Time to harmonize mitochondrial syndrome nomenclature and classification: A consensus from the North American Mitochondrial Disease Consortium (NAMDC).

OBJECTIVE: To harmonize terminology in mitochondrial medicine, we propose revised clinical criteria for primary mitochondrial syndromes. METHODS: The North American Mitochondrial Disease Consortium (NAMDC) established a Diagnostic Criteria Committee comprised of members with diverse expertise. It included clinicians, researchers, diagnostic laboratory directors, statisticians, and data managers. The Committee conducted a comprehensive literature review, an evaluation of current clinical practices and diagnostic modalities, surveys, and teleconferences to reach consensus on syndrome definitions for mitochondrial diseases. The criteria were refined after manual application to patients enrolled in the NAMDC Registry. RESULTS: By building upon published diagnostic criteria and integrating recent advances, NAMDC has generated updated consensus criteria for the clinical definition of classical mitochondrial syndromes. CONCLUSIONS: Mitochondrial diseases are clinically, biochemically, and genetically heterogeneous and therefore challenging to classify and diagnose. To harmonize terminology, we propose revised criteria for the clinical definition of mitochondrial disorders. These criteria are expected to standardize the diagnosis and categorization of mitochondrial diseases, which will facilitate future natural history studies and clinical trials.

Regulatory environment for novel therapeutic development in mitochondrial diseases.

At present, there is just one approved therapy for patients with mitochondrial diseases in Europe, another in Japan, and none in the United States. These facts reveal an important and significant unmet need for approved therapies for these debilitating and often fatal disorders. To fill this need, it is critical for clinicians and drug developers to work closely with regulatory agencies. In the United States, mitochondrial disease patients and clinicians, the United Mitochondrial Disease Foundation, and pharmaceutical industry members have engaged with the Food and Drug Administration to educate each other about these complex and heterogeneous diseases and about regulatory requirements to obtain approvals for novel therapies. Clinical development of therapies for rare diseases has been facilitated by the 1983 US Orphan Drug Act (ODA) and similar legislation in Japan and the European Union. Further legislation and regulatory guidance have expanded and refined regulatory flexibility. While regulatory and financial incentives of the ODA have augmented involvement of pharmaceutical companies, clinicians, with patient advocacy groups and industry, need to conduct natural history studies, develop clinical outcome measures, and identify potential supportive surrogate endpoints predictive of clinical benefit, which together are critical foundations for clinical trials. Thus, the regulatory environment for novel therapeutic development is conducive and offers flexibility for mitochondrial diseases. Nevertheless, flexibility does not mean lower standards, as well-controlled rigorous clinical trials of high quality are still required to establish the efficacy of potential therapies and to obtain regulatory agency approvals for their commercial use. This process is illustrated through the authors' ongoing efforts to develop therapy for thymidine kinase 2 deficiency.

Treating and reusing wastewater generated by the washing operations in the non-hazardous plastic solid waste recycling process: Advanced method vs. conventional method.

The effectiveness of an advanced treatment of wastewater generated by non-hazardous plastic solid waste (PSW) washing, based on the Sequencing Batch Biofilter Granular Reactor (SBBGR), was assessed in terms of gross parameters, removal efficiencies and sludge production. The proposed treatment was also compared with the conventional treatment, which was based on primary and secondary treatments, using the activated sludge process, performed by Recuperi Pugliesi, a leading company in the plastic recycling industry located in Bari, Italy. The company produces low-density polyethylene (LDPE) regenerated granules from PSW used in agricultural and floricultural greenhouse activities and industrial packaging after a washing stage in the aqueous phase. The latter generates large volumes of wastewater, the conventional treatment of which is characterised by large quantities of sludge and the associated disposal problems. Under steady-state conditions, the SBBGR provided impressive removal efficiencies regarding the main gross parameters (over 90% for COD and TKN, over 99% for BOD5, TSS, VSS and NH3, and over 80% for TN) with a statistically better effluent quality than that of the conventional treatment. The SBBGR effluent quality was modelled in terms of washing water characteristics by using generalized additive models (GAMs). The SBBGR treatment was characterised by a specific sludge production five times lower than that of the conventional treatment (0.21 kg TSS vs. 1.0 kg TSS per m3 of wastewater treated). Compared with the conventional treatment, the proposed process showed a five-fold reduction in the cost of sludge disposal, which saved 50% of the operating cost.

