2. DIAGNOSIS AND MANAGEMENT OF YOUNG WOMEN WITH TURNER SYNDROME (TS) Carolyn Bondy, MD National Institute of Child Health, National Institutes of Health (NIH) Center for Human Reproduction April 9, 2013

3. I have no declarations and no conflict of interest in regards to this presentation

4. Objectives Criteria for diagnosis of Turner syndrome (TS) Epidemiology and chromosomal origins Diagnostic tests and screening evaluation Potential for endogenous fertility Outcomes of spontaneous and OD pregnancies Medical management Psychosocial concerns X chromosome genomic imprinting and ischemic heart disease

5. Monosomy X or TS: combined clinical & genetic dx Absent or fragmented 2 nd sex chromosome – Phenotypic female – Early ovarian failure – Short stature – Congenital heart defects – Hypothyroidism – Hearing loss – Osteoporosis Patients have given written authorization for use of photos

6. NICHD TS PROTOCOL 2001-2012 400 participants 7-67 yrs Recent focus on cardiovascular evaluation ~100 participants in longitudinal follow-up studies focused on aortic complications and ischemic heart disease STUDY PARTICIPANTS BY AGE GROUP

7. Epidemiology 1/200 conceptions 1/2500 live births <50% adults with TS are diagnosed Sporadic Not clearly related to maternal age All races & regions Only monosomy compatible with life Stochholm K et al. JCEM 2006;91:3897-3902

8. Chromosomal Origins Non-disjunction of sex chromosomes during meiosis – 45X Non-disjunction during early embryonic mitoses – Cell line mosaicism: 45X/46XX; 45X/47XXX; 45X/46XY – Abundance of normal cell line typically attenuates phenotype Fragmentation of a sex chromosome during meiosis – 46XiXq, 46XdelXp, 46XdelY – Loss of the fragmented X or Y in some cells during embryonic development  cell line mosaicism 45X/46XiXq, etc

9. The Lyon Hypothesis In cells with multiple X- chromosomes, all but one are inactivated during embryogenesis X-inactivation leads to clumped chromatin, termed Barr bodies, considered inert (Lyonization) So is the 2 nd X totally superfluous?

10. Why a 45,X phenotype ? Pseudoautosomal genes – Present on both X & Y – Not inactivated SHOX – short stature ??Congenital heart disease Genomic imprinting of X- linked genes 46,XX females are 50:50 mosaic for X M & X P – 45,X females are monosomic for X M OR X P – ??Ischemic heart disease Pseudoautosomal region 1 (PAR1) PAR2 X Y Trimethylation of H3 at Lys4; Khalil et al, 2007

11. Presented age 27 - infertility 45,X[45]/46,XrX[5] OCP to regulate menses age 15 Cont. til age 25 Father 6’, mother 5’5” Her predicted ht 5’6” High-arched palate Low posterior hairline Cubitus valgus

12. Clues from PMH, FH and PE Chronic otitis, shorter than sisters, less than mid-parental height (F-5” + M)/2; history Coarc repair, other CHD Mx nevi, high-arched palate, short 4 th MCP or MTP, short or web neck, low hair line, low set ears

13. To confirm the diagnosis of TS 20-30 cell peripheral blood karyotype is gold standard 3-5 ml blood in heparinized tube at room T˚ Cytogenetics referral lab 2-3 wk turnaround & costly New methods Whole genome snp chips

14. Affymetric Cytosnp Log R ratio B allele frequency Karyotype = 46,X, del (Xp11)

15. Illumina Hapmap Bead Chip 46,X,idic(X)(p11)

16. ring marker Troublesome karyotypes Further study to identify Y-material- FISH, PCR or array

17. Concern about Y chromosome material ~12% risk for gonadoblastoma large primordial germ cells, immature granulosa or Sertoli cells, stromal cells Unknown risk for conversion to malignancy, e.g., dysgerminoma Anecdotal evidence Standard of care at present – laparascopic bilateral gonadectomy Psychological counseling Consider preservation of ovarian follicles

18. Fertility Preservation in Girls with Turner Syndrome Borgström Birgit, Hovatta Outi -Karolinska Institutet, JCEM 2009 Borgström BirgitHovatta Outi 15/56 biopsied girls had ovarian follicles Recommend laparascopic harvesting/freezing of juvenile ovarian tissue for later fertility

