Clinical, pathological, imaging, and genetic characterization in a Taiwanese cohort with limb-girdle muscular dystrophy.

BACKGROUND: Limb-girdle muscular dystrophy (LGMD) is a genetically heterogeneous, hereditary disease characterized by limb-girdle weakness and histologically dystrophic changes. The prevalence of each subtype of LGMD varies among different ethnic populations. This study for the first time analyzed the phenotypes and genotypes in Taiwanese patients with LGMD in a referral center for neuromuscular diseases (NMDs). RESULTS: We enrolled 102 patients clinically suspected of having LGMD who underwent muscle biopsy with subsequent genetic analysis in the previous 10 years. On the basis of different pathological categories, we performed sequencing of target genes or panel for NMDs and then identified patients with type 1B, 1E, 2A, 2B, 2D, 2I, 2G, 2 N, and 2Q. The 1B patients with LMNA mutation presented with mild limb-girdle weakness but no conduction defect at the time. All 1E patients with DES mutation exhibited predominantly proximal weakness along with distal weakness. In our cohort, 2B and 2I were the most frequent forms of LGMD; several common or founder mutations were identified, including c.1097_1099delACA (p.Asn366del) in DES, homozygous c.101G > T (p.Arg34Leu) in SGCA, homozygous c.26_33dup (p.Glu12Argfs*20) in TCAP, c.545A > G (p.Tyr182Cys), and c.948delC (p.Cys317Alafs*111) in FKRP. Clinically, the prevalence of dilated cardiomyopathy in our patients with LGMD2I aged > 18 years was 100%, much higher than that in European cohorts. The only patient with LGMD2Q with PLEC mutation did not exhibit skin lesions or gastrointestinal abnormalities but had mild facial weakness. Muscle imaging of LGMD1E and 2G revealed a more uniform involvement than did other LGMD types. CONCLUSION: Our study revealed that detailed clinical manifestation together with muscle pathology and imaging remain critical in guiding further molecular analyses and are crucial for establishing genotype-phenotype correlations. We also determined the common mutations and prevalence for different subtypes of LGMD in our cohort, which could be useful when providing specific care and personalized therapy to patients with LGMD.

Correction: Crosstalk from Non-Cancerous Mitochondria Can Inhibit Tumor Properties of Metastatic Cells by Suppressing Oncogenic Pathways.

[This corrects the article DOI: 10.1371/journal.pone.0061747.].

Comprehensive target capture/next-generation sequencing as a second-tier diagnostic approach for congenital muscular dystrophy in Taiwan.

PURPOSE: Congenital muscular dystrophy (CMD) is a heterogeneous disease entity. The detailed clinical manifestation and causative gene for each subgroup of CMD are quite variable. This study aims to analyze the phenotypes and genotypes of Taiwanese patients with CMD as the epidemiology of CMD varies among populations and has been scantly described in Asia. METHODS: A total of 48 patients suspected to have CMD were screened and categorized by histochemistry and immunohistochemistry studies. Different genetic analyses, including next-generation sequencing (NGS), were selected, based on the clinical and pathological findings. RESULTS: We identified 17 patients with sarcolemma-specific collagen VI deficiency (SSCD), 6 patients with merosin deficiency, two with reduced alpha-dystroglycan staining, and two with striking lymphocyte infiltration in addition to dystrophic change on muscle pathology. Fourteen in 15 patients with SSCD, were shown to have COL6A1, COL6A2 or COL6A3 mutations by NGS analysis; all showed marked distal hyperlaxity and normal intelligence but the overall severity was less than in previously reported patients from other populations. All six patients with merosin deficiency had mutations in LAMA2. They showed relatively uniform phenotype that were compatible with previous studies, except for higher proportion of mental retardation with epilepsy. With reduced alpha-dystroglycan staining, one patient was found to carry mutations in POMT1 while another patient carried mutations in TRAPPC11. LMNA mutations were found in the two patients with inflammatory change on muscle pathology. They were clinically characterized by neck flexion limitation and early joint contracture, but no cardiac problem had developed yet. CONCLUSION: Muscle pathology remains helpful in guiding further molecular analyses by direct sequencing of certain genes or by target capture/NGS as a second-tier diagnostic tool, and is crucial for establishing the genotype-phenotype correlation. We also determined the frequencies of the different types of CMD in our cohort which is important for the development of a specific care system for each disease.

