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.

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.

Inducible expression of the human interferon beta 1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells.

A 1.6-kilobase DNA segment of the genomic human interferon beta 1 (IF-beta 1) gene was inserted into each of two possible orientations at the single HindIII site of a recombinant plasmid pBPV69T, consisting of the 69% transforming region of the bovine papilloma virus type 1 (BPV-1) and a modified SalI-SalI fragment of plasmid pBR322. After cleavage of the pBR322 sequences from this recombinant, BPV69T-IF-beta 1 hybrid DNAs were transfected onto C127 mouse cells by the standard calcium precipitation technique. Mouse cells transformed by this hybrid DNA produced low levels of human IF-beta 1 constitutively and responded to induction with either inactivated Newcastle disease virus or polyriboinosinic acid-polyribocytidylic acid. The BPV69T-IF-beta 1 hybrid DNA was nonintegrated in the transformed mouse cells but had acquired DNA sequences as a result of the transfection. Accurate transcripts of the IF-beta 1 mRNA were detected in cells only after induction. When the IF-beta 1 gene was oriented in the plasmid in the same direction of transcription as the BPV-1 genome, transcription was promoted from within the BPV-1 sequences. These results indicate that the regulatory sequences responsible for the inducible expression of the human IF-beta 1 gene are present in the 1.6-kilobase genomic segment and that these sequences can function in a free extrachromosomal state linked to BPV-1 sequences.

Life or death by NFκB, Losartan promotes survival in dy2J/dy2J mouse of MDC1A.

Inflammation and fibrosis are well-defined mechanisms involved in the pathogenesis of the incurable Laminin α2-deficient congenital muscular dystrophy (MDC1A), while apoptosis mechanism is barely discussed. Our previous study showed treatment with Losartan, an angiotensin II type I receptor antagonist, improved muscle strength and reduced fibrosis through transforming growth factor beta (TGF-ß) and mitogen-activated protein kinases (MAPK) signaling inhibition in the dy(2J)/dy(2J) mouse model of MDC1A. Here we show for the first time that Losartan treatment up-regulates and shifts the nuclear factor kappa B (NFκB) signaling pathway to favor survival versus apoptosis/damage in this animal model. Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-ß protein level, indicating NFκB activation. Moreover, NFκB anti-apoptotic target genes TNF receptor-associated factor 1 (TRAF1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were increased following Losartan treatment. Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased. Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay. Thus, along with TGF-ß and MAPK signaling, NFκB serves as an important regulatory pathway which following Losartan treatment promotes survival in the dy(2J)/dy(2J) mouse model of MDC1A.

Physical and transcriptional map of the hereditary inclusion body myopathy locus on chromosome 9p12-p13.

Hereditary inclusion body myopathy (HIBM) is a group of neuromuscular disorders characterised by adult-onset, slowly progressive distal and proximal muscle weakness and typical muscle pathology. Previously, we have mapped the gene responsible for a recessive form of HIBM to chromosome 9p1 and narrowed the interval to one single YAC clone of 1 Mb in size. As a further step towards the identification of the HIBM gene, we have constructed a detailed physical and transcriptional map of this region. A high resolution BAC contig that includes the HIBM critical region, flanked by marker 327GT4 and D9S1859, was constructed. This contig allowed the precise localisation of 25 genes and ESTs to the proximal region of chromosome 9. The expression pattern of those mapped genes and ESTs was established by Northern blot analysis. In the process of refining the HIBM interval, 13 new polymorphic markers were identified, of which 11 are CA-repeats, and two are single nucleotide polymorphisms. Certainly, this map provides an important integration of physical and transcriptional information corresponding to chromosome 9p12-p13, which is expected to facilitate the cloning and identification not only of the HIBM gene, but also other disease genes which map to this region.

Integration and transcription of human papillomavirus type 6 recombinant DNA in mouse cells.

Human papillomavirus (HPV) DNA is found in nature mostly in an episomal form. However, in permanent cell lines established from cervical carcinomas in which HPV sequences are present, they are usually integrated in the host genome. In vitro studies of HPV have been hampered because of the difficulty of stably maintaining HPV sequences in transfected cells. We have cloned the entire HPV6 genome into three vectors: pML2, a derivative of pBR322 lacking the "poison sequences"; p142-6, a plasmid containing the complete bovine papillomavirus type 1 (BPV-1) genome which usually remains extrachromosomal in transfected mouse cells; and p302-3, a plasmid containing the bacterial gene conferring resistance to neomycin. Each of the three constructs has been transfected onto C127 mouse cells. In the case of the recombinant molecules cloned into pML2 and into p42-6, the transfection had been done together with the p302-3 plasmid. G418 resistant colonies were selected and analyzed for their DNA content. In all cases (5/5) the transfected molecules were present and integrated in the cellular genome. No free episomal DNA was detected, even in the cells transfected with the BPV-1 containing plasmid. One junction with the flanking cellular sequences is located in the late open reading frames region of the HPV6 genome and there is a disruption of the early region, as has been described for the HPV18 genome integrated into the cervical carcinoma cell line HeLa. Transcription analysis revealed transcripts of heterogeneous sizes, spanning the distal early region (E2S4E5) of the HPV6 genome, as happens in vivo in Condylomata acuminata, although in these genital lesions the HPV6 genome is maintained as a non-integrated episome.

