Tommaso Biagini

High-confidence assessment of functional impact of human mitochondrial non-synonymous genome variations by APOGEE

24,189 are all the possible non-synonymous amino acid changes potentially affecting the human mitochondrial DNA. Only a tiny subset was functionally evaluated with certainty so far, while the pathogenicity of the vast majority was only assessed in-silico by software predictors. Since these tools proved to be rather incongruent, we have designed and implemented APOGEE, a machine-learning algorithm that outperforms all existing prediction methods in estimating the harmfulness of mitochondrial non-synonymous genome variations. We provide a detailed description of the underlying algorithm, of the selected and manually curated training and test sets of variants, as well as of its classification ability.

Near full length hepatitis C virus genome reconstruction by next generation sequencing based on genotype-independent amplification

BACKGROUND Deep sequencing has a deep impact on the study of rapidly mutating RNA viruses, such as hepatitis C virus, proving to be an invaluable tool for analyzing virus diversity and evolution. AIM Genotype-independent high-throughput pyrosequencing was used to obtain near full length hepatitis C virus genome sequence reconstruction directly from clinical samples. METHODS Samples from hepatitis C virus infected subjects harbouring different subtypes (1a, 1b, 2c) were analyzed (viral load range: 1.2-20.8 × 10(6)IU/ml). Data were generated with a modified sequence-independent single primer amplification method followed by 454 sequencing. RESULTS the extent of reconstructed hepatitis C virus genome varied from 79.95% to 99.64%. No correlation between extent of genome reconstruction and either viral load (r=0.4857, p=0.3556) or number of HCV reads (r=0.08571, p=0.9194) was observed. CONCLUSION This study describes a protocol for obtaining whole genome sequences from different hepatitis C virus patients with different genotypes in a single sequencing run.

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50)

Abstract Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2n = 50) and the swamp (2n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1.

Between SCA5 and SCAR14: delineation of the SPTBN2 p.R480W-associated phenotype

We read with interest the article by Elsayed et al. [1] describing a family with autosomal recessive congenital ataxia due to a homozygous 5-bp deletion in the β3-spectrin gene (SPTBN2). In-frame heterozygous variants of this gene had previously been identified as the cause of autosomal dominant adult-onset SCA5 (MIM#600224) (Suppl.Tab.1) [2–4]. The authors postulated the existence of SPTBN2 genotype-phenotype correlates, suggesting that loss of function mutations would act recessively, producing a severe congenital ataxic phenotype associated with cognitive impairment and variable additional neurological signs. This hypothesis was partially supported by three subsequent studies, reporting homozygous missense, nonsense and splicing SPTBN2 variants, all resulting in an analogous congenital ataxia syndrome, defined as SCAR14 (MIM#615386) (Suppl.Tab.1) [5–7]. Elsayed and colleagues [1] also mentioned a previously reported patient affected by a severe infantile-onset cerebellar ataxia with developmental delay, carrying the heterozygous missense variant c.1438C>T (p.R480W) in the SPTBN2 gene [8]. Based on previous literature data on heterozygous SPTBN2 mutation carriers (presenting the typical SCA5 phenotype of adult-onset, slowly progressive pure cerebellar ataxia), they speculated that either a second-site SCA5 modifier or an undetected SPTBN2 variant in trans (e.g., deep intronic or in a non-coding regulatory region) should contribute to the phenotypic manifestation. In this regard, we would like to report an additional case of congenital severe cerebellar ataxia and intellectual impairment carrying the same SPTBN2 p.R480W variant. This is a 2-year-old girl, the third child of healthy nonconsanguineous parents. She was born by cesarean section after an uncomplicated full-term pregnancy and was referred to the neuropsychiatric clinic because of generalized hypotonia, global developmental delay, and alternating esotropia. At age 12 months, she said her first words and got head control, but could not sit without support. Two months later, her developmental quotient was calculated to be 56. She progressively developed a cerebellar syndrome with gait ataxia and dysarthria. Brain MRI performed at age 1 year 10 months showed global cerebellar hypoplasia with enlarged interfolial spaces, in the absence of any brainstem or supratentorial abnormalities (Suppl.Fig.1A). Molecular analysis consisted of a next-generation sequencing (NGS) panel of 50 genes causative of different forms of nonprogressive cerebellar ataxia (with the exclusion of Joubert Syndrome genes), using TruSeq Custom Amplicon (TSCA) technology on a MiSeq platform (Illumina, San Diego, CA, USA). As in the case reported by Jacob et al. [8], this child was heterozygous for SPTBN2 p.R480W missense variant (NM_006946). This variant was not detected in either of the unaffected parents, suggesting de novo occurrence (Suppl. Fig.1B). Potential heterozygous deletions involving one or more exons were ruled out using a custom script tool aimed at detecting significant differences in read depth from NGS data. The p.R480W variant (rs397514749) is not present in population databases, affects a highly conserved amino acid, and multiple in silico tools consistently predict it as Sara Nuovo and Alessia Micalizzi contributed equally to this work.

