MAGI-ACMG: Algorithm for the Classification of Variants According to ACMG and ACGS Recommendations.

We have developed MAGI-ACMG, a classification algorithm that allows the classification of sequencing variants (single nucleotide or small indels) according to the recommendations of the American College of Medical Genetics (ACMG) and the Association for Clinical Genomic Science (ACGS). The MAGI-ACMG classification algorithm uses information retrieved through the VarSome Application Programming Interface (API), integrates the AutoPVS1 tool in order to evaluate more precisely the attribution of the PVS1 criterion, and performs the customized assignment of specific criteria. In addition, we propose a sub-classification scheme for variants of uncertain significance (VUS) according to their proximity either towards the "likely pathogenic" or "likely benign" classes. We also conceived a pathogenicity potential criterion (P_POT) as a proxy for segregation criteria that might be added to a VUS after posterior testing, thus allowing it to upgrade its clinical significance in a diagnostic reporting setting. Finally, we have developed a user-friendly web application based on the MAGI-ACMG algorithm, available to geneticists for variant interpretation.

Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing.

BACKGROUND: The filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated. METHODS AND RESULTS: For the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification. CONCLUSIONS: Taken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients' disorders.

Next-Generation Sequencing of a Large Gene Panel for Outcome Prediction of Bariatric Surgery in Patients with Severe Obesity.

Obesity is a chronic disease in which abnormal deposition of fat threatens health, leading to diabetes, cardiovascular diseases, cancer, and other chronic illnesses. According to the WHO, 19.8% of the adult population in Italy is obese, and the prevalence is higher among men. It is important to know the predisposition of an individual to become obese and to respond to bariatric surgery, the most up-to-date treatment for severe obesity. To this purpose, we developed an NGS gene panel, comprising 72 diagnostic genes and 244 candidate genes, and we sequenced 247 adult obese Italian patients. Eleven deleterious variants in 9 diagnostic genes and 17 deleterious variants in 11 candidate genes were identified. Interestingly, mutations were found in several genes correlated to the Bardet-Biedl syndrome. Then, 25 patients were clinically followed to evaluate their response to bariatric surgery. After a 12-month follow-up, the patients that carried deleterious variants in diagnostic or candidate genes had a reduced weight loss, as compared to the other patients. The NGS-based panel, including diagnostic and candidate genes used in this study, could play a role in evaluating, diagnosing, and managing obese individuals, and may help in predicting the outcome of bariatric surgery.

PacMAGI: A pipeline including accurate indel detection for the analysis of PacBio sequencing data applied to RPE65.

Third generation sequencing methods, like PacBio, provide information about structural variants, introns, enhancers and promoters. We developed an automated pipeline, called PacMAGI, including quality control, alignment, SNV, INDELs, structural variant calling, phasing, annotation and variant interpretation, for the analysis of PacBio data for any target region. Bi-allelic mutations in the RPE65 gene are associated with different inherited retinal dystrophies, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Diagnostic panel-based NGS analysis is performed on coding regions and intron/exon junctions of genes. To obtain a more conclusive diagnosis, we applied PacMAGI to obtain a second hit on RPE65 in LCA or RP patients who showed a single heterozygous variant by NGS. We used PacBio to sequence the full gene and identify putative second-hits in intronic, problematic and promoter regions. All variants identified in the diagnostic setting with NGS were correctly detected by the pipeline, and thanks to our custom algorithm for INDELs, a previously undetected 'Pathogenic' frameshift variant was found in a RP patient already identified to carry a 'Likely Pathogenic' variant.

Variant Selection and Interpretation: An Example of Modified VarSome Classifier of ACMG Guidelines in the Diagnostic Setting.

