Instability of the mitochondrial alanyl-tRNA synthetase underlies fatal infantile-onset cardiomyopathy.

Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.

MYOD1 as a prognostic indicator in rhabdomyosarcoma.

BACKGROUND/OBJECTIVES: Rhabdomyosarcoma (RMS) is characterized by the expression of the myogenic regulatory protein MYOD1. Histologic types include alveolar, embryonal (ERMS), and spindle cell sclerosing RMS (SRMS). SRMS harbors MYOD1 mutations in a subset of adult cases in association with poor prognosis. DESIGN/METHODS: To study the level of MYOD1 protein expression and its clinical significance, we have analyzed variable numbers of pediatric (<18 years of age) and adult (age range ≥18 to 35 years) ERMS and SRMS cases for presence or absence of MYOD1 immunoreactivity in correlation with clinical outcome and MYOD1 L122R mutations. RESULTS: Lack of MYOD1 immunoreactivity, identified in 23.8% of nonalveolar RMS (non-ARMS) cases, was more prevalent in SRMS (44%) than ERMS (17.2%) and was significantly associated with low overall survival and unfavorable tumor sites (p < .05). Lack of MYOD1 immunoreactivity was not associated with MYOD1 L122R mutations, which were identified in 3/37 (8%) cases including only two of 31 (6.5%) pediatric cases, one of 11 or 9% pediatric SRMS, and one case of infant ERMS. CONCLUSION: These studies highlight the prognostic role of MYOD1 in non-ARMS. Lack of MYOD1 immunoreactivity is associated with poor prognosis in ERMS and SRMS. MYOD1 gene mutations are generally infrequent in pediatric RMS. Although mutations are predominant in SRMS, they may exceptionally occur in infantile ERMS.

Arthrogryposis and pterygia as lethal end manifestations of genetically defined congenital myopathies.

Arthrogryposis multiplex congenita affects approximately 1 in 3,000 individuals of different ethnic backgrounds and displays an equal incidence in males and females. The underlying mechanism for congenital contracture of the joints is decreased fetal movement during intrauterine development. This disorder is associated with over 400 medical conditions and 350 known genes that display considerable variability in phenotypic expression. In this report, four fetal or perinatal autopsy cases of arthrogryposis were studied by gross morphology, microscopic histopathologic examination, and whole genome sequencing of postmortem DNA. Two stillborn sibling fetuses with arthrogryposis, pterygia, and amyoplasia had compound heterozygous pathogenic variants in NEB. A neonate with a histopathologic diagnosis of nemaline myopathy had a heterozygous de novo pathogenic variant in ACTA1. Another stillborn infant with pterygia and arthrogryposis had a heterozygous de novo likely pathogenic variant in BICD2. These cases demonstrate the utility of whole genome sequencing as the principal diagnostic method of lethal forms of skeletal muscle disorders that present with arthrogryposis and muscle amyoplasia/hypoplasia. Molecular diagnosis provides an opportunity for studying patterns of inheritance and for family counseling concerning future pregnancies.

NanoString Digital Molecular Profiling of Protein and microRNA in Rhabdomyosarcoma.

PURPOSE: Rhabdomyosarcoma (RMS) exhibits a complex prognostic algorithm based on histologic, biologic and clinical parameters. The embryonal (ERMS) and spindle cell-sclerosing RMS (SRMS) histologic subtypes warrant further studies due to their heterogenous genetic background and variable clinical behavior. NanoString digital profiling methods have been previously highlighted as robust novel methods to detect protein and microRNA expression in several cancers but not in RMS. METHODS/PATIENTS: To identify prognostic biomarkers, we categorized 12 ERMS and SRMS tumor cases into adverse (n = 5) or favorable (n = 7) prognosis groups and analyzed their signaling pathways and microRNA profiles. The digital spatial profiling of protein and microRNA analysis was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue using NanoString technology. RESULTS: The detectable expression of several component members of the PI3K/AKT, MAPK and apoptosis signaling pathways was highlighted in RMS, including INPP4B, Pan-AKT, MET, Pan-RAS, EGFR, phospho-p90 RSK, p44/42 ERK1/2, BAD, BCL-XL, cleaved caspase-9, NF1, PARP and p53. Compared to cases with favorable prognosis, the adverse-prognosis tumor samples had significantly increased expression of INPP4B, which was confirmed with traditional immunohistochemistry. The analysis of microRNA profiles revealed that, out of 798 microRNAs assessed, 228 were overexpressed and 134 downregulated in the adverse prognosis group. Significant over-expression of oncogenic/tumor suppressor miR-3144-3p, miR-612, miR-302d-3p, miR-421, miR-548ar-5p and miR-548y (p < 0.05) was noted in the adverse prognosis group. CONCLUSION: This study highlights the utility of NanoString digital profiling methods in RMS, where it can detect distinct molecular signatures with the expression of signaling pathways and microRNAs from FFPE tumor tissue that may help identify prognostic biomarkers of interest. The overexpression of INPP4B and miR-3144-3p, miR-612, miR-302d-3p, miR-421, miR-548y and miR-548ar-5p may be associated with worse overall survival in ERMS and SRMS.