Insights From Molecular Characterization of Adult Patients of Families With Multigenerational Diabetes

Multigenerational diabetes of adulthood is a mostly overlooked entity, simplistically lumped into the large pool of type 2 diabetes. The general aim of our research in the past few years is to unravel the genetic causes of this form of diabetes. Identifying among families with multigenerational diabetes those who carry mutations in known monogenic diabetes genes is the first step to then allow us to concentrate on remaining pedigrees in which to unravel new diabetes genes. Targeted next-generation sequencing of 27 monogenic diabetes genes was carried out in 55 family probands and identified mutations verified among their relatives by Sanger sequencing. Nine variants (in eight probands) survived our filtering/prioritization strategy. After likelihood of causality assessment by established guidelines, six variants were classified as “pathogenetic/likely pathogenetic” and two as “of uncertain significance.” Combining present results with our previous data on the six genes causing the most common forms of maturity-onset diabetes of the young allows us to infer that 23.6% of families with multigenerational diabetes of adulthood carry mutations in known monogenic diabetes genes. Our findings indicate that the genetic background of hyperglycemia is unrecognized in the vast majority of families with multigenerational diabetes of adulthood. These families now become the object of further research aimed at unraveling new diabetes genes.

Multifaceted enrichment analysis of RNA–RNA crosstalk reveals cooperating micro-societies in human colorectal cancer

Alterations in the balance of mRNA and microRNA (miRNA) expression profiles contribute to the onset and development of colorectal cancer. The regulatory functions of individual miRNA-gene pairs are widely acknowledged, but group effects are largely unexplored. We performed an integrative analysis of mRNA–miRNA and miRNA–miRNA interactions using high-throughput mRNA and miRNA expression profiles obtained from matched specimens of human colorectal cancer tissue and adjacent non-tumorous mucosa. This investigation resulted in a hypernetwork-based model, whose functional backbone was fulfilled by tight micro-societies of miRNAs. These proved to modulate several genes that are known to control a set of significantly enriched cancer-enhancer and cancer-protection biological processes, and that an array of upstream regulatory analyses demonstrated to be dependent on miR-145, a cell cycle and MAPK signaling cascade master regulator. In conclusion, we reveal miRNA-gene clusters and gene families with close functional relationships and highlight the role of miR-145 as potent upstream regulator of a complex RNA–RNA crosstalk, which mechanistically modulates several signaling pathways and regulatory circuits that when deranged are relevant to the changes occurring in colorectal carcinogenesis.

