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A pharmacogenetic ‘gene chip’ test kit produced by Swiss pharmaceutical firm Roche has received approval from the Food and Drug administration (FDA) for use in the US along with the Affymetrix GeneChip Microarray Instrumentation System (see press release). The AmpliChip Cytochrome P450 was launched in 2003 by Swiss pharmaceutical firm Roche Diagnostics, but withdrawn from sale in the US in November 2003 due to regulatory issues. The test became the first DNA microarray based test to receive EU approval in September 2004 (see newsletter item). The device detects common polymorphisms in the CYP2C19 and CYP2D6 genes, which encode two key enzymes involved in the metabolism of many sorts of drugs including common anti-depressants, painkillers and beta-blockers. Certain common variants of these genes affect the rate at which drugs are metabolized by an individual and hence the response to the drugs. On the basis of the AmpliChip CYP450 results, individuals are classified as poor, intermediate, extensive, or ultra-rapid drug metabolisers, allowing doctors to select the most appropriate drug and dose for the patient’s genotype with respect to drug metabolism. The FDA approval paves the way for the marketing of similar microarray-based genetic tests in the US.
The Human Fertilisation and Embryology Authority (HFEA) has announced that is will implement a new policy that it says will streamline the approval process for applications for pre-implantation genetic diagnosis (PGD) embryo screening for clinics. In the future, if a clinic applies for a license to screen for a certain condition, and that screening process is already been licensed at other clinics and carried out successfully, the HFEA will approve the license without having to go through the full HFEA license committee process. The clinic must have proven expertise in performing embryo biopsies and the techniques and methods used must be the same as at the previously licensed clinics.
PGD is currently used to screen for many conditions such as:
• Cancer disposition (e.g. Familial Adenomatous polyposis coli)
• Autosomal dominant disorders (e.g. Huntington’s disease)
• Autosomal recessives diseases (e.g. cystic fibrosis)
• Haemoglobinopathies (e.g. sickle cell anaemia)
• X-linked conditions (e.g. Duchenne muscular dystrophy)
However, applications for specialised applications of PGD will not follow this new policy and will still need to be considered by the HFEA license committee. These include: licensing for new conditions, PGD/HLA tissue typing, HLA on its own and late onset conditions or susceptibility genes
Regardless of the new policy, applications by patients requesting PGD for any licensed purpose will still need to be considered and approved by the clinic staff on an individual basis. The HFEA Code of Practice (6th edition) states that many factors would expect to be taken under consideration when considering an application. These include the view of the people seeking the treatment, their previous reproductive history, their family circumstances and the likely degree of suffering associated with the condition.
While patients’ procedures will remain the same, the HFEA expects this new policy to relax the burden on clinics seeking new licenses. As Suzi Leather, Chair of the HFEA, has stated that “…whilst PGD is still a specialised procedure, which can only be carried out by a qualified embryo biopsy practitioner, it should be straightforward for those clinics with a proven track record in the appropriate techniques to be able to carry out screening for any of the conditions currently approved.”
A service that provides advice, assessment and support from specialist cancer genetics nurses for people from families with inherited forms of cancer is to be expanded. Pilot projects are already based in GP practices in South West London, Yorkshire and the North East, and now Oldham, Poole and Somerset Coast Primary Care Trusts will also provide the service in 2005 (see BBC news report). Family members can ring and speak to a cancer genetics nurse to discuss their personal risk; in many cases they may only need reassurance that this risk is extremely low, but in other cases the nurses may suggest further assessment for possible genetic testing and advice on preventative interventions, should the individuals be found to have a strong genetic predisposition towards developing a form of cancer. The expansion of family cancer services is part of the £50 million investment in genetics in the National Health Service set out by the Government in the Genetics White Paper of 2003, in particular the commitment to provide funding for new initiatives in primary care genetics. For more information on cancer genetics, see the disease profiles section.
