Recommendations for pre-symptomatic genetic testing for hereditary transthyretin amyloidosis in the era of effective therapy: a multicenter Italian consensus.

Hereditary transthyretin amyloidosis (ATTRv, v for variant) is a late-onset, autosomal dominant disease caused by progressive extracellular deposition of transthyretin amyloid fibrils, leading to organ damage and death. For other late-onset fatal diseases, as Huntington's disease, protocols for pre-symptomatic genetic testing (PST) are available since decades. For ATTRv, limited experience has been reported to date, mostly gathered before the availability of approved therapies. We aimed at developing recommendations for a safe and feasible PST protocol in ATTRv in the era of emerging treatments, taking also into account Italian patients' characteristics and healthcare system rules. After an initial survey on ongoing approaches to PST for ATTRv in Italy, two roundtable meetings were attended by 24 experts from 16 Italian centers involved in the diagnosis and care of this disease. Minimal requirements for PST offer and potential critical issues were highlighted. By November 2019, 457 families affected by ATTRv with 209 molecularly confirmed pre-symptomatic carriers were counted. The median age at PST was 41.3 years of age, regardless of the specific mutation. Half of the Italian centers had a multidisciplinary team, including a neurologist, an internist, a cardiologist, a medical geneticist and a psychologist, although in most cases not all the specialists were available in the same center. A variable number of visits was performed at each site. Experts agreed that PST should be offered only in the context of genetic counselling to at risk individuals aged 18 or older. Advertised commercial options for DNA testing should be avoided. The protocol should consist of several steps, including a preliminary clinical examination, a pre-test information session, an interval time, the genetic test and a post-test session with the disclosure of the test results, in the context of an experienced multidisciplinary team. Recommendations for best timing were also defined. Protocols for PST in the context of ATTRv can be refined to offer at risk individuals the best chance for early diagnosis and timely treatment start, while respecting autonomous decisions and promoting safe psychological adjustment to the genetic result.

Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP-AON complexes.

Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropil-acrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2-AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2-AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as alpha-, beta-, gamma- and delta-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials.

Transcriptional behavior of DMD gene duplications in DMD/BMD males.

DMD gene exons duplications account for up to 5-10 % of Duchenne (DMD) and up to 5-19% of Becker (BMD) muscular dystrophies; as for the more common deletions, the genotype-phenotype correlation and the genetic prognosis are generally based on the "reading frame rule". Nevertheless, the transcriptional profile of duplications, abridging the genomic configuration to the eventual protein effect, has been poorly studied. We describe 26 DMD gene duplications occurring in 33 unrelated patients and detected among a cohort of 194 mutation-positive DMD/BMD patients. We have characterized at the RNA level 16 of them. Four duplications (15%) behave as exception to the reading frame rule. In three BMD cases with out-of-frame mutations, the RNA analysis revealed that exon skipping events occurring in the duplicated region represent the mechanism leading to the frame re-establishment and to the milder phenotype. Differently, in a DMD patient carrying an in-frame duplication the RNA behaviour failed to explain the clinical phenotype which is probably related to post-transcriptional-translational mechanisms. We conclude that defining the RNA profile in DMD gene duplications is mandatory both for establishing the genetic prognosis and for approaching therapeutic trials based on hnRNA modulation.

Intronic breakpoint definition and transcription analysis in DMD/BMD patients with deletion/duplication at the 5' mutation hot spot of the dystrophin gene.

Dystrophin mutations occurring at the 5' end of the gene frequently behave as exceptions to the "frame rule," their clinical severity being variable and often not related to the perturbation of the translation reading frame. The molecular mechanisms underlying the phenotypic variability of 5' dystrophin mutations have not been fully clarified. We have characterized the genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of dystrophin exons 3-7, 3-6 and duplication of exons 2-4. Our findings indicate that the occurrence of intronic cryptic promoter as well as corrective splicing events are unlikely to play a role in exons 3-7 deleted patients phenotypic variability. Our data suggest that re-initiation of translation could represent a major mechanism responsible for the production of a residual dystrophin in some patients with exons 3-7 deletion. Furthermore, we observed that the out-of-frame exon 2a is almost constantly spliced into a proportion of the dystrophin transcripts in the analysed patients. In the exons 2-4 duplicated DMD patient, producing both in-frame and out-of-frame transcripts, this splicing behaviour might represent a critical factor contributing to the severe phenotype. In conclusion, we suggest that multiple mechanisms may have a role in modulating the outcome of 5' dystrophin mutations, including recoding mechanisms and unusual splicing choices.

Atypical familial motor neuropathy in patients with mutant TTR Ile68Leu.

Two sisters from an Italian family shared progressive motor symptoms, preceding the onset of sensory and autonomic disturbances. The familial occurrence of axonal and slowly progressive polyneuropathy led us to consider these patients as candidates for TTR molecular analysis. We found a missense mutation causing Ile68Leu TTR substitution in both. The aims of this work are to report the possibility of a motor onset of amyloid polyneuropathy and to suggest the search for TTR mutations in familial cases of axonal polyneuropathy. Second, to stress the possible occurrence of amyloid within the spinal canal as the potential pathogenesis and responsible for motor presentation.