USMG5 Ashkenazi Jewish founder mutation impairs mitochondrial complex V dimerization and ATP synthesis.

Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.

Retrospective natural history of thymidine kinase 2 deficiency.

BACKGROUND: Thymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy. OBJECTIVE: To perform a retrospective natural history study of a large cohort of patients with TK2 deficiency. METHODS: The study was conducted by 42 investigators across 31 academic medical centres. RESULTS: We identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion. CONCLUSIONS: In TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder.

Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2-/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. METHODS: To test these hypotheses, we assessed two therapies in Tk2-/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. RESULTS: We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2-/- animals compared to dCMP+dTMP. INTERPRETATION: Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.

Lack of aprataxin impairs mitochondrial functions via downregulation of the APE1/NRF1/NRF2 pathway.

Ataxia oculomotor apraxia type 1 (AOA1) is an autosomal recessive disease caused by mutations in APTX, which encodes the DNA strand-break repair protein aprataxin (APTX). CoQ10 deficiency has been identified in fibroblasts and muscle of AOA1 patients carrying the common W279X mutation, and aprataxin has been localized to mitochondria in neuroblastoma cells, where it enhances preservation of mitochondrial function. In this study, we show that aprataxin deficiency impairs mitochondrial function, independent of its role in mitochondrial DNA repair. The bioenergetics defect in AOA1-mutant fibroblasts and APTX-depleted Hela cells is caused by decreased expression of SDHA and genes encoding CoQ biosynthetic enzymes, in association with reductions of APE1, NRF1 and NRF2. The biochemical and molecular abnormalities in APTX-depleted cells are recapitulated by knockdown of APE1 in Hela cells and are rescued by overexpression of NRF1/2. Importantly, pharmacological upregulation of NRF1 alone by 5-aminoimidazone-4-carboxamide ribonucleotide does not rescue the phenotype, which, in contrast, is reversed by the upregulation of NRF2 by rosiglitazone. Accordingly, we propose that the lack of aprataxin causes reduction of the pathway APE1/NRF1/NRF2 and their target genes. Our findings demonstrate a critical role of APTX in transcription regulation of mitochondrial function and the pathogenesis of AOA1 via a novel pathomechanistic pathway, which may be relevant to other neurodegenerative diseases.

Update on diagnostics of metabolic myopathies.

PURPOSE OF REVIEW: This review aims to highlight the most relevant clinical and laboratory findings, regarding acute and progressive metabolic myopathies, and to develop an algorithm addressing clinicians to clinical practice. RECENT FINDINGS: Although diagnosis of metabolic myopathies remains still challenging, the recent identification of new disorders has increased the number of patients requiring specific investigations. Nowadays, a more detailed characterization of the clinical spectrum of metabolic myopathies improved awareness as well as a deeper knowledge on their natural history or multisystem involvement. Diagnostic procedures, as first-line screening tests are necessary for an earlier and more accurate diagnostic work up, not only in infantile cases, but also in adults with suspected metabolic myopathies. New generation diagnostic techniques such as NGS (Next Generation Sequencing) and whole exome/genome sequencing have emerged as innovative tools to extensively evaluate either known genes variants or new candidate genes as possible causes of metabolic myopathies. SUMMARY: Diagnosis of metabolic myopathies is still challenging for clinicians because of rarity and clinical heterogeneity which is often overlapping with other neuromuscular disorders. Detailed algorithms supported by advanced laboratory investigations may be helpful to timely reach a diagnosis, so allowing an earlier therapeutic decision.

Functional cellular analyses reveal energy metabolism defect and mitochondrial DNA depletion in a case of mitochondrial aconitase deficiency.

Defects in the tricarboxylic acid cycle (TCA) are associated with a spectrum of neurological phenotypes that are often difficult to diagnose and manage. Whole-exome sequencing (WES) led to a rapid expansion of diagnostic capabilities in such disorders and facilitated a better understanding of disease pathogenesis, although functional characterization remains a bottleneck to the interpretation of potential pathological variants. We report a 2-year-old boy of Afro-Caribbean ancestry, who presented with neuromuscular symptoms without significant abnormalities on routine diagnostic evaluation. WES revealed compound heterozygous missense variants of uncertain significance in mitochondrial aconitase (ACO2), which encodes the TCA enzyme ACO2. Pathogenic variants in ACO2 have been described in a handful of families as the cause of infantile cerebellar-retinal degeneration syndrome. Using biochemical and cellular assays in patient fibroblasts, we found that ACO2 expression was quantitatively normal, but ACO2 enzyme activity was <20% of that observed in control cells. We also observed a deficiency in cellular respiration and, for the first time, demonstrate evidence of mitochondrial DNA depletion and altered expression of some TCA components and electron transport chain subunits. The observed cellular defects were completely restored with ACO2 gene rescue. Our findings demonstrate the pathogenicity of two VUS in ACO2, provide novel mechanistic insights to TCA disturbances in ACO2 deficiency, and implicate mitochondrial DNA depletion in the pathogenesis of this recently described disorder.