Differential expression of HPV types 6 and 11 in condylomas and cervical preneoplastic lesions.

Tumor biopsies from exophytic and flat condylomas at different locations on the genital epithelium (10 cases) and in cervical intraepithelial neoplasia (CIN) grades 1-2 (6 cases) were analysed for HPV types 6 and 11 DNA and RNA. The presence of mRNA species which could encode the E6, E7, E1M, E2, E2C, E4, E5 and L1 proteins was determined using the RNA polymerase chain reaction (PCR) technique with primers that flank previously mapped or predicted splice sites. The state of the viral DNA in the tumor biopsies was established by Southern blot analysis. We could detect the various mRNA species in biopsies from condylomas associated with both HPV types. The size of the RNA PCR products were in agreement with the previously mapped splice sites of mRNAs recovered from an experimental condyloma induced by HPV11. The major viral transcript encoding the E1i--E4 protein was detected in all the tumor biopsies. From the rare transcripts the rate of detection of mRNA species encoding the E1M, E2C proteins was the highest. In 2 of 6 CIN biopsies analysed only the major viral transcript was detected. The overall results of this study suggest that early gene products of HPV types 6 and 11 may be important in the induction of cellular proliferation and condylamatous differentiation but these possibly may not be required for the development of the HPV-associated cervical neoplasia.

Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation.

Mutations in dysferlin were recently described in patients with Miyoshi myopathy, a disorder that preferentially affects the distal musculature, and in patients with Limb-Girdle Muscular Dystrophy 2B, a disorder that affects the proximal musculature. Despite the phenotypic differences, the types of mutations associated with Miyoshi myopathy and Limb-Girdle Muscular Dystrophy 2B do not differ significantly. Thus, the etiology of the phenotypic variability associated with dysferlin mutations remains unknown. Using genetic linkage and mutation analysis, we identified a large inbred pedigree of Yemenite Jewish descent with limb-girdle muscular dystrophy. The phenotype in these patients included slowly progressive, proximal, and distal muscular weakness in the lower limbs with markedly elevated serum creatine kinase (CK) levels. These patients had normal development and muscle strength and function in early life. Muscle biopsies from 4 affected patients showed a typical dystrophic pattern but interestingly, in 2, an inflammatory process was seen. The inflammatory infiltrates included primarily CD3 positive lymphocytes. Associated with this phenotype, we identified a previously undescribed frameshift mutation at nucleotide 5711 of dysferlin. This mutation produced an absence of normal dysferlin mRNA synthesis by affecting an acceptor site and cryptic splicing. Thus, splice site mutations that disrupt dysferlin may produce a phenotype associated with inflammation.

Transmissibility and treatment failures of different types of human papillomavirus.

In order to evaluate the transmissibility and treatment failures of different types of human papillomavirus (HPV), we obtained biopsy specimens from genital lesions related to HPV in 113 women prior to laser therapy. Sixty-eight had condylomata acuminata, 14 had flat condyloma, and 31 had cervical intraepithelial neoplasia with or without condylomata. Of 76 male sexual partners examined colposcopically, 58 (76%) had HPV-related lesions. All biopsy specimens were analyzed for specific type of HPV DNA by Southern blot hybridization with probes for HPVs 6, 11, 16, and 18. Sixty-one women had HPV 6 or 11 (6/11), 40 had HPV 16 or 18 (16/18), and in 12 the analysis was negative for these types of viral DNA. Deoxyribonucleic acid analysis of biopsy specimens from recurrent lesions showed the same type of viral DNA as in the primary lesion. Significantly more male partners (28 of 47) with the same HPV type were found among women with HPV 6/11 than among women with HPV 16/18 (P less than .005). Patients with HPV 16/18 had significantly more recurrences (14 of 40) after laser therapy than patients with HPV 6/11 (six of 60) (P less than .001).

Monopaternal superfecundation of quintuplets after transfer of two embryos in an in vitro fertilization cycle.

