Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

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.

Ablation of collagen VI leads to the release of platelets with altered function.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI-related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI-null mouse model (Col6a1-/-), we found that collagen VI-null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1-/- megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1-/- mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1-/- mice. Peripheral blood platelets of patients affected by collagen VI-related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI-related diseases.

Molecular Epidemiology in 591 Italian Probands With Nonsyndromic Retinitis Pigmentosa and Usher Syndrome.

Purpose: To describe the molecular epidemiology of nonsyndromic retinitis pigmentosa (RP) and Usher syndrome (US) in Italian patients. Methods: A total of 591 probands (315 with family history and 276 sporadics) were analyzed. For 155 of them, we performed a family segregation study, considering a total of 382 relatives. Probands were analyzed by a customized multigene panel approach. Sanger sequencing was used to validate all genetic variants and to perform family segregation studies. Copy number variants of selected genes were analyzed by multiplex ligation-dependent probe amplification. Four patients who tested negative to targeted next-generation sequencing analysis underwent clinical exome sequencing. Results: The mean diagnostic yield of molecular testing among patients with a family history of retinal disorders was 55.2% while the diagnostic yield including sporadic cases was 37.4%. We found 468 potentially pathogenic variants, 147 of which were unpublished, in 308 probands and 66 relatives. Mean ages of onset of the different classes of RP were autosomal dominant RP, 19.3 ± 12.6 years; autosomal recessive RP, 23.2 ± 16.6 years; X-linked RP, 13.9 ± 9.9 years; and Usher syndrome, 18.9 ± 9.5 years. We reported potential new genotype-phenotype correlations in three probands, two revealed by TruSight One testing. All three probands showed isolated RP caused by biallelic variants in genes usually associated with syndromes such as PERCHING and Senior-Loken or with retinal dystrophy, iris coloboma, and comedogenic acne syndrome. Conclusions: This is the largest molecular study of Italian patients with RP in the literature, thus reflecting the epidemiology of the disease in Italy with reasonable accuracy.

Genetic test for the prescription of diets in support of physical activity.

Owing to the fields of nutrigenetics and nutrigenomics today we can think of devising approaches to optimize health, delay onset of diseases and reduce its severity according to our genetic blue print. However this requires a deep understanding of nutritional impact on expression of genes that may result in a specific phenotype. The extensive research and observational studies during last two decades reporting interactions between genes, diet and physical activity suggest a cross talk between various genetic and environmental factors and lifestyle interventions. Although considerable efforts have been made in unraveling the mechanisms of gene-diet interactions the scientific evidences behind developing commercial genetic tests for providing personalized nutrition recommendations are still scarce. In this scenario the current mini-review aims to provide useful insights into salient feature of nutrition based genetic research and its commercial application and the ethical issue and concerns related to its outcome.

Somatic Variant Analysis Identifies Targets for Tailored Therapies in Patients with Vascular Malformations.

Vascular malformations include various disorders characterized by morphological, structural and/or functional alterations of blood and lymph vessels. Most are sporadic, due to somatic mutations. Here, we report a cohort of patients with sporadic and/or unifocal vascular malformations, in whom we carried out next generation sequencing analysis of a panel of genes associated with vascular malformations. The 115 patients analyzed were from different clinical centres. In 37 patients (32%), we found pathogenic mutations: most of these were gain-of-function mutations in PIK3CA (18%, 21/115) and TEK (13/115, 11%). We also found mutations in GNAQ, CCM2 and PTEN. Identifying pathogenic variants in patients with vascular malformations can help improve management, particularly in cases with activating mutations that cause an increase in cell proliferation. Personalized pharmacological treatment, if possible, is now considered preferable to surgery and can help prevent recurrences, i.e., long-term complications of residual malformation or regrowth of tumors. For instance, rapamycin is currently being investigated for the treatment of various vascular malformations associated with hyperactivation of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway.

CRB1-Related Cystic Maculopathy in Twins Conceived Through Heterologous Fertilization With Variant-Carrying Oocytes.

