Wilhelm Johansen Symposium: the impact of deep sequencing on the gene, genotype and phenotype concepts
21-23 March, 2011
Copenhagen, Denmark

Organisers:

Draft Report

Summary

Some of the most commonly used genetic terms (gene, genotype, phenotype) was coined in the early 20th Century by the Danish geneticist Wilhelm Johannsen in 1909. Since then, genetics has become one of the most central disciplines in biology and medicine, facilitated by the first biological big science achievement: the completion of the draft sequence of the human genome a decade ago. Now, the development of high-throughput or next generation sequencing (NGS) technologies promises a new revolution in genetics, molecular biology and medicine. This will lead to a growing understanding of the complex interaction between genes and gene products, the functional importance of genome structure, and the heterogeneity and complex inheritance of both rare and
common human traits and disorders. The symposium with more than 100 scientists from 15 countries, and speakers from 7 countries, celebrated the century of Wilhelm Johannsen’s terms and the establishment of the Wilhelm Johannsen Centre for Functional Genome Research (WJC) in 2001 by the Danish National Research Foundation. The programme gave a broad overview of the latest developments and impact of highthroughput sequencing technologies, how the technology will improve our
dissection of the functional organisation and modification of genes, extend our knowledge about genetic factors in human disease from mostly rare Mendelian disorders to complex disorders with complex inheritance patterns, and mediate the establishment of novel genotype-phenotype relationships. The symposium also included talks and discussions about the involvement of non-coding RNAs including microRNAs in disease, and illustrated amply how the enormous power of next generation sequencing for experimental data-generation will create an ever growing need for bioinformatics and in silico systems biology modelling. Both the social and the scientific content of the Symposium was of excellent quality, and gave the participants both an insight into the field, and a social network which especially the young scientists will find important for their future carreer.

Scientific Content

The introductory talk of the symposium was given by the Director of The Danish National Research Foundation (DNRF), Thomas Sinkjær, who showed that longterm funding of centres-of-excellence like WJC has provided high-impact results beyond the investment. Thus, DNRF sponsors 2% of the public research budget, but 20% of all Danish publications in Science and Nature originated from DNRFfunded centres. There was a discussion about how this strategy resembled foreign systems, e.g. the Mac Planck Society in Germany and the Howard Hughes Medical Institute in USA.

Wang Jun from Beijing Genome Institute, China, gave an impressive presentation of the present and future possibilities of NGS. BGI is by far the largest sequencing facility/company in the world, with over 150 next generation sequencing machines, and over 5.000 employees. The strategy of BGI is to sequence everything of value – including medical, cultural or economic values. Examples included the first sequencing of an ancient human, from DNA extracted from a 4.000 year old hairlock detected in Greenland, the sequencing of important crops like rice, of vegetables like the cucumber, of the plan to identify 1.000 disease genes related to Mendelian disorders, to the sequencing of multiple single cells in tumours, revealing their evolutionary history, with likely clinical and therapeutical implications. An important lesson and discussion theme
in the auditorium was that the bioinformatic part of sequencing will become the primary bottleneck in the future. It is now cheaper to obtain the data than to analyse them. The young scientists who have studied bioinformatics will have golden opportunities in the future.

Many applications and technical challenges of NGS were touched upon over the 3 days of the symposium. Its application for molecular testing of genetic disorders, exemplified by Leber Congenital Amaurosis (Elfride De Baere, Belgium). How the technology has revolutionised the sequencing of bacterial genomes with circular chromosomes, and made it possible to reveal all types of mutations, including large structural rearrangements like duplications and inversions (Ole Skovgaard, Denmark). One of the young scientists, Christina Halgren (Denmark), demonstrated that next generation sequence mate pair mapping of prenatally detected de novo translocations will greatly improve genetic counceling and hence decisions in this classical counceling dilemma with
profound implications for the families involved. This exemplifies the ultimate in personal medicine, where next generation sequencing results may lead to life-or-death decisions.

The rapid technological advances which have developed in the slip-stream of the Human Genome Project, including next generation sequencing, has greatly facilitated the reductional approach to identify genetic factors associated with a range of human disorders. However, to be able to understand how these genetic factors relates to phenotypes, both at cellular and organismal level, a more holistic approach with a focus on complex interactions in biological systems are direly needed. Søren Brunak (Denmark) gave an overview of how Systems Biology, ranging from the study of protein-protein-interactions (PPI) to the extraction and integration of medical data from patient files, have developed and how this will become an ever more important field in the future. This was exemplified by Lars Allan Larsen (Denmark), who had identified 19 spatiotemporal protein networks driving the formation of different anatomical structures
across many stages of heart development. Congenital Heart Disease (CHD) provided a general model for the understanding of the interplay between different genetic and environmental factors in complex disorders. The detected PPInetworks participating in normal heart development can now be used as a scaffold for filtering of whole-exome sequence data from large cohorts with CHD.