OBJECTIVE: To present the first genetically proven identity of quintuplets in an IVF treatment cycle after transferring only two embryos. DESIGN: Case report. SETTING: IVF unit and obstetrics department of university-affiliated general hospital. PATIENT(S): Twenty-five-year-old patient undergoing IVF treatment for unexplained infertility. INTERVENTION(S): In vitro fertilization with intracytoplasmic sperm injection performed on 50% of oocytes, resulting in successful production of nine early-cleavage embryos. Transfer of two embryos on day 3 and freezing of the remaining embryos. MAIN OUTCOME MEASURE(S): Development of five separate embryonic sacs. Fetal reduction of three embryos at 12 weeks of gestation. RESULT(S): Successful completion of the twin pregnancy and full genetic analysis of the three embryos and the twins that were born at term. CONCLUSION(S): Despite transferring only two embryos, superfecundation occurred, resulting in five embryos. Genetic analysis can be used to determine paternity and identity of all the embryos.

GNE protein expression and subcellular distribution are unaltered in HIBM.

Mutations in GNE encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) cause hereditary inclusion body myopathy (HIBM). To define the role of GNE mutations in HIBM pathogenesis, GNE protein expression was analyzed. GNE protein is expressed at equal levels in HIBM patients and normal control subjects. Immunofluorescence detection of GNE did not reveal any mislocalization of GNE in skeletal muscle. We conclude that impaired GNE function, not lack of expression, may be the key pathogenic factor in HIBM. For diagnostic purposes, direct genetic analysis of the GNE gene in patients with IBM will remain the mainstay and is not aided by immunohistochemistry or immunoblotting using antibodies against the GNE protein.

Multimarker RT-PCR assay for the detection of minimal residual disease in sentinel lymph nodes of breast cancer patients.

The presence of metastases in lymph nodes is the most powerful prognostic factor in breast cancer patients. Routine histological examination of lymph nodes has limited sensitivity for the detection of breast cancer metastases. The aim of the present study was to develop a multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of minimal residual disease in sentinel nodes of breast cancer patients. RNA was extracted from 30 sentinel lymph nodes (SLN) obtained from 28 patients, three primary breast cancers (positive controls), three lymph nodes from patients with benign diseases, and peripheral blood lymphocytes of 10 healthy volunteers (negative controls). RT-PCR was performed using the following markers; cytokeratin (CK)-19, NY-BR-1 and mammaglobin B. RT-PCR results were compared to enhanced histopathologic examination and immunohistochemistry (IHC). All three positive controls showed strong PCR amplification for all three markers. None of the 13 negative controls was amplified by any of the three markers. Among the 30 SLN analysed, breast cancer metastases were detected in six SLNs by routine histology, in eight by IHC and in 15 by RT-PCR. We conclude that a multimarker RT-PCR assay probing for NY-BR-1, mammaglobin-B, and CK-19 is more sensitive compared to enhanced pathologic examination. This method may prove to be of value in breast cancer staging and prognosis evaluation.

Human papillomaviruses and the diagnosis of genital microorganisms.

Considerable evidence points to certain types of human papillomaviruses (HPV) as high risk factors in the malignant progression mechanism in lesions of the lower genital tract. Since infection of the genital mucosa by papillomaviruses includes a wide variety of viral types, it is essential to recognize the specific HPV type eventually present in a genital lesion to ensure an appropriate follow-up of the patient. We have developed a polymerase chain reaction (PCR) based assay which differentiates in a single amplification reaction the "malignant" HPV genital types from the "benign" ones. We report also the use of PCR for the detection of other frequent sexually transmitted agents, such as herpes simplex virus and Chlamydia trachomatis. We show that it is possible to design and use a PCR-based assay for the simultaneous detection of various genital agents from a single genital swab. This assay can be a model for the development of additional multidetection systems of microorganisms potentially involved in similar pathologies.

Use of a stable bovine papillomavirus vector to study inducible genes.

The human beta interferon (hIFN beta 1) gene has been cloned into a bovine papillomavirus type 1 (BPV-1)/pML2 vector. The recombinant DNAs can be maintained as stable plasmids in mouse cells, with no detectable rearrangements. The linked hIFN beta 1 gene is inducible with little line-to-line variability in independently transformed cell lines. The hIFN beta 1 gene is equally induced when it is inserted adjacent to or away from the BPV-1 enhancer. The great stability of the BPV-1/pML2 vector has allowed us to study the regulation of the hIFN beta 1 gene in mouse cells, with the construction of deleted and hybrid hIFN beta 1 genes. Using this strategy, we have confirmed that the 5' sequences of the hIFN beta 1 gene are involved in the induction mechanism. The entire hIFN beta 1 regulatory element is able to express heterologous genes with no induction, suggesting that the negative regulatory sequences present in the 3' region of the hIFN beta 1 regulatory element are not fully active on heterologous genes.