Intracranial arterial abnormalities in patients with late onset Pompe disease (LOPD).

BACKGROUND AND OBJECTIVES: Pompe disease is a rare metabolic disorder due to lysosomal alpha-glucosidase (GAA) deficiency. It is considered as a multi-systemic disease since, although glycogen accumulation is largely prominent in heart, skeletal and respiratory muscles, other organs can also be affected. As regards the vascular system, few reports have documented cerebrovascular malformations in Pompe patients. The aim of this study was to define the presence and type of intracranial arterial abnormalities in a cohort of late onset Pompe disease (LOPD) patients. METHODS: We have studied 21 LOPD patients with cerebral CT angiography (CTA), using maximum intensity projection and volume rendering technique for 3D-image reconstruction. RESULTS: We found intracranial arterial abnormalities in 13/21 patients (62 %), of whom: 2/21 patients (9.5 %) showed an unruptured intracranial aneurysm (respectively 2 and 4 mm), 10/21 (47 %) had a vertebrobasilar dolichoectasia (VBD) and 1/21 a basilar artery fenestration. Signs of lacunar encephalopathy (insular, capsular and frontal subcortical lesions) were detected in 13/21 patients (62 %) and this correlated with the presence of respiratory impairment (p = 0.017). CONCLUSIONS: These findings differ from what has been previously observed in healthy, aged-matched populations and confirm that cerebral arteries abnormalities, mainly involving the posterior circle, are not so rare in LOPD patients and are often accompanied by a lacunar encephalopathy that might represent a hypoxic-ischemic origin. A CTA or an MRA is recommended, in LOPD patients, for early detection of cerebrovascular malformations as they could lead to life-threatening events such as sub-arachnoid haemorrhage or brainstem compression.

Similarity indices of meteo-climatic gauging stations: definition and comparison.

Space-time dependencies among monitoring network stations have been investigated to detect and quantify similarity relationships among gauging stations. In this work, besides the well-known rank correlation index, two new similarity indices have been defined and applied to compute the similarity matrix related to the Apulian meteo-climatic monitoring network. The similarity matrices can be applied to address reliably the issue of missing data in space-time series. In order to establish the effectiveness of the similarity indices, a simulation test was then designed and performed with the aim of estimating missing monthly rainfall rates in a suitably selected gauging station. The results of the simulation allowed us to evaluate the effectiveness of the proposed similarity indices. Finally, the multiple imputation by chained equations method was used as a benchmark to have an absolute yardstick for comparing the outcomes of the test. In conclusion, the new proposed multiplicative similarity index resulted at least as reliable as the selected benchmark.

Metabolic Myoglobinuria.

One large group of hereditary myopathies characterized by recurrent myoglobinuria, almost invariably triggered by exercise, comprises metabolic disorders of two main fuels, glycogen and long-chain fatty acids, or mitochondrial diseases of the respiratory chain. Differential diagnosis is required to distinguish the three conditions, although all cause a crisis of muscle energy. Muscle biopsy may be useful when performed well after the episode of rhabdomyolysis. Molecular genetics is increasingly the diagnostic test of choice to discover the underlying genetic basis.

Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy.

Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches.

Late-onset Pompe disease (LOPD): correlations between respiratory muscles CT and MRI features and pulmonary function.