Cystic maculopathy has been associated with genetic disorders such as retinitis pigmentosa, X-linked retinoschisis, cone dystrophy, and foveal retinoschisis. Familial foveal retinoschisis was recently described as a rare disease caused by CRB1 variants. The authors report the phenotype-genotype pattern of a pair of dizygotic twins with early-onset cystic maculopathy due to CRB1 pathogenic variants. The twins were conceived by heterologous fertilization with variant-carrying oocytes. The probands were monitored for a period of 4 years. Next generation sequencing of a panel of genes responsible for retinal dystrophies was performed. Both children carried three pathogenic variants in CRB1: a novel heterozygous truncating variant p.(Val855*) inherited from the father and two known heterozygous missense variants, p.[(Phe144Val; Thr745Met)], inherited from the oocyte donor. The findings confirm that CRB1 variants can be responsible for foveal retinoschisis with variable clinical expressivity ranging from schitic macular alteration to early-onset forms of cystic maculopathy. The authors highlight the importance of exome analysis of gamete donors to assess the likelihood of recessively inherited disorders by means of a prediction algorithm able to combine parent and donor exome data. [J Pediatr Ophthalmol Strabismus. 2020;57:e19-e24.].

Male Infertility Diagnosis: Improvement of Genetic Analysis Performance by the Introduction of Pre-Diagnostic Genes in a Next-Generation Sequencing Custom-Made Panel.

Background: Infertility affects about 7% of the general male population. The underlying cause of male infertility is undefined in about 50% of cases (idiopathic infertility). The number of genes involved in human spermatogenesis is over two thousand. Therefore, it is essential to analyze a large number of genes that may be involved in male infertility. This study aimed to test idiopathic male infertile patients negative for a validated panel of "diagnostic" genes, for a wide panel of genes that we have defined as "pre-diagnostic." Methods: We developed a next-generation sequencing (NGS) gene panel including 65 pre-diagnostic genes that were used in 12 patients who were negative to a diagnostic genetic test for male infertility disorders, including primary spermatogenic failure and central hypogonadism, consisting of 110 genes. Results: After NGS sequencing, variants in pre-diagnostic genes were identified in 10/12 patients who were negative to a diagnostic test for primary spermatogenic failure (n = 9) or central hypogonadism (n = 1) due to mutations of single genes. Two pathogenic variants of DNAH5 and CFTR genes and three uncertain significance variants of DNAI1, DNAH11, and CCDC40 genes were found. Moreover, three variants with high impact were found in AMELY, CATSPER 2, and ADCY10 genes. Conclusion: This study suggests that searching for pre-diagnostic genes may be of relevance to find the cause of infertility in patients with apparently idiopathic primary spermatogenic failure due to mutations of single genes and central hypogonadism.

Monogenic hypertension.

Hypertension is a significant public health problem. Thirty percent of cases are caused by a single genetic mutation. Hypertension is the predominant and usually the only manifestation in monogenic hypertension Monogenic hypertension may involve mineralcorticoid-dependent or -independent increase in Na+ transport. Diagnosis is based on routine physical examination, blood pressure measurement and laboratory analysis of renin, aldosterone, cortisol and potassium. Genetic testing is useful for confirming diagnosis and for differential diagnosis. Monogenic hypertension has autosomal dominant or autosomal recessive inheritance.

The Link of the Prion Protein with Ca2+ Metabolism and ROS Production, and the Possible Implication in Aß Toxicity.

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aß oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.

Vascular anomalies: molecular bases, genetic testing and therapeutic approaches.

INTRODUCTION: Vascular anomalies encompass an extremely heterogeneous group of congenital abnormalities of the vascular system. They include vascular tumors and malformations and have a prevalence of 4.5%. Vascular anomalies are frequently sporadic and associated with somatic mutations and/or a double-hit mechanism and are characterized by considerable phenotypic and genetic heterogeneity. The aim of this review was to provide a genetic description of vascular anomalies, the sequencing technologies used for their diagnosis and the drugs that may potentially be used for their treatment. EVIDENCE ACQUISITION: PubMed, OMIM, Orphanet, Genetic Testing Registry and ClinicalTrials.gov were searched for monogenic vascular anomalies in order to evaluate the genetic tests (based on sequencing) currently used for their diagnosis, and for any drugs that could be useful to treat them. EVIDENCE SYNTHESIS: From the search of the clinical synopsis section of OMIM and PubMed for vascular anomalies we selected 19 disorders with a known molecular etiology. From the search for pharmacological trials and therapies in the ClinicalTrials.gov and PubMed databases we selected 87 drugs. CONCLUSIONS: Most genetic tests with validated clinical utility are based on a next generation sequencing (NGS) approach. Targeted NGS is indeed the best approach for the analysis of disorders with complex phenotypes and genetics and involvement of somatic mutations. Genetic diagnosis provides data for determine genotype-phenotype correlations, segregation and recurrence risk in families, and new targets for gene- or mutation-specific pharmacological therapies. Improvement of diagnostic techniques is needed to offer patients appropriate care, more focused follow-up, and hopefully drugs to treat their disorders.