Molecular monitoring of 8p11 myeloproliferative syndrome in an infant.

The 8p11 myeloproliferative syndrome is a rare hematologic malignancy derived from a pluripotent hematopoietic stem cell associated with rearrangements involving the fibroblast growth factor receptor 1 (FGFR1) gene located on chromosome 8p11. The most common translocation, t(8;13) (p11;q13), results in a ZNF198-FGFR1 fusion gene and constitutively active FGFR1 tyrosine kinase activity. Typical pathologic findings include myeloid hyperplasia, lymphadenopathy, precursor T-lymphoblastic lymphoma, and eosinophilia. The disease is usually associated with an aggressive course and progression to acute myeloid leukemia is frequent. We report here the first case of 8p11 myeloproliferative syndrome in an infant and demonstrate the value of molecular testing in the diagnosis and minimal disease monitoring of this rare disease.

Potential Value of YAP Staining in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a common malignancy of soft tissue, subclassified as alveolar (ARMS), pleomorphic (PRMS), spindle cell/sclerosing (SRMS), and embryonal (ERMS) types. The Yes-associated protein (YAP) is a member of the Hippo pathway and a transcriptional regulator that controls cell proliferation. We have studied the immunohistochemical expression of YAP in different RMSs, arranged in tissue microarray (TMA) and whole slide formats. Pertinent clinical data including patient age, gender, tumor location, and clinical stage were collected. Out of 96 TMA cases, 30 cases (31%) were pleomorphic, 27 (28%) were embryonal, 24 (25%) alveolar, and 15 (16%) spindle cell. Positive nuclear YAP staining was seen in the PRMS (17/30, 56.7%), SRMS (7/15, 46.7%), ERMS (19/27 or 70%), and less in ARMS (37.5%). YAP nuclear staining was significantly more prevalent in ERMS than ARMS ( p=0.02). Of the 41 whole slide cases, nuclear staining was detected in all ARMS but was restricted in distribution to <30% of the cells, in contrast to ERMS and SRMS, which had diffuse or >30% staining. These results highlight the role of YAP in RMS tumorigenesis, a fact that can be useful in engineering targeted therapy. Restricted nuclear YAP staining (<30% of cells) may be of value in the diagnosis of ARMS.

The Rare Association of Spina Bifida and Extrarenal Wilms Tumor: A Case Report and Review of the Literature.

BACKGROUND: The diagnosis of nephroblastoma outside of the kidneys, in the absence of a renal primary tumor, is known as extrarenal Wilms tumor (ERWT). ERWT is an uncommon entity that typically involves the embryonic path of the developing kidneys and gonads. The occurrence of ERWT in a dysraphic spine is uncommon, with no reported cases of preoperative diagnosis, with all cases diagnosed at pathology. These tumors are malignant and ideally should be completely excised. Thus, preoperative diagnosis would be highly desirable. CASE DESCRIPTION: A newborn female was found to have a lumbar lipoma. Magnetic resonance imaging (MRI) was performed to rule out lipomyelomeningocele. The MRI showed a dorsal lipoma on the terminal spinal cord, as well as a 2 × 2 cm uniformly enhancing mass abutting the bifid posterior elements of L5. The lesion was completely excised, and the pathological diagnosis was ERWT. We report this case with a review of the literature to raise awareness of this association, illustrate the key imaging findings, and document the clinical outcome. CONCLUSIONS: The lack of pathognomonic radiologic features makes the preoperative diagnosis extremely difficult, but a diagnosis of ERWT should be considered in the context of a soft tissue mass without the typical imaging features of a hemangioma or teratoma.

Increase in bile flow and biliary excretion of glutathione-derived sulfhydryls in rats by drug-metabolizing enzyme inducers is mediated by multidrug resistance protein 2.

Glutathione (GSH) is an important cellular constituent for normal liver homeostasis. Certain drug-metabolizing enzyme inducers (i.e., phenobarbital [PB] and pregnenolone-16alpha-carbonitrile [PCN]) increase biliary excretion of GSH-derived sulfhydryls (SH) as well as bile flow, whereas other drug-metabolizing enzyme inducers (i.e., 3-methylcholanthrene [3MC] and benzo(a)pyrene [BaP]), do not. The purpose of the study was to determine whether rat multidrug resistance protein 2 (Mrp2) is the inducible transporter responsible for increasing biliary SH excretion and bile flow. Sprague-Dawley (SD) rats were injected ip daily for 4 days with PB, PCN, 3MC, BaP, or vehicle; Mrp2-null Eisai hyperbilirubinemic (EHBR) rats were injected ip daily for 4 days with PCN or vehicle. Although no drug-metabolizing enzyme inducer altered hepatic GSH in SD rats, PB and PCN significantly increased the rate of biliary SH excretion and bile flow. Neither 3MC nor BaP affected the biliary SH excretion rate or bile flow. In control EHBR rats, despite elevated hepatic GSH, the rate of biliary SH excretion was almost completely eliminated and bile flow was dramatically reduced compared with SD rats. Furthermore, PCN treatment did not affect bile flow or the biliary SH excretion rate in EHBR rats. PB and PCN also increased Mrp2 protein levels, but 3MC and BaP did not. None of the drug-metabolizing enzyme inducers tested significantly increased Mrp2 mRNA levels. PCN increased Mrp2 protein, but not Mrp2 mRNA, in a time-dependent manner. In conclusion, Mrp2 is the inducible efflux transporter responsible for increased biliary SH excretion and bile flow after administration of some drug-metabolizing enzyme inducers.