Late onset Pompe disease (LOPD) is a rare muscle disorder often characterized, along the disease course, by severe respiratory failure. We describe herein respiratory muscles and lung abnormalities in LOPD patients using MR imaging and CT examinations correlated to pulmonary function tests. Ten LOPD patients were studied: 6 with a limb-girdle muscle weakness, 1 with myalgias, 2 with exertional dyspnoea and 1 with isolated hyperckemia. Respiratory function was measured using forced vital capacity (FVC) in both upright and supine positions, maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP) and peak cough flow (PCF) tests. The involvement (atrophy) of diaphragms, abdominal respiratory muscles and intercostal muscles was ranked by CT and MRI examinations using appropriate scales. Height of lungs and band-like atelectasis presence were also recorded. Seven out of 10 patients showed a functional diaphragmatic weakness (FVC drop percentage >25%). In 8 out of 10 patients, involvement of both diaphragms and of other respiratory muscles was seen. The mean height of lungs in patients was significantly reduced when compared to a control group. Marked elevation of the diaphragms (lung height < 15 cm) was also seen in 6 patients. Multiple unilateral or bilateral band-like atelectasis were found in 4 patients. Statistically significant correlations were found between diaphragm atrophy grading, evaluated by MRI and CT, and FVC in supine position, FVC drop percentage passing from upright to supine position, PCF and MIP. Our data showed that diaphragm atrophy, often associated to reduced lung height and band-like atelectasis, can be considered the CT-MRI hallmark of respiratory insufficiency in LOPD patients. Early recognition of respiratory muscles involvement, using imaging data, could allow an early start of enzyme replacement therapy (ERT) in LOPD.

SBBGR technology for reducing waste sludge production during plastic recycling process: Assessment of potential increase in sludge hazardousness.

Sludge production in the wastewater treatment sector is consistently increasing and represents a critical environmental and economic issue. This study evaluated an unconventional approach for treating wastewater generated from the cleaning of non-hazardous plastic solid waste during the plastic recycling process. The proposed scheme was based on sequencing batch biofilter granular reactor (SBBGR) technology, which was compared with the activated sludge-based treatment currently in operation. These treatment technologies were compared regarding sludge quality, specific sludge production, and effluent quality to highlight whether the reduced sludge production shown by SBBGR corresponded to an increase in the concentration of hazardous compounds in the sludge. The SBBGR technology showed remarkable removal efficiencies (TSS, VSS, and NH3 > 99 %; COD >90 %; TN and TP > 80 %) and a sludge production six-fold lower than the conventional plant (in terms of kgTSS/kg CODremoved). Biomass from the SBBGR did not show a significant accumulation of organic micropollutants (i.e., long-chain hydrocarbons, chlorinated pesticides and chlorobenzenes, PCB, PCDD/F, PAH, chlorinated and brominated aliphatic compounds, and aromatic solvents), whereas a certain accumulation of heavy metals was observed. Furthermore, an initial attempt to compare the operating costs of the two treatment approaches revealed that the SBBGR technology would provide 38 % savings.

Auditory system involvement in late onset Pompe disease: a study of 20 Italian patients.

Glycogen storage disease type II (GSD II), also known as Pompe disease, is an autosomal recessive inherited disorder caused by a reduced activity of acid alpha glucosidase (GAA). Two different clinical entities have been described: rapidly fatal infantile and late onset forms. Hearing loss has been described in classic infantile Pompe patients but rarely in late onset cases. The main purpose of this study was to investigate the involvement of the auditory system in a cohort of Italian patients with late onset GSD II. We have enrolled 20 patients, 12 males and 8 females. The auditory system assessment included speech and pure tone audiometry, impedance audiometry and auditory brainstem responses (ABR). A combined interpretation of those tests allowed us to define the origin of the hearing impairment (sensorineural, conductive or mixed). Clinically, all patients but one denied subjective hearing disturbances. On the other hand, audiological evaluation revealed that 21/40 patient ears (52.5%) had a hearing impairment: 57% had a sensorineural deficit, 33% showed a conductive hearing loss whereas 10% presented with a mixed pattern. Our study revealed that, in this group of GSDII late onset patients, the auditory system impairment was more frequently present than thought with a prominent cochlear involvement. Our results emphasize the importance of a routinely auditory function evaluation in all forms of Pompe disease.

A methodology for rapid assessment of the environmental status of the shallow aquifer of "Tavoliere di Puglia" (Southern Italy).

