Wednesday, 9 July 2014

Non-invasive pre-natal testing for chromosomal abnormalities: bioinformatics and the UK NHS process


NIPT signal associated with a fetal micro
deletion on chromosome 22 causing
 DiGeorge syndrome.
Kitty Lo, a postdoctoral researcher in my group at UCL, just published a new bioinformatics application note and an associated R package called RAPIDR (available on CRAN) to streamline the bioinformatics of non-invasive pre-natal testing (NIPT). It is an opportunity to advertise the package and say a bit more on NIPT and what is happening in the UK with regard to this relatively new technology.
What is non-invasive pre-natal testing (NIPT) and how does it work?
Much has been written on the impact of DNA sequencing technologies on people’s lives, and some fields like genetic diagnosis for rare disorders clearly have been radically changed. But (thankfully) this type of application only concerns a small fraction of the general population. There is one application, however, that has the potential to affect thousands of people: the ability to diagnose chromosomal abnormalities, such as Down syndrome, from the blood of the pregnant mother.
While the technical aspects are clearly non trivial, the concept is relatively simple. It turns out that there are bits of DNA floating in the plasma of the mother. Some of these DNA fragments originate from the fetus (typically 10%, but this number varies quite a lot).  If the fetus has an extra chromosome 21, there will be more reads mapping to chromosome 21 than for a normal fetus. Statistics can detect this, and generate a firm diagnosis for the parents.
The key advantage of this non-invasive test of course is to avoid the invasive test, which carries a small but significant risk of miscarriage. It should be possible to test more women, hence increase the ability to detect Down syndrome while avoiding the risk associated with existing methodologies. There are also fierce legal and ongoing battles around the intellectual property associated with these tests but I am not best placed to comment on this.
NIPT in the UK: the RAPID project
Much of the development of NIPT for aneuploidy has been driven by US private companies. As a consequence, a large proportion of tests currently performed in the UK simply ship samples to the US for an analysis. Furthermore, it is currently only available through the private sector in the UK. Implementing this into routine clinical practice in a public sector health service will require more evaluation to see where in the care pathway it might fit, how we educate women and health professionals, the health economic aspects etc. The RAPID programme, which is funded by the NIHR PGfAR, is now evaluating some of these aspects and will report to the National Screening Committee in the next year.
As part of this project, my group (and in particular postdoctoral researcher Kitty Lo) collaborates with the chief investigator of the RAPID programme Prof Lyn Chitty in the development of the bioinformatics and statistical aspects of the RAPID project.
What women participate to the RAPID evaluation?
The RAPID project is working with selected maternity units located in the South of England and, more recently, Dundee. As traditionally done in the NHS, all pregnant women who opt for Down syndrome screening receive a risk based on the routine 12-week scan. Current NHS guidelines offer the option of an invasive test to women for which the estimated risk is greater than 1/150 (about 3% of pregnancies). In the context of the NIPT evaluation study NIPT is being offered to women undergoing Down syndrome screening who receive a risk of 1/1,000 or greater, we estimate that this will be around 12% of pregnancies. A blood test is then obtained, sent to the Regional Genetics Laboratory where cell free DNA is extracted and sequenced. Research midwives give the result of the NIPT to the mother. If NIPT suggests a chromosomal abnormality, an invasive test is recommended as there are a number of reasons why there may be discordance between the result obtained from maternal blood and the karyotype of the fetus.  
Does it work?
Remarkably well. It is already well understood that the science behind NIPT is robust and reliable but, as discussed above, the NHS needs data that goes beyond laboratory performance before making decisions if and how to implement this on a large scale. The study seems to be going well and is welcomed by women. An exhaustive description of the outcome of the study will be published in a few months.
Bioinformatics and the next steps
The bioinformatics are relatively simple but nevertheless need to be performed correctly. We think that an open source R package that researchers can use and modify is a useful step toward making the technique reliable and widespread.
A key challenge is the noise of sequencing assays, with parameters such as GC content of the DNA sequence creating noise and potential false positive. The RAPIDR package has been designed to correct for this using several previously published strategies. It is also designed to generate quality control measurements to identify unreliable samples, also a source of false positive. The package is freely available on CRAN, we are keen to see it used, and please get in touch if you are interested in trying it out. Ongoing work tackles the much more challenging issues of smaller abnormalities (partial chromosome deletions or duplications). These are harder to detect but are nevertheless important for the parents to make informed choices.

There is seems little doubt, based on worldwide experience, that NIPT is the future for pre-natal diagnosis. Implementation into routine maternity care will no doubt take time but hopefully our ongoing work (in the lab as well as for the bioinformatics) will help expedite these changes.

Sunday, 26 January 2014

Three jobs @UCL

With clinical colleagues at UCL, we are looking for motivated applicants to take on three job opportunities with a significant computational/statistical/bioinformatics component. I am involved in each of these projects but the main lead is clinical.Two are at the postdoctoral level and one at the PhD. The closest deadline is the PhD application (January 31st, which is this coming Friday), the two postdocs deadlines are further in time and about to be advertised. Do not hesitate to contact either myself or one of the other listed investigators directly if interested.