The Scottish Executive is to provide £4.4 million in funding for a three-year proof of principle collaborative project between Scotland’s five medical schools and other bodies (see BBC news report). The Genetic Health Initiative (GHI) will assess genetic predisposition to diseases with major public health impacts in Scotland: depression and schizophrenia, osteoporosis, cardiovascular disease, stroke and diabetes. Researchers will recruit 15,000 individuals aged 35-55 in the first instance (with a view to enlarging this cohort to 50,000) and their close relatives, such as siblings. Demographic and lifestyle information will be combined with healthcare records and medical data such as heart and blood vessel function. Blood samples will also be tested for genetic variants, in an attempt to determine the extent to which ill health is an inherited trait. Anonymous lifestyle and healthcare information will be linked to genetic profiles.
Project co-ordinator Dr Blair Smith, from Aberdeen University, commented: "It's the next exciting step following the mapping of the human genome", whilst Professor Anna Dominiczak of the University of Glasgow said: "This unique cross-institutional, interdisciplinary endeavour will make Scotland internationally competitive in human genetics of common complex diseases" (see press release). Members of the GHI consortium are:
26 January 2005The European Patent Office, after conducting opposition hearings earlier this month, has announced that it will amend a BRCA1 gene patent (EP 705903) held by Myriad Genetics. The original patent related to 34 mutations in the BRCA1 gene sequenced from the human genome and diagnostic methods for detecting these mutations to show predisposition to breast cancer. The amended patent, according to the EPO, “…now relates to a gene probe of a defined composition for the detection of a specific mutation in the breast- and ovarian cancer susceptibility gene and no longer includes claims for diagnostic methods.” Full details of the decision will be published shortly on the EPO website.
The patent, ‘17q-linked breast and ovarian cancer susceptibility gene’ was originally granted on 23 May 2001. Oppositions to the patent were filed by six different groups: the Institut Curie; Assistance publique – Hopitaux de Paris; the Institut Gustave Roussy; the Vereninging van Stichtingen Klinische Genetica, Leiden, the Netherlands; the Netherlands represented by the Ministry of Health; and Greenpeace Germany. Their concern was that the patent, with the others that had been granted to Myriad for the BRCA1 and BRCA2 genes), were too restrictive, giving a virtual monopoly on genetic testing to Myriad. Myriad were requiring that all samples be sent to their laboratories in the US for analysis at a fee of over $2600 (approximately £1380). European laboratories had developed their own methods of BRCA1 testing and did not want to have to pay Myriad for analysis. In addition some argued that Myriad’s test was not completely effective in finding large DNA deletions or rearrangements [Benowitz S (2002) JNCI 94(2):80-81].
Whether in response to these arguments or other factors, the EPO has been backing off from its original decisions. Last year, the EPO revoked Myriad’s first patent (EP 699754) for methods to diagnose a predisposition to breast or ovarian cancer using the normal BRCA1 sequence (as opposed to any mutated sequences) (see newsletter article May 2004). In February 2004, EPO granted a patent to Cancer Research UK for the BRCA2 gene, although Myriad also has a BRCA2 patent (see newsletter article February 2004). Now it appears that the broader community will be able to provide genetic tests for BRCA1 mutations without challenge, although Myriad is entitled to contest the opposition division's decision in this case. Opposition hearings on another Myriad patent, EP 705902, were also held earlier this month and a decision is expected soon. Details on that decision will be posted on their website and at www.epoline.org as part of their public file inspection service.
19 January 2005The NIH has revised its initial plan to encourage researchers to publish results of publicly-funded research studies on the internet within six months of publication [Zerhouni EA Science 2004 1106929]. Now, according to the Washington Post, officials are recommending the time-limit be extended to one year, responding to concerns that such a move would jeopardise the existence of some journals by undercutting their subscriber base.
