A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings.

The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the APOE locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing.

HIV-1 TAT-mediated protein transduction of human HPRT into deficient cells.

Lesch-Nyhan disease (LND) is a severe and incurable X-linked genetic syndrome caused by the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT), resulting in severe alterations of central nervous system, hyperuricemia and subsequent impaired renal functions. Therapeutic options consist in supportive care and treatments of complications, but the disease remains largely untreatable. Enzyme replacement of the malfunctioning cytosolic protein might represent a possible therapeutic approach for the LND treatment. Protein transduction domains, such as the TAT peptide derived from HIV TAT protein, have been used to transduce macromolecules into cells in vitro and in vivo. The present study was aimed to the generation of TAT peptide fused to human HPRT for cell transduction in enzyme deficient cells. Here we document the construction, expression and delivery of a functional HPRT enzyme into deficient cells by TAT transduction domain and by liposome mediated protein transfer. With this approach we demonstrate the correction of the enzymatic defect in HPRT deficient cells. Our data show for the first time the feasibility of the enzyme replacement therapy for the treatment of LND.

Analysis of the HPRT1 gene in 35 Italian Lesch-Nyhan families: 45 patients and 77 potential female carriers.

BACKGROUND: Lesch-Nyhan (LND) disease is an inborn error of purine metabolism which results from deficiency of the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT). In the classical form of the disease the activity of the enzyme is completely deficient and the patient has cognitive impairment, spasticity, dystonia and self-injurious behaviour, as well as elevated concentrations of uric acid in blood and urine that leads to consequences such as nephropathy, urinary tract calculi and tophaceous gout. There are disease variants without self-injurious behaviour. In these cases neurological manifestations may vary widely. The HPRT1 gene is located on the X chromosome in position Xq26-27.2, and mutations have been found in quite a large number of patients. OBJECTIVE: Documenting our experience with the diagnosis of LND in 45 Italian patients from 35 nonrelated families and 77 females at risk of being carriers of the condition. DESIGN: Internal review. SETTING: An institute devoted to the investigation and care of patients with rare diseases. RESULTS: In 94% of the LND families gDNA sequencing of the patients was informative while in 6% a cDNA study was required. For the carrier females gDNA sequencing was informative in 71% of the families, 23% required qPCR studies and 6% required segregation studies combined with enzymatic activity testing. Classical cDNA studies proved to be unreliable in carrier females as there is a significant risk of failure to detect the mutated allele. Four novel HPRT1 mutations were found: c.145C>T (p.Leu49Phe), c.112C>T (p.Pro38Ser), c.89_96dup8 (p.Glu33Argfs) and c.506dupC (p.Arg170Thrfs). CONCLUSION: In the diagnosis of LND it is very important to consider all the possible alterations of the HPRT1 gene when searching for mutations especially if no affected male is available. Biochemical assessment of the enzymatic activity of HPRT in an affected male is the ideal starting point for molecular analysis of the gene.

Shedding light on dark genes: enhanced targeted resequencing by optimizing the combination of enrichment technology and DNA fragment length.

The exome contains many obscure regions difficult to explore with current short-read sequencing methods. Repetitious genomic regions prevent the unique alignment of reads, which is essential for the identification of clinically-relevant genetic variants. Long-read technologies attempt to resolve multiple-mapping regions, but they still produce many sequencing errors. Thus, a new approach is required to enlighten the obscure regions of the genome and rescue variants that would be otherwise neglected. This work aims to improve the alignment of multiple-mapping reads through the extension of the standard DNA fragment size. As Illumina can sequence fragments up to 550 bp, we tested different DNA fragment lengths using four major commercial WES platforms and found that longer DNA fragments achieved a higher genotypability. This metric, which indicates base calling calculated by combining depth of coverage with the confidence of read alignment, increased from hundreds to thousands of genes, including several associated with clinical phenotypes. While depth of coverage has been considered crucial for the assessment of WES performance, we demonstrated that genotypability has a greater impact in revealing obscure regions, with ~1% increase in variant calling in respect to shorter DNA fragments. Results confirmed that this approach enlightened many regions previously not explored.

Insulin-driven translational capacity is impaired in primary fibroblasts of Prader Willi.

Prader-Willi (PW) syndrome is a rare genetic disorder characterized by hypothalamic-pituitary abnormalities and severe hypotonia, hyperphagia, behavioural and psychiatric problems. Absence of satiety leads to severe obesity and frequently to diabetes. Furthermore, adult patients suffer from a severe loss of muscle mass, which severely impacts their quality of life. The mechanisms underlying alterations in muscle growth in PW remain to be clarified. In this study we explored the hypothesis that, in PW cells, alterations of protein synthesis are determined by dysfunctions in the promotion of cell growth. In order to study the molecular changes leading to dysfunction in protein translation, primary fibroblasts derived from four PW patients and five control subjects were used to study the insulin-mediated signaling pathway implicated in the control of protein synthesis by immunoblotting. Here we present, for the first time, evidences that the protein translation response to insulin is impaired in PW fibroblasts. Insulin alone has a major upregulatory effect on protein kinase B (AKT), glycogen synthase kinase (GSK3beta), while phosphorylation of p70S6K1 protein elongation factor controlled by mammalian target of rapamycin complex I (mTORC1) is reduced. In addition, we provide data that the response to insulin in PW cells can be restored by previous treatment with the amino acid L-Leucine (L-Leu). Our experiments in primary cell cultures demonstrate an impairment of insulin signaling that can be rescued by supplementation with the branched aminoacid L-Leu, indicating a possible therapeutic approach for alleviating muscle mass loss in PW patients.

Proteome changes induced by knock-down of the deubiquitylating enzyme HAUSP/USP7.

Modification of proteins by ubiquitin plays a major role in a broad array of biological processes. Reversal of this process through deubiquitylation likely represents an important regulatory step in the maintenance of cellular homeostasis. However, the biological functions of deubiquitylating enzymes still remain poorly characterized. To investigate the biological role of the herpes virus-associated ubiquitin-specific protease HAUSP/USP7, we have generated stably transfected cells carrying inducible shRNA expression plasmids. USP7 mRNA and protein were strongly down-regulated 48-72 h after shRNA induction. We used a selected clone to compare whole-cell proteomes by 2D-SDS-PAGE before and after knockdown of USP7. Alterations in 36 proteins were detected and their identities were revealed by mass spectrometry analysis. Components of the replication machinery, DNA/RNA binding proteins, enzymes involved in apoptosis and metabolism were found to be down-regulated upon USP7 removal, representing proteins that are either more rapidly turned over or synthesized less efficiently in the absence of USP7-mediated deubiquitylation. Alix/HP95, a protein implicated in endosomal organization and virus budding, was confirmed by immunoblotting to become down-regulated when USP7 levels were reduced. Our results extend the current list of USP7-dependent biological processes and suggest a role for this enzyme not only in transcriptional regulation but also in DNA replication, apoptosis, and possibly endosomal organization.