2%. Focal adhesion and regulation of actin cytoskeleton pathways, were frequently disrupted (14.1% of cases) thus suggesting potential novel therapeutic strategies to prevent disease progression. Notably BARD1, CHEK2 and AXIN2 were enriched in rare, potentially pathogenic, germline variants. In summary, whole exome and deep targeted sequencing identified novel cancer genes of clinically aggressive neuroblastoma. Our analyses show pathway-level implications of infrequently mutated genes in leading neuroblastoma progression.

Combinations of genetic data in a study of neuroblastoma risk genotypes.

Analysis of combinations of genetic changes that occur exclusively in patients may be a supplementary strategy to the single-locus strategy used in many genetic studies. The genotypes of 16 SNPs within susceptibility loci for neuroblastoma (NB) were analyzed in a previous study. In the present study, combinations of these genotypes have been analyzed. The theoretical number of combinations of 3 SNP genotypes taken from 16 SNPs is 15,120. Of these, 14,307 were found in 370 patients and 803 controls; 12,772 combinations were common to both patients and controls; 1,213 were found in controls only; and 322 combinations were found in patients only. Among the latter, a cluster of 24 combinations was found to be significantly associated with NB (P < 0.00001).

Biphenyl Modulates the Expression and Function of Respiratory Oxidases in the Polychlorinated-Biphenyls Degrader Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs) using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(c)aa3 type oxidases (Caa3 and Ccaa3), two cbb3 type oxidases (Cbb31 and Cbb32), and one bd type cyanide-insensitive quinol oxidase (CIO). While the activity and expression of both Cbb31 and Cbb32 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb32 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb31 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb32 and C(c)aa3 deletion mutants. These results, together, reveal that both function and expression of cbb3 and caa3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.