Promotion of thyroid tumors in rats by pregnenolone-16alpha-carbonitrile (PCN) and polychlorinated biphenyl (PCB).

Pregnenolone-16alpha-carbonitrile (PCN) and Aroclor 1254 (PCB) both reduce serum thyroid hormone levels in rats, but only PCN consistently produces an increase in serum thyrotropin (TSH). PCN-mediated increases in TSH result in increased thyroid follicular cell proliferation and hyperplasia, which may represent early events on a morphological continuum leading to neoplasia. The purpose of this study was to assess whether PCN, a compound that increases serum TSH, and PCB, which does not increase TSH, promote thyroid tumors in a two-stage carcinogenesis model. Male SD rats were administered the thyroid tumor initiator diisopropanolnitrosamine (2.5 g/kg, sc), and after seven days were fed control diet, diet containing 1000 ppm PCN, or diet containing 100 ppm PCB for 19 weeks. Body weights were unaffected by PCN treatment, but were reduced 21% after 19 weeks of PCB treatment compared to control. PCN treatment significantly reduced serum T4 through week 3 before returning to control concentrations, whereas T4 levels following PCB treatment fell below detection limits by week 3 and remained drastically reduced through week 19. TSH concentrations in PCN-treated rats increased three-fold at week 2, then declined to near control values at week 19. After one week of PCB treatment, TSH concentrations reached nearly twice that of controls, and were sustained until week 6. The incidence of thyroid follicular cell proliferative lesions, including cystic and follicular hyperplasia, cystic and follicular adenoma, and follicular carcinoma, was significantly increased following PCN treatment, but not following PCB treatment. PCB treatment caused an increase in thyroid carcinomas (4 of 22 rats) not associated with the proliferative-type lesions produced by PCN, despite an increase in TSH serum concentrations. In conclusion, PCN appears to promote thyroid tumors in a manner consistent with known effects of excessive TSH stimulation. However, thyroid carcinomas stemming from PCB treatment indicate that separate mechanisms exist for the production of thyroid cancer in rodents by chemicals classically considered microsomal enzyme inducers.

Testicular toxicity of di-(2-ethylhexyl)phthalate in young Sprague-Dawley rats.

Di-(2-ethylhexyl)phthalate (DEHP), used widely in the manufacture of plastics, is a well-known reproductive toxicant. It causes apoptosis and loss of spermatogenic cells, resulting in testicular atrophy. Reports are scarce in the literature on the progression of apoptosis following repeated doses of phthalates. DEHP's mechanism of inducing testicular atrophy has been associated with depletion of zinc in the testis. ZnT-1 is a zinc transporter that is highly expressed in the testis. Thus, DEHP might exert its toxic effects on the testis by altering the expression of ZnT-1. In this regard, 25-day old Sprague-Dawley rats were given vehicle (5 ml corn-oil/kg, po) for 2, 7 and 14 days, or DEHP (2 g/5 ml corn-oil/kg, po) daily, for 1, 2, 3, 5, 7, 10 and 14 days. Zinc content in testes was determined by atomic absorption spectrophotometry, and ZnT-1 mRNA was quantified by the branched DNA signal amplification method. Body weight gain and testicular weight (absolute and relative) were significantly lower in DEHP-treated rats. DEHP produced morphological changes in the testis, including apoptosis, necrosis, and loss of spermatogenic cells, which resulted in testicular atrophy. Apoptotic index (AI: the percentage of apoptotic cells in seminiferous tubules), determined using the TUNEL technique, was markedly increased after 1 day (AI: 2.9%, control AI: 0.1-0.3%) followed by a peak at 3 days (AI: 11.5%) and a gradual decrease till 10-14 days (AI: 7-9%). Zinc content in testis was not changed 1 day after DEHP administration, but decreased significantly at later time points. No difference was found in ZnT-1 mRNA expression between control and DEHP-treated animals until day 14. Our results suggest that apoptosis, along with necrosis, plays an important role in the mechanism of testicular atrophy by DEHP. In addition, ZnT-1 mRNA expression was not altered by DEHP and therefore, it appears that ZnT-1 cannot account for the decrease in testicular Zn content. Pathological lesions and apoptosis occurred prior to the loss of zinc in testis, suggesting that zinc depletion might be a secondary effect of DEHP-induced testicular toxicity, rather than the cause.