Deafness-associated tRNAPhe mutation impaired mitochondrial and cellular integrity.

Defects in mitochondrial RNA metabolism have been linked to sensorineural deafness that often occurs as a consequence of damaged or deficient inner ear hair cells. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAPhe 593T > C mutation that changed a highly conserved uracil to cytosine at position 17 of the DHU-loop. The m.593T > C mutation altered tRNAPhe structure and function, including increased melting temperature, resistance to S1 nuclease-mediated digestion, and conformational changes. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced by decreases in levels of ND1, ND5, CYTB, CO1, and CO3 harboring higher numbers of phenylalanine. These alterations resulted in aberrant assembly, instability, and reduced activities of respiratory chain enzyme complexes I, III, IV, and intact supercomplexes overall. Furthermore, we found that the m.593T > C mutation caused markedly diminished membrane potential, and increased the production of reactive oxygen species in the mutant cell lines carrying the m.593T > C mutation. These mitochondrial dysfunctions led to the mitochondrial dynamic imbalance via increasing fission with abnormal mitochondrial morphology. Excessive fission impaired the process of autophagy including the initiation phase, formation, and maturation of the autophagosome. In particular, the m.593T > C mutation upregulated the PARKIN-dependent mitophagy pathway. These alterations promoted an intrinsic apoptotic process for the removal of damaged cells. Our findings provide critical insights into the pathophysiology of maternally inherited deafness arising from tRNA mutation-induced defects in mitochondrial and cellular integrity.

Optimized allotopic expression of mitochondrial ND6 transgene restored complex I and apoptosis deficiencies caused by LHON-linked ND6 14484T > C mutation.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mutations in mitochondrial DNA. However, there is no effective treatment for this disease. LHON-linked ND6 14484T > C (p.M64V) mutation caused complex I deficiency, diminished ATP production, increased production of reactive oxygen species (ROS), elevated apoptosis, and impaired mitophagy. Here, we investigated if the allotopic expression of human mitochondrial ND6 transgene corrected the mitochondrial dysfunctions due to LHON-associated m.14484T > C mutation. METHODS: Nucleus-versions of ND6 was generated by changing 6 non-universal codons with universal codons and added to mitochondrial targeting sequence of COX8. Stable transfectants were generated by transferring human ND6 cDNA expressed in a pCDH-puro vector into mutant cybrids carrying the m.14484T > C mutation and control cybrids. The effect of allotopic expression of ND6 on oxidative phosphorylation (OXPHOS) was evaluated using Blue Native gel electrophoresis and extracellular flux analyzer. Assessment of ROS production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analyses for apoptosis and mitophagy were undertaken via flow cytometry, TUNEL and immunofluorescence assays. RESULTS: The transfer of human ND6 into the cybrids carrying the m.14484T > C mutation raised the levels of ND6, ND1 and ND4L but did not change the levels of other mitochondrial proteins. The overexpression of ND6 led to 20~23% increases in the assembly and activity of complex I, and ~ 53% and ~ 33% increases in the levels of mitochondrial ATP and ΔΨm in the mutant cybrids bearing m.14484T > C mutation. Furthermore, mutant cybrids with overexpression of ND6 exhibited marked reductions in the levels of mitochondrial ROS. Strikingly, ND6 overexpression markedly inhibited the apoptosis process and restored impaired mitophagy in the cells carrying m.14484T > C mutation. However, overexpression of ND6 did not affect the ND6 level and mitochondrial functions in the wild-type cybrids, indicating that this ND6 level appeared to be the maximum threshold level to maintain the normal cell function. CONCLUSION: We demonstrated that allotopic expression of nucleus-versions of ND6 restored complex I, apoptosis and mitophagy deficiencies caused by the m.14484T > C mutation. The restoration of m.14484T > C mutation-induced mitochondrial dysfunctions by overexpression of ND6 is a step toward therapeutic interventions for LHON and mitochondrial diseases.