The rationale behind the original plan was to enable free and easy access to the results of research funded from tax-payer money by asking researchers to publish their work on the US National Library of Medicine's PubMed Central, after an initial six month embargo period. The extension to one year has angered those who advocate greater accessibility of research publications. They believe that the NIH bowed to those with monetary interests who fear that a six month publication scheme would damage their profits, which in some cases have increased by 30 percent or more recently. As NIH-funded research makes up only a small proportion of material in the scientific journals, they argue, there should be little concern that subscriptions will be cancelled.
Elias A. Zerhouni, NIH Director, responding to opponents, has said, “The new policy continues to call for release of information as soon as possible after publication, but it really leaves it in the hands of the scientists to decide when. What’s important is that we’re creating a precedent in which the agency that funds medical research is establishing a public database containing all its scientific output.” Advocates of open access agree that even a voluntary programme will help to increase the information freely available to the public.
21 January 2005Australian doctors have reportedly used pre-implantation genetic diagnosis (PGD) to screen embryos for their blood group, to ensure that the embryo selected for implantation shared a rhesus negative blood group with the mother (see BBC news report). Women who are rhesus negative but have rhesus positive babies will produce an immune response termed alloimmunization against the ‘foreign’ rhesus blood cell markers when the maternal and fetal blood mixes, mostly during labour. The severity of this response increases with each subsequent rhesus-positive pregnancy, and can cause severe and potentially fatal anaemia in the new baby. In the vast majority of cases (around 99%) this situation can be prevented by giving rhesus negative mothers an anti-Rhesus injection during pregnancy to prevent the production of potentially dangerous antibodies; babies affected before birth can also be treated by blood transfusion in the womb. In England and Wales, around 62,000 such births occur each year; around 500 babies develop haemolytic disease of the newborn and up to 30 of these babies will die, with a similar number having developmental problems (see European Society of Human Reproduction & Embryology press release).
Dr Sean Seeho of the Royal North Shore Hospital in Sydney where the procedure was carried out said: "A couple who have had a significantly affected pregnancy are faced with the dilemma of whether or not to attempt further pregnancies". In this instance PGD was used to select an embryo for a couple whose second child had suffered from severe haemolytic disease due to a maternal alloimmunization response. A subsequent healthy rhesus-negative child, implanted following the PGD selection procedure, was born in 2003. The BBC claims that the use of PGD in this case “enabled a couple to have a healthy child who might otherwise have died” but it should be noted that even without PGD this couple could still have had a perfectly healthy child; a naturally conceived fetus would have had a 50% probability of being Rhesus negative anyway. The technique is also only viable for couples where the father was heterozygous for the Rhesus blood group; where the father is homozygous for the Rhesus blood group, all his offspring would be Rhesus positive and so PGD to select for a Rhesus negative fetus would be futile.
19 January 2005UK Health Minister John Hutton has announced that patients will have the right not to have their medical records stored electronically as part of the new NHS IT database (see BBC news report). An alternative option would allow sensitive data to be restricted for emergency access only, but Mr Hutton reportedly said that he thought few people would choose to opt-out of the system. However, it has also been reported that the NHS National Programme for IT has qualified Mr Hutton’s statement, saying instead that only in extreme circumstances would an individual be able to opt-out of electronic storage of their medical records, although they would be able to choose to restrict sharing of medical information to emergency use only. The ‘extreme circumstances’ would require a patient to demonstrate that having their information held electronically on NHS databases will cause them or someone else unwarranted substantial damage or distress (see zdnet report).
Concerns about the electronic database of patient records have been raised over issues of confidentiality and security. Richard Granger, NHS director general of IT, said the new system would be an improvement over the old, paper-based records system in this respect, saying: “The system will log every person who accesses a patient’s information…Currently there is no record of who has accessed what so it is very difficult to control who picks notes up”. Individuals choosing to restrict access to their records will be warned of the potential risks, such as staff making mistakes due to lack of relevant information about previous medical conditions or allergic reactions.