Variant interpretation is challenging as it involves combining different levels of evidence in order to evaluate the role of a specific variant in the context of a patient's disease. Many in-depth refinements followed the original 2015 American College of Medical Genetics (ACMG) guidelines to overcome subjective interpretation of criteria and classification inconsistencies. Here, we developed an ACMG-based classifier that retrieves information for variant interpretation from the VarSome Stable-API environment and allows molecular geneticists involved in clinical reporting to introduce the necessary changes to criterion strength and to add or exclude criteria assigned automatically, ultimately leading to the final variant classification. We also developed a modified ACMG checklist to assist molecular geneticists in adjusting criterion strength and in adding literature-retrieved or patient-specific information, when available. The proposed classifier is an example of integration of automation and human expertise in variant curation, while maintaining the laboratory analytical workflow and the established bioinformatics pipeline.

Genetics of pain: From rare Mendelian disorders to genetic predisposition to pain.

BACKGROUND AND AIM OF THE WORK: Pain is defined by the International Association for the Study of Pain as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage". In this mini-review, we focused on the Mendelian disorders with chronic pain as the main characteristic or where pain perception is disrupted, and on the polymorphisms that can impart susceptibility to chronic pain. METHODS: We searched PubMed and Online Mendelian Inheritance in Man (OMIM) databases and selected only syndromes in which pain or insensitivity to pain were among the main characteristics. Polymorphisms were selected from the database GWAS catalog (https://www.ebi.ac.uk/gwas/home). RESULTS: We retrieved a total of 28 genes associated with Mendelian inheritance in which pain or insensitivity to pain were the main characteristics and 70 polymorphisms associated with modulation of pain perception. CONCLUSIONS: This mini-review highlights the importance of genetics in phenotypes characterized by chronic pain or pain insensitivity. We think that an effective genetic test should analyze all genes associated with Mendelian pain disorders and all SNPs that can increase the risk of pain.

Comparison between American and European legislation in the therapeutical and alimentary bacteriophage usage.

Bacteriophages, though discovered a century ago, still lag behind in the race of antimicrobials due to scarce information about their biology, pharmacology, safety and suitability as therapeutic agents. Although they possess several capabilities of practical utility in medicine, they are still unable to satisfy the regulatory standards set by the regulatory authorities in both United States (US) and European Union (EU). Bacteriophages and their products (lysins) are considered as drugs, therefore they should follow the same route of the chemical drugs in order to achieve regulatory approvals for commercial production and application. However, lack of definitive guidelines and regulations has rendered bacteriophages less attractive to pharmaceutical companies and funding agencies, making it difficult for clinicians and researchers to set up wide scale clinical trials in order to prove efficacy, safety and stability of bacteriophages and their products. In this review, we will discuss the current regulations for developing phages and phage-based products for therapeutic purposes in the US and EU.

Monogenic hyperlipidemias.

Monogenic hyperlipidemias are a group of inherited disorders characterized by elevated plasma concentrations of lipids and lipoproteins. High plasma concentrations of lipids are the most frequent risk factor for cardiovascular disease. Monogenic hyperlipidemias are a minor cause with respect to multifactorial hyperlipidemias. Diagnosis is based on clinical findings and lipid panel measurements. Genetic testing is useful for confirming diagnosis and for differential diagnosis, recurrence risk calculation and prenatal diagnosis in families with a known mutation. Monogenic hyperlipidemias can have either autosomal dominant or recessive inheritance.

Hypothyroidism and hyperthyroidism.

Congenital hypothyroidism is a condition in which the thyroid gland does not produce enough thyroid hormones. It occurs in 1:2000-4000 newborns. Common clinical features include decreased activity and increased sleep, feeding difficulty, constipation, prolonged jaundice, myxedematous facies, large fontanels (especially posterior), macroglossia, distended abdomen with umbilical hernia, and hypotonia. Slow linear growth and developmental delay are usually apparent by 4-6 months of age. Without treatment, congenital hypothyroidism leads to severe intellectual deficit and short stature. Congenital hyperthyroidism occurs when the thyroid gland produces too much of the hormone thyroxine, which can accelerate body metabolism, causing unintentional weight loss and a rapid or irregular heartbeat. Hyperthyroidism is very rare and its prevalence is unknown. Common clinical features include unintentional weight loss, tachycardia, arrhythmia, palpitations, anxiety, tremor and sweating. Here we summarize the genes involved in congenital hypo- and hyperthyroidism and the tests we use for genetic analysis.