Leber's hereditary optic neuropathy-associated ND6 14484T > C mutation caused pleiotropic effects on the complex I, RNA homeostasis, apoptosis and mitophagy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mitochondrial DNA (mtDNA) mutations. LHON-linked ND6 14484T > C (p.M64V) mutation affected structural components of complex I but its pathophysiology is poorly understood. The structural analysis of complex I revealed that the M64 forms a nonpolar interaction Y59 in the ND6, Y59 in the ND6 interacts with E34 of ND4L, and L60 of ND6 interacts with the Y114 of ND1. These suggested that the m.14484T > C mutation may perturb the structure and function of complex I. Mutant cybrids constructed by transferring mitochondria from lymphoblastoid cell lines of one Chinese LHON family into mtDNA-less (ρo) cells revealed decreases in the levels of ND6, ND1 and ND4L. The m.14484T > C mutation may affect mitochondrial mRNA homeostasis, supported by reduced levels of SLIRP and SUPV3L1 involved in mRNA degradation and increasing expression of ND6, ND1 and ND4L genes. These alterations yielded decreased activity of complex I, respiratory deficiency, diminished mitochondrial ATP production and reduced membrane potential, and increased production of reactive oxygen species in the mutant cybrids. Furthermore, the m.14484T > C mutation promoted apoptosis, evidenced by elevating Annexin V-positive cells, release of cytochrome c into cytosol, levels in apoptotic proteins BAX, caspases 3, 7, 9 and decreasing levels in anti-apoptotic protein Bcl-xL in the mutant cybrids. Moreover, the cybrids bearing the m.14484T > C mutation exhibited the reduced levels of autophagy protein LC3, increased levels of substrate P62 and impaired PINK1/Parkin-dependent mitophagy. Our findings highlighted the critical role of m.14484T > C mutation in the pathogenesis of LHON.

Mitochondrial tRNA variants in 811 Chinese probands with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is the maternal inheritance of eye disorder. LHON-linked mitochondrial DNA (mtDNA) mutations affect the ND1, ND4 or ND6 genes encoding essential subunits of complex I. However, the role of mitochondrial tRNA defects in the pathogenesis of LHON is poorly understood. In this report, Sanger sequence analysis of 22 mitochondrial tRNA genes identified 139 variants in a cohort of 811 Han Chinese probands and 485 control Chinese subjects. Among these, 32 (4 known and 28 novel/putative) tRNA variants in 71 probands may contribute to pathogenesis of LHON, as these exhibited (1) present in < 1% of controls; (2) evolutionary conservation; (3) potential and significance of structural and functional modifications. Such variants may have potentially compromised structural and functional aspects in the processing of tRNAs, structure stability, tRNA charging, or codon-anticodon interactions during translation. These 32 variants presented either singly or with multiple mutations, with the primary LHON-linked ND1 3640G > A, ND4 11778G > A or ND6 14484 T > C mutations in the probands. The thirty-eight pedigrees carrying only one of tRNA variants exhibited relatively low penetrances of LHON, ranging from 5.7% to 42.9%, with an average of 19%. Strikingly, the average penetrances of optic neuropathy among 33 Chinese families carrying both a known/putative tRNA variant and a primary LHON-associated mtDNA mutation were 40.1%. These findings suggested that mitochondrial tRNA variants represent a significant causative factor for LHON, accounting for 8.75% cases in this cohort. These new insights may lead to beneficial applications in the pathophysiology, disease management, and genetic counseling of LHON.

Atypical Giant Suprasellar Prolactinoma Presenting With Visual Field Changes in the Absence of Symptoms of Hyperprolactinemia.

Prolactinomas are benign tumors that make up the majority of all pituitary adenoma cases and present most commonly in women. Prolactinomas presenting in adolescents and children, however, are extremely rare. We report a case of a 17-year-old male who presented with a six-month history of headaches and a previously unrecognized visual field deficit on examination. Neuroimaging revealed a large suprasellar tumor with imaging, more characteristic of a craniopharyngioma or suprasellar low-grade glioma impinging, on the left intracranial optic nerve causing right-sided hemianopsia. Due to the extensive mass effect and bitemporal hemianopsia on examination, the decision to proceed with initial surgical debulking was made following informed consent. A subtotal resection was performed where the pathology was consistent with a prolactinoma that correlated with markedly elevated prolactin (PRL) levels obtained pre and post-operatively that have not resulted until five days post procedure. The patient was subsequently treated with dopamine agonist (DA) cabergoline therapy and is now five-years disease-free with normal neurological examination and no residual tumor on neuroimaging. DA therapy has shown high clinical efficacy and should be considered prior to any surgical intervention; however, extensive mass effect may appropriate surgical debulking to increase therapy efficacy. Our case highlights an atypical appearance of a giant prolactinoma that may mimic other more common suprasellar tumors, a presentation associated with unrecognized visual field deficits, and the importance of rapid turnaround testing for serum PRL that may aid in the upfront diagnosis and management of prolactinomas.

Assocation Between Leber's Hereditary Optic Neuropathy and MT-ND1 3460G>A Mutation-Induced Alterations in Mitochondrial Function, Apoptosis, and Mitophagy.

