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8 June 2005

The Department of Health (DH) has published the Report of the Ad Hoc Advisory Group on the Operation of NHS Research Ethics Committees. In November 2004 Health Minister Lord Warner announced he had formed an advisory group to give “…independent advice on the operation of the research ethics committee system, including the Central Office for National Health Service Research Ethics Committee [COREC].” Research ethics committees (RECs) are independent groups of lay and expert members who review proposals for research projects to ensure that, if the research were to happen, that the dignity, rights, safety and well being of the participants are protected. All proposed research studies involving NHS patients must be reviewed before it can begin. This report reviews the current practice of RECs in England and gives recommendations for the more efficient management of the ethics review process.

The Advisory Group’s recommendations include:

• Reorganising RECs into a ‘nationally networked system’ with fewer committees meeting more often
• Considering creating ‘professional’ RECs in the future, with members being paid for their work, as opposed to the present volunteer system
• Reviewing the on-line application form with a view to further improvements
• Appointing Scientific Officers who could be called on to review research proposals for scientific quality, allowing RECs to concentrate on ethics review
• Providing greater training for REC members, in order to reduce inconsistencies between committees
• Creating a UK-wide system of ethics review in the future

The Advisory Group heard complaints from critics who claim that ethics review procedures unnecessarily slow the research process, however the report recognises that COREC staff have done much work to reduce this problem. But more is needed and the report suggests, in particular, striving for better interaction between research ethics and research governance submissions. The report also suggests that researchers need to more aware of the ethical issues in their research so they can better describe the ethical arguments in favour of their proposed research in the application form. The report has been given to the National Patient Safety Agency, under which COREC was placed earlier this year, which will consider how these recommendations can be implemented.

15 June 2005Alain Pompidou, President of the European Patent Office (EPO), has said that the EPO will, for the time-being, stop making decisions on patent applications involving human embryonic stem cell technologies [Schubert, S. Nature 435:720-1]. The announcement, made during a presentation of the EPO’s 2004 Annual Report, did not include an expectation of when this decision will change. Pompidou gave the reason that “…there are too many ethical aspects that have not been resolved at the political level". This may refer to the fact that the European Commission is still in the process of deciding whether or not to fund embryonic stem cell research under the upcoming 7th Framework Programme.

It is reported that three applications involving embryonic stem cells are currently being considered by the EPO. One is before the EPO’s technical appeal board with a decision expected in October. If this board cannot settle the case on technical grounds it could be sent to the Enlarged Board of Appeal, EPO’s highest appeals board. If so, any decision made by the Enlarged Board may set a precedent about the patentability of inventions involving human embryonic stem cell technologies. Other countries already patent such inventions, such as the United States and South Korea, and European scientists worry that refusing patents on these inventions will cause economic repercussions. The UK Patent Office (UKPTO) has stated in its guidance, Inventions Involving Human Embryonic Stem Cells, that, in regards to human embryonic pluripotent stem cells, it “…is ready to grant patents for inventions involving such cells provided they satisfy the normal requirements for patentability". The UKPTO describes pluripotent stem cells as "...not hav[ing] the potential to develop into an entire human being". Processes for obtaining stem cells from human embryos and human totipotent cells (those that do have the potential to develop into a human being) are not patentable.

28 June 2005The US Food and Drug Administration (FDA) has approved the first ever drug marketed for use solely in a specific ethnic group. BiDil, produced by US company NitroMed, has been approved for the treatment of heart failure in self-identified black patients, having been through clinical trials for the treatment of African Americans (see November 2004 newsletter item). This decision has been made amid considerable controversy; critics say that using race or ethnicity as a marker for genetic differences is crude and inaccurate, and fear that approval of BiDil lends weight to notions of race as a distinct biological marker. Others, such as the Association of Black Cardiologists, who sponsored the Nitromed trials, welcomed the decision.

