 It's a pleasure being here, and I wanted to share with you a patient that's gone through our site, and I think it illustrates many of the points that were highlighted today, how to transfer the UDP process to the extramural world, the goals of the border UDN, and hopefully it's a fruitful case for discussion. First let me acknowledge the team of over 25 individuals, but specific for this case, Joe Marquery, who's our lead coordinator, Shwetadar, who's our lead adult medical geneticist, Shuk Balazs of Romania, who's our internal medicine lead, and also endocrinology, and then Mahim Jain and Lindsay Berridge, who are physician scientists who lead our clinical site analysis pipeline, which was also very important in this case. So let me underline sort of the approach to patients, the aim at Baylor, which is different from site to site, is to try to use clinical resources as much as possible prior to using research funds to help as many patients as possible, and then there's variability, as we discussed in the setting up of the UDN. Some sites are not using this model and using primarily research funds, and primarily what I mean is that we use a hybrid billing model using clinically indicated tests built first to insurance, and then research evaluations covered by the grant, and we use a nonprofit third source to try to cover and overcome challenges of coinsurance when that becomes an issue. As many of you know, that can be a problem even in clinically indicated tests are covered by insurance. So our mix of patients reflect the whole payer landscape. We have privately insured patients, we have publicly insured patients, and obviously we have uninsured patients. This research subject, which I will discuss, is part of our soft launch, and so this is a local patient, and the history was reviewed prior to the UDN opening for applications, and then obviously subsequently went through the gateway. We did the pre-evaluation sequencing. This was in-person consent during the visit of the pro-band. We obtained phone consent from the father, the mother was deceased and unavailable. Because of the nature of the phenotype as you saw, we decided to do whole genome sequencing in this case, primarily because of the potential to pick up structural and copy number variations in the genomic data. And this was sent two and a half weeks prior to the clinical evaluation. This was sent to Hudson Alpha. The data was received in a timely fashion with about a four-week turnaround time, and the patient was publicly insured, so obviously there are challenges with regards to getting genetic testing in that context. So there was a pre-UDN evaluation when this patient was seen in a genetics clinic, and the individual was an adult in this case. It's a 30-year-old West African male referred for evaluation of Kline-Felter syndrome. And the clinical history was where the patient was first seen in the primary care clinic, and where he was reported with a known diagnosis of Kline-Felter at approximately age 16 years old. And currently, he reports being sexually active with reduced libido, but no problems with ejaculation. Genetic testing of a carry type was unavailable at the time, and he had endocrine, baseline endocrine evaluation which showed that follicle-stimulating hormone and luteinizing hormone were high while testosterone levels were low. So in terms of the past medical history, at 16 years of age, which brought the Kline-Felter diagnosis to the forefront initially, he was found to have undescended testes and small testes, and the serum testosterone was found to be low, and he was started on supplementation empirically, and he was on and off this until age 24 years. He did have pubic care and facial and chest care, and he started shaving around 18 to 19 years of age. And on physical exam, he was of normal stature. He had a BMI of 32, otherwise testes were present bilaterally, and the GU exam was otherwise normal. So there was no evidence of underlying genetic syndrome that was a dysmorphology on the evaluation. This is the pedigree, and red is our individual when he was initially seen on the pre-UDM workup. And of note, on the paternal side of the family, the ethnicity and origins from Nigeria and African-American on the maternal side of the family, and this would be relevant in some of the genomic informatics analysis that we did. Unfortunately, the mother passed away and was unavailable for this analysis. So the clinical workup that was available to us when we evaluated the case was that he did have a karyotype and it was 46XX, was repeated and confirmed. There was fish for SRY, and this was negative, and he had a CMA, which did not show any copy number variations that were suggested to be involved in the phenotype, chromosome microarray. He happened to be vitamin D deficient. He had a semen analysis which showed that he was as a spermic. And on imaging, he had a DEXA scan which showed that he was osteopenic. A pelvic ultrasound was normal, and abdominal ultrasound did not show any evidence of intra-abdominal gonads or any residual malaria in structures. So he did have an opportunity for repeat endocrine evaluation, and again, the FSH and the LH were both elevated. And the testosterone was, again, low. So the impression on the review was that this was a classic form of hypogonadotropic hypogonadism. He was SRY negative on fish, but there was a question of whether maybe he had gonadomosaicism for SRY. There was a question of, eventually he might need a fish carried out on testicular biopsy. SRY being the master gene for male sex differentiation. We also thought about the possibility of mutation of genes downstream of SRY. If, in fact, this were SRY negative form of hypogonadotropic hypogonadism. You can have loss of function of DAX1. You can have alterations in SOX9, which is immediately downstream of SRY. This was part of the rationale for getting whole genome sequencing, as it's well known that there's a very large multi-megabase regulatory region for SOX9. And then there's this hypothesized Z gene, which is a repressor of the male pathway. A way of explaining exactly the situation, XX males who are SRY negative thought to be due to mutation of some type of repressor. And so for these reasons, we really thought this was an interesting case as a potential of identifying, first, a novel physiology or a mechanism in a situation where you had non-syndromic form of 46XX sex reversal. Just as a quick physiology reminder, the LH and FSH were up-regulated. And this is, of course, the hypogonadotropic component of the hypogonadism, in which we had evidence of low testosterone. And clinically, we had evidence of low testosterone and the function related to testosterone. We didn't know at the time what the status of Sertoli cell function was. So it was proposed to the UDN. As I said, this was a case of a 46XX male, which occurs in the population. But the majority are, in fact, SRY positive, often due to a transplication or residual of this gene, which then stimulates the male sex differentiation pattern in an otherwise female carrier type. So he was accepted based on, A, he had the objective finding, B, there was a potential for identifying a rare molecular pathology to lucidate on some downstream mechanistic process of sex determination. And so that was the basis of the acceptance. And so the UDN evaluation was performed primarily in the outpatient setting. And so at Baylor, we've tried to do almost all of our evaluations in the outpatient settings in terms of many of these individuals. And he first received endocrine testing at the evaluation to evaluate Sertoli cell function. He received inhibit B measurements. He received sex hormone binding globulin measurements. He also received an ACG stimulation test to assess latex cell function. And then we also assessed adrenofunction with agglucocorticoids and androgens. He also had a measurement of anti-malarion hormone. Imaging included MRI of the brain to assess centrally whether there was a pituitary issue and then CT of the abdomen to value it at higher resolution than any residual malarion structures that may have occurred. And we also did peripheral blood PCR for SRY to try to obtain a higher resolution assay for SRY. We obtained a biopsy for skin fibroblasts. At Baylor, we've added to our protocol whole blood for RNA-seq for the potential of really to assess the role of this technology, not only in determining the underlying genetic defect, but also potentially understanding mechanistically in the context where that may be useful. And then as I said, in this situation, we requested whole genome sequencing because of the potential for non-coding variant or copy number or structural alterations upstream of Fox 9. As I mentioned, it's well been established in the sex differentiation literature that SRY drives Fox 9 in this important process. So here is the results of the UDM workup. The anti-malarion hormone was in fact low, though we think that at least during development, given the absence of malarion structures that there was a time period where there was anti-malarion hormone, and even B was low, but basically the rest of the endocrine workup, looking at adrenal function was basically normal. And so this was sort of the outcome of the workup. During, for the beta-HCG stimulation, we found that basically, again, latex cell function was deficient here after stimulation. So you can see after stimulation, you see elevated HCG, given that we gave it exogenously. And in spite of this, testosterone was low, while angiostin diome was low normal. The SRY peripheral blood PCR was negative. The MRI of the brain was unremarkable. Pituitary was normal. CT of the abdomen clearly showed absence of any malarion structures. And at that time, the whole blood RNA sequence pending. We do have the initial analysis of it, which we could talk about. It's actually quite interesting. So in terms of the post-UDN visit summary, we had a 30-year-old 46XX male, bilaterally small testes and azospermia, no evidence of malarion structures, and by all technologies, SRY negative. And so the hypothesis, molecularly at the time, was that there could be potentially a structural change or regulatory variant downstream of SRY that led to autonomous activation of the male-sex differentiation. And again, trying to identify what would be one of the first causes of a non-syndromic 46XX6 reversal case. And so Mahim Jain and Lindsay Burge led the genomic analysis on the data that was deposited by Hetzel Nalfa. We received FASQ and BAM files, and we used the Baylor-UDN alignment pipeline. This was a 30X coverage of about 1.3 billion reads mapped to the genome. The average insert size was about 288 base pairs. We applied a structural variant analysis, combining breakdancer and SV detect. We looked especially for genes, tendon duplication of genes that were known to be in the male-sex differentiation pattern like SOX9, like regulatory regions upstream of SOX9. There were many filtering of over 120,000 possible CNVs to about 217, which were manually curated. And what was nice in this case was that we were able to actually correlate the copy number changes in this group with findings that were in this clinical CMA that was done. And so that helped to sort of give us some QA with regard to the whole genome sequencing. With regards to single nucleotide variants and indels, the virtual exome, which we derived from the data, showed about 11 and a half thousand variants using our standard filters. So I'm not going to go through all this analysis, but basically there were no candidate variants on the structural analysis. And so with regards to, for example, duplication of SOX9 or some, initially we were hoping to find some alteration in the promoter or enhancer regions that regulate SOX9 and it was negative. On the review of SNVs and about 11,000 such variants in the virtual exome, we found what we now know we think to be the causative variant. So this, we found a arginine to tryptophan change in NR5A1, nuclear receptor subfamily 5 group A member 1. This was not present in the father who we had was available and was not present in the exact database. And it's very, very well conserved among vertebrates. We'll talk more about this variant in a little bit. Now keep in mind we did not have the mother, so we were unable to establish that this variant was de novo directly by sequencing. And this is where I think the expansion of the informatic analysis was very, very helpful. We observed in the genome data that in fact this variant was phased with a rare intronic variant and the father was also heterozygous for this rare intronic variant. Now this rare intronic variant when we pulled down actually all the data from 1,000 genomes was present. It wasn't present in the process data but when we went back and look at the unprocessed data it was in fact present. And this was very important in terms of supporting that this was in fact a de novo change on informatics analysis. So we took a haplotype analysis strategy. So here is the family structure and here are the different haplotypes. P1, P2 for the maternal, M1, M2 for the mom, P1 and M1 for the affected individual. And what we found was that in fact the P1 haplotype was what carried the mutation based on the phasing of the intronic variant with the mutation and we were able to infer the P2 haplotype directly by sequence analysis because again we had to hold genome on these two individuals. Now by reviewing the 1,000 genomes data especially for the African population, remember the father is from Nigeria, the mother was African-American from the Southwest U.S., we were able to basically identify the M1 haplotypes based on the frequencies across these populations, again from the reprocessed 1,000 genome data. We found that the intronic variant which occurred again in this case on the same allele as the NR5A mutation was limited to the P1 haplotype. And so based on this we were able to derive that there was very high likelihood that the NR5A1 variant was in fact a de novo mutation on the paternal allele. And basically I'm not going to go through all of this but by analyzing the allele frequencies in the African-American Southwest population of the P1 and P2 and presumed M1 haplotypes versus the Nigerian allele frequencies, that led us to this conclusion that this was likely a de novo variant on the paternal allele. So let me just cover then what we think is happening and then how this then has actually by actually the nature of the network led us to I think clear evidence that this is in fact the pathogenic variant. So NR5A1 is a very important transcriptional factor that occurs at multiple set stages of test development. It can occur, act on SOI, it can especially affect SOX9 in cooperation with DAX1 and this balance regulates the downstream activation of male differentiation target genes. And so we have this point mutation that is in NR5A1. So it turns out that this exact position, the R92 position when substituted for a glutamine was actually previously reported in a case of 46XY sex reversal. So the other opposite situation and it's well known that this region of the protein is called the A-box and it is important for stabilizing transcription factor DNA binding. So the hypothesis is that in fact the variant likely affects interaction with its target DNA binding. Now this was where I think the story I think really nicely illustrates the power of the network. We presented this at our recent Houston meeting of the UDN network and in fact I talked to Eric Villain, Eric of course is a world leader in sex differentiation about this case. And in fact he had seen the same variant R92W in a patient of his and in fact another two cases was also currently found in a consortium. And in fact these cases were being studied and have been studied and was being ready for report. And so we had this opportunity where we had one allele, one case and now we have four cases of the exact same mutation R92W which basically causes what we think to be this non-syndromic 46XX sex reversal. I think the mechanism is still unclear, Eric can comment on it. I think that there are some in vitro data that consortium have generated that supports certainly the possibility that NR5A1 can act in a context of dependent function whether you're in an XY or an XX carotypic background. And that in this situation one hypothesis is that the mutation is causing a derepression of SOX9 primarily because of its function or its action on DAX1. And I think that that's certainly feasible. You know I think the alternative is whether this variant itself because it can act at multiple levels could function in a gain of function fashion directly on SOX9. You know this is where again I think the model organism core will be extremely important. It turns out that NR5A1 exists in fruit flies and the approaches that the model organism core is taking is asking first whether human genes can rescue the deficiency in fruit flies and if then yes, how does the mutant allele operate in fruit flies. So this is one potential option that could be taken. But actually working with John Postwhite and Monty in the fish core they're actually going to generate the knock-in of this variant. Part of the reason is that in the zebra fish there are actually two copies of NR5A1 and this has sort of diverged the central versus the gonadal functions of NR5A1 allowing actually for a unique opportunity to study the variant. And so they're actually pursuing this with regards to generating a knock-in model of this. We are at the same time generating the knock-in model for CRISPR-Cas in the mouse and we've really tried to get at this novel functional aspect of it. So let me end by sort of getting back to what the UDN goals are. The first is improve the level of diagnosis and care of undiagnosed disease and so I think this clearly is a case where the experts coming together bringing the genomic technologies and the informatics technologies were able to come to a diagnosis for this individual. I think facilitating the research is key. You know I think one of the big areas that the UDN will be able to deliver on in the future is exactly situations like this where there are not clear simple loss of function or gain of function mutations but these alleles which are recurrent in the human population that have an enormous opportunity to teach us about structure function and thresholds of activity. Remember this patient does not have any of the other syndromic features that you would think about when you think about this pathway. Sox9 is very active in many other tissues. So I think this and the downstream studies with regards to fish and mice would be extremely powerful and then of course the collaborative and integrated research community. In fact we would not have been able to prove this case without finding this out with Eric as he was working on this case even outside of the UDN and of course the greater consortium of these two other groups with these two other cases. So I think clearly that this has been a very nice example of engaging the broader research community. So I will end there and take any questions. Thank you. Thanks Brendan. And so we're pretty close to right on time so if we just have any quick questions Judy. I couldn't help noticing he was a monosygotic twin. Is this twin? No he was not monosygotic. Not monosygotic. Sorry. Sorry. That line wasn't there. Okay. Thanks Brendan.