Purpose: To investigate the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation. Methods: Cybrid cell models were generated by fusing mitochondrial DNA-less ρ0 cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis. Results: Nineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy. Conclusions: Our findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.

Leber's Hereditary Optic Neuropathy Arising From the Synergy Between ND1 3635G>A Mutation and Mitochondrial YARS2 Mutations.

Purpose: To investigate the mechanism underlying the synergic interaction between Leber's hereditary optic neuropathy (LHON)-associated ND1 and mitochondrial tyrosyl-tRNA synthetase (YARS2) mutations. Methods: Molecular dynamics simulation and differential scanning fluorimetry were used to evaluate the structure and stability of proteins. The impact of ND1 3635G>A and YARS2 p.G191V mutations on the oxidative phosphorylation machinery was evaluated using blue native gel electrophoresis and enzymatic activities assays. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analysis of effect of mutations on autophagy was undertaken via flow cytometry for autophagic flux. Results: Members of one Chinese family bearing both the YARS2 p.191Gly>Val and m.3635G>A mutations exhibited much higher penetrance of optic neuropathy than those pedigrees carrying only the m.3635G>A mutation. The m.3635G>A (p.Ser110Asn) mutation altered the ND1 structure and function, whereas the p.191Gly>Val mutation affected the stability of YARS2. Lymphoblastoid cell lines harboring both m.3635G>A and p.191Gly>Val mutations revealed more reductions in the levels of mitochondrion-encoding ND1 and CO2 than cells bearing only the m.3635G>A mutation. Strikingly, both m.3635G>A and p.191Gly>Val mutations exhibited decreases in the nucleus-encoding subunits of complex I and IV. These deficiencies manifested greater defects in the stability and activities of complex I and complex IV and overproduction of ROS and promoted greater autophagy in cell lines harboring both m.3635G>A and p.191Gly>Val mutations compared with cells bearing only the m.3635G>A mutation. Conclusions: Our findings provide new insights into the pathophysiology of LHON arising from the synergy between ND1 3635G>A mutation and mitochondrial YARS2 mutations.

Leiomyosarcoma in Birt-Hogg-Dubé Syndrome.

Birt-Hogg-Dubé syndrome (BHDS) is an autosomal dominant genodermatosis characterized by benign cutaneous tumors (fibrofolliculomas, trichodiscomas, and acrochordons), basal lung cysts, pneumothoraces, and a 20% to 30% lifetime risk for renal cancer. There are isolated cases of other cancers in BHDS reported in the literature, including oncocytoma, rhabdomyoma, melanoma, thyroid cancer, meningioma, colon cancer, and breast cancer, but only the increased renal cancer risk has been substantiated. This is the case of a 9-year-old girl who presented with a leiomyosarcoma whose tumor genetic analysis showed FLCN c.365_372del, p.Arg122Leufs*8. She was diagnosed with BHDS when the same mutation was confirmed in her germline lymphocytes. This is the second known reported case of leiomyosarcoma in BHDS.

Undescended retropharyngeal parathyroid adenoma with adjacent thymic tissue in a 13-year-old boy with primary hyperparathyroidism.

We describe a rare presentation of a symptomatic parathyroid adenoma located in an ectopic retropharyngeal position in a 13-year-old boy. Preoperative CT scan and MRI demonstrated the ectopic location of the parathyroid adenoma. The patient underwent successful parathyroidectomy with cure of his hyperparathyroidism. On pathologic exam, the specimen was made up of a parathyroid adenoma and adjacent thymic tissue, indicating that it was likely an undescended lower parathyroid gland arising from the third pharyngeal pouch. Ectopic retropharyngeal parathyroid adenomas are very rare and to our knowledge, none have been previously described in adolescents.

Relationship between Rosai-Dorfman disease and IgG4-related disease: study of 32 cases.

OBJECTIVES: To assess the association between Rosai-Dorfman disease (RDD) and IgG4-related disease (IgG4-RD). METHODS: We studied the number of IgG4-positive plasma cells and the IgG4/IgG ratio in 32 biopsy specimens (13 nodal, 19 extranodal) from 29 patients with RDD and compared the findings with those in IgG4-RD of the pancreas and reactive lymph nodes. We also assessed the number of FOXP3-positive regulatory T cells (Tregs) since they were reported to be increased in IgG4-RD. RESULTS: We found that RDD cases had much lower numbers of IgG4-positive plasma cells and lower IgG4/IgG ratios compared with IgG4-RD but were similar to reactive lymph nodes. Furthermore, RDD had lower numbers of FOXP3-positive Tregs than did IgG4-RD. There were no significant differences in the number of IgG4-positive plasma cells and the IgG4/IgG ratio between the nodal and extranodal RDD cases. CONCLUSIONS: Our study suggests that RDD does not belong in the spectrum of IgG4-RD.