Multimodal Imaging in Autosomal Dominant Cone-Rod Dystrophy Caused by Novel CRX Variant.

AIM: To characterize by multimodal approach the phenotype of patients from a 3 generations pedigree, affected by autosomal dominant cone-rod dystrophy (CRD), found to carry a novel pathogenic variant in the cone-rod homeobox-containing (CRX) gene. METHODS: Examination of the adult patients included the following tests: visual acuity, multicolour imaging, spectral domain optical coherence tomography (SD-OCT), fundus autofluorescence (FAF) and OCT angiography (OCT-A) recordings. In a 2.5-year-old child, cycloplegic refraction, fundoscopy, ocular motility evaluation and electrophysiological exams were performed. Next Generation Sequencing of patients' DNA has been carried out. RESULTS: A novel CRX pathogenic variant has been identified in our patients. The 2.5-year-old child in the third generation was found to have inherited the variant, with no clinical signs of the condition, but electroretinographic abnormalities in the scotopic component. In the adult patients, diffuse atrophy of the retinal pigment epithelium/photoreceptor complex in the macular region was evident at the OCT and FAF, while OCT-A showed choriocapillaris density reduction. CONCLUSIONS: Multimodal study allowed the characterization of a peculiar form of CRD. The novel pathogenic variant seems to have a different effect on the phenotype if compared with a previously described similar one, giving an insight into the pathogenic mechanism of CRX-related retinal dystrophies and offering valuable information that could lead to the development of possible future therapies.

Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion- and epilepsy-associated brain tissue.

The chloride channel 2 (CLCN2) gene codes for a protein organized in N- and C-terminal regions with regulatory functions and a transmembrane region which forms the ring of the pore. Mutations in the gene have previously been described in patients with idiopathic familial epilepsy. In this study we looked for new isoforms of CLCN2 and we estimated expression levels by real time PCR in brain tissue containing epileptic foci. Samples used in this study were first analyzed and selected to exclude mutations in the coding region of the gene. Four isoforms (skipping exons 3, 16, 22 and 6/7) were identified and quantified by Real Time PCR and compared with total expression of the gene. Expression of the region common to all CLCN2 isoforms was 50% less in epilepsy-associated brain tissue than in controls. The ratio of the various isoforms was slightly greater in epileptic than control tissue. The greatest difference was recorded in the temporal lobe for the isoform with skipped exon 22. Analysis of these isoforms in brain tissue containing epileptic foci suggests that CLCN2 could be implicated in epilepsy, even in the absence of mutations.

Study of the adenosinergic system in the brain of HPRT knockout mouse (Lesch-Nyhan disease).

BACKGROUND: Lesch-Nyhan disease (LND), an X-linked genetic disease caused by complete deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), is characterized by hyperuricemia and psychiatric disturbance, mainly self-aggressiveness. Literature dates support the hypothesis that dopaminergic deficit and serotonergic excess in the circuit of basal ganglia are responsible for the aggressive behavior. Altered adenosine transport across the membrane of HPRT-deficient lymphocytes has been reported, suggesting adenosine involvement in LND. METHODS: The expression of several genes related to the adenosinergic system (ADORA1A, ADORA2A, ADORA2B) were studied in the brain of the murine model of LND by real time PCR. Nucleotide levels and enzyme activities possibly involved in adenosine release were also measured. RESULTS: Studies performed by real time PCR showed 95% increase in ADORA1A expression, 15% decrease in ADORA2A expression, and no change in ADORA2B expression in knockout mice compared to controls. No significant differences were found in the level of nucleotides or enzyme activities between control and mutant mice. CONCLUSIONS: Our results suggest that adenosine neurotransmission might be involved in the specific neurobehavioral features of LND by increased expression of adenosine A1 receptors.