27 January 2005The Department of Health has launched a new campaign, ‘Give Life, Give Hope’, to encourage gamete donations from men aged 28-45 and women aged 28-35. Leaflets, posters and business cards will be distributed to 95 fertility clinics in order to raise awareness about donation and spread the message that donors will have no financial or legal responsibilities to any related offspring. This comes in advance of the new rule banning anonymity. From April 2005, donors must provide personal information so that any child conceived from their donation, at age 18, will be able to access information on their genetic parent. There are fears that this new rule and lack of knowledge about what it does and does not entail will inhibit individuals from donating. Donations are already low; ministers have said that while 500 sperm donors are needed each year, only 250 currently donate. While 1,100 women donate their eggs, 1,500 donations are needed. The campaign hopes to improve these numbers. It will also promote the National Gamete Donation Trust’s website and confidential helpline that can help those with questions about sperm and egg donation.
In a related story (see BBC news), researchers from Leiden University Medical Center in the Netherlands have found that couples seeking fertility treatment from poorer backgrounds are more inclined to want anonymous donors than those in better circumstances. This suggests that banning anonymous donors will negatively impact poorer couples. The Netherlands is facing the same concern regarding a decrease in donors, as they banned anonymous donation last year. The Dutch team interviewed 105 heterosexual and lesbian couples. A majority of heterosexual couples (40 out of 64) chose an identifiable donor. This choice was greater in lesbian couples where 40 out of 41 couples chose this way. 39% of couples did not choose an identifiable donor; most of these were from a poorer social background. The researchers believe their study shows that issues around male fertility and non-genetic parenthood were still seen as taboo by poorer social groups and that education is needed to help with this problem. -
A new publication in the advance online version of Science journal reports on variable susceptibility to HIV infection among individuals depending on the number of copies of the CCL3L1 gene [Gonzalez E et al. Science 2005: 1101160]. The CCL3L1 protein blocks HIV infection by interacting with the cellular receptor CCR5, a major receptor for viral entry to target human blood cells. Researchers at the US National Institutes of Health (NIH) analysed blood from over 4300 African-Americans, Europeans and Hispanic-Americans, both HIV-positive and HIV-negative, and determined the average number of copies of the CCL3L1 gene for each group. These were four, two and three respectively for HIV-negative individuals. Individuals with fewer than the average number of CCL3L1 genes for their ethnic group were found to be more susceptible to HIV infection, and at an increased risk of between 39% and 260% of rapid progression to AIDS following infection. Individuals with more than the average number of CCL3L1 copies were reportedly less susceptible; each additional gene copy was said to be associated with a 4.5-10.5% reduction in the probability of HIV infection. Researchers also looked at CCR5 gene variants known to be associated with different rates of AIDS progression, and found that individuals who possessed both disease-accelerating CCR5 variants and lower than average CCL3L1 copy numbers had an even higher risk of infection and disease progression.
Dr Anthony Fauci, director of National Institute of Allergy and Infectious Diseases (NIAID), commented: "This important study identifies genetic factors of particular groups that either mitigate or enhance one's susceptibility to infection and disease onset" whilst Dr Carl Dieffenbach of the NIAID said: “…this study further emphasizes the significance of defining all existing types of genetic variation and the impact that these variations may have on human susceptibility to infectious diseases” (see press release). The researchers reportedly hope that their findings could lead to a screening test to determine someone's susceptibility to HIV/AIDS, which might have an application in the treatment of the disease (see BBC news report).6 January 2005Rett syndrome (RTT) is a complex and serious neurological disorder that affects at least one in every 10,000 female births, making it the second most common cause of severe and profound learning disability in girls; clinical features appear 6-18 months after birth. A very small number of boys are also affected by the condition. In the majority of cases, Rett syndrome is caused by heterozygous mutations in the MECP2 gene on the X chromosome. Methyl-CpG binding protein 2 (Mecp2) is thought to selectively bind methyl-CpG islands in the mammalian genome and to function as a repressor of gene expression. It has previously been postulated that Mecp2 functions in imprinting, a process whereby certain genes are chemically modified (typically by methylation of CpG islands) according to whether they are maternal or paternal in origin. This is important because for normal development and function these genes are expressed primarily from the maternal or paternal alleles. Disruption of normal imprinting can cause disease due to altered and inappropriate gene expression; mutations in the MECP2 gene have been suggested to act in this way. Previous studies failed to find any loss of imprinting in RTT, looking at well known imprinted genes, but a new paper in Nature Genetics reports the loss of imprinting of the DLX5 gene in mouse models of RTT and human cell lines derived from individuals with the disease [Horike S et al. (2005) Nat. Gen. 37, 31-40].
