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Background: Hemoglobin A (Hb A) (α2β2) in the normal adult subject constitutes 96–98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the β-thalassemia carrier. Methods: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. Results: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1–2% during the screening tests in our laboratories. Conclusions: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

Background Due to the limited availability of mRNA analysis data, the number of exonic variants resulting in splicing impairment is underestimated although aberrant splicing correction is a promising therapeutic option to treat monogenic diseases, including choroideremia (CHM), a rare X‐linked eye disorder arising from sequence alteration of the CHM gene. Herein we report an exonic frameshift variant associated with an mRNA splicing alteration that leads to a CHM hypomorphic allele. Methods Total RNA and genomic DNA were extracted from peripheral blood of a patient affected by a mild form of CHM. The CHM gene was analyzed by PCR‐based methods and Sanger sequencing. Results Besides the known c.1335dup frameshift variant, mRNA analysis revealed a splicing alteration that restored the reading frame of the mutant transcript, likely leading to an aberrant protein with residual activity. Bioinformatic analyses identified novel putative exonic splicing enhancer elements and provided clues that also pre‐mRNA secondary structure should be taken into account when exploring splicing mechanisms. Conclusion A careful molecular characterization of the c.1335dup variant's effect explains the relationship between genotype and phenotype severity in a CHM patient and provides new perspectives for the study of therapeutic strategies based on splicing correction in human diseases., The number of exonic variants resulting in splicing impairment is underestimated because they are primary considered “changing‐code” mutations. We reveal a pathogenic CHM frameshift variant associated with an exon skipping that restores the gene reading frame. The mRNA alteration transforms a potential null allele into a hypomorphic disease‐allele that mitigates the phenotype severity.

Background A considerable minority of patients on waiting lists for kidney transplantation either have no diagnosis (and fall into the subset of undiagnosed cases) because kidney biopsy was not performed or histological findings were non-specific, or do not fall into any well-defined clinical category. Some of these patients might be affected by a previously unrecognised monogenic disease. Methods Through a multidisciplinary cooperative effort, we built an analytical pipeline to identify patients with chronic kidney disease (CKD) with a clinical suspicion of a monogenic condition or without a well-defined diagnosis. Following the stringent phenotypical and clinical characterization required by the flowchart, candidates meeting these criteria were further investigated by clinical exome sequencing followed by in silico analysis of 225 kidney-disease-related genes. Results By using an ad hoc web-based platform, we enrolled 160 patients from 13 different Nephrology and Genetics Units located across the Piedmont region over 15 months. A preliminary “remote” evaluation based on well-defined inclusion criteria allowed us to define eligibility for NGS analysis. Among the 138 recruited patients, 52 (37.7%) were children and 86 (62.3%) were adults. Up to 48% of them had a positive family history for kidney disease. Overall, applying this workflow led to the identification of genetic variants potentially explaining the phenotype in 78 (56.5%) cases. Conclusions These results underline the importance of clinical exome sequencing as a versatile and highly useful, non-invasive tool for genetic diagnosis of kidney diseases. Identifying patients who can benefit from targeted therapies, and improving the management of organ transplantation are further expected applications.

Background During the twentieth century, frontotemporal dementia (FTD) was often misdiagnosed, confused with Alzheimer's disease or psychiatric disorders, jeopardizing care and research. Objective To analyze the FTD genes in the DNA samples of patients belonging to families clinically classified as probable Alzheimer's disease (FAD) in the early 1990s and not carrying mutation in the three main genes linked to FAD (Presenilin 1, Presenilin 2, and Amyloid precursor protein). Methods The genetic screening was performed on 63 probands diagnosed as FAD before the early 2000s. Results Four patients out of the 63 studied (4/63, 6.3%) resulted as carrying four different GRN genetic variations: p.T272SfsX10, p.R110X, p.C149LfsX10, and p.W304C. The first two mutations (p.T272SfsX10, p.R110X) are the most frequent ones in Italy in FTD patients; the latter two (p.C149LfsX10 and p.W304C) are not described in the scientific literature. Conclusion Our data suggest that it can be important to re-examine FAD patients diagnosed when the FTD spectrum was not well recognized and the causative FTD genes had not yet been identified. Moreover, we propose initially analyzing genes associated with the first form of suspected dementia and, if the results are negative, studying genes implicated in the other form of dementia.

Pathogenic germline variants in the BAP1 tumor suppressor gene can cause a cancer syndrome called BAP1 tumor predisposition syndrome (BAP1-TPDS), which is characterized by predisposition to mesothelioma, melanoma, renal cell carcinoma, basal cell carcinoma, and other tumors. Other genes that may predispose to mesothelioma are CDKN2A and DNA repair genes. Asbestos exposure has often been reported in patients with malignant pleural mesothelioma (MPM) and germline variants in BAP1, but this exposure has never been quantified. We aimed to search for germline variants in BAP1 among 25 new Italian probands with suspected BAP1-TPDS, summarize the prevalence of these variants in 39 Italian patients with familial MPM and other tumors recruited over a 5-year period, and compare cumulative asbestos exposure in 14 patients with MPM and pathogenic germline variants in BAP1, CDKN2A, or DNA repair genes with that of 67 patients without germline variants in 94 cancer-predisposing genes. We report here a new pathogenic germline variant in BAP1: c.783 + 2 T > C. The prevalence of pathogenic germline variants in BAP1 was 7.7% among patients with familial MPM (3/39). Patients with pathogenic germline variants in BAP1, CDKN2A, or DNA repair genes showed lower cumulative asbestos exposure than patients without germline variants in 94 cancer-predisposing genes (P = .00002). This suggests an interaction between genetic risk factors and asbestos in the development of mesothelioma.

The detection of somatic mutations in a tumor represents a valuable tool for tumor characterization and provides the clinicians with information for setting up the most appropriate therapy. KRAS mutations in codons 12 and 13 are important biomarkers routinely analyzed in the clinic for the management of anti-EGFR treatment in colorectal carcinoma (CRC). Here we report a sensitive and inexpensive assay for KRAS mutations based on a PNA-mediated PCR clamping. The assay displays very high sensitivity (0.7%) and specificity (96.7%) when compared to traditional sequencing (SS) and pyrosequencing (PS), two of the most commonly and routinely used methods employed today by diagnostic laboratories. Furthermore, the PNA assay requires only basic and low-cost laboratory equipment, in contrast with all the most recent PCR-based technologies, which are highly sensitive but also much more expensive. Finally, despite the PNA assay does not allow for the definition of specific mutations, it is the cheapest and easiest screening method to firstly stratify wild-type and mutated patients, information that is strictly necessary to clinicians for the management of CRC and anti-EGFR treatment.

Summary Birt-Hogg-Dube syndrome (BHD) is an autosomal dominant condition characterised clinically by skin fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax, and renal cancer. The condition is caused by germline mutations in the FLCN gene, which encodes folliculin; the function of this protein is largely unknown, although FLCN has been linked to the mTOR pathway. The availability of DNA-based diagnosis has allowed insight into the great variation in expression of FLCN , both within and between families. Patients can present with skin signs and also with pneumothorax or renal cancer. Preventive measures are aimed mainly at early diagnosis and treatment of renal cancer. This Review gives an overview of current diagnosis and management of BHD.