Stella Mitrani-Rosenbaum

The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM; OMIM 600737) is a unique group of neuromuscular disorders characterized by adult onset, slowly progressive distal and proximal weakness and a typical muscle pathology including rimmed vacuoles and filamentous inclusions. The autosomal recessive form described in Jews of Persian descent is the HIBM prototype. This myopathy affects mainly leg muscles, but with an unusual distribution that spares the quadriceps. This particular pattern of weakness distribution, termed quadriceps-sparing myopathy (QSM), was later found in Jews originating from other Middle Eastern countries as well as in non-Jews. We previously localized the gene causing HIBM in Middle Eastern Jews on chromosome 9p12–13 (ref. 5) within a genomic interval of about 700 kb (ref. 6). Haplotype analysis around the HIBM gene region of 104 affected people from 47 Middle Eastern families indicates one unique ancestral founder chromosome in this community. By contrast, single non-Jewish families from India, Georgia (USA) and the Bahamas, with QSM and linkage to the same 9p12–13 region, show three distinct haplotypes. After excluding other potential candidate genes, we eventually identified mutations in the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene in the HIBM families: all patients from Middle Eastern descent shared a single homozygous missense mutation, whereas distinct compound heterozygotes were identified in affected individuals of families of other ethnic origins. Our findings indicate that GNE is the gene responsible for recessive HIBM.

Distinctive patterns of microRNA expression in primary muscular disorders

The primary muscle disorders are a diverse group of diseases caused by various defective structural proteins, abnormal signaling molecules, enzymes and proteins involved in posttranslational modifications, and other mechanisms. Although there is increasing clarification of the primary aberrant cellular processes responsible for these conditions, the decisive factors involved in the secondary pathogenic cascades are still mainly obscure. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs regulated during the degenerative process of muscle to gain insight into the specific regulation of genes that are disrupted in pathological muscle conditions. We describe 185 miRNAs that are up- or down-regulated in 10 major muscular disorders in humans [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophies types 2A and 2B, Miyoshi myopathy, nemaline myopathy, polymyositis, dermatomyositis, and inclusion body myositis]. Although five miRNAs were found to be consistently regulated in almost all samples analyzed, pointing to possible involvement of a common regulatory mechanism, others were dysregulated only in one disease and not at all in the other disorders. Functional correlation between the predicted targets of these miRNAs and mRNA expression demonstrated tight posttranscriptional regulation at the mRNA level in DMD and Miyoshi myopathy. Together with direct mRNA–miRNA predicted interactions demonstrated in DMD, some of which are involved in known secondary response functions and others that are involved in muscle regeneration, these findings suggest an important role of miRNAs in specific physiological pathways underlying the disease pathology.

Mutations and polymorphisms of the skeletal muscle alpha-actin gene (ACTA1).

The ACTA1 gene encodes skeletal muscle α‐actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease‐causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease‐causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core‐like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype–phenotype correlations, we have developed a locus‐specific database for ACTA1 variations (http://waimr.uwa.edu.au). Hum Mutat 30:1–11, 2009.

Colon cancer associated transcript‐1: A novel RNA expressed in malignant and pre‐malignant human tissues

Early detection of colorectal cancer (CRC) is currently based on fecal occult blood testing (FOBT) and colonoscopy, both which can significantly reduce CRC‐related mortality. However, FOBT has low‐sensitivity and specificity, whereas colonoscopy is labor‐ and cost‐intensive. Therefore, the discovery of novel biomarkers that can be used for improved CRC screening, diagnosis, staging and as targets for novel therapies is of utmost importance. To identify novel CRC biomarkers we utilized representational difference analysis (RDA) and characterized a colon cancer associated transcript (CCAT1), demonstrating consistently strong expression in adenocarcinoma of the colon, while being largely undetectable in normal human tissues (p < 000.1). CCAT1 levels in CRC are on average 235‐fold higher than those found in normal mucosa. Importantly, CCAT1 is strongly expressed in tissues representing the early phase of tumorigenesis: in adenomatous polyps and in tumor‐proximal colonic epithelium, as well as in later stages of the disease (liver metastasis, for example). In CRC‐associated lymph nodes, CCAT1 overexpression is detectable in all H&E positive, and 40.0% of H&E and immunohistochemistry negative lymph nodes, suggesting very high sensitivity. CCAT1 is also overexpressed in 40.0% of peripheral blood samples of patients with CRC but not in healthy controls. CCAT1 is therefore a highly specific and readily detectable marker for CRC and tumor‐associated tissues.

Oestrogen Stimulates Differential Transcription of Human Papillomavirus Type 16 in SiHa Cervical Carcinoma Cells

Human papillomavirus (HPV) type 16 is highly associated with cervical cancer, but it seems that cofactors such as hormones affect its potential oncogenicity. We have analysed the HPV-16 gene expression in response to sex hormones and glucocorticoids in SiHa cells, a human cervical carcinoma cell line. An eightfold induction of HPV-16 transcripts was obtained in oestrogen-treated SiHa cells. Of the five HPV-16 transcripts detected in these cells only the two major ones, the 4.6 kb and the 4.1 kb mRNA species, were affected by oestrogen. Since the five transcripts span the E6 and E7 open reading frames of the HPV-16 genome, these results suggest that the expression of the various transcripts is differentially controlled, as oestrogen regulates only two of them. We have identified in the HPV-16 genome seven different regions with a high degree of similarity to the oestrogen-responsive element consensus sequence (GGTCANNNTGACC). These sequences are located throughout the entire HPV-16 genome. Progesterone or dexamethasone had no detectable stimulatory effect on the various transcripts of HPV-16 in SiHa cells, up to 24 h after treatment of the cells. Since the E6 and E7 open reading frames have been associated with the oncogenic potential of HPV-16, the effect of oestrogen on the transcription of these viral genes may be of biological relevance in the malignant transformation of HPV-16-infected cervical cells.