Salvage therapy of refractory hemophagocytic lymphohistiocytosis with alemtuzumab.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome that remains difficult to treat. Even with current standard HLH therapy, only approximately half of patients will experience complete resolution of disease, and early mortality remains a significant problem. Salvage therapies have been described only in limited case reports, and there are no large studies of second-line therapies. PROCEDURE: We reviewed the charts of 22 pediatric and adult patients who received alemtuzumab for the treatment of refractory HLH at our center or in consultation with our group. RESULTS: Patients had received conventional therapies for a median of 8 weeks (range: 2-70) prior to alemtuzumab, and treatment immediately prior to alemtuzumab included dexamethasone (100%), etoposide (77%), cyclosporine (36%), intrathecal hydrocortisone ± methotrexate (23%), methylprednisolone (9%), and rituximab (14%). Patients received a median dose of 1 mg/kg alemtuzumab (range: 0.1-8.9 mg/kg) divided over a median of 4 days (range: 2-10). Fourteen patients experienced an overall partial response, defined as at least a 25% improvement in two or more quantifiable symptoms or laboratory markers of HLH 2 weeks following alemtuzumab (64%). Five additional patients had a 25% or greater improvement in a single quantifiable symptom or laboratory marker of HLH (23%). Seventy-seven percent of patients survived to undergo allogeneic hematopoietic cell transplantation. Patients experienced an acceptable spectrum of complications, including CMV and adenovirus viremia. CONCLUSION: Alemtuzumab appears to be an effective salvage agent for refractory HLH, leading to improvement and survival to HCT in many patients. Prospective trials to define optimal dosing levels, schedules, and responses are needed.

Focal nodular hyperplasia in children, adolescents, and young adults.

BACKGROUND: Focal nodular hyperplasia (FNH) is a benign hepatic tumor that is rare in children. In order to understand whether there are differences in the etiology or appearance of FNH in children, we analyzed the clinical information and imaging of pathologically proven cases. MATERIALS AND METHODS: A pathology database was used to identify all cases of FNH diagnosed at our institution. Each patient's imaging was evaluated for the characteristics of FNH lesions. Clinical information was obtained on each patient. RESULTS: Thirteen patients with FNH were identified (7 male/6 female, mean age 14.3 years, range 1-27 years). Seven patients (5 male/2 female) had a remote history of childhood malignancy. The time interval between the diagnoses of malignancy and FNH ranged from 9 to 27 years (mean 14.4 years). On imaging, all seven cancer survivors had multiple liver lesions. In the remaining six patients (2 male/4 female), there was no history of malignancy and all but one of these patients had a solitary FNH. CONCLUSION: Half of the patients with FNH in this study were long-term cancer survivors and each of these patients had multiple masses. Recognizing the features of FNH will aid in diagnosis and appropriate management.

Immature cell populations and an erythropoiesis gene-expression signature in systemic juvenile idiopathic arthritis: implications for pathogenesis.

INTRODUCTION: Previous observations suggest that active systemic juvenile idiopathic arthritis (sJIA) is associated with a prominent erythropoiesis gene-expression signature. The aim of this study was to determine the association of this signature with peripheral blood mononuclear cell (PBMC) subpopulations and its specificity for sJIA as compared with related conditions. METHODS: The 199 patients with JIA (23 sJIA and 176 non-sJIA) and 38 controls were studied. PBMCs were isolated and analyzed for multiple surface antigens with flow cytometry and for gene-expression profiles. The proportions of different PBMC subpopulations were compared among sJIA, non-sJIA patients, and controls and subsequently correlated with the strength of the erythropoiesis signature. Additional gene-expression data from patients with familial hemophagocytic lymphohistiocytosis (FHLH) and from a published sJIA cohort were analyzed to determine whether the erythropoiesis signature was present. RESULTS: Patients with sJIA had significantly increased proportions of immature cell populations, including CD34+ cells, correlating highly with the strength of the erythropoiesis signature. The erythropoiesis signature strongly overlapped with the gene-expression pattern in purified immature erythroid precursors. The expansion of immature cells was most prominently seen in patients with sJIA and anemia, even in the absence of reticulocytosis. Patients with non-sJIA and anemia did not exhibit the erythropoiesis signature. The erythropoiesis signature was found to be prominent in patients with FHLH and in a published cohort of patients with active sJIA, but not in patients with inactive sJIA. CONCLUSIONS: An erythropoiesis signature in active sJIA is associated with the expansion of CD34+ cells, also is seen in some patients with FHLH and infection, and may be an indicator of ineffective erythropoiesis and hemophagocytosis due to hypercytokinemia.