FDA Associate Director of Medical Policy Dr Robert Temple said the drug was "a striking example of how a treatment can benefit some patients even if it does not help all patients”, adding that they hoped in the future “to discover characteristics that identify people of any race who might be helped by Bidil" (see FDA press release). However, some fear that marketing of a race-specific drugs is likely to be driven by financial motives irrespective of the validity of using ethnicity as a marker for genetic variation between individuals; BiDil is a combination of two pre-existing drugs, isosorbide dinitrate and hydralazine, for which the patent covering general use will expire in 2007; patent protection for the use of BiDil in black patients will extend until 2020 (see BBC news report)

14 June 2005The Italian people have stayed away from the polls, choosing not to vote in a referendum that asked if changes should be made in Italy’s strict fertility treatment law [Willey, D., BBC news, 13 June 2005]. The law, which came into effect in 2004, placed tough restrictions on fertility treatment services, giving embryos the same rights as citizens (see PHGU newsletter article February 2004). The law banned research using embryos; the freezing of embryos (only three embryos can be created, all of which have to be implanted into the woman); stem cell research; and the use of donor eggs and sperm and surrogacy. The four-part referendum, which took place on 12-13 June, gave Italians the chance to vote whether or not to overturn any or all of these four strict measures. In order for the referendum to be valid, over 50% of the Italian population had to vote; according to reports, just over a quarter of the population voted. Although 80-90% of those who did vote voted yes to overturn the strict measures, the referendum will have no legal effect.

Analysts have pointed to the intervention of the Roman Catholic Church as a deciding factor in the failure of people to vote. The Pope, Benedict XVI, gave his personal support to clergy to encourage people to boycott the referendum. During their Sunday sermons, it is said, priests continued to preach against the referendum using the slogan of the campaign, “Life cannot be put to a vote.” However, others have said that the low turnout was due to apathy or the fact that the issues on the referendum were complex [Popham, P., The Independent, 14 June 2005].

It has been reported that the fertility law has adversely affected those who have sought treatment [Smith, T., BBC news, 9 August 2004]. Success rates have dropped from one-in-four to one-in-nine. Because no embryos can be frozen, if treatment is not successful, a woman has to undergo ovarian stimulation for every attempt, a procedure that is painful and has potential serious side effects. Screening for genetic disease is also banned, but abortion is not, which some say is a contradiction. Whether there will be further consideration of this law after this dramatic boycott remains to be seen.

6 June 2005Hwang Woo-suk, who with his researchers created the first patient-matched embryonic stem cell lines, has announced that he plans to open a stem cell bank in Korea. In an interview with the Associated Press [Kim. J., Washington Post, 1 June 2005], Hwang discussed his plans for a stem cell bank to consolidate access to existing stem cell lines. He envisages a bank where researchers from around the world could come to seek out stem cells that would match a patient in need, a process like that used to find organ transplants. He expects the bank to open as early as later this year; management of the bank would be given eventually to an international agency. The Korea Times reports [Kim, T. Korea Times, 25 May 2005] that the Korean government will be supporting Hwang with an increase in his research budget and a new research facility at Seoul National University.

Hwang and his colleagues announced their groundbreaking work creating genetically matched stem cell lines in May 2005 [see Hwang, W.S. et al, Science, 19 May 2005]. He and his team derived 11 stem cell lines that genetically matched the patients from whom skins cells used in the procedure were taken. The patients suffered from disease or spinal cord injury. Last year, Hwang and his colleagues announced they had cloned a human embryo [see PHGU newsletter item February 2004]. Now he plans to move to the next phase of his research plans. He told the Korea Times, “We already have some globally organised research teams. After concluding the worldwide framework this year, we will push for more progress. I hope to lower the curtain of the first act (of the stem cell play) by as early as next autumn.” The Associated Press reports that he next plans to move into growing specific tissues and organs from stem cells.

One international collaboration being planned is with Dr Douglas Melton at the Harvard Stem Cell Institute in Harvard, Massachusetts, according to a Bloomberg report [2 June 2005]. Melton is seeking to use embryonic stem cells in treatments for diabetes. He has already isolated 17 stem cell lines from unneeded IVF embryos, using private funding. The Massachusetts legislature recently overrode a veto by the state’s governor of a state law that allows somatic cell nuclear transfer or therapeutic cloning, as long as the research has ethics approval and does not result in the cloning of a human being. This puts Massachusetts, according to Senator Edward Kennedy, “…at the forefront of science, innovation and discovery.” The report states that a link with Harvard might help with Hwang’s goal of creating the stem cell bank in Korea.