The authors searched for Mecp2 target genes in the mouse brain by identifying and sequencing genomic Mecp2 binding sites (MBSs), working on the assumption that target genes would be close to such binding sites. 100 binding sites identified by chromatin immunoprecipitation, were sequenced; two MBSs were found to map to a cluster of imprinted genes on chromosome 6 (homologous to a region on human chromosome 7) which included the genes Dlx5 and Dlx6. To determine whether Mecp2 deficiency could cause dysregulation of these (and other) genes, the researchers used quantitative RT-PCR to compare their expression in the brains of wild-type and Mecp2 knockout mice. Expression of the Dlx5 and Dlx6 genes was found to be around two times higher in Mecp2-null mice than in normal mice.
To investigate whether this increase in Dlx5 expression was due to altered imprinting, Mecp2-null and control mouse lines were created with a single-nucleotide polymorphism (SNP) in Dlx5, which was used as a marker to monitor parental allele-specific transcription; there was no equivalent SNP available for analysis of the imprinted status of Dlx6. In the absence of Mecp2 there was complete loss of normal maternal imprinting of the Dlx5 gene in the mouse brain. The human gene, DLX5, is imprinted in normal human brain cells and lymphoblasts; the authors therefore examined lymphoblastoid cells from individuals with RTT who had mutations in MECP2 and found loss of imprinting in three out of four such samples.
Next, the researchers examined the methylation status of CpG islands in the DLX5 gene in both humans and mice, and found that they were unmethylated, suggesting that, despite the ability of Mecp2 to bind to these islands, regulation of DLX5 expression must be mediated by a different mechanism. A key factor in gene silencing and activation is chromatin structure; higher-order loops are associated with chromatin silencing. The authors investigated the role of Mecp2 in the formation of higher-order loops using a chromosome conformation capture technique combined with primers to test for MBSs brought together to form loops. They demonstrated that Mecp2 mediates the formation of an 11kb higher-order chromatin loop at the Dlx5-Dlx6 locus associated with silencing of the Dlx5 gene, via interaction with histone deacetylase 1 (Hdac1). In Mecp2-null mice, this region loses the ability to bind Hdac1 whereas in wild-type mice, this region is associated with a higher-order chromatin loop structure. This finding provides a potential mechanism for the loss of DLX5 imprinting in RTT.
Comment: Taken together, these results suggest a novel role for the loss of imprinting of the Mecp2 target gene DLX5 in the pathology of Rett syndrome, and provide avenues for further research into the underlying molecular mechanisms of the disease. DLX genes are expressed by neurons that respond to the neurotransmitter GABA in the brain; GABA neurotransmission has been linked to other neurodevelopmental disorders, including Angelman syndrome, which shares some clinical features with RTT and is also associated with a loss of imprinting. A degree of cross-regulation between different members of the DLX gene family has been previously reported; the authors postulate that small increases in levels of DLX5 expression due to a loss of maternal imprinting could significantly affect levels of other DLX genes leading to neurological pathology. A commentary accompanying the report queries whether DLX5 is in fact the key factor in pathogenesis of RTT, suggesting that it is probably only one of several gene targets regulated by Mecp2 [Pescucci C et al. (2005) Nat. Gen. 37, 10-11]. However, the targets and mechanisms of Mecp2 mediated gene expression are likely to be an important research area for unravelling the cause of RTT.