Transcriptomic Analysis of Single Isolated Myofibers Identifies miR-27a-3p and miR-142-3p as Regulators of Metabolism in Skeletal Muscle.

Skeletal muscle is composed of different myofiber types that preferentially use glucose or lipids for ATP production. How fuel preference is regulated in these post-mitotic cells is largely unknown, making this issue a key question in the fields of muscle and whole-body metabolism. Here, we show that microRNAs (miRNAs) play a role in defining myofiber metabolic profiles. mRNA and miRNA signatures of all myofiber types obtained at the single-cell level unveiled fiber-specific regulatory networks and identified two master miRNAs that coordinately control myofiber fuel preference and mitochondrial morphology. Our work provides a complete and integrated mouse myofiber type-specific catalog of gene and miRNA expression and establishes miR-27a-3p and miR-142-3p as regulators of lipid use in skeletal muscle.

Alisporivir rescues defective mitochondrial respiration in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle disease of known etiology without effective, or generally applicable therapy. Mitochondria are affected by the disease in animal models but whether mitochondrial dysfunction is part of the pathogenesis in patients remains unclear. We show that primary cultures obtained from muscle biopsies of DMD patients display a decrease of the respiratory reserve, a consequence of inappropriate opening of the permeability transition pore (PTP). Treatment with the cyclophilin inhibitor alisporivir - a cyclosporin A derivative that desensitizes the PTP but does not inhibit calcineurin - largely restored the maximal respiratory capacity without affecting basal oxygen consumption in cells from patients, thus reinstating a normal respiratory reserve. Treatment with alisporivir, but not with cyclosporin A, led to a substantial recovery of respiratory function matching improved muscle ultrastructure and survival of sapje zebrafish, a severe model of DMD where muscle defects are close to those of DMD patients. Alisporivir was generally well tolerated in HCV patients and could be used for the treatment of DMD.

Forty years later: Mitochondria as therapeutic targets in muscle diseases.

The hypothesis that mitochondrial dysfunction can be a general mechanism for cell death in muscle diseases is 40 years old. The key elements of the proposed pathogenetic sequence (cytosolic Ca2+ overload followed by excess mitochondrial Ca2+ uptake, functional and then structural damage of mitochondria, energy shortage, worsened elevation of cytosolic Ca2+ levels, hypercontracture of muscle fibers, cell necrosis) have been confirmed in amazing detail by subsequent work in a variety of models. The explicit implication of the hypothesis was that it "may provide the basis for a more rational treatment for some conditions even before their primary causes are known" (Wrogemann and Pena, 1976, Lancet, 1, 672-674). This prediction is being fulfilled, and the potential of mitochondria as pharmacological targets in muscle diseases may soon become a reality, particularly through inhibition of the mitochondrial permeability transition pore and its regulator cyclophilin D.

Melanocytes from Patients Affected by Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy have Dysfunctional Mitochondria That Can be Rescued with Cyclophilin Inhibitors.

Ullrich congenital muscular dystrophy and Bethlem myopathy are caused by mutations in collagen VI (ColVI) genes, which encode an extracellular matrix protein; yet, mitochondria play a major role in disease pathogenesis through a short circuit caused by inappropriate opening of the permeability transition pore, a high-conductance channel, which causes a shortage in ATP production. We find that melanocytes do not produce ColVI yet they bind it at the cell surface, suggesting that this protein may play a trophic role and that its absence may cause lesions similar to those seen in skeletal muscle. We show that mitochondria in melanocytes of Ullrich congenital muscular dystrophy and Bethlem myopathy patients display increased size, reduced matrix density, and disrupted cristae, findings that suggest a functional impairment. In keeping with this hypothesis, mitochondria (i) underwent anomalous depolarization after inhibition of the F-ATP synthase with oligomycin, and (ii) displayed decreased respiratory reserve capacity. The non-immunosuppressive cyclophilin inhibitor NIM811 prevented mitochondrial depolarization in response to oligomycin in melanocytes from both Ullrich congenital muscular dystrophy and Bethlem myopathy patients, and partially restored the respiratory reserve of melanocytes from one Bethlem myopathy patient. These results match our recent findings on melanocytes from patients affected by Duchenne muscular dystrophy (Pellegrini et al., 2013), and suggest that skin biopsies may represent a minimally invasive tool to investigate mitochondrial dysfunction and to evaluate drug efficacy in ColVI-related myopathies and possibly in other muscle wasting conditions like aging sarcopenia.