28 June 2005

MicroRNAs (miRNAs) are a recently discovered group of small (21-25 nucleotides long), non-coding RNAs that act as negative regulators of gene expression in a range of organisms, including plants. MicroRNAs work via RNA interference complexes (‘RNA-induced silencing complexes’ or RISCs), targeting them to messenger RNAs where they either inhibit translation or direct destructive cleavage [see Meltzer PS (2005) Nature 435, 745-746]. They are known to be involved in the control of key cellular processes such as cellular proliferation and apoptosis. More than 200 miRNAs have been identified in humans, but their precise functions have not been well established. However, three new papers in the latest edition of Nature demonstrate a role for miRNAs in the formation of human tumours.

The first paper reports investigation of a cluster of miRNAs on chromosome 13, the mir-17-92 polycistron, that is present in a region of DNA known to be amplified in forms of human B-cell lymphoma [He L et al. (2005) Nature 435, 828-833]. The researchers compared levels of miRNAs from this cluster in four lymphoma B-cell lines, normal B-cell lines and also five leukaemia and lymphoma cell lines in which the region containing mir-17 is not amplified. It was shown that expression levels of miRNAs from the cluster were high in the lymphoma cells.They also looked at mir-17 expression in lymphoma and colorectal tumour samples, and found significant overexpression in 65% of the lymphoma samples compared with 15% of the colorectal samples; levels of expression were also higher in the lymphoma than colorectal tumours.

To test whether mir-17 contributes to tumour formation, they used a mouse model of human B-cell lymphoma and used a viral vector to transfect animals with the miRNA polycistron. The mice normally develop tumours by 4-6 months, but those in which the miRNAs were overexpressed developed tumours much faster, in an average of 51 days. All had developed tumours within 90 days, compared with less than 30% of control animals transfected with ‘dummy’ vectors. The authors conclude that the mir-17-92 cluster is a potential oncogene.

The second paper reports the use of a bead-based system for miRNA expression profiling, whereby polystyrene beads impregnated with fluorescent dyes were linked to oligonucleotides complementary to the miRNAs of interest. Analysis of fluorescence following passage of miRNAs over the beads revealed the identity and abundance of miRNAs captured on the beads [Lu J et al. (2005) Nature 435, 834-838]. The research team looked at expression of all known mammalian miRNAs in a total of 334 tumour samples. They found that almost all miRNAs were expressed, and that characteristic expression patterns were associated with specific tumour cell lineages. For example, the profiles from colon, liver, pancreas and stomach cancers clustered together, which the authors propose reflects their shared developmental lineage from the embryonic endoderm. These patterns were not reproduced by mRNA profiles based on the expression of 16,000 mRNAs.

It was then decided to compare miRNA expression in human cancer samples and normal tissues; it was found to be generally much lower in the tumour samples than the corresponding normal tissue, leading the authors to hypothesise that global miRNA expression reflects the state of cellular differentiation. They report that miRNA profiling of cell lines showed induction of multiple miRNAs when cells were stimulated to differentiate. They conclude that downregulation or other abnormalities of miRNA expression may contribute to tumour formation and maintenance, and that the use of miRNA expression profiling may prove to be valuable in cancer diagnosis.

The third paper reports that c-Myc, the product of a proto-oncogene commonly dysregulated in human cancers, activates expression of the mir-17 miRNA cluster [O'Donnell KA et al. (2005) Nature 435, 839-843]. The researchers used oligonucleotide arrays to analyse miRNA expression profiles in human B-cells in which expression of c-Myc (a transcription factor) was repressed or normal. Several miRNAs from two clusters, one of which was the mir-17 cluster on chromosome 13, were found to be consistently upregulated in cells with normal levels of c-Myc. Using different cell lines, they found that expression of the mir-17 miRNAs was repressed in the absence of c-Myc and restored by its presence. Immunoprecipitation experiments suggested that c-Myc binds to a specific region just upstream of the mir-17 locus.

To investigate the effects of c-Myc induction of the mir-17 region miRNAs, the team examined expression of the E2F1 gene, which is known to be induced by c-Myc and thought to be a target of the miRNAs in question, miR-17-p and miR-20a. They used human HeLa cells transfected with constructs that bind to and inhibit the miRNAs, and found that this increased E2F1 protein (but not mRNA) levels four-fold. Conversely, the use of a construct overexpressing the mir-17 cluster in HeLa cells produced a two-fold decrease in E2F1 protein (but not mRNA) levels. The authors conclude that c-Myc targets miRNAs, and that the mir-17 region regulates c-Myc mediated cellular proliferation by affecting E21F expression levels.