Transcription pattern of human herpesvirus 8 open reading frame K3 in primary effusion lymphoma and Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) is found in immunoblastic B cells of patients with multicentric Castleman's disease (MCD) and, predominantly in a latent form, in primary effusion lymphoma (PEL) cells and Kaposi's sarcoma (KS) spindle cells. Recent studies have shown that upon reactivation, HHV-8 expresses factors that downregulate major histocompatibility class I proteins and coactivation molecules and that may enable productively infected cells to escape cytotoxic T lymphocytes and natural killer cell responses. One of these viral factors is encoded by open reading frame (ORF) K3. Here we show that in PEL cells, ORF K3 is expressed through viral transcripts that are induced very early upon virus reactivation, including bicistronic RNA molecules containing coding sequences from viral ORFs K3 and 70. Specifically, we found that a bicistronic transcript was expressed in the absence of de novo protein synthesis, thereby identifying a novel HHV-8 immediate-early gene product. Several features of the RNA molecules encoding the K3 product, including multiple transcriptional start sites, multiple donor splicing sites, and potential alternative ATG usage, suggest that there exists a finely tuned modulation of ORF K3 expression. By contrast, ORF K3 transcripts are not detected in the majority of cells present in KS lesions that are latently infected by the virus, suggesting that there are other, as-yet-unknown mechanisms of immune evasion for infected KS spindle cells. Nevertheless, because HHV-8 viremia precedes the development of KS lesions and is associated with the recrudescence of MCD symptoms, the prompt expression of ORF K3 in productively infected circulating cells may be important for virus pathogenesis. Thus, molecules targeting host or viral factors that activate ORF K3 expression or inactivate the biological functions of the K3 product should be exploited for the prevention or treatment of HHV-8-associated diseases in at-risk individuals.

Transfer of human chromosome 3 to an ovarian carcinoma cell line identifies regions involved in ovarian cancer.

Earlier studies with somatic cell hybrids had clearly shown that when malignant cells were fused with normal cells, the resulting hybrid cells were nontumorigenic and that reexpression of tumorigenicity was often associated with the loss of specific chromosomes derived from the normal parental cells (1). A more direct approach to identify chromosomes carrying tumor suppressor genes is the introduction of specific chromosomes into tumor cells by microcell monochromosome transfer (MMCT) (2). The key feature of this technique is that the transferred chromosome is retained as a complete structural unit in succeeding generations of recipient cells, unlike the technique of metaphase chromosome transfer, where the transferred chromosome is rapidly degraded. To allow for transfer and selective retention of single specific chromosomes, dominant selectable markers such as the bacterial neo gene, which encodes an aminoglycoside 3'phosphotransferase (APH), are integrated into individual human chromosomes via plasmid DNA transfection or retroviral infection. Chromosomes tagged with dominant selectable markers can then be transferred from normal cells into cancer cells previously shown to have deletions in specific chromosome regions (3). The MMCT procedure is schematically outlined in Fig. 1. MMCT was used successfully with cell lines from tumors of different histotypes to detect chromosomal regions containing tumor suppressor genes and to identify chromosomes involved in the tumorigenic phenotype.

A human melanoma metastasis-suppressor locus maps to 6q16.3-q23.

Loss, deletion or rearrangement along large portions of the long arm (q-arm) of chromosome 6 occurs in >80% of late-stage human melanomas, suggesting that genes controlling malignant characteristics are encoded there. Metastasis, but not tumorigenicity, was completely suppressed in the human melanoma cell line C8161 into which an additional intact chromosome 6 had been introduced by microcell-mediated chromosome transfer. Our objective was to refine the location of a putative metastasis suppressor gene. To do this, we transferred an intact (neo6) and a deletion variant [neo6qdel; neo6(del)(q16.3-q23)] of neomycin-tagged human chromosome 6 into metastatic C8161 subclone 9 (C8161.9) by MMCT. Single cell hybrid clones were selected in G-418 and isolated. Following verification that the hybrids retained the expected regions of chromosome 6 using a panel of polymorphic sequence-tagged sites, the hybrids were tested for tumorigenicity and metastasis in athymic mice. As reported previously, intact, normal chromosome 6 suppressed metastasis whether tumor cells were injected i.v. or into an orthotopic (i.e., intradermal) site. In contrast, metastasis was not suppressed in the neo6qdel hybrids. Tumorigenicity was unaffected in hybrids prepared with either chromosome 6 donor. These data strongly suggest that a human melanoma metastasis suppressor locus maps between 6q16.3-q23 ( approximately 40 cM).

Multicenter comparison of PCR assays for detection of human herpesvirus 8 DNA in semen.

Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors (n = 30) and human immunodeficiency virus (HIV)-infected patients (n = 7) plus positive (n = 4) and negative (n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods.

Alpha interferon inhibits human herpesvirus 8 (HHV-8) reactivation in primary effusion lymphoma cells and reduces HHV-8 load in cultured peripheral blood mononuclear cells.

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-alpha), we analyzed the effects of IFN-alpha or anti-IFN-alpha antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-alpha inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-alpha inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-alpha was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-alpha Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-alpha Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-alpha reduced the HHV-8 load in PBMC. Thus, IFN-alpha appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-alpha may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.