Very mild features of dysequilibrium syndrome associated with a novel VLDLR missense mutation

Dysequilibrium syndrome (DES) is a non-progressive congenital ataxia characterized by severe intellectual deficit, truncal ataxia and markedly delayed, quadrupedal or absent ambulation. Recessive loss-of-function mutations in the very low density lipoprotein receptor (VLDLR) gene represent the most common cause of DES. Only two families have been reported harbouring homozygous missense mutations, both with a similarly severe phenotype. We report an Italian girl with very mild DES caused by the novel homozygous VLDLR missense mutation p.(C419Y). This unusually benign phenotype possibly relates to a less disruptive effect of the mutation, falling within a domain (EGF-B) not predicted as crucial for the protein function.

A single-center study on 140 patients with cerebral cavernous malformations: 28 new pathogenic variants and functional characterization of a PDCD10 large deletion

Cerebral cavernous malformation (CCM) is a capillary malformation arising in the central nervous system. CCM may occur sporadically or cluster in families with autosomal dominant transmission, incomplete penetrance, and variable expressivity. Three genes are associated with CCM KRIT1, CCM2, and PDCD10. This work is a retrospective single‐center molecular study on samples from multiple Italian clinical providers. From a pool of 317 CCM index patients, we found germline variants in either of the three genes in 80 (25.2%) probands, for a total of 55 different variants. In available families, extended molecular analysis found segregation in 60 additional subjects, for a total of 140 mutated individuals. From the 55 variants, 39 occurred in KRIT1 (20 novel), 8 in CCM2 (4 novel), and 8 in PDCD10 (4 novel). Effects of the three novel KRIT1 missense variants were characterized in silico. We also investigated a novel PDCD10 deletion spanning exon 4–10, on patients fibroblasts, which showed significant reduction of interactions between KRIT1 and CCM2 encoded proteins and impaired autophagy process. This is the largest study in Italian CCM patients and expands the known mutational spectrum of KRIT1, CCM2, and PDCD10. Our approach highlights the relevance of seeking supporting information to pathogenicity of new variants for the improvement of management of CCM.

Systematic Analysis of Mouse Genome Reveals Distinct Evolutionary and Functional Properties Among Circadian and Ultradian Genes

In living organisms, biological clocks regulate 24 h (circadian) molecular, physiological, and behavioral rhythms to maintain homeostasis and synchrony with predictable environmental changes, in particular with those induced by Earth’s rotation on its axis. Harmonics of these circadian rhythms having periods of 8 and 12 h (ultradian) have been documented in several species. In mouse liver, harmonics of the 24-h period of gene transcription hallmarked genes oscillating with a frequency two or three times faster than circadian periodicity. Many of these harmonic transcripts enriched pathways regulating responses to environmental stress and coinciding preferentially with subjective dawn and dusk. At this time, the evolutionary history of genes with rhythmic expression is still poorly known and the role of length-of-day changes due to Earth’s rotation speed decrease over the last four billion years is totally ignored. We hypothesized that ultradian and stress anticipatory genes would be more evolutionarily conserved than circadian genes and background non-oscillating genes. To investigate this issue, we performed broad computational analyses of genes/proteins oscillating at different frequency ranges across several species and showed that ultradian genes/proteins, especially those oscillating with a 12-h periodicity, are more likely to be of ancient origin and essential in mice. In summary, our results show that genes with ultradian transcriptional patterns are more likely to be phylogenetically conserved and associated with the primeval and inevitable dawn/dusk transitions.

Molecular dynamics recipes for genome research

&NA; Molecular dynamics (MD) simulation allows one to predict the time evolution of a system of interacting particles. It is widely used in physics, chemistry and biology to address specific questions about the structural properties and dynamical mechanisms of model systems. MD earned a great success in genome research, as it proved to be beneficial in sorting pathogenic from neutral genomic mutations. Considering their computational requirements, simulations are commonly performed on HPC computing devices, which are generally expensive and hard to administer. However, variables like the software tool used for modeling and simulation or the size of the molecule under investigation might make one hardware type or configuration more advantageous than another or even make the commodity hardware definitely suitable for MD studies. This work aims to shed lights on this aspect.