The Undiagnosed Diseases Network: Future of Precision Medicine in Endocrinology. Ashok Balasubramanyam, MD Baylor College of Medicine

Transcription

1 The Undiagnosed Diseases Network: Future of Precision Medicine in Endocrinology Ashok Balasubramanyam, MD Baylor College of Medicine

2 The Undiagnosed Diseases Program: Rationale Personalized, precision medicine Scientific knowledge Decrease (emotional and financial) costs of diagnostic odysseys Lack of a diagnosis Creates concern or suspicion Challenges patient physician relationship Gahl et al JAMA

3 Goals of the UDP Provide patients who have been extensively evaluated with an accurate diagnosis Use new genomic technologies to aid in diagnosis Assess phenotyping approaches Identify novel diseases Uncover new information about the causes of disease Identify potential therapeutic targets

4 History of the UDP : Received over 10,000 inquiries Reviewed records of over 3300 applicants 800 patients evaluated 40% pediatric 30% adults with disease onset in childhood 30% with disease noted in adulthood ~6% receive diagnosis of novel disease; ~12% diagnosis of rare disease Tifft & Adams 2014 Curr Opin Pediatr

5 Types of diagnoses Extremely rare diseases Ehlers Danlos syndrome, musculocontractural type I [CHST14] Early onset myopathy, areflexia, respiratory distress, and dysphagia [MEGF10] Rare presentations of known diseases Blended phenotypes of multiple diseases Novel diseases Calcification of joints and arteries [NT5E] Familial distal myopathy [HINT3] Gahl et al Genet Med

6 Lessons learned at the UDP Whole exome sequencing can be most economical when multiple gene candidates are being considered. Sequencing of family members can be a powerful tool to help filter and interpret variants found on whole exome sequencing. Accurate and meticulous phenotyping is essential. Multidisciplinary collaborations are critical to making diagnoses. It takes a network. Gahl et al Genet Med

7 Who is in the Network? 7 Clinical sites: Baylor College of Medicine Duke Medical Center Harvard affiliated hospitals NIH Undiagnosed Diseases Program Stanford Medical Center UCLA Medical Center Vanderbilt University Medical Center 2 Sequencing cores: Baylor College of Medicine HudsonAlpha 1 Coordinating center: Harvard Medical School

8 Additional resources Central biorepository: Vanderbilt University Medical Center Model organisms screening center: Baylor College of Medicine University of Oregon Metabolomics core: Battelle Pacific Northwest National Laboratories Oregon Health Sciences University

9 The UDN process Patient applies online, supplying a physician s referral letter Patient is assigned to a clinical site, where application is reviewed and medical records are gathered Patient is informed of decision of acceptance into study Patient travels to clinical site for study visit: up to 5 days of consultations and clinically indicated tests Diagnosis, therapy, and/or further basic research

10 Process Y1: Organization, SOP s, review panels Y2: Start up (through April 2016) 35 patients per clinical site Y3 4: 50 patients per year per clinical site No costs to patient for travel, stay or research tests. Standardized triage and review process, locally and for network.

11 An Undiagnosed Patient CC: Impotence, low energy 30 year old African American male Initially evaluated by physician in Austin, TX Diagnosed with Klinefelter syndrome at age 16 years No chest or facial hair Some pubic and axillary hair Small testes Low testosterone

12 Case History Started testosterone supplementation at age 16 Began shaving age 19 Genetic testing had been done, but results were unavailable

13 Nigerian (Yoruba) Pedigree African American (Southwest US) 69y d.50y gastric ulcer/sle 7y 32y 29y 29y 30y

14 Exam Ht 5 11 ; BMI 32; Vital Signs: normal Normal male exam except for following: Small gynecomastia Minimal chest and facial hair Long limbs, short trunk Testicles present and descended bilaterally, L>R (<5cc) Phallus 5 6 cm length, 2 3 cm width

15 Labs Follicle Stimulating Hormone (FSH) 23 & 25 ( miu/ml) Luteinizing Hormone (LH) 15 & 15 ( miu/ml) Testosterone 44 & 80 ( ng/dl)

