ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections.

Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.

Exosomes from Plasma of Neuroblastoma Patients Contain Doublestranded DNA Reflecting the Mutational Status of Parental Tumor Cells.

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available.

Evidence for expression and functionality of FSH and LH/hCG receptors in human endometrium.

PURPOSE: Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mediate intracellular functions by binding their specific protein G-coupled gonadotrophin receptor, respectively FSH receptor (FSHR) and LH/choriogonadotrophin receptor (LHCGR). Whereas the expression of FSHR and LHCGR in mammals was considered gonad-specific and cell-specific, studies identified gonadotrophin receptors in human female extragonadal reproductive tissues. This study aims to demonstrate that gonadotrophin receptors are expressed in endometrium and mediates intracellular functions. METHODS: Collected endometria (n = 12) from healthy patients (mean age of 36 ± 6) were primary cultured for 24 h. The presence of gonadotrophin receptors was evaluated by RT-PCR followed by the sequencing of the resulted amplicons and by immunohistochemistry in original samples. Endometrial primary cultures were treated with increasing concentration (range 0-100 ng/ml) of either recombinant human LH (rhLH) or recombinant human FSH (rhFSH). Endometria controls had gonadotrophin replaced by the same volume of the culture medium. In gonadotrophin-treated samples, it was evaluated the intracellular cyclic adenosine monophosphate (cAMP) content by enzymatic immunoassay and the expression of steroidogenic genes by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). RESULTS: The sequencing of the RT-PCR amplicons confirmed the presence of both gonadotrophin receptors and immunohistochemistry localized them on the membrane of endometrial glands cells throughout the glandular epithelium. The gonadotrophin-receptor complex was able to increase the intracellular cAMP in a dose-response and time-course manner and to induce steroidogenic genes expression. CONCLUSION: This study demonstrates that both gonadotrophin receptors are expressed along the glandular epithelium of endometria and they mediate the effects of gonadotrophins on intracellular functions.

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing.

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ~50 kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif, and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype-phenotype correlations for the better stratification of DM2 patients in clinical trials.

Whole-exome sequencing of the mummified remains of Cangrande della Scala (1291-1329 CE) indicates the first known case of late-onset Pompe disease.

Mummified remains of relevant historical figures are nowadays an important source of information to retrace data concerning their private life and health, especially when historical archives are not available. Next-generation-sequencing was proved to be a valuable tool to unravel the characteristics of these individuals through their genetic heritage. Using the strictest criteria currently available for the validation of ancient DNA sequences, whole-genome and whole-exome sequencing were generated from the mummy remains of an Italian nobleman died almost 700 years ago, Cangrande della Scala. While its genome sequencing could not yield sufficient coverage for in depth investigation, exome sequencing could overcome the limitations of this approach to achieve significantly high coverage on coding regions, thus allowing to perform the first extensive exome analysis of a mummy genome. Similar to a standard "clinical exome analysis" conducted on modern DNA, an in-depth variant annotation, high-quality filtering and interpretation was performed, leading to the identification of a genotype associated with late-onset Pompe disease (glycogen storage disease type II). This genetic diagnosis was concordant with the limited clinical history available for Cangrande della Scala, who likely represents the earliest known case of this autosomal recessive metabolic disorder.