Differential cooperation of a carcinogen with human papillomavirus type 6 and 16 DNAs in in vitro oncogenic transformation.

In contrast to the strong association of human papillomavirus (HPV) type 16 with genital malignancies, HPV 6 has been found essentially in benign genital lesions. In these studies we show that HPV type 6 and 16 DNAs behave differently also in their ability to transform NIH 3T3 cells in cooperation with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although we could show that both HPV-6- and HPV-16-transfected genomes were integrated and expressed in NIH 3T3 cells, only the NIH 3T3 cells which contained the HPV 16 genome became fully transformed after MNNG treatment, as assessed by their ability to form colonies in soft agar and to induce tumors in nude mice. NIH 3T3 cells containing the HPV 6 genome and treated with MNNG did not show this potential. Furthermore, we could detect an increased expression of the E7 gene of HPV 16 in the carcinogen-treated cells containing the HPV 16 genome. These studies indicate that the presence of the HPV 16 genome specifically is also an essential step to in vitro cellular transformation.

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.

Induction of Epstein-Barr virus nuclear antigen and DNA synthesis in a human epithelial cell line after Epstein-Barr virus infection.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma is supported by the presence of EBV genomes in the epithelial elements of the tumor and by elevated antibody titers to EBV-specific antigens in the patients; the levels of these titers are related to the clinical course of the disease. However, since most laboratory data suggest that EBV is a B-lymphotropic virus, it is unclear how the virus becomes associated with the epithelial elements of the nasopharynx. The purpose of the present work was to find a human model system to study this association. A human epithelial line (U) was found that could be directly infected by EBV, and viral functions, the induction of EBV nuclear antigen and cellular DNA synthesis, were demonstrated. The U line was established in 1957 by the late H. J. Van Kooten (Kok-Doorschodt at the University of Utrecht), and although it is no longer diploid, it exhibits density inhibition. When U cells were infected with EBV, EBV nuclear antigen was expressed in 6 to 16% of the cells, 1 and 2 days after infection with B95-8 virus, but not with the P3HR-1 strain. No evidence for virus replication was obtained; immunofluorescence staining for early antigens and virus capsid antigens gave negative results. Quantitative adsorption experiments for EBV indicated that the adsorption capacity of U cells is significant (60% of Raji cells). The present results also demonstrated that infection with the virus overcomes block(s) in cellular DNA synthesis caused by 5-fluorodeoxyuridine. The induction of DNA synthesis was determined by increased incorporation of [3H]thymidine into the cells. The highest level of isotope incorporation was observed at about 15 h after infection and thereafter decreased. Analysis of the induced DNA indicated that it was of cellular origin.

Genetics of inclusion body myopathies.

We review the current knowledge about the genetic susceptibility to develop inflammatory inclusion body myositis, especially in relation to the increased presence of the HLA DR3 allele in patients with familial and sporadic forms, indicating an autoimmune predisposition. The main focus of the review is the clinical and genetic presentations of the various hereditary inclusion body myopathies. Criteria for diagnosis and classification of these myopathies are presented. The spectrum of the recessive forms of hereditary inclusion body myopathies currently linked to chromosome 9p1-q1 is described, with emphasis on the up-to-date status of the gene search for these forms.

Presence of infective Epstein-Barr virus in the urine of patients with infectious mononucleosis.

The presence of Epstein-Barr virus (EBV) in the blood and urine of 20 patients with infectious mononucleosis (IM) was investigated together with the clinical course of the disease, and in 9 patients up to 2-7 months after recovery. EBV DNA, analyzed by the polymerase chain reaction (PCR), was detected in the blood of all 20 patients from the first sample obtained and detected between 3 to 42 days from the beginning of symptoms and up to 2-3 months after recovery. In the urine, EBV DNA was detected in 15 out of 16 (93%) patients in the first sample obtained and detected between 3 to 50 days during the clinical course of the disease. In four patients EBV DNA was detected in the urine up to 3 months after full recovery. Seventeen out of 26 (65%) urine samples including 3 which were obtained 2-7 months after recovery infected B cells as assessed by PCR. Nine out of 12 (75%) urine samples tested induced Epstein-Barr nuclear antigen (EBNA) in the infected B-cell line. In addition to the persistence of EBV in the blood of IM patients, these studies show for the first time the presence of infective EBV in the urine during the clinical course of the disease and up to 7 months after full clinical recovery.