Section on Growth and Metabolism, Section on Women's Health Research and Unit on Turner's Syndrome

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gender-specific differences indisease susceptibility

Carolyn Bondy, MD, Head, Section on Growth and Metabolism, Section on Women’s Health Research, and Unit on Turner’s Syndrome

Vladimir Bakalov, MD, Staff Clinician

Clara Cheng, PhD, Senior Fellow

Alastair Smith, PhD, Visiting Fellow

Jian Zhou, MD, PhD, Staff Scientist

Jie Wang, MD, Biologist

Eileen Lange, RN, Research Nurse

Porsche Brown, BS, Postbaccalaureate Fellow

Phillip Van, BS, MS, Postbaccalaureate Fellow

Jose Arraztoa, MD, Guest Researcher^a

Constantine Dimitrakakis, MD, PhD, Guest Researche^b

Judith Ross, MD, Guest Researcher^c

In addition to the obvious phenotypic differences between the sexes, there are differences in cognitive, immunological, and metabolic functions as well as in longevity. Our goal is to elucidate the genetic and physiological mechanisms underlying these important but poorly understood differences. Previously, it was believed that the genetic contribution to sexual dimorphism begins and ends with the action of genes specifying gonads during early embryonic development, with all phenotypic differences occurring subsequent to gonadal development attributed to sex steroid effects. We now realize that many X-chromosome genes may be asymmetrically expressed in normal women and men due to escape from X inactivation or to genomic imprinting, thus contributing to gender differences by dosage effects. We aim to identify and define the function of X-chromosome genes involved in the differential development and function of brain, reproductive, metabolic, and immune systems in women and men. The study of monosomy X, or Turner’s syndrome (TS), provides a unique opportunity to elucidate X-chromosome gene dosage effects. The research will enhance our understanding of disease processes such as the increased susceptibility to autoimmune disease in women and increased risk for coronary disease in men.

The X chromosome and longevity

Bakalov, Van, Bondy

The major reason for women’s greater longevity is their relative protection, across all age groups, from ischemic heart disease (IHD). The traditional idea that estrogen protects women against IHD has recently been called into question. To investigate the potential contribution of X-chromosome gene(s) to the protection against IHD, we examined IHD risk factors in women with TS. TS is characterized by short stature, premature ovarian failure, cardiovascular anomalies, and premature IHD. To control for ovarian failure in TS, we compared glucose tolerance, lipid metabolism, and blood pressure in lean young women with TS and age- and body composition–matched women with 46,XX premature ovarian failure (POF).

Diabetes mellitus (DM) is a major cardiac risk factor. We have shown that, while most girls and women with TS have normal fasting glucose and insulin, the glycemic response to a glucose challenge is dramatically abnormal and consistent with diabetes in about 40 percent of such girls and women and significantly exceeds the POF control group in all women with TS. Interestingly, the glucose intolerance in these girls and young lean women with TS is not explained by insulin resistance but rather by a novel insulin secretory defect. In all women with TS, the insulin response to an oral or IV glucose challenge is significantly lower than in women with POF or normal controls. It thus appears that the Turner “metabolic syndrome” is not secondary to obesity or hypogonadism as previously thought. Rather, it is a distinct entity characterized by decreased insulin secretion reminiscent of mature onset diabetes of the young (MODY) syndromes, which is caused by haploinsufficiency for autosomal genes involved in pancreatic development, suggesting that haploinsufficiency for unknown X-chromosome gene(s) impairs beta cell function and predisposes to DM in TS.

We have also found that LDL cholesterol and triglycerides are all significantly increased in TS compared with age- and body mass index–matched women with POF. Moreover, NMR spectroscopy revealed a concentration of smaller, denser HDL and LDL lipid particles in women with TS. The data show a distinctly atherogenic lipid profile in otherwise healthy, nonobese young women with TS.

Evidence from the study of women with monosomy X, or TS, suggests that dosage-sensitive X-chromosome genes may contribute to normal women’s relative protection against IHD by suppressing atherogenic lipids, independent of gonadal effects. Normal young, 46,XX women also have lower average blood pressure (BP) values than men, suggesting that the second X chromosome might also contribute to BP modulation in women and thus add to their protection from IHD. Confirming this hypothesis, we found that systolic and diastolic BPs were about 10 percent higher and heart rate 17 percent higher in women with TS compared with women with 46,XX POF. Higher BP in TS could not be attributed to adiposity or congenital heart or renal defects. To evaluate parental imprinting as a source of asymmetry in X-gene dosage, we compared BP within the TS group after identifying the parental source of the subjects’ single normal X chromosome. We found that the X_mat group had a greater systolic and diastolic BP and heart rate than the X_pat group.