Comment: These papers investigate different aspects of the role of microRNAs in cancer, but taken together provide a clear demonstration that miRNAs are indeed involved in tumour formation. Commenting on the research, Dr Paul Meltzer, of the US National Human Genome Research Institute, said: "These studies change the landscape of cancer genetics" (see BBC news report). The precise nature of this involvement may prove to be highly complex, and it is likely that a great deal of research will be necessary to elucidate the pathways and mechanisms by which miRNAs work in normal and cancerous cell development. However, besides basic cancer research, some of the potential applications of this research are already clear, such as improved diagnostic and perhaps prognostic tools.

7 June 2005

Early embryonic development involves the sequential expression of a number of different genes, directing cells towards specific pathways that eventually lead to the formation of different cell types and tissues. A key way in which gene expression is regulated is through epigenetic mechanisms. These mechanisms, such as DNA methylation or histone modification, work by altering DNA and its associated proteins without changing the DNA sequence. For example, methylation of the nucleotide cytosine results in the physical displacement of transcription factors that normally bind to the sequence and the compaction of chromatin around the site, which together lead to gene silencing. Imprinted genes are subject to regulation by epigenetic modifications, and are expressed from either maternally or paternally inherited chromosomes. Imprinted domains on the two parental chromosomes have different epigenetic modifications that control expression from each one. These genes are expressed in the developing embryo and placenta, and in the prenatal and postnatal brain, and are involved in the control of normal growth and development. To date around seventy imprinted genes have been identified, and imprinting defects can cause several neurological and developmental disorders such as Prader-Willi and Beckwith-Wiedemann Syndromes. It has also been suggested that individuals born via assisted reproductive technologies may also have a higher incidence of imprinting-related disorders.

Human embryonic stem cells are derived from the blastocyst stage of embryonic development and could therefore be subject to epigenetic instability or variation, resulting in the inappropriate expression of imprinted genes. A recent paper in Nature Genetics has examined the epigenetic stability of six imprinted genes in cultured human embryonic stem (hES) cells. The researchers at Cambridge University cultured four hES cell lines to middle or high passage under standard conditions and used RT-PCR to detect the presence of specific transcripts from six imprinted genes. The four cells lines carried polymorphisms between the maternal and paternal alleles of these genes, and therefore direct sequencing of the RT-PCR products was able to determine whether the transcripts were as a result of maternal or paternal expression. The imprinted genes, IGF2, IPW and KCNQ1OT1, are normally paternally expressed, and were found to be strictly monoallelically expressed in the four lines. The maternally expressed genes, SLC22A18, NESP55 and H19, were also investigated. Expression of SLC22A18 and NESP55 were predominantly monoallelic with low expression of the paternal allele. H19 was initially monoallelically expressed, but biallelic expression increased during prolonged passage (p66-76). The non-imprinted gene TSSC4 was studied as a control and was found to be consistently biallelically expressed.

The epigenetic mechanisms responsible for imprinting were also investigated. Two differentially methylated regions (DMRs), linked to the genes KCNQ1OT1 and SNRPN, normally have maternal-specific methylation that is acquired during gametogenesis. Bisulphite sequencing was used to analyse the methylation status of these key imprinting control regions. The investigators detected equal proportions of fully methylated and unmethylated DNA, which was consistent with the maintenance of normal differential methylation. Similar analysis of the paternally methylated H19 DMR also identified differential methylation. The H19 promoter region of the paternal allele is not normally methylated during gametogenesis but after implantation. It was found that all the cell lines analysed possessed differential methylation at this region, but the levels were more variable than for the primary DMRs. Previous work on mouse embryos has often demonstrated inappropriate regulation of H19, with altered methylation at the DMR. The investigators suggest that as methylation appears to be retained in the human cell lines alternative mechanisms, such as chromatin modification, may be circumventing the germline methylation signal. The authors conclude that although further work is required on the methylation status of imprinted genes and their expression, this study demonstrates that hES cell lines are epigenetically stable at imprinted regions and that epigenetic status should not act as a barrier to the use of these cells for therapeutic purposes.