Anticipating the European Water Framework Directive (2000/60/EC), the Italian Government issued Legislative Decree n.152/99 which sets out rules for classifying the environmental status of national water bodies in order to achieve specific qualitative objectives by 2016. The most recent European Groundwater Directive (2006/118/EC), which was only recognized by Italy in early 2009 (Legislative Decree 30/09), requires such resources to be characterized from a qualitative standpoint and the risk of their being polluted by individual pollutants or groups of pollutants to be evaluated. This paper reports a simple methodology, based on easy-to-apply rules, for the rapid classification of groundwater, and the results of its application to the shallow aquifer of the plain of Tavoliere delle Puglie located in south Italy. Data collected during well-water monitoring campaigns carried out from 2002 to 2003 made it possible to assess the environmental status of the Tavoliere which, unfortunately, was found to be characterized by "significant anthropic pressures on quality and/or quantity of groundwater and necessitating specific improvement actions".

Effects of Weed Control Practices on Plant Diversity in a Homogenous Olive-Dominated Landscape (South-East of Italy).

Olive groves represent an important economic, agro-ecological, and cultural resource in the Mediterranean Basin. Weed management plays a fundamental role in their sustainable management. The aim of this work was to characterize and assess the plant diversity associated with different weed control practices, in a homogeneous olive-dominated landscape in the South-East of Italy. Sixty-five vegetation plots were sampled in orchards treated with different weed control practices: mowing, tillage, and use of chemical herbicides. The multi-response permutation procedure was used to test the hypothesis of no difference among the treatments. The relationships between plots were visualized by means of non-metric multidimensional scaling ordination. A generalized linear mixed model was used to analyze the relationships between weed control practices and life forms, chorotypes, and diversity indexes. The results showed that the three weed control practices determined slightly different plant communities. Chemically weeded orchards showed an impoverished floristic composition and the lowest diversity, whereas mowing and tillage yielded similar values. These latter two treatments differed for the percentages of hemicryptophytes and therophytes. Moreover, different from other studies, we did not find plant species of particular concern for biodiversity conservation. We hypothesize that this result is due to the monotonous structure of the agro-landscape we investigated, where natural elements are almost lacking. From this point of view, a correct management of agro-districts should consider both the agronomic practices at the level of the individual olive groves and the structure of the agro-landscape.

A methodology for treating missing data applied to daily rainfall data in the Candelaro River Basin (Italy).

Environmental time series are often affected by the "presence" of missing data, but when dealing statistically with data, the need to fill in the gaps estimating the missing values must be considered. At present, a large number of statistical techniques are available to achieve this objective; they range from very simple methods, such as using the sample mean, to very sophisticated ones, such as multiple imputation. A brand new methodology for missing data estimation is proposed, which tries to merge the obvious advantages of the simplest techniques (e.g. their vocation to be easily implemented) with the strength of the newest techniques. The proposed method consists in the application of two consecutive stages: once it has been ascertained that a specific monitoring station is affected by missing data, the "most similar" monitoring stations are identified among neighbouring stations on the basis of a suitable similarity coefficient; in the second stage, a regressive method is applied in order to estimate the missing data. In this paper, four different regressive methods are applied and compared, in order to determine which is the most reliable for filling in the gaps, using rainfall data series measured in the Candelaro River Basin located in South Italy.

Mitochondrial diseases in North America: An analysis of the NAMDC Registry.

OBJECTIVE: To describe clinical, biochemical, and genetic features of participants with mitochondrial diseases (MtDs) enrolled in the North American Mitochondrial Disease Consortium (NAMDC) Registry. METHODS: This cross-sectional, multicenter, retrospective database analysis evaluates the phenotypic and molecular characteristics of participants enrolled in the NAMDC Registry from September 2011 to December 2018. The NAMDC is a network of 17 centers with expertise in MtDs and includes both adult and pediatric specialists. RESULTS: One thousand four hundred ten of 1,553 participants had sufficient clinical data for analysis. For this study, we included only participants with molecular genetic diagnoses (n = 666). Age at onset ranged from infancy to adulthood. The most common diagnosis was multisystemic disorder (113 participants), and only a minority of participants were diagnosed with a classical mitochondrial syndrome. The most frequent classical syndromes were Leigh syndrome (97 individuals) and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (71 individuals). Pathogenic variants in the mitochondrial DNA were more frequently observed (414 participants) than pathogenic nuclear gene variants (252 participants). Pathogenic variants in 65 nuclear genes were identified, with POLG1 and PDHA1 being the most commonly affected. Pathogenic variants in 38 genes were reported only in single participants. CONCLUSIONS: The NAMDC Registry data confirm the high variability of clinical, biochemical, and genetic features of participants with MtDs. This study serves as an important resource for future enhancement of MtD research and clinical care by providing the first comprehensive description of participant with MtD in North America.