At least two major mechanisms are involved whereby a second X chromosome in women could contribute to moderation of BP; certain X-chromosome genes involved in BP regulation may escape inactivation and thus normally be active in two copies in 46,XX women. Alternatively or additionally, parental imprinting of X-chromosome genes involved in BP may have favorable effects in women. For example, a gene that exerts a moderating effect could be imprinted or silenced on the maternal X (X_mat) but active from the paternal X allele. Given that only men receive the X_mat, they would not experience the moderating effects on BP, but normal women with random X inactivation would express the X_patallele in about 50 percent of their cells.

Our novel findings implicating haploinsufficiency for X-chromosome genes in dyslipidemia, diabetes, and high BP explain the increased risk for IHD in women with TS and may account for some of the increased risk for IHD among normal XY men compared with women. The identification of these genes is clearly of great clinical importance.

Bakalov VK, Axelrod L, Baron J, Hanton L, Nelson LM, Reynolds JC, Hill S, Troendle J, Bondy CA. Selective reduction in cortical bone mineral density in Turner syndrome independent of ovarian hormone deficiency. J Clin Endocrinol Metab 2003;88:5717-5722.

Bakalov VK, Chen ML, Baron J, Hanton L, Stratakis C, Axelrod L, Bondy CA. Bone mineral density and fractures in Turner Syndrome. Am J Med 2003;115:259-264.

Bakalov VK, Cooley MM, Quon MJ, Luo ML, Yanovski JA, Nelson LM, Sullivan G, Bondy CA. Impaired insulin secretion in the Turner metabolic syndrome. J Clin Endocrinol Metab 2004;89:3516-3520.

Cooley M, Bakalov V, Bondy CA. Lipid profiles in women with 45,X vs 46,XX primary ovarian failure. JAMA 2003;290:2127-2128.

RossJL, Stefanatos GA, Kushner H, Bondy CA, Nelson L, ZinnA, RoeltgenD. The effect of genetic differences: intact cognitive function in adult women with premature ovarian failure versus Turner syndrome. J Clin Endocrinol Metab 2004;89:1817-1822.

Testosterone and breast cancer

Zhou, Dimitrakakis, Bondy

The normal ovary produces abundant quantities of testosterone in addition to estradiol, but usual hormone “replacement” treatment (HRT) for ovarian failure consists of estrogen and progesterone for most women with a uterus or estrogen alone for smaller numbers of hysterectomized women. The risk of breast cancer, however, is increased in menopausal women with such treatment, thereby limiting HRT’s usefulness. We have previously shown that androgens have antimammogenic effects and inhibit estrogen’s mitogenic effects on the mammary epithelium. In some countries, including Australia, testosterone is often prescribed for menopausal women in addition to usual HRT. The rationale for testosterone supplementation has been that estrogen treatment reduces residual ovarian androgen production in post-menopausal women and may lead to sequestration of available testosterone by increasing sex hormone–binding globulin, resulting in symptoms of asthenia and loss of libido in some women.

We therefore undertook a systematic review of breast cancer incidence in an Australian clinic population where women are routinely treated with testosterone along with usual HRT. Breast cancer status was ascertained by mammography at the beginning of testosterone treatment and biannually thereafter with mean duration of follow-up equal to 5.8 ± 2.5 years. The mean age of the women at the start of observation was 56.4 yrs. Within this observation period, seven invasive breast cancer cases were diagnosed among these women, resulting in an incidence of 239 per 100,000 woman-years. Notably, six of the seven cases and the only death occurred in the estrogen/progestin group. These rates are substantially lower than those reported for age-matched women receiving conventional hormone treatment. For example, the Women’s Health Initiative study reported a rate of 380 per 100,000 women-years in women receiving estrogen plus progestin, and the Million Woman Study reported 430 cases per 100,000 woman-years for current HRT users compared with 283 per 100,000 for never users. The prevalence of a positive family history was rather high in our group, suggesting a higher risk for breast cancer at baseline, making the present observations of breast cancer rates similar to untreated post-menopausal women all the more remarkable. These observations suggest that the addition of testosterone to conventional HRT for post-menopausal women does not increase, and may indeed reduce, the HRT-induced breast cancer risk, returning the incidence to normal rates of the order of those observed in the general untreated population. Follow-up studies are under way using Genechip and protein arrays to detect the molecular effects of testosterone on the primate mammary epithelium.

Bondy CA, Arraztoa JA. Insulin like growth factors and ovarian follicular growth and function. In: O’Neill K, Richards J, eds. The Physiology of Reproduction, in press.