NIM811, a cyclophilin inhibitor without immunosuppressive activity, is beneficial in collagen VI congenital muscular dystrophy models.

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.

Differences in visceral fat and fat bacterial colonization between ulcerative colitis and Crohn's disease. An in vivo and in vitro study.

Crohn's disease (CD) is notably characterized by the expansion of visceral fat with small adipocytes expressing a high proportion of anti-inflammatory genes. Conversely, visceral fat depots in ulcerative colitis (UC) patients have never been characterized. Our study aims were a) to compare adipocyte morphology and gene expression profile and bacterial translocation in omental (OM) and mesenteric (MES) adipose tissue of patients with UC and CD, and b) to investigate the effect of bacterial infection on adipocyte proliferation in vitro. Specimens of OM and MES were collected from 11 UC and 11 CD patients, processed and examined by light microscopy. Gene expression profiles were evaluated in adipocytes isolated from visceral adipose tissue using microarray and RTqPCR validations. Bacteria within adipose tissue were immuno-detected by confocal scanning laser microscopy. Adipocytes were incubated with Enterococcus faecalis and cells counted after 24 h. Morphology and molecular profile of OM and MES revealed that UC adipose tissue is less inflamed than CD adipose tissue. Genes linked to inflammation, bacterial response, chemotaxis and angiogenesis were down-regulated in adipocytes from UC compared to CD, whereas genes related to metallothioneins, apoptosis pathways and growth factor binding were up-regulated. A dense perinuclear positivity for Enterococcus faecalis was detected in visceral adipocytes from CD, whereas positivity was weak in UC. In vitro bacterial infection was associated with a five-fold increase in the proliferation rate of OM preadipocytes. Compared to UC, visceral adipose tissue from CD is more inflamed and more colonized by intestinal bacteria, which increase adipocyte proliferation. The influence of bacteria stored within adipocytes on the clinical course of IBD warrants further investigations.

Molecular and morphologic characterization of superficial- and deep-subcutaneous adipose tissue subdivisions in human obesity.

OBJECTIVE: Human abdominal subcutaneous white adipose tissue (SAT) is composed of two different subcompartments: a "superficial" SAT (SSAT), located between the skin and a fibrous-fascia plane; and a deeper SAT, located under this fibrous fascia plane, indicated as "deep" SAT (DSAT). DESIGN AND METHODS: In order to investigate whether SSAT and DSAT have different molecular and morphological features, paired SSAT/DSAT biopsies were collected from 10 female obese patients and used for microarray and morphologic analysis. The stroma-vascular fraction cells were also isolated from both depots and cultured in vitro to assess the lipid accumulation rate. RESULTS: SSAT and DSAT displayed different patterns of gene expression, mainly for metabolic and inflammatory genes, respectively. Detailed gene expression analysis indicated that several metabolic genes, including adiponectin, are preferentially expressed in SSAT, whereas inflammatory genes are over-expressed in DSAT. Despite a similar lipid accumulation rate in vitro, in vivo SSAT showed a significant adipocyte hypertrophy together with a significantly lower inflammatory infiltration and vascular vessel lumen mean size, when compared to DSAT. CONCLUSIONS: These data show that, SSAT and DSAT are functionally and morphologically different and emphasize the importance of considering independent these two adipose depots when investigating SAT biology and obesity complications.

The sleeve gastrectomy intervention to treat morbid obesity in a Parkinson's disease patient.

BACKGROUND & AIMS: Some Parkinson's disease patients may develop morbid obesity, on account of the reduction in exercise and/or of the appearance of compulsive food intake in the first years after diagnosis. The prescription of central appetite suppressants is actually not recommended in Parkinson's disease patients. To the best of our knowledge, no cases of morbidly obese Parkinson's disease patients submitted to bariatric surgery procedures have been reported in literature before. METHODS: We here describe for the first time the outcome of a sleeve gastrectomy intervention in a morbidly obese Parkinson's disease patient, resistant to several non-surgical weight-loss treatments. RESULTS: The outcome of the sleeve gastrectomy intervention was satisfactory in terms of body weight-loss, long term weight stabilization and improvement of cardioprotective circulating factors, including adiponectin. Furthermore, the antiparkinson therapy (levodopa) was reduced by 25%. CONCLUSIONS: These observations suggest that morbidly obese Parkinson's disease patients, who are resistant to other dietary treatments, might be candidated for sleeve gastrectomy.

Melanocytes--a novel tool to study mitochondrial dysfunction in Duchenne muscular dystrophy.

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal-epidermal junction. In addition the full-length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments.