 Good morning. I am Jennifer Puck. I'm one of the leaders of the end-site project at the University of California in San Francisco and our project is called newborn sequencing or NB seek and I'm joined by Dr. Poo Yon Kwok and Barbara Koenig who will talk to you just after I do in the leadership of this project. So our site which won one of the competitions for funding aims to explore potential applications of whole exome sequencing which I'm going to abbreviate WES in this presentation and we're looking at how this could be applied to public health newborn screening. We want to develop whole exome sequencing from archived blood spots or DBS as you'll see on the next slides and California is unique in that it has saved residual dried blood spots used for newborn testing and under very rigorous controlled circumstances can make these available for research. So we are using them to look at currently screened metabolic disorders and primary immune deficiencies. We want to build on our experience with California screening for severe combined immunodeficiency or SCID also popularly known as the bubble boy disease and the screening program has identified infants with low T lymphocytes some of whom have SCID and many who have had a gene diagnosis now made by whole exome sequencing. We also will study parents views and values regarding newborn screening and that part of the project is run by Barbara Koenig. So here are some of our methodologic questions. We want to know what are the sensitivity and specificity of whole exome sequencing for metabolic disorders and our first naive question here was can whole exome sequencing replace the tend of mass spectroscopy that is now used and I think we have come to the conclusion already that the exome sequencing that we are able to do today is actually not going to replace this. But then again maybe the sequence analysis can augment the information that we derive from metabolic screening. In other words, we'd like to know could it identify patients who might have an abnormal screen and yet be asymptomatic during their lifetime or in contrast patients who would need early intervention and special diets for example. We also want to know would addition of deep sequencing shorten the time to a definitive diagnosis and relieve parents of the anxiety of waiting to see what their child really has. For infants and children with immune system defects, early diagnosis is essential for optimum treatment and outcome and so here we want to ask different in a different set of patients can whole exome sequencing detect pre-symptomatic immune disorders that would otherwise not be found before onset of serious infectious complications. So these are the methods and samples that we are using. We have an IRB approved collaboration with the California Department of Public Health Genetic Disease Laboratory. And through this collaboration, we are obtaining archived dried blood spots from which all identifying information has been removed, but the phenotype or clinical data regarding the metabolic diagnosis is still available. We are also getting archived dried blood spots from identified subjects with known or suspected immune system disorders and these are obtained following written informed consent. Our pilot DNA extraction and whole exome sequencing and analysis have been optimized with very old anonymous samples and also samples from our immune deficient patient cohort. We've worked on sequence annotation and variation calling for metabolic disorders and we're looking really at a restricted part of the genome here to a particular list of metabolic disease genes and gene pathways and we are using informatics that has been custom designed in collaboration with our partners at Berkeley and Tata Consultancy Services led by Stephen Brenner. We also have an immunodeficiency variant calling system using the whole exome. So how do we isolate our DNA? This is a picture of a large DNA robot called Otogen 965 which we use and it uses a 96 well deep plate in which a tiny punch three millimeters in diameter from the newborn blood spot is placed and the method involves digestion of proteins in this dried blood spot sample and then precipitation of the DNA and we actually find that we can isolate plenty of DNA this way to do a whole exome sequence that is to sequence all the genes in this sample of human DNA. We always look at the quality of our DNA and this is a photograph of an electrophoresis and you can see that in the top line there's one sample that maybe I can point to well no I can't that has perhaps a little bit of degradation but all the other samples from these newborn dried blood spots show very nice yields of intact DNA. So although we don't for example California does not save DNA from dried blood spots we do have the capability when we have permission to use these spots of making adequate DNA. And one way to assess whether our DNA is adequate is to go ahead and do the whole exome sequencing and here we're comparing a dried blood spot DNA sample to a sample obtained from fresh blood and what is depicted in these two graphs is the exome coverage or number of individual reads that cover these regions of one particular gene that is on our metabolic panel and you can see that the dried blood spot and the fresh blood give essentially comparable results. The immune deficiency part of our project relates to newborn screening which has already been instituted for the most severe immune deficiency and we test for that in every baby in California using a biomarker called T cell receptor excision circles or trex and as you can see in the cartoon which is a diagram of the T cell receptor this gene gets rearranged in developing T cells and the leftover pieces which here are shown in brown are