11 January 2005Osteoarthritis, the most common form of arthritis and the most frequent cause of joint disease, affects more than 5% of all adults worldwide. The disease involves the progressive loss of cartilage around the joints, leading to pain and dysfunction; the joints are normally protected by the balanced production and degredation (homeostasis) of cartilage extracellular matrix (ECM) components. If cartilage is degraded more rapidly than new tissue is formed, the ends of bones that meet in joints are inadequately protected and can rub together, causing inflammation. Osteoarthritis is known to be a polygenic disease influenced by both genetic and environmental factors. A Japanese study published in Nature Genetics identifies a new association between a gene and susceptibility to osteoarthritis, and also provides evidence of the functional link between mutations in the gene and development of the disease [Kizawa H et al. Nat Genet. 2005; advance online publication].
Researchers from Japan's Institute of Physical and Chemical Research (RIKEN) looked at the ASPN gene, which encodes asporin, a recently identified ECM protein that binds to TGF-ß, a key growth factor in cartilage metabolism, and to other cartilage ECM components. Microarray analysis of asporin mRNA expression in cartilage from 5 individuals with osteoarthritis and 3 controls showed that expression of asporin was abundant in the osteoarthritis group but negligible in controls. Real-time quantitative PCR analysis of a further 8 affected and 9 unaffected individuals reproduced this observation. Having sequenced the ASPN gene and identified 8 polymorphisms with a minor allele frequency of over 5%, the team moved on to examine the disease-gene association, using 134 patients with osteoarthritis of the knee and 234 unaffected control patients.
One gene variant, an aspartic acid (D) repeat polymorphism, showed a modest positive association with the disease. Within the cohort as a whole, there were a number of variants of this polymorphism with 12-18 D residues, the most common being D13. However, within the osteoarthritis group, the D13 variant had a significantly lower frequency whilst the D14 variant had a significantly higher frequency. The association was tested in a second independent cohort of 393 knee osteoarthritis patients and 374 controls, and the D14 variant again found to be significantly over-represented among affected individuals relative to the D13 variant. Similarly, the D14 variant was also over represented among a total of 593 individuals with hip osteoarthtitis, when compared with the control population. The frequency of individuals with the D14 variant was found to increase with severity of the knee osteoarthritis phenotype (as indicated by radiological grade) among both the cohort and general populations.
Kizawa et al. then investigated the role of asporin in cellular processes related to cartilage metabolism. They over-expressed the D13 or D14 ASPN gene variants in ATDC5 cell lines that model chondrogenesis (cartilage formation), and examined the expression of key cartilage marker genes AGC1 and COL2A1. Expression of the marker genes was reportedly suppressed and cartilage ECM formation inhibited for both D13 and D14; this effect was also observed when cell lines were treated with TGF-ß, which normally induces expression of the AGC1 and COL2A1 genes as part of chondrogenesis. A transient assay system was used to examine the effect of transient expression of asporin variants (D13, 14, 16 and 17) on TGF-ß induced cartilage gene expression in the ATDC5 cells; all forms of asporin suppressed induction of the cartilage matrix genes, but the D14 variant had the strongest inhibitory effect. Expression levels of TGF-ß were not found to be affected, so the researchers looked at whether the inhibitory effect of asporin was mediated via binding to TGF-ß. In vitro assays showed that both asporin D13 and D14 bound specifically to TGF-ß, leading to the proposal that this interaction mediated the negative regulation of chrondrogenesis induced by asporin.
The authors suggest that regulation of TGF-ß by asporin is the key mechanism in the pathogenesis of osteoarthritis, and propose that the TGF-ß/ECM system represent potential targets for therapeutic intervention against the disease. How the D-repeat polymorphic asporin variants may e