Identification of KLHL40 mutations by targeted next-generation sequencing facilitated a prenatal diagnosis in a family with three consecutive affected fetuses with fetal akinesia deformation sequence.

BACKGROUND: Fetal akinesia deformation sequence (FADS) refers to a broad spectrum of disorder with the absent fetal movement as the unifying feature. The etiology of FADS is heterogeneous, and the majority remains unknown. Prenatal diagnosis of FADS because of neuromuscular origin has relied on clinical features and fetal muscle pathology, which can be unrevealing. The recent advance of next-generation sequencing (NGS) can provide definitive molecular diagnosis effectively. METHODS AND RESULTS: An 18-week-old fetus presented with akinesia and multiple contractures of joints. The mother had two previously aborted similarly affected fetuses. Clinical diagnosis of FADS was made. Molecular diagnosis using cord blood by NGS of genes related to neuromuscular diseases revealed two compound heterozygous mutations; c.602G > A(p.W201*) and c.1516A > C(p.T506P), in the Kelch-like 40 (KLHL40) gene. Based on this information, prenatal diagnosis was performed on the CVS of the subsequent pregnancy that resulted in an unaffected female baby, heterozygous for the c.1516A > C(p.T506P) mutation. CONCLUSION: Identification of KLHL40 mutations in one of the aborted fetuses provided a confirmative diagnosis of FADS, facilitating the prenatal diagnosis of the subsequent pregnancy. This report underscores the importance of target NGS in providing FADS families with an affordable, precise molecular diagnosis for genetic counseling and options of prenatal diagnosis. © 2016 John Wiley & Sons, Ltd.

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer.

Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple-negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid ß oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1A (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2.

Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3' region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome.

Recurrent ACADVL molecular findings in individuals with a positive newborn screen for very long chain acyl-coA dehydrogenase (VLCAD) deficiency in the United States.

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.

Hepatic SRC-1 activity orchestrates transcriptional circuitries of amino acid pathways with potential relevance for human metabolic pathogenesis.

Disturbances in amino acid metabolism are increasingly recognized as being associated with, and serving as prognostic markers for chronic human diseases, such as cancer or type 2 diabetes. In the current study, a quantitative metabolomics profiling strategy revealed global impairment in amino acid metabolism in mice deleted for the transcriptional coactivator steroid receptor coactivator (SRC)-1. Aberrations were hepatic in origin, because selective reexpression of SRC-1 in the liver of SRC-1 null mice largely restored amino acids concentrations to normal levels. Cistromic analysis of SRC-1 binding sites in hepatic tissues confirmed a prominent influence of this coregulator on transcriptional programs regulating amino acid metabolism. More specifically, SRC-1 markedly impacted tyrosine levels and was found to regulate the transcriptional activity of the tyrosine aminotransferase (TAT) gene, which encodes the rate-limiting enzyme of tyrosine catabolism. Consequently, SRC-1 null mice displayed low TAT expression and presented with hypertyrosinemia and corneal alterations, 2 clinical features observed in the human syndrome of TAT deficiency. A heterozygous missense variant of SRC-1 (p.P1272S) that is known to alter its coactivation potential, was found in patients harboring idiopathic tyrosinemia-like disorders and may therefore represent one risk factor for their clinical symptoms. Hence, we reinforce the concept that SRC-1 is a central factor in the fine orchestration of multiple pathways of intermediary metabolism, suggesting it as a potential therapeutic target that may be exploitable in human metabolic diseases and cancer.

Crosstalk from non-cancerous mitochondria can inhibit tumor properties of metastatic cells by suppressing oncogenic pathways.