Three year postdoc: Transcriptome analysis in blood and iPS cells of retinitis pigmentosa (RP) patients
Funding: RP fighting blindness
Investigators/Collaborators; Andrew Webster, Tony Moore, Michel Michealides, Shomi Bhattacharya (inherited retinal disorders), Pete Coffey (induced pluripotential stem cell technology) and myself
This project has the strongest mol bio/wet lab component but the applicant should either have or will gain familiarity with the analysis of RNA sequencing data. It will investigates retinitis pigmentosa (RP), a Mendelian disorder that causes retinal degeneration and blindness. It is due to many specific genes that affect the development and/or maintenance of rod photoreceptors, the most abundant light-transducing cell in the human retina. Although many genes have already been identified, discovering the biological link between mutant or absent protein on the one hand, and specific dysfunction and death of rod photoreceptors on the other, is challenging. Without understanding the detailed molecular pathology, novel treatments will not be possible.

The project benefits from a large resource of such patients and families managed at Moorfields Eye Hospital and investigates the pathophysiology of those affected by mutations in splicing-factors. These comprise a significant proportion of affected patients including RP11, RP9, RP13 and RP18. The underlying genes encode proteins that make up the spliceosome, and are expressed in all eukaryotic cells. One enigmatic feature of these disorders is the fact that heterozygous mutation (they are all autosomal dominant disorders) causes specific problems with the retina and not, as far as is known, any other organ or cell-type. One further interesting feature of these disorders is the manifestation of variability, and sometimes non-penetrance in gene-carriers. By investigating this further, ameliorating factors might be identified.

The project plans to address these questions by the thorough analysis of the transcriptome of affected gene-carriers compared to those gene-carriers without disease and controls (ethinically matched non-carriers). We intend to use RNA-Seq experiments to explore this comprehensively, in collaboration with Dr Vincent Plagnol and colleagues at UCL Genetics Institute. Secondly, we intend to generate iPS cells from patients’ skin biopsies and thereafter retinal pigment epithelial cells and photoreceptor progenitor cells to explore their transcriptomes and phenotypes. This will be under the supervision of Professor Pete Coffey and team at UCL Institute of Ophthalmology.

This represents an opportunity to understand eukaryotic splicing and its aberration in human retinal disease. It will require a willingness to generate and work with RNA-Seq data, understand human inherited disease and be able to develop iPS and derived cells in cell culture. Hence the successful candidate will gain a wide experience in molecular and cell biology as well as bioinformatics.



Three year postdoc: Genetics of epilepsy
Investigator: Sanjay Sisodiya, UCL Institute of Neurology and myself
Funding: Wellcome Trust, European Union, MRC

Epilepsy refers a complex and heterogeneous set of disorders, and its etiology remains hard to elucidate. Sanjay Sisodiya at the UCL Institute of Neurology (IoN) leads a successful research program that investigates the genetic basis of these diseases and how this genetic basis affects response to treatments. Cases that are part of the study are very deeply phenotyped, including novel experimental techniques and extensive long term follow-up. High throughput sequence data (a lot of exome sequences, > 200 whole genome sequences) are being generated and are now becoming available. We are looking for an postdoctoral researcher to work on these data. There will be plenty of scope to develop new bioinformatics/statistical methods. The size of the dataset continues to increase and a lot of additional information about these patients is also available, which provides an opportunity to tackle pharmacogenomics questions.



Four year PhD studentship: System biology for graft versus host disease (GVHD)
Investigators: Ronjon Chakraverty, Clare Bennett (UCL Cancer Institute) and myself
Funding: Gordon Piller PhD studentship, Leukaemia and Lymphoma research
Deadline: January 31

This project uses a systems biology approach and mouse models to investigate the basis of graft-vs-host disease, i.e. the process of donor immune cells attacking the host organs, a severe complication following bone marrow transplant. As part of our research program, we are tracking clonal or polyclonal donor T cell responses across multiple sites in pre-clinical models of GVHD and evaluating transcriptional profiles and/or T cell receptor (TCR) sequences of purified cell populations. The student will use computational methods in a ‘dry lab’ environment (in collaboration with V Plagnol) to assess these microarray and TCR sequence data and to evaluate the extent to which GVHD is driven by TCR repertoire-dependent or independent factors. Systematic methods will be used to compare differentially expressed genes in our models with (1) multiple chipSeq maps that provide a genome-wide view of transcription factor binding sites and (2) extensive, publicly available gene expression datasets for human and murine T cells in other inflammatory conditions. These approaches will be used to identify novel regulatory or downstream effector pathways implicated in GVHD. Extensive cross talk between ‘dry’ and ‘wet’ lab researchers in our LLR program will permit further experimentation or data analysis as new hypotheses are formulated and tested.