Several of the lectures focused on how next generation sequencing will allow us to dissect the enormous genetic heterogeneity and phenotypic overlap associated with many complex disorders, including immune related phenotypes (Cisca Wijmenga, The Netherlands), intellectual disability (Hans-Hilger Ropers, Germany), epilepsy (Ingo Helbig, Germany) and a range of neurodevelopmental, cognitive and neurological disorders which co-segregate in large families (Niels Tommerup, Denmark). These presentations stimulated discussions about how next generation sequencing will provide the molecular data that can explain classical genetic terms like genetic variability and incomplete penetrance. Oluf B. Pedersen (Denmark) discussed how the last five years humongous genome wide association studies (GWAS) have identified numerous genetic susceptibility loci for complex disorders, exemplified by type 2 diabetes and obesity, but combined
these susceptibility loci only explain a small proportion of the heritability in these disorders. The hypothesis is now that the hidden heritability may be rare variants which have not been included in the GWAS so far, which have only tested SNPs which are common (>5%) in the population. The protein coding part of the genome occupy les than 1.5% of the human genome, or about 40 Mb. This is exploited in next generation sequencing by the development of capturing technologies by which the coding part, the Exome, can be enriched for by hybridisation prior to sequencing. Next generation sequencing is the method of choice for detecting these rare variants. The so far largest exome sequencing effort encompassing 2000 Danes were presented. The discussion included how this approach will not only transform many aspects of clinical genetics, but also many other areas of medicine.

Until recently, most genes were believed to be protein-coding. However, other significant targets are now emerging, including a vast repertoire of non-coding RNA genes, which are now being detected by next generation sequencing. Among these are more than 1.000 microRNA genes, which are involved in the posttranslational regulation of mRNA stability. Bart de Strooper (Belgium) gave a intruiging lecture about the emerging role of microRNAs in neurodegenerative disorders.

The NGS technology has not only revolutionised the detection of rare genomic variants associated with disease, but has also revolutionized the functional analysis of DNA and RNA. Henk Stunnenberg (The Netherlands) provided an overview of how the appr. 20.000 genes/genome present in each of our cells, despite being genetically identical, are modified by epigenetic mechanisms to establish cell specific expression programmes resulting in at least 200 diffferent cell types in the human body. NGS has revolutionised the analysis and mapping of the involved modifications at DNA level (DNA methylation) and protein level (histone modifications), as well as the global analyses of the transcriptomes associated with these modifications. Henk Stunnenberg delineated the
systematic international efforts to characterize the Epigenome in all cell types in the human being. A detailed understanding of the molecular basis and stability of the Epigenome will obtain a therapeutical dimension in the area of regenerative medicine, which have rapidly emerged in the slip-stream of stem cell research and the succesful reprogramming of one cell type to another (Juan Carlos Izpisua-Belmonte, Spain/USA).

Next generation sequence based analyses will become a standard approach for the characterisation of specialised cell types. A whole subsection of the symposium was devoted the one of these cell types, the sperm cells. This included the proteomics of the human sperm cells (Rafael Oliva, Spain), and presentation of studies of RNA expression and chromatin organisation in human sperm cells (Meritxell Jodar, Spain; Tanya Vavouri, Spain). The latter two also illustrated another important aspect of the symposium: The active participation of young investigators, who submitted abstracts when applying. All posters were on display during the whole meeting, and all coffee- and lunch breaks took place among the posters to stimulate discussions. The best of the abstracts were selected for oral presentation, ranging from the sperm lectures to technical notes on multiplex targeted sequencing in leukemia (Agata Weselowska, Poland).

Although we have a detailed knowledge of our genome, we know very little about how this genome is positioned within the cell nucleus. Wouter de Laat (The Netherlands), showed how next generation sequencing has revolutionised the detection of DNA-DNA interactions in the nucleus by 4C and HiC techniques based on proximity-based ligation of DNA strands, and how these cis- and transinteractions are intimately related to elements that regulate the expression of specific genes. Many of these interactions involve evolutionary high conserved non-genic elements (CNE), and Stefan Mundlos (Germany) gave an overview of the many different genetic conditions where point mutations, microdeletions or microduplications of CNEs have resulted in congenital malformations. It is conceivable that full genome sequencing or targeted resequencing of CNEs in the future will detect many new mutations of this type, and the discussion
included which types of disorders would be expected to be associated with such
mutations.