Characterization of a 4-Mb region at chromosome 6q21 harboring a replicative senescence gene.

A 4-Mb region containing a senescence gene was defined at 6q21 by fluorescence in situ hybridization and deletion mapping after transfer of a normal human chromosome 6 to a BK virus-transformed mouse cell line. By screening three different yeast artificial chromosome (YAC) libraries, a YAC contig was constructed that covers the deleted region at 6q21. The contig is composed of 18 overlapping YACs with a size of 250-1800 kb and contains 3 CpG islands and 10 expressed sequence tags. By sequencing YACs and P1 artificial chromosomes, nine new sequence tagged sites and three new expressed sequence tags were detected that enrich the genetic resources of the region. The contig may also contain a fragile site, FRA6F, located close to a CpG island, which could be a landmark to localize the senescence gene. This YAC contig will be used to detect expressed sequences to clone and characterize the senescence gene at 6q21.

Growth arrest and suppression of tumorigenicity of bladder-carcinoma cell lines induced by the P16/CDKN2 (p16INK4A, MTS1) gene and other loci on human chromosome 9.

Wild-type P16/CDKN2 (p16INK4A, MTS1) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder-carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild-type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over-expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2-gene expression is modulated by the physiological control of chromosomal regulatory sequence. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder-carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32-33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor-suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder-carcinoma cells when it is over-expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder-cancer progression.

A BK virus episomal vector for constitutive high expression of exogenous cDNAs in human cells.

A BK virus (BKV) episomal vector (pRPneoCMV) was constructed for expression of cDNAs under control of the cytomegalovirus (CMV) immediate-early promoter. Transfection of pRPneoCMV for expression of the chloramphenicol acetyltransferase (CAT) gene in several human cell lines showed that the CMV promoter is more efficient than the HIV-1 and RSV LTRs in directing gene expression from episomal vectors. In 293 human cells pRPneoCMV/CAT is twenty times more active in CAT expression than the well known pSV2CAT vector in COS7 cells. Stable expression of the gene of the herpes simplex virus type 1 and type 2 glycoprotein G, cloned into pRPneoCMV, was obtained in 293 cells. This vector will allow direct cloning of newly synthesized cDNAs whose expression can be monitored in human cells.

Transfer of human chromosome 3 to an ovarian carcinoma cell line identifies three regions on 3p involved in ovarian cancer.

The molecular pathogenesis of ovarian carcinoma involves altered expression of growth factors, activation of oncogenes and loss of tumor suppressor genes. Loss of heterozygosity on chromosomes 3p, 6q, 11p, 17 and 18q was reported as a significant alteration in ovarian cancer. However, no functional proof has been provided of tumor suppressor activity located in these chromosomal regions. We therefore introduced normal human chromosomes 3 and 11 into an ovarian carcinoma cell line by microcell mediated chromosome transfer. Transfer of chromosome 3 induced senescence and growth arrest as well as suppression of tumorigenicity. Tumors induced by chromosome 3 monochromosomic hybrids consistently lost three small regions on 3p, two of which located in 3p23-24.2 and one located in 3p21.1-21.2, suggesting that these chromosomal regions are important for suppression of tumorigenicity of ovarian carcinoma cells. Transfer of chromosome 11 reduced the in vitro growth properties of ovarian cancer cells but did not significantly affect tumorigenicity. These results provide functional evidence for chromosome 3 tumor suppressor activity in ovarian cancer and define the chromosomal regions on 3p involved in the pathogenesis of this tumor. This experimental system, based on functional effects, may be useful for further delimitation and isolation of critical regions on 3p involved in tumor suppression.

Induction of senescence and control of tumorigenicity in BK virus transformed mouse cells by human chromosome 6.

Viral transformation models may be useful to detect and map human tumor suppressor genes. BK virus (BKV), a human papovavirus, readily transforms rodent cells but is unable to transform human cells, suggesting that oncosuppressive functions expressed in human cells control BKV oncogenic activity. We have transferred human chromosome 6 to BKV-transformed mouse pRPcT1ss1 cells. The great majority of the colonies growing in selective medium degenerated by senescence. Only five hybrid pRPcT1ss1/H6 clones maintained the immortalized phenotype of the recipient cell line. All the immortalized clones had two common regions of deletion involving bands 6q21-22 and the SOD2 gene in 6q25. Senescent colonies carried an intact chromosome 6. A specific human sequence in 6q21-22 was amplified by PCR in senescent cells, suggesting that this region harbors a gene inducing senescence. The SOD2 deletion confirms recent data on the role of the Mn-dependent superoxide dismutase in inhibition of proliferation. The monochromosomic hybrids bearing a deleted chromosome 6 showed a reverted phenotype in vitro and a significantly longer latency period before they were tumorigenic in nude mice, indicating the presence of a tumor suppressor gene in the residual regions of chromosome 6. Molecular mapping suggests that this gene is located in 6q27. The BKV transformation model detects genes inducing senescence and tumor suppressor genes on human chromosome 6 and may represent a useful system to isolate and clone such genes.