16 Labs Semen analysis: azoospermia Genetic testing Karyotype: 46, XX (repeated & confirmed)

17 Imaging Pelvic u/s: normal Abd CT: no evidence of intra abdominal gonads or Mullerian structures DXA: osteopenia

18 Assessment Hypergonadotropic hypogonadism Atrophic testicles Azoospermia 46 XX male Bilateral, descended testicles No Mullerian structures

19 Novel Phenotype? 46, XX male 1 in 25,000 people 90% of these patients are SRY+ FISH for SRY negative Peripheral blood PCR for SRY negative Rare syndrome with even more rare molecular pathology Opportunity to elucidate mechanistic processes downstream from SRY

20 Pre UDN Hypothesis Some structural change or regulatory variant downstream of SRY led to autonomous activation of male sex differentiation Mutation of downstream SRY genes: 1) DAX1 deletion 2) SOX9 duplication Best guess was SOX9 duplication based on our findings until this point and the literature.

21 UDN Evaluation Endocrine testing: Adrenal function Anti Mullerian Hormone (AMH) Inhibin B hcg stimulation test

22 Results Value Reference range ACTH pg/ml Cortisol μg/dl

23 Results Value Reference range ACTH pg/ml Cortisol μg/dl Aldosterone ng/dl Renin ng/ml/hr

24 Results Value Reference range ACTH pg/ml Cortisol μg/dl Aldosterone ng/dl Renin ng/ml/hr 17 Hydroxyprogesterone 60.9 <138 ng/dl 17 Hydroxypregnenolone 369 <442 ng/dl DHEA S μg/dl

25 Results Value Reference range Anti Mullerian Hormone ng/ml Inhibin B < pg/ml

26 hcg Stimulation Test Baseline Day 4 LH ( miu/ml) 16.2 FSH ( miu/ml) 21.2 Sex Hormone Binding Globulin ( nmol/l) β hcg (0 5 miu/ml) 93 Testosterone ( ng/dl) Androstenedione ( ng/ml) Dihydrotestosterone ( pg/ml) 45 55

27 Genomic / genetic Analysis

28 Chromosomal Microarray (CMA) Confirmed 46, XX status 11q23.3 deletion involving SCN4B gene (missense mutations associated with Long QT syndrome with incomplete penetrance) No history of syncope; no family history of cardiac arrhythmia or sudden cardiac death EKG: normal sinus rhythm with QTc of 406

29 Whole Genome Sequence Analysis Structural variant analysis revealed no candidate variants (including SOX9) SNV/INDEL review > 11,500 variants reviewed in virtual exome : 217 manually reviewed 4 suspects NR5A1 (SF1) leading candidate Gene Variant/Domain EXAC Present in Father SIFT/POLYPHEN2 Conservation NR5A1 (SF1) Nuclear Receptor Subfamily 5, included) Previous implication in regulation of SOX9 Group A, Member 1 c.274c>t p.arg92trp A box domain N/A (~117,000 alleles Previously implicated in regulation of SOX9 No D/D Well conserved among vertebrates

30 WGS Analysis Summary Unable to establish this variant as de novo directly (proband s mother unavailable). In the proband, the candidate variant is phased with a rare intronic variant. The father is heterozygous for the rare intronic variant. The variant is not present in 1000 genomes processed data.

31 Haplotype Analysis Strategy Identify P1 haplotype (paternally inherited), infer M1 haplotype (maternally inherited), infer P2 haplotype (paternal non inherited) from review of proband/father whole genome sequencing. Review 1000 genomes African populations for P1, M1 and P2 haplotypes for (1) presence of phased intronic variant (2) frequencies across populations. P1 P2 M1 M2 P1 M1

32 Findings: 1) All individuals with P1 have the intronic variant. 2) No individuals with M1 or P2 have the intronic variant. 3) P1 is a rare haplotype but highest in Nigerian population (consistent with paternal ancestry) 4) M1 is rare but highest in ASW population (consistent with maternal ancestry) Haplotype Analysis Results African Americans from SW US n=61 P1: AF=0 M1: AF=0.025 P2: AF=0.057 Afro Caribbean in Barbados n=97 P1: AF=0.010 M1: AF=0.021 P2: AF=0.124 Mende in Sierra Leone n=85 P1: AF=0.012 M1: AF=0.018 P2: AF=0.200 Gambian Western District n=131 P1: AF=0.004 M1: AF=0.019 P2: AF=0.080 Luhya in Webuye, Kenya n=99 P1: AF=0.005 M1: AF=0.020 P2: AF=0.157 Esan and Yoruban Nigerian n=102 P1: AF= M1: AF=0.005 P2: AF=0.162