The term next generation sequencing should actually be termed 2nd generation or“now generation” sequencing, since the next or 3rd generation sequencing is already emerging. This includes single molecule, real time (SMRT) sequencing, and computer chip based sequencing (e.g. Ion Torrent). Despite powerful potential none of these technologies can sequence DNA molecules beyond the kilobase range. As a finale to the symposium, Henrik Flyvbjerg (Denmark) showed how Single-Molecule Denaturation-Mapping of DNA may provide new avenues for large scale sequence information on DNA molecules in the megabase range.

Assessment of the results & impact of the event

The symposium has highlighted Denmark as an important and forefront player in the field of next generation sequencing, and have brought together Danish scientists with international experts within the field. The scientific content of the symposium was very diverse and of excellent quality, and gave the participants both an updated insight into the present status of the field of next generation sequencing, and how this field will rapidly change by the emergence of new applications and technologies.

By gathering experts from many different disciplines, both at national and international level, the symposium with its intimate size have provided a basis for networking. One immediate effect of this are the novel international collaborations, mainly at the European level, which were established and strengthened by the physical meeting of scientists from different countries. This has been especially important for the many young participants.

This networking was supported by the social activities: The symposium was hosted by Faculty of Health Sciences at the University of Copenhagen. The University also hosts the oldest and largest Medical Museum (Museion) in Northern Europe, situated in the restored 17th Century building of the first public hospital in the city. The welcoming reception of the symposium took place in this historical setting, within a public exhibition of the impact of genetics and genomics which very timely had just opened. The exhibition included “Illuminatum”, a light installation sculpture of 800 genomic arrays which have been used for genotyping Danish patients with metabolic syndrome.

The University of Copenhagen also provided the venue for the symposium dinner, in the old historical University building. One of the highlights of the dinner was the surprise lecture about Ancient DNA by Eske Willerslev (Denmark), who gave an intruiging insight into how DNA sequencing will change our concept of human evolution, variation, migration and history.

Programme

Monday 21st March 2011
8:30 Registration and Breakfast/Coffee
9:30 Welcome. The Danish National Research Foundation – Thomas Sinkjær
10:00 Sequencing, sequencing – Wang Jun.
11:00 Structural rearrangements in circular chromosomes - Ole Skovgaard.
11.30 Detection of translocations and inversions by mate pair analysis - Christina Halgren.
12:00 Photos
12:10 Lunch
13:00 Functional organization of the genome – Wouter de Laat.
14:00 Multiplex targeted sequencing in leukemia - Agata Weselowska.
14.20 Leber Congenital Amaurosis: molecular test using NGS. Elfride De Baere.
14.40 Analysis of metagenomic sequencing datasets – Josef Voght.
15:00 Coffee break/posters
15:45 Epigenetics – Henk Stunnenberg.
16:30 Closing
18:15 Reception at Museion

Tuesday 22nd March 2011: Phenotype and genotype
8:30 Breakfast/Coffee
09:00 Genetic heterogeneity - Hans-Hilger Ropers.
10:00 Phenotypes and the regulome - Stefan Mundlos.
11:00 microRNAs in neurodegenerative disorders – Bart de Strooper.
12:00 Lunch
13:00 Systems Biology – Søren Brunak.
14:00 Systems Biology of Congenital Heart Disease – Lars Allan Larsen.
14:45 Coffee break/posters
15:30 Exome sequencing in metabolic syndrome - Oluf B. Pedersen.
16:30 Closing
19:00 Galla-Dinner, surprise lecture, poster awards
Surprise Lecture: Ancient DNA. Eske Willerslev.

Wednesday 23rd March 2011 - Phenotype and genotype models
9:00 Breakfast/Coffee
9:30 Genetic sharing of immune-related phenotypes – Cisca Wijmenga.
10:30 Genetics of epilepsies – Ingo Helbig.
11:30 Lunch
12:30 Cognitive Comorbidity – a model for variability and penetrance – Niels Tommerup.
13.15 Proteomics of the human sperm cells - Rafael Oliva.
14.00 RNA expression in human sperms - Meritxell Jodar.
14.15 Chromatin organization in sperm - Tanya Vavouri.
14:35 Coffee break.
15:00 Stem cell models - Juan Carlos Izpisua-Belmonte.
16.00 Single-Molecule Denaturation-Mapping of DNA - Henrik Flyvbjerg.
16:30 Closing of Symposium