19. Ovarian Failure in TS Fetal ovaries develop normally but experience accelerated follicular atresia Biphasic FSH levels – difficulty in predicting spontaneous puberty (Conte et al., 1975) Spontaneous menarche in ~16% (Pasquino et al., 1997) More common in 45,X/46,XX 2  amenorrhea or irregular cycles in ~half of these girls within 2 yrs Spontaneous pregnancy in 1-2% NICHD, NIH, DHHS

20. Illustrative Case Swedish woman with menarche and full pubertal development age 13, irregular menses Premature menopause diagnosis ~age 32 At least two unsuccessful IVF w/donated oocytes Spontaneous pregnancy age 36 w/ NSVD 2 nd spontaneous pregnancy age 38 – Htn, chest pain, aortic dissection 7 th month – Emergency C-section and aortic repair Endocrine clinic- 45,X/46,X,delY—DX TS Prophylactic gonadectomy age 40 Journal of Assisted Reproduction and GeneticsJournal of Assisted Reproduction and Genetics 21;229-230,

21. Comments… Consider the diagnosis of TS – Hypergonadotropic hypogonadism Do not assume infertility if Y chromosome material is detected – Oocyte preservation Serious cardio complications in natural as well as ART pregnancies

22. Illustrative case(2) DG- classic presentation of TS dx’d at birth Participated in pediatric GH Rx protocol in 1990’s. – Started on estradiol at age 12 per protocol; dose escalated to full pubertal development by age 15, lost to f/u Spontaneous full term pregnancy age 18 – Evaluated in our TS protocol at age 20 Height 4’7”, single kidney, BAV, FSH 40, no visible ovaries Prescribed OCP for HRT – Returned for protocol f/u age 25 - 3 mo pregnant Delivered 2 nd healthy son at full term and reportedly resumed OCP – PBC karyotype 200/200 cells 45,X; skin bx 45,X

23. Comments (2) Patients, parents and care-givers of girls with full blown TS dx’d in infancy may assume pregnancy is not possible or that the girl will not sexually active Importance of patient/parent sex education in this special situation

24. X chromosome genes and fertility Gene dose Short Stature BAV POF (>25 yo) Autoimmunity 45,X1 Xp, 1 Xq100%32%95%32% 46,X,delXp1 Xp, 2 Xq100%27%90%20% 46,X,i(Xq)1 Xp, 3 Xq100%22%95%38% 46,X,del(Xq)2 Xp, 1 Xq10%0100%5% 46,XX2 Xp, 2 Xq1%<0.5%1%5% SS, BAV and autoimmunity clearly related to haploinsufficiency for Xp genes POF seems equally prevalent in Xp deletions with normal or even extra Xq complement, and in Xq deletions with normal Xp complement

25. NIH study, Hadnott et al Of 276 women with Turner syndrome >25 yr: – 88% had no children – 9% adopted children – 3% spontaneous or assisted pregnancy (1) Participants had a total of 13 pregnancies and 14 live births One child had cerebral palsy; the others were chromosomally and developmentally normal. One pre-eclampsia One (mother of twins) has dilation of asc. aorta Fertility and Sterility Volume 95, Volume 95, 2011, 2251–2256

26. Oocyte donation in TS Karnis et al 2003 estimated ~2% maternal mortality (F&S) – Aortic rupture or dissection Chevalier et al 2011 < 35% of women pre- screened for CVS disease – Poor fetal and maternal outcomes; 2/93 patients died due to aortic rupture (JCEM) Hagman et al 2013 reviewed 122 deliveries- no fatalities and generally good fetal outcomes

27. Aortic diameters in a TS pregnancy 30 yo married social worker had IVF, referred to cardiologist at 12 wks Initial echo showed probable BAV and ? aortic dilation Followed monthly until 24 wks when ↑ in AA diameter noted; beta- blocker initiated Elective C-section at 37 wks Follow-up echos return to baseline? Fatal aortic dissection 2 yrs later C-section Week Pregnancy STJ AA

28. ASRM pages Increased maternal cardiovascular mortality associated with pregnancy in women with Turner syndrome Practice Committee of the American Society for Reproductive Medicine Received 22 November 2011. Accepted 29 November 2011. Available online 21 December 2011. TS a relative contra-indication to pregnancy Comprehensive cardiological and maternal-fetal medicine eval. before considering pregnancy by oocyte donation. Cardiac MRI revealing any significant abnormality and/or ASI >2 cm/m2 (ascending aortic diameter/BSA) represents an absolute contraindication for attempting pregnancy in a woman with Turner syndrome. Women with normal cardiac MRI and evaluation who decide to attempt pregnancy after thorough counseling are still at much higher risk for associated morbidity and mortality and require careful observation and frequent formal reevaluation throughout gestation and postpartum.