Pathology of the liver in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis is a rare, rapidly progressive disorder characterized by an activation of the immune system resulting in a systemic proliferation of lymphocytes and histiocytes. The disease is genetically heterogeneous and maps to at least 4 loci including the gene encoding perforin, a protein critical for the cytotoxic and regulatory functions of T lymphocytes and natural killer (NK) cells. Hepatic dysfunction often occurs early in the clinical course, but the pathology of the liver is not well characterized. The clinical history, laboratory data, and pathologic material (25 hepatic specimens) from 19 children (11 boys, 7 girls, 1 unknown, 12 d to 11 mo of age, median 3 mo) with FHL were reviewed. Routine and immunohistochemical stains were carried out in all cases, and perforin gene sequencing in a subset. Common to all specimens was a portal and sinusoidal infiltrate of CD3, CD8, granzyme B+ lymphocytes admixed with CD68, CD1a- histiocytes that exhibited hemophagocytosis. There was endothelialitis of portal and central veins and lymphocyte-mediated bile duct injury. The degree of portal and sinusoidal lymphohistiocytic infiltrate and endothelialitis varied from mild to marked and correlated with clinical severity. In some specimens, histiocytic cells predominated and in others, there was extensive hepatocellular giant cell transformation. Accordingly, 4 histopathologic patterns were observed: (1) chronic hepatitis-like, (2) leukemia-like, (3) histiocytic storage disorder-like, and (4) neonatal giant cell hepatitis-like. Two siblings homozygous for a 50delT nucleotide deletion had no perforin immunoreactive cells, 1 compound heterozygote for a deletion and missense mutation had cells with markedly diminished perforin expression, and 1 infant hemizygous for a perforin missense mutation had intact expression. Recognizing the morphologic changes in the liver and the immunophenotypic features of the infiltrate are critical for a rapid diagnosis and a prompt institution of treatment.

Pediatric hepatic angiosarcoma: case report and review of the literature.

Pediatric hepatic angiosarcoma (PHAS) is a rare tumor, which usually presents as a rapid enlargement of the liver. To date, surgery, chemotherapy, and radiotherapy have not improved the poor prognosis of PHAS with only three survivors reported. The histology of PHAS is distinct from adult angiosarcoma, because PHAS displays hypercellular whorls of sarcomatous cells, or "kaposiform" spindle cells, in addition to the general features of angiosarcoma. We report a case of PHAS that was treated with vascular ablation, chemotherapy, and liver transplantation. Lung metastases occurred 14 months posttransplant.

MALT Lymphoma in Children: Case Report and Review of the Literature.

Mucosa-associated lymphoid tissue (MALT) lymphoma predominantly occurs in adults, and is rare in children. We report a case of MALT lymphoma involving minor salivary gland of the lip in an otherwise healthy 12-year-old boy. This is the second case report of MALT lymphoma of minor salivary gland in an immunocompetent child. Of 24 cases of MALT lymphomas in children reported in the English literature, parotid MALT lymphomas in human immunodeficiency virus (HIV) patients and H. pylori infection-associated gastric MALT lymphomas are the most common. As in adult cases, most MALT lymphomas in the pediatric age group are localized and follow an indolent clinical course, respond well to therapy, and have an excellent outcome.

GLUT1 endothelial reactivity distinguishes hepatic infantile hemangioma from congenital hepatic vascular malformation with associated capillary proliferation.

Hepatic vascular lesions in pediatric patients have overlapping definitions and a plethora of confusing terminology. The so-called hepatic infantile hemangioendothelioma (IHE) frequently coexists and shares some biological features with cutaneous juvenile hemangioma (CJH). To clarify the nature of hepatic vascular lesions in pediatric patients and to investigate the association between IHE and CJH, we reviewed the clinical features, imaging findings and histopathology of 19 cases of hepatic vascular lesions diagnosed at our institution over the last 33 years. Immunohistochemical stains for a battery of endothelial markers, including GLUT1, were performed. Our results indicate that there are two fundamentally different hepatic vascular lesions in infants and young children: GLUT1-positive hepatic infantile hemangioma (HIH) and GLUT1-negative hepatic vascular malformation with capillary proliferation (HVMCP). The finding of consistent GLUT1 immunoreactivity of endothelial cells in HIH not only offers a powerful tool to distinguish HIH from HVMCP, but also provides immunophenotypic evidence of the similar biological origins of CJH and HIH.