Mitochondrial-nucleus cross talks and mitochondrial retrograde regulation can play a significant role in cellular properties. Transmitochondrial cybrid systems (cybrids) are an excellent tool to study specific effects of altered mitochondria under a defined nuclear background. The majority of the studies using the cybrid model focused on the significance of specific mitochondrial DNA variations in mitochondrial function or tumor properties. However, most of these variants are benign polymorphisms without known functional significance. From an objective of rectifying mitochondrial defects in cancer cells and to establish mitochondria as a potential anticancer drug target, understanding the role of functional mitochondria in reversing oncogenic properties under a cancer nuclear background is very important. Here we analyzed the potential reversal of oncogenic properties of a highly metastatic cell line with the introduction of non-cancerous mitochondria. Cybrids were established by fusing the mitochondria DNA depleted 143B TK- ρ0 cells from an aggressive osteosarcoma cell line with mitochondria from benign breast epithelial cell line MCF10A, moderately metastatic breast cancer cell line MDA-MB-468 and 143B cells. In spite of the uniform cancerous nuclear background, as observed with the mitochondria donor cells, cybrids with benign mitochondria showed high mitochondrial functional properties including increased ATP synthesis, oxygen consumption and respiratory chain activities compared to cybrids with cancerous mitochondria. Interestingly, benign mitochondria could reverse different oncogenic characteristics of 143B TK(-) cell including cell proliferation, viability under hypoxic condition, anti-apoptotic properties, resistance to anti-cancer drug, invasion, and colony formation in soft agar, and in vivo tumor growth in nude mice. Microarray analysis suggested that several oncogenic pathways observed in cybrids with cancer mitochondria are inhibited in cybrids with non-cancerous mitochondria. These results suggest the critical oncogenic regulation by mitochondrial-nuclear cross talk and highlights rectifying mitochondrial functional properties as a promising target in cancer therapy.

Mitochondrial myopathy due to novel missense mutation in the cytochrome c oxidase 1 gene.

We report a novel heteroplasmic mutation p.Y440C in the mitochondrial DNA-encoded subunit I of the cytochrome c oxidase (COX) gene in a patient with late onset progressive painless weakness. Her muscle biopsy showed scattered COX-negative fibers and several small collections of inflammatory cells. The mutation was detected in the patient's muscle but not in her blood. The low mutant load in muscle could explain the patient's late onset of the myopathy and milder phenotype when compared to the previously published cases with MTCO1 mutations.

Ablation of steroid receptor coactivator-3 resembles the human CACT metabolic myopathy.

Oxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of ß-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.

Individualizing antimetabolic treatment strategies for head and neck squamous cell carcinoma based on TP53 mutational status.

BACKGROUND: Mutations in the tumor protein 53 (TP53) tumor suppressor gene are common in head and neck squamous cell carcinoma (HNSCC) and correlate with radioresistance. Currently, there are no clinically available therapeutic approaches targeting p53 in HNSCC. In this report, the authors propose a strategy that uses TP53 mutational status to individualize antimetabolic strategies for the potentiation of radiation toxicity in HNSCC cells. METHODS: Glycolytic flux and mitochondrial respiration were evaluated in wild-type (wt) and mutant (mut) TP53 HNSCC cell lines. Sensitivity to external-beam radiation (XRT) was measured using a clonogenic assay. RESULTS: HNSCC cells that expressed mutTP53 demonstrated radioresistance compared with HNSCC cells that expressed wtTP53. Glycolytic inhibition potentiated radiation toxicity in mutTP53-expressing, but not wtTP53-expressing, HNSCC cells. The relative sensitivity of mutTP53 HNSCC cells to glycolytic inhibition was caused by a glycolytic dependence associated with decreased mitochondrial complex II and IV activity. The wtTP53-expressing cells maintained mitochondrial reserves and were relatively insensitive to glycolytic inhibition. Inhibition of respiration using metformin increased glycolytic dependence in wtTP53-expressing cells and potentiated the effects of glycolyic inhibition on radiation toxicity. CONCLUSIONS: TP53 mutation in HNSCC cells was correlated with a metabolic shift away from mitochondrial respiration toward glycolysis, resulting in increased sensitivity to the potentiating effects of glycolytic inhibition on radiation toxicity. In contrast, wtTP53-expressing cells required inhibition of both mitochondrial respiration and glycolysis to become sensitized to radiation. Therefore, the authors concluded that TP53 mutational status may be used as a marker of altered tumor cell metabolism to individualize HNSCC treatment selection of specific, targeted metabolic agents that can overcome cellular resistance to radiation therapy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)-like phenotype: an expanded clinical spectrum of POLG1 mutations.