29. Medical Management PrevalenceScreenTXF/U Short stature99%Predicted htNA Ovarian fail.95%Hx, PE, FSHEstrogen/progAnnual Heart50%Cardiol./CMR Abn-per cardiol WNL- q3-5 yr Thyroid40-50%TSH, absThyroxineAnnual Hearing loss30-50%AudiometryAmplificationQ3 yr or prn HTN30-40%BP, 24h monitor StandardAnnual Renal anom30%UltrasoundCorrect obstr.PRN Osteoporosis25%DXA/size*HRT, standardAdm, q3-5 yr Metabolic25-30% Lipids, CRP, HA1c StandardAnnual Liver30%LFTs, USAnnual LFTs Celiac5%tTG, EMAGluten freePrn

30. Gradual induction of sexual maturation beginning by age 12 Full dose by age 18 through ~ age 40 – OC, 2 mg estradiol, 100 mg transdermal estradiol – Best choice for cycling is unknown – Adherence is paramount Beginning age 35-40 – lower dose, plan to taper and dc by age 50

31. HRT and Bone Health in TS Age 30, 45X HRT since age 12 Age 30, 45X HRT age 12-18, off 18-30yrs

32. SCREENING FOR CHD IN TS

33. Normal and Coarcted Aortas

34. Aortic Valve: Bicuspid or tricuspid? TAV BAV

35. A B Cardiac magnetic resonance angiography (CMRA) showing a dilated ascending aorta (solid arrow) and normal descending aorta (open arrow). This patient has a distinctive squaring off and elongation of the transverse aortic arch with a prominent kink at the transition to descending aorta (arrowhead) termed ETA. Although the actual diameter of the ascending aorta in this woman was only 3.7 cm, which is considered at the upper limit of normal for women of her age, it is almost 3-fold larger than the descending aorta (normal ascending/descending ratio is < 1.5). 3.7 cm Dilation of Ascending Aorta

36. Value of CMR in TS Diagnose occult but clinically significant – Coarctation - dissection – Anomalous pulmonary veins Detects abnormal aortic valve in 15% of cases not visualized on cardiac echo Visualize entire aortic arch revealing ascending dilatation relative to descending Ho et al, Circulation, 2004; Matura et al, Circulation 2007; Sachdev et al, JACC 2008

37. Shortcomings of Cardiac Echo 28 yo Latina Routine GH and ERT Hashimoto’s thyroiditis Cardiac echo age 18 – mild coarc Cardiac echo at 25 “stable” BP 125/85; Ht 4’10” neck webbing; 3/6 diastolic murmur BAV w/ AR; Asc ao aneurysm 4 cm Descending ao aneurysm 6.1 cm R

38. Illustrative Case 3 62 yo woman referred for osteoporosis – PMH -Coarc repair age 12, always short, no puberty, Cochlear implants, no medications – Recent back pain prompted plain Xrays (aortic calcifications) and DXA (diffuse osteoporosis) – PE- Many signs of TS, appearing considerably older than 62. Disparity in right and left arm BP (R>L). Bilat carotid bruits. Tanner 1 breast and T2 pubic hair. What test(s) would you order?

39. L. subclavian a. Coarc repair Aortic dissection True lumen False lumen R. Kidney CT Angiography Proximal stenosis of left subclavian artery Atherosclerotic plaque with ulceration and dissection of the abdominal aorta, occlusion of l. renal a. Calcified coronary plaques

40. Final comments It is never too late to diagnose TS There is an excess of atherosclerosis as well as congenital heart disease

41. QOL Issues in TS Carel et al in JCEM 2005-2006 – disappointment with GH treatment for SS. Self-esteem and social adjustment in young women with TS more influenced by pubertal management and sexuality NIH – major concerns related to TS (Sutton et al 2005) Infertility Short stature Sexual development and function Health

42. Importance of “Truth Telling” During interview sessions addressing concerns about living with TS, 30/97 participants spontaneously mentioned that all or part of their TS diagnosis had been withheld from them Fifteen girls and women from whom a secret had been kept knew every aspect of their diagnosis except the infertility component – “The doctor finished his exam and I got dressed and went in. And he just kind of stopped, put his pen down and went, ‘you know you’re not having kids ever, don't you?' So that was the hardest time.” (Diagnosed at 17, learned of infertility at 19.) – “I did get upset with my mother [when I found out]…. She… didn't explain. She said, ‘Well, what’s the big deal?' I said, ‘I think it would have helped me to understand myself a little bit better. To tell—to fill in the gaps to tell you more of the story.’” (Diagnosed at 17, learned infertile at 28) – “There I am at this sterile place. And I found out that since the age of 9 to 16, everyone knew something I had no knowledge of about me that was pretty darn important. I was told [I was infertile] in such a horrendous way. And all I knew was that the people that I trusted forever were willing to betray me.” (Diagnosed at 9, learned infertile at 16). Sutton et al, Peds 2006