Dimitrakakis C, Jones RA, Liu A, Bondy CA. Breast cancer incidence in menopausal women using testosterone in addition to usual hormone therapy. Menopause 2004;11:531-535.

Dimitrakakis C, Zhou J, Wang J, Belanger A, LaBrie F, Cheng C, Powell D, Bondy C. A physiologic role for testosterone in limiting estrogenic stimulation of the breast. Menopause 2003;10:292-297.

Zhou J, Wang J, Penny D, Bondy CA. IGF Binding Protein 4 expression parallels follicle selection and luteinization in the primate ovary. Biol Reprod 2003;69:22-29.

IGF1’s role in normal brain development

Cheng, Wang, Smith, Bondy

We have shown that, during postnatal development, endogenous brain IGF1 plays an insulin-like role in promoting neuronal glucose utilization and hence growth. We have also shown that brain growth in Igf1 null mice falls behind that of normal littermates by almost 40 percent during the postnatal period when brain IGF1 expression is normally most abundant. Further, we have demonstrated that brain glucose uptake and utilization are profoundly reduced in the Igf1 null brain during this period. Our studies have implicated IGF1-induced phosphorylation of Akt/PKB in translocation of glucose across the neuronal membrane and IGF1-induced phosphorylation of GSK3b in neuronal glycogenesis, suggesting that IGF1 augments neuronal glucose uptake and storage by familiar, insulin-like pathways. We have investigated IGF1’s role in neuronal generation, survival, growth, and morphogenesis. While neuronal cell numbers are preserved throughout most brain structures in the Igf1 null brain, there is a significant reduction in the hippocampal

dentate granule cell number as a result of increased cell death in the Igf1 null dentate germinal zone. Neuronal numbers were preserved in the Igf1 null frontoparietal cortex, but morphometric analysis showed that pyramidal neuron soma size was reduced by about 10 percent; Golgi staining showed a significant reduction in pyramidal dendritic length and complexity in Igf1 null mice (see Figure 3.2). In addition, the density of dendritic spines and presumably synaptic contacts declined by 16 percent in the Igf1 null brain. Taken together, these findings illustrate the multifaceted roles of IGF1 in postnatal brain development and explain why individuals with IGF1 gene deletions demonstrate mental retardation in addition to short stature.

Bondy CA, Cheng CM. Signaling by insulin-like growth factor 1 in brain. Eur J Pharmacol 2004;490:25-31.

Cheng CM, Hicks K, Wang J, Eagles DA, Bondy CA. Caloric restriction augments brain glutamic acid decarboxylase-65 and -67 expression. J Neurosci Res 2004;77:270-276.

Wang J, Cheng CM, Zhou J, Smith A, Weickert CS, Periman WR, Becker KG, Powell D, Bondy CA. Estradiol alters transcription factor gene expression in primate prefrontal cortex. J Neurosci Res 2004;76:306-314.

^aSantiago University, Chile

^bAthens University, Greece

^cThomas Jefferson University, Philadelphia, PA

COLLABORATORS

Jeff Baron, MD, Developmental Endocrinology Branch, NICHD, Bethesda, MD

Barbara Biesecker, MS, Medical Genetics Branch, NIHGR, Bethesda, MD

Harry Deitz, MD, The Johns Hopkins University, Baltimore, MD

Andrew Griffith, MD, PhD, Neuro-Otology Branch, NIDCD, Bethesda, MD

Suvimol Hill, MD, Department of Radiology, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD

Vince Ho, MD, Department of Radiology, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD

Robert A. Jones, MD, Memorial Medical Center, North Adelaide, Australia

Aiyi Liu, PhD, Biometry and Mathematical Statistics Branch, NICHD, Bethesda, MD

Lawrence Nelson, MD, Developmental Endocrinology Branch, NICHD, Bethesda, MD

Mike Quon, MD, PhD, Laboratory of Clinical Investigation, NCAM, Bethesda, MD

James Reynolds, MD, Nuclear Medicine, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD

Douglas Rosing, MD, Cardiovascular Branch, NHLBI, Bethesda, MD

David Rubinow, MD, Behavioral Endocrinology Branch, NIMH, Bethesda, MD

Peter Schmidt, MD, Behavioral Endocrinology Branch, NIMH, Bethesda, MD

Constantine Stratakis, MD, Developmental Endocrinology Branch, NICHD, Bethesda, MD

James Troendle, PhD, Biometry and Mathematical Statistics Branch, NICHD, Bethesda, MD

Jack A. Yanovski, MD, PhD, Developmental Endocrinology Branch, NICHD, Bethesda, MD

Andrew Zinn, MD, PhD, University of Texas, Southwestern Medical School, Dallas, TX

For further information, contact bondyc@mail.nih.gov