33 Haplotype Analysis Strategy If intronic variant is limited to P1 haplotype, there is a high likelihood the NR5A1 variant is de novo on the paternal allele P1 P2 M1 M2 P1 M1

34 NR5A1 mutations and human phenotypes 46,XY Sex Reversal and Adrenal Failure 46,XY Disorder of Sexual Development 46,XX Primary Ovarian Insufficiency 46,XX Gonadal Dysgenesis AR and AD modes of inheritance Incomplete penetrance

35 Proposed Mechanism

36 Embryology Germ cell migration at ~6 weeks gestation SRY expressed by pre Sertoli cells and induce Sertoli cell differentiation Sertoli cells present by 6 7 weeks NR5A1 expressed by 6 7 weeks Sertoli cells envelop germ cells to form seminiferous cords by 7 9 weeks in XY gonad Testicular descent begins after weeks

37 NR5A1 NR5A1

38 Functions of NR5A1 A. NR5A1 drives male sex development up regulates SRY and SOX9 expression NR5A1 and SOX9 expression precede AMH expression TESCO = testis specific enhancer of SOX9 core NR5A1 + SRY cooperate to activate TESCO and increase SOX9 expression SOX9 expression then maintained by combination of SRY/NR5A1/SOX9 B. NR5A1 possibly blocks female sex development

39 Ono, M. & Harley, V. R. Nat. Rev. Endocrinol. 9, (2013)

40 Published Mutations in NR5A1 (46XY DSD or 46XX ovarian insufficiency )

41 Homozygous Arg92Gln (R92Q) Mutation in NR5A1: Causes 46XY Sex Reversal This region of protein called Abox or FTZf1 box stabilizes protein/dna binding Achermann et al., 2001 Ferraz de Souza et al., 2011

42 Genetic Analysis Summary p.arg92trp mutation affects a highly conserved amino acid in the Abox of NR5A1 Motif interacts with the minor groove of DNA to stabilize binding by NR5A1 Mutation replaces charged arginine with a bulky tryptophan downstream effects produce XX male phenotype (Mutation replacing arginine in the same position with polar glutamine promotes XY female phenotype!) How??

43 Hypothesis Variants at aa 92 in NR5A1 can act as a switch in human sex development NR5A1 R92W is a gain of function mutation leading to activation of the male sexual differentiation program

44 Ongoing / Future Studies

45 In Vitro Studies Immunostaining: mouse NR5A1 is mostly in early testis with only trace expression in early ovary DNA binding ability: R92W mutant protein failed to bind the known consensus target sequence Reporter assay for testing the transactivation activity: reduced ability of the R92W mutant protein to activate reporter activity using the Tesco enhancer element (Sox9). Reporter assay: both WT and R92Q proteins could synergise with b catenin to upregulate Wnt signal activity, while R92W showed a reduced ability

46 NR5A1 NR5A1

47 In Vivo Studies Model Organisms Drosophila (ftz-f1) Experiment to engineer over expression (Hugo Bellen lab) Mouse (CRISPR Cas9) Knock in and breeding ongoing (Brendan Lee lab) Will these mice be able to breed?

48 Pending studies Skin biopsy for fibroblasts RNA Seq

49 BCM UDN clinical team Genetics Brendan Lee (PI) Carlos Bacino Bill Craigen Shweta Dhar Seema Lalani Daryl Scott Jenn Posey Neurology Paolo Moretti Lisa Emrick Gary Clark Medicine Ashok Balasubramanyam Ophthalmology Dick Lewis Immunology Jordan Orange Pediatrics Jan Drutz Clinical research staff Jill Mokry Alyssa Tran Mahshid Azamian Mercedes Alejandro Wren Pratt Ada McGowan Sequence analysis Lindsay Burrage Mahim Jain Fan Xia Narayan Mulukutla