The aim of the study was to determine the prevalence of MNGIE-like phenotype in patients with recessive POLG1 mutations. Mutations in the POLG1 gene, which encodes for the catalytic subunit of the mitochondrial DNA polymerase gamma essential for mitochondrial DNA replication, cause a wide spectrum of mitochondrial disorders. Common phenotypes associated with POLG1 mutations include Alpers syndrome, ataxia-neuropathy syndrome, and progressive external ophthalmoplegia (PEO). Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, PEO and/or ptosis, peripheral neuropathy, and leukoencephalopathy. MNGIE is caused by TYMP mutations. Rare cases of MNGIE-like phenotype have been linked to RRM2B mutations. Recently, POLG1 mutations were identified in a family with clinical features of MNGIE but no leukoencephalopathy. The coding regions and exon-intron boundaries of POLG1 were sequence analyzed in patients suspected of POLG1 related disorders. Clinical features of 92 unrelated patients with two pathogenic POLG1 alleles were carefully reviewed. Three patients, accounting for 3.3% of all patients with two pathogenic POLG1 mutations, were found to have clinical features consistent with MNGIE but no leukoencephalopathy. Patient 1 carries p.W748S and p.R953C; patient 2 is homozygous for p.W748S, and patient 3 is homozygous for p.A467T. In addition, patient 2 has a similarly affected sibling with the same POLG1 genotype. POLG1 mutations may cause MNGIE-like syndrome, but the lack of leukoencephalopathy and the normal plasma thymidine favor POLG1 mutations as responsible molecular defect.

Mitochondrial DNA content varies with pathological characteristics of breast cancer.

Changes in mitochondrial DNA (mtDNA) content in cancers have been reported with controversial results, probably due to small sample size and variable pathological conditions. In this study, mtDNA content in 302 breast tumor/surrounding normal tissue pairs were evaluated and correlated with the clinico-pathological characteristics of tumors. Overall, mtDNA content in tumor tissues is significantly lower than that in the surrounding normal tissues, P < 0.00001. MtDNA content in tumor tissues decreased with increasing tumor size. However, when the tumor is very large (>50 cm(3)), mtDNA content started to increase. Similarly, mtDNA content decreased from grades 0 and I to grade II tumors, but increased from grade II to grade III tumors. Tumors with somatic mtDNA alterations in coding region have significantly higher mtDNA content than tumors without somatic mtDNA alterations (P < 0.001). Tumors with somatic mtDNA alterations in the D-Loop region have significantly lower mtDNA content (P < 0.001). Patients with both low and high mtDNA content in tumor tissue have significantly higher hazard of death than patients with median levels of mtDNA content. mtDNA content in tumor tissues change with tumor size, grade, and ER/PR status; significant deviation from the median level of mtDNA content is associated with poor survival.

Mitochondria of highly metastatic breast cancer cell line MDA-MB-231 exhibits increased autophagic properties.

Autophagy is a cellular housekeeping process that removes damaged or unwanted cellular components and recycles them to build new constituents. It is essential for tumor growth under adverse environment. Mitochondria play an important role in the formation of autophagosome and its subsequent docking and fusion with lysosome. To understand the contribution of mitochondria to the regulation of homeostatic autophagy in cancer cells, we used the transmitochondrial cytoplasmic hybrid (cybrid) model. Cybrid system allowed us to compare mitochondria from different cell types including highly metastatic breast cancer cell line MDA-MB-231 (c231), less metastatic breast cancer cell lines: MDA-MB-436 (c436) and MDA-MB-468 (c468), as well as non-cancerous mammary epithelial cell MCF-10A (c10A) in a defined nuclear background. The c231 exhibited lower LC3-II levels but higher ratio of LC3-II/LC3-I than c436, c468 and c10A. In addition, c231 displayed more punctate LC3-positive cells and had lower levels of sequestosome 1 (p62/SQSTM1) than other cybrids. These suggested that mitochondria could contribute to the increased autophagy and autophagic flux in metastatic cancer. This increased autophagy was found to be non-selective autophagy instead of selective mitophagy since LC3 puncta in c231 did not co-localize with mitochondria labeled by Mitotracker red or Tomm 20. The promotion of mitochondrial permeability transition (MPT) in c231 also contributed to increased autophagy. Block of MPT by the inhibition of low-conductance stage of MPT pores resulted in a decrease of LC3 puncta in c231. These results suggested that mitochondria from highly metastatic breast cancer cell line MDA-MB-231 can promote homeostatic autophagy of cancer through opening low-conductance MPT pores.