deleted from what will eventually be the mature gene and turned into circles and then a PCR reaction across the joint of that circle can reveal whether T cells are being made appropriately or the absence of these circles suggests that a baby might have a serious immune disorder so that is the basis of the universal newborn screening for SCID and in cases that are identified by screening this way some of them have a quite readily detectable definitive diagnosis but others do not and so of 34 of the cases without a good diagnosis that we have looked at we've been able to diagnose 13 after doing whole exome sequencing and again this is with the patient's consent so this is a 38% rate of getting a definitive diagnosis and we have three more under study because each good candidate has to be confirmed in an immunology lab to see whether that really is the cause of disease and if all three of these cases pan out then the rate of success would be 47% which is actually a very high success rate compared to other projects using whole exome sequencing that run about 25 to 33% success so we do believe that this is a very good way to approach patients identified with a possible immune deficiency but without a definitive diagnosis and just to show you which states are doing newborn screening for SCID this is something that started in 2008 in Wisconsin and Louisiana had a pilot then Massachusetts and California was one of the earliest adopters in 2010 but this has been spreading very very rapidly to other states so that now over three quarters of the babies in the country are being screened with this Trek biomarker and we anticipate that almost all babies will be screened by the end of 2016 what we're finding in general from this is quite different from what you would find if you open any old textbook about SCID or read any of the literature coming from SCID bone marrow transplant centers as shown on the left side of this slide and you can see compared to that on the right side are patients in California with SCID identified by newborn screening and the sections of this pie chart are quite different showing that the proportions when you look at an unbiased newborn screen sample are not the same as the cases that in the past got all the way to a referral center for treatment and that's not because we have fewer for example x-linked SCID cases with the IL-2RG gene it's because we're finding more cases with autosomal recessive disorders and we believe that in the past these babies died of infections before their condition was recognized and another important thing to note on this slide is the little section of that's colored tan that shows the unknown cases so before newborn screening we thought we had solved the underlying diagnosis in almost all cases but now that we have newborn screening we're showing in California 12 percent of our cases don't have one of the SCID genes we already knew about and that means we have a lot more work to do so these are the infants who are going to be enrolling with consent with identifiers into our immune deficiency program and here's an example of one of the unknown cases that turned out not to have SCID but very low T cells we enrolled this family and did the whole exome sequence and you can see as we filtered away the variants that had low quality or were not part of the immune system or did not change a protein we were left with actually very few and in this case we documented that the infant had ataxia telangiectasia this is actually not a treatable condition at this time although there are research protocols considering ways to treat it but the family was able to take advantage of this information because this is also a breast cancer susceptibility gene so we were able to inform the parents and we were also able to give them anticipatory guidance for caring for the child and reproductive counseling meanwhile there are many immune disorders that are not detectable by trek screening and I've listed some on this slide anything that involves T cells beyond the stage at which the T cell receptor rearranges would have a normal trek biomarker even if the T cells don't function properly and those are the conditions listed on the top here there are also many syndromes that have variable amounts of T cell deficiency and these can be very severe and then there are also immune defects that don't involve T cells but involve other aspects of the immune system such as antibody production which is missing in x-linked agama globulinemia or chronic granulomatous disease and it would be very useful and very important to be able to detect these conditions early so that we could get the children on prophylactic antibiotics before they experience life-threatening infections we just don't have good markers for that now like the trek marker and therefore deep sequencing is probably going to be necessary to look at these and so a part of this project as shown in bold here is could newborn screening whole exome sequencing identify actionable primary immunodeficiency conditions prior to the onset of infectious complications and in order to do this study we are enrolling patients from our immune deficiency clinic after the time their diagnosis has been obtained and of course we have to take patients who are born in California so that their archived dried blood spots will be accessible and then with informed consent we fish out the leftover dried blood spots and do whole exome sequencing so far we've enrolled 10 individuals and plan to enroll 50 and as I showed earlier we expect a rate of finding a gene from the newborn dried blood spot of up to up to above 30 to even 40 percent so I'm going to now turn the screen over to Barbara Koenig who's going to talk about the ethical program in our UCSF new beseek