Comment: This paper goes some way to address the concerns regarding the epigenetic stability of hES cell lines, and the potential impact of this on their suitability for therapeutic use. Previous research on sheep and mouse embryos, and mouse stem cells has shown that they are prone to epigenetic variation and the loss of normal imprinted gene expression, when cultured in vitro. This study demonstrates that at low passage hES cell lines are both appropriately imprinted and epigenetically stable. However, the investigators also highlight the fact that methylation profile does not always relate to normal gene expression, and that both must be studied for a definitive assessment of epigenetic stability during development. Additionally, this study only examined the expression of six imprinted genes, a small proportion of the total number of imprinted genes that have been identified to date. However, this study does establish hES cells as a potential model in which to study the expression of imprinted genes and early development and also indicates that epigenetic stability should not be an obstacle to their use in stem cell therapies.

The full research article can be found at Rugg-Gunn, P.J. et al (2005) Nature Genetics 37 585-587 (requires full text access).

8 June 2005A paper published in the new Royal Society journal Biology Letters reports on a classical twin study to investigate genetic factors in female orgasmic dysfunction. Sexual dysfunction among women is widely reported in the UK, but there is no consensus as to how far this represents a genuine medical problem, as opposed to a construct of sociological factors such as a changing cultural environment. The authors of the new study report that female sexual function is the subject of far less research than male sexual function, possibly because of the view that it plays no direct role in reproductive ability [Dunn KM et al. (2005) Biol. Lett. Early Online Publication 10.1098/rsbl.2005.0308]. Monozygotic (MZ) twins are genetically identical, whilst dizygotic (DZ) twins have 50% of their genes in common, on average; assuming the family environment of twins brought up together is broadly equivalent, significantly closer similarities with respect to the factor of interest observed in MZ twin pairs than DZ twin pairs are taken to be indicative of genetic influences on that factor. For this study, surveys with questions about sexual problems were completed by a total of 4037 adult women aged between 19 and 83 (average age 50), comprising 683 MZ and 714 DZ pairs of female twins. The MZ and DZ twin groups did not differ significantly with respect to average age (50), number of sexual partners (5) or reported incidence of having been heterosexual and sexually active ‘at some point’ (98%).

Among those who were or had ever been sexually active, the frequency of orgasm was assessed by the use of seven categories, ranging from ‘never’ to ‘always’. Overall, 32% of respondents reported sexual dysfunction (defined as ‘never or infrequently’ achieving orgasm) during intercourse, and 21% during masturbation. There was no difference in the proportion of MZ and DZ twins in either of these categories. However, the authors report – without presenting their data - that the relative frequencies of orgasm during intercourse and masturbation were found to be higher for MZ twin pairs (31% and 39%, respectively) than for DZ pairs (10% and 17%), an observation suggestive of a genetic influence. They estimate a heritability factor of 34% for difficulty reaching orgasm during intercourse, and 45% during masturbation, which the authors conclude represents a ‘significant genetic influence’ on orgasmic dysfunction in females. They call for further research into the biological basis of the phenomenon (see also BBC news report).

Comment: If valid, this study would suggest that female orgasmic dysfunction represents a complex condition with both environmental (psychosocial) and genetic components, which could have implications for both research and treatment. However, there was in fact no significant difference in the proportions of MZ and DZ twins in the groups that ‘never or infrequently’ reached orgasm by either sexual intercourse or masturbation (12% and 13%, respectively). The observed differences between the MZ and DZ groups used to infer a genetic component were based on inherently inaccurate self-reported measures of respondents’ overall frequency of orgasm throughout their life, relying on arbitrary selection of categories such as ’about 50%’ and ’51-75%’. Moreover, the data used for this calculation were not provided, which could have caused publication bias. Whilst the use of any self-reporting survey method has certain limitations, this study cannot be very accurate, and its conclusions are not really warranted by the data presented. Further, more robust studies using better surveys might yield more valuable data. However, even definitively establishing the existence of a degree of genetic influence over women’s ability to reach orgasm would not necessarily mean that identification of relevant genes would follow. As with all complex conditions, there are probably scores or even hundreds of contributing genetic variants.