Development of a MicroRNA-Based Molecular Assay for the Detection of Papillary Thyroid Carcinoma in Aspiration Biopsy Samples

BACKGROUND Although thyroid nodules are common and diagnosed in over 5% of the adult population, only 5% harbor malignancy. Patients with clinically suspicious thyroid nodules need to undergo fine-needle aspiration biopsy (FNAB). The main limitation of FNAB remains indeterminate cytopathology. Only 20%-30% of the indeterminate nodules harbor malignancy, and therefore up to 80% of patients undergo unnecessary thyroidectomy. The aim of this study was to identify and validate a panel of microRNAs (miRNAs) that could serve as a platform for an FNAB-based diagnostic for thyroid neoplasms. METHODS The study population included 27 consecutive patients undergoing total thyroidectomy for FNAB-based papillary thyroid cancer (n = 20) and benign disorders (n = 7). Aspiration biopsy was performed from the index lesion and from the opposite lobe normal tissue in all study patients at the time of operation. RNA was extracted from all aspiration biopsy samples. Quantitative polymerase chain reaction on a panel of previously selected miRNAs was performed. Polymerase chain reaction results were compared with final histopathology. miRNA from tumor tissues was amplified using the highest value of each miRNA expression in normal tissue as a threshold for malignancy detection. RESULTS Diagnostic characteristics were most favorable for mir-221 in differentiating benign from malignant thyroid pathology. mir-221 was overexpressed in 19 patients (p < 0.0001) with a sensitive yield of 95%. Specificity, negative and positive predictive value, and accuracy of the miRNA panel were 100%, 96%, 100%, and 98%, respectively. CONCLUSIONS miRNA quantification for differential diagnosis of thyroid neoplasms within aspiration biopsy samples is feasible and may improve the accuracy of FNAB cytology.

Hereditary inclusion body myopathy The Middle Eastern genetic cluster

Background: Recessively inherited hereditary inclusion body myopathy (HIBM) with quadriceps sparing was initially described only in Jews originating from the region of Persia. The recent identification of the gene responsible for this myopathy and the common “Persian Jewish mutation” (M712T) enabled the re-evaluation of atypical phenotypes and the epidemiology of HIBM in various communities in the Middle East. Objective: To test for the M712T mutation in the DNA from HIBM patients in the Middle East. Methods: DNA from all suspected HIBM patients was tested for the M712T mutation. Unaffected members of families with genetically proven HIBM were studied too. In the majority of families, haplotype construction with markers spanning the 700-kb region of the HIBM gene was performed. Results: One hundred twenty-nine HIBM patients of 55 families (Middle Eastern Jews, Karaites, and Arab Muslims of Palestinian and Bedouin origin) were homozygous for the M712T mutation, and all carried the same haplotype. Five clinically unaffected subjects were also homozygous for the common mutation and haplotype, including two older adults (ages 50 and 68 years). Atypical features with this same mutation were marked quadriceps weakness in five patients, proximal weakness only in two patients, facial weakness in three patients, and a muscle biopsy showing perivascular inflammation in one patient. Conclusions: The phenotypic spectrum of recessive HIBM is wider than previously described, and the diagnostic criteria for this myopathy must be changed. The Middle Eastern cluster is the result of a founder mutation, with incomplete penetrance, that is approximately 1,300 years old and is not limited to Jews.

The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine‐712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N‐acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP‐N‐acetylglucosamine 2‐epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.

UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) Binds to Alpha-Actinin 1: Novel Pathways in Skeletal Muscle?

Background Hereditary inclusion body myopathy (HIBM) is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle. Methodology/Principal Findings We used a Surface Plasmon Resonance (SPR)-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed α-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and α-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. Conclusions/Significance The interaction of GNE with α-actinin 1 might point to its involvement in α-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of α-actinin, α-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of α-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with α-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of α-actinin 1, and to the muscle-restricted pathology of HIBM.

Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events.

Hereditary inclusion body myopathy (HIBM) is a unique muscular disorder caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. GNE encodes a bi-functional enzyme acting in the biosynthetic pathway of sialic acid. Since the underlying myopathological mechanism leading to the disease phenotype is poorly understood, we have established human myoblasts cultures, derived from HIBM satellite cells carrying the homozygous M712T mutation, and identified cellular and molecular characteristics of these cells. HIBM and control myoblasts showed similar heterogeneous patterns of proliferation and differentiation. Upon apoptosis induction, phosphatidylserine externalization was similar in HIBM and controls. In contrast, the active forms of caspase-3 and -9 were strongly enhanced in most HIBM cultures compared to controls, while pAkt, downregulated in controls, remained high in HIBM cells. These results could indicate impaired apoptotic signaling in HIBM cells. Since satellite cells enable partial regeneration of the post-mitotic muscle tissue, these altered processes could contribute to the muscle mass loss seen in patients. The identification of survival defects in HIBM affected muscle cells could disclose new functions for GNE in muscle cells.