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Introduction: Mitochondrial DNA (mtDNA) is a 16.6 kb, double-stranded, circular molecule that contains 37 genes coding for 13 subunits of the respiratory chain plus two rRNAs and 22 tRNAs, required to synthetize them. Mutations in these genes have been identified in patients with a variety of disorders affecting every system in the body. Mitochondrial diseases are clinically heterogeneous, so both diagnosis and treatment are extremely difficult. Traditional molecular diagnostic approach was limited to testing the most frequent mtDNA point mutations related to well-known mitochondrial diseases like MELAS, MERRF, NARP, LHON. The advent of next generation sequencing technologies has provided the whole mtDNA sequencing, allowing the identification of disease-causing pathogenic variants in a single platform. Aim of the study: The aim of this study was to develop an NGS strategy, by using an amplicon-based method to sequence the entire mitogenome, in order to identify disease-causing pathogenic variants in patients affected by mitochondrial diseases.Materials and Methods: The whole mtDNA coming from 20 patients was analyzed using an amplicon-based approach (Precision ID mtDNA Whole Genome Panel - ThermoFisher Scientific). The enriched libraries were sequenced on the Personal Genome Machine (PGM) from ThermoFisher Scientific. Primary bioinformatic analysis was carried out using the Ion Torrent suite v. 5.6. Annotation of the variants was made by the Variant Annotation Integrator from the UCSC browser. Results: After bioinformatics analysis and filtering, 100% coverage of the targeted regions was obtained with at least 500-fold mean depth. The size distribution of the amplicons ranged from 118 to 150 bp. The generated reads were 100% on target, with a uniformity of 99.8%. After variant calling, we were able to detect a mean of 35 SNVs for sample. Conclusions: The analysis of the whole mtDNA is a useful strategy for researchers studying human diseases linked to variations present in this type of genome. The NGS approach, compared to the standard methods, is a more reliable and time-cost reducing strategy to detect all the variants present in the mitogenome possibly associated with mitochondrial diseases.

La sindrome di McLeod è una rara malattia multisistemica, caratterizzata clinicamente da sintomi neurologici e psichiatrici e da manifestazioni neuromuscolari. Dalle indagini di laboratorio risulta alterazione della morfologia dei globuli rossi (acantocitosi), assenza dell'antigene Kx e debole espressione degli antigeni Kell (fenotipo McLeod) ed inoltre elevati livelli di creatin fosfochinasi (CK). La malattia è causata da mutazioni nel gene XK (Xp21.1) che codifica per la proteina XK, legata covalentemente alla glicoproteina Kell sulla superficie dei globuli rossi. Noi presentiamo il caso di un paziente di 52 anni che a partire dall'età di 45 anni aveva sviluppato gradualmente movimenti coreici con conseguente instabilità posturale, distonia orolinguale, moderata disartria, ipotonia degli arti e atrofia muscolare con ridotto riflesso tendineo. Egli presentava una storia familiare di corea, convulsioni e depressione di natura non determinata riferita al nonno materno. Le indagini di laboratorio mostravano una lieve anemia normocitica, moderato aumento degli enzimi epatici ed elevati valori di CK. La RMN rivelava atrofia del caudato, mentre l'elettromiografia e lo studio della velocità di conduzione nervosa fornivano indicazioni di una polineuropatia assonale sensorimotoria degli arti inferiori. Nel sangue periferico erano presenti acantociti suggestivi di una sindrome da neuroacantocitosi. Successive indagini immunoematologiche rivelavano una notevole riduzione della proteina Kell. L'analisi genetica, effettuata sequenziando i tre esoni del gene XK, individuava la presenza della mutazione emizigote da delezione c.856_860delCTCTA ) (precedentemente conosciuta come c.938_942delCTCTA) nell'esone 3. Questa mutazione, che crea un codone di stop prematuro in posizione amminoacidica 301, è stata precedentemente descritta in pochi pazienti affetti da sindrome di McLeod. Questo è il primo caso riportato in un paziente italiano. La sindrome di McLeod risulta spesso indistinguibile dalla corea di Huntington e la sua frequenza è quindi probabilmente sottostimata. Non vi è una chiara correlazione genotipo-fenotipo, ma la caratterizzazione genetica risulta utile per una ulteriore conferma diagnostica.

summaryIn order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in south-east Sicily, we have analysed the G6PD gene in 25 unrelated males with abnormal G6PD activityand}or electrophoretic mobility, by using the analysis of the appropriate PCR-amplified fragmentof DNA and subsequent digestion by appropriate restriction-enzymes, looking for the presence ofcertain known G6PD mutations. We amplified the entire G6PD coding sequence into eightfragments,followedbysingle-strandconformationpolymorphism(SSCP)analysisandsequencingofthose individual fragments that were found to be abnormal by SSCP. Through these methods wefound a total of twelve G6PD Mediterranean variants with the association of a silent mutation 1311(alsoknownaspolymorphicsiteBcl I),oneG6PDMediterraneanwithoutthisassociation,fourG6PDA− Val 68 and two G6PD Santamaria and five G6PD Chatham. In a subject with normal activitya mutation was found in exon 5, designated as G6PD Sao Borja. This is the first report on themolecular analysis of G6PD mutations in Sicily and we have obtained evidence for four distinctclasses of variants.introductionGlucose-6-phosphate dehydrogenase (G6PD;EC 1.1.1.49) is a ‘housekeeping’ enzyme and itsubiquitous distribution suggests its fundamentalrole in the intracellular metabolism; it is an X-linked enzyme that catalyzes the first step of thepentose phosphate pathway. The G6PD locus,mappedonthelongarmoftheXchromosomeatband Xq28, is extremely polymorphic. Many oftheover3OOdifferentG6PDvariantshavebeendescribedonthebasisofbiochemical parameters(Luzzatto&Metha,1989).Aftertheintroductionof molecular biology techniques, many of thesebiochemicallydifferentvariantshavebeenshown

The unlikely high frequency of G6PD deficiency which we found in a mountain village (San Giovanni in Fiore: province of Cosenza — Calabria, Southern Italy), with respect to the mean frequency of the other mountain villages in the same province, led us to carry out some investigations on the population of this village to understand this difference. The historical analysis, together with the social-demographic, endogamic and isonymyc study revealed the isolation of this village; while, the analysis of the allele frequencies of AK, ADA, 6PGD, PGM1, ABO and Rh markers revealed no significant differences if compared to the rest of the province. But the AK*5 variant which we found in ten unrelated subjects with a frequency of 0.0104, allows us to hypothesise together with the high G6PD frequency a random genetic drift effect on this village.