43. Shyness, Social Anxiety, and Impaired Self-Esteem in Young Women with Ovarian Failure NIH Study – Women with 46,XX with normal ovarian function (NC) – Women with 46,XX premature ovarian failure (POF) – Women with TS Schmidt et al., JAMA 2006

44. Psychosocial Outcomes in TS Adults w/ TS 25 yr and older in NIH Study* – N=261 – Average age 38 yr; average height 147 cm – 65% had a baccalaureate degree (vs. 25% US women) – 6% had advanced degree (vs. 3% US women) – 80% employed (vs. 55% US women) Similar findings in recent Danish registry study- suggesting ascertainment bias not crucial (Stochholm et al EJE, 2012) *Gould et al, J Women’s Health 2013

45. X Chromosome Imprinting Genomic imprinting of X-linked genes – Men monosomic for X M – Because or random X-inactivation, women are ~50:50% mosaic X M & X P – Genes imprinted or silenced on X M will be expressed in ~50% of female but in no male cells – Genes imprinted on X P will be expressed in ~50% of female but suppressed in male cells To test hypothesis that X imprinting causes gender differences in body composition and IHD risk, we compare groups of women monosomic for X M vs. X P 46,X M X P 46,X M Y XMXM XPXP

46. XMXM XPXP Body Composition & IHD Risk Factors We compared – BMI – Total body fat (by DXA) – Visceral and sc abdominal fat by CT – Lipids In age-matched women monosomic for X M vs. X P Both groups had ovarian failure and very low levels endogenous sex steroids

47. Visceral Fat and IHD Risk Factors Selectively Increased in X M Women XMXM XPXP XPXP XMXM P BMI 26 (6)27 (6)0.15 BF% 36 (8)37 (8)0.55 VAT 14 (8)25 (19)<0.001 TGs 101 (42)132 (60)0.02 LDL-Ch 103 (41)137 (44)0.005 Van et al., JAMA, 2005

48. Incidence of coronary disease related to parental origin of the single X Further study to determine actual incidence of coronary atherosclerosis – >age 34 - CT Ca++ score – “pure” 45X – Parental DNA for POO genotyping

49. X Chromosome Origin and Atherosclerosis Parental Origin Of X-chromosome P Paternal X (n=15)Maternal X (n=35) Age; years (SD)42.6 (7.7)45.6 (7.0)0.155 BMI; kg/m 2 (SD)28.4 (7.9)28.6 (6.6)0.81 Estrogen Index % (SD)74.5 (34.3)75.4 (31.1)0.89 Family CAD history n (%)2 (13.3%)5 (14.3%)0.93 Obesity n (%)6 (40%)15 (42.9%)0.85 Dyslipidemia n (%)7 (47%)21 (60%)0.39 Hypertension4 (27%)19 (54%)0.068 Diabetes n (%)3 (20%)7 (20%)>0.99 Tobacco use n (%)1 (6.7%)3 (8.6%)0.82 CORONARY ATHEROSCLEROSIS Prevalence n (%)0140.0021* Calcium score; AU (SD)091.7 (312.4)0.0052 $

50. Coronary atherosclerosis determined by CT Ca++ Comparing healthy population data from Framingham

51. X M Linked to Abdominal Adiposity and IHD Risk Normal 46,X M Y men and 45,X M women – Greater visceral adiposity, atherogenic lipids & Inflammation compared to 46, X M X P women – This is not due to sex steroids 46,XX protection may be due to imprinting of unknown X-linked genes resulting in: – Selective expression high risk genes from X M – Selective expression of protective genes from X P 46,X M X P 46,X M Y 45,X M XMXM XPXP

52. Gratitude NICHD – Vladimir Bakalov – Harley Gould – Liat Gutin – Clara Cheng – Jian Zhou – Helen Hougen – Rachel Poliquin – Kateri McCarthy NHLBI – Douglas R Rosing – Andrew Arai – Vandana Sachdev NIH CC Radiology – Vincent Ho – Ahmed Gharib Girls and women with TS and their familes