Heterogeneous patterns of tissue injury in NARP syndrome.

Point mutations at m.8993T>C and m.8993T>G of the mtDNA ATPase 6 gene cause the neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome, a mitochondrial disorder characterized by retinal, central and peripheral neurodegeneration. We performed detailed neurological, neuropsychological and ophthalmological phenotyping of a mother and four daughters with NARP syndrome from the mtDNA m.8993T>C ATPase 6 mutation, including 3-T brain MRI, spectral domain optical coherence tomography (SD-OCT), adaptive optics scanning laser ophthalmoscopy (AOSLO), electromyography and nerve conduction studies (EMG-NCS) and formal neuropsychological testing. The degree of mutant heteroplasmy for the m.8993T>C mutation was evaluated by real-time allele refractory mutation system quantitative PCR of mtDNA from hair bulbs (ectoderm) and blood leukocytes (mesoderm). There were marked phenotypic differences between family members, even between individuals with the greatest degrees of ectodermal and mesodermal heteroplasmy. 3-T MRI revealed cerebellar atrophy and cystic and cavitary T2 hyperintensities in the basal ganglia. SD-OCT demonstrated similarly heterogeneous areas of neuronal and axonal loss in inner and outer retinal layers. AOSLO showed increased cone spacing due to photoreceptor loss. EMG-NCS revealed varying degrees of length-dependent sensorimotor axonal polyneuropathy. On formal neuropsychological testing, there were varying deficits in processing speed, visual-spatial functioning and verbal fluency and high rates of severe depression. Many of these cognitive deficits likely localize to cerebellar and/or basal ganglia dysfunction. High-resolution retinal and brain imaging in NARP syndrome revealed analogous patterns of tissue injury characterized by heterogeneous areas of neuronal loss.

Functional effects of cancer mitochondria on energy metabolism and tumorigenesis: utility of transmitochondrial cybrids.

Reprogramming of energy metabolism is one of the hallmarks of cancer. In normal conditions, cells rely on mitochondrial oxidative phosphorylation to provide energy for cellular activities. Cancer cells are characterized by increased glycolysis and reduced mitochondrial respiratory function. In the past decade, somatic mitochondrial DNA alterations are found to be common in all types of cancers. However, the functional significance of the altered cancer mitochondria is largely unknown. This is because the bulk of cancer properties are regulated by nuclear encoded genes. To overcome this problem, the transmitochondrial cybrid system, which allows the study of the effect of cancer mitochondria in a common nuclear background, has been used. Here we review the accumulating evidence that altered cancer mitochondria affect the respiratory chain function and oncogenic properties in vitro and in vivo using cybrid technologies.

Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis.

The molecular and cellular mechanisms underlying nephropathic cystinosis, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. Renal biopsies from patients with this disorder can reveal abnormally large mitochondria, but the relevance of this and other ultrastructural abnormalities is unclear. We studied the ultrastructure of fibroblasts and renal proximal tubular epithelial cells from patients with three clinical variants of cystinosis: Nephropathic, intermediate, and ocular. Electron microscopy revealed the presence of morphologically abnormal mitochondria and abnormal patterns of mitochondrial autophagy (mitophagy) with a high number of autophagic vacuoles and fewer mitochondria (P < 0.02) in nephropathic cystinosis. In addition, we observed increased apoptosis in renal proximal tubular epithelial cells, greater expression of LC3-II/LC3-I (microtubule-associated protein 1 light chain 3), and significantly more autophagosomes in the nephropathic variant. The autophagy inhibitor 3-methyl adenine rescued cell death in cystinotic cells. Cystinotic cells had increased levels of beclin-1 and aberrant mitochondrial function with a significant decrease in ATP generation and an increase in reactive oxygen species. This study provides ultrastructural and functional evidence of abnormal mitophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.