Update in Genetics

From Obstetrics and Gynecology Update: What Does the Evidence Tell Us?, presented by the University of California,
San Francisco, Department of Obstetrics, Gynecology, and Reproductive Sciences

Educational Objectives

The goal of this program is to improve obstetric outcomes through an understanding of the principles of epigenetics and noninvasive prenatal diagnostic (NIPDx) testing. After hearing and assimilating this program, the clinician will be better able to:

1.   Recognize the correlation between diet in pregnancy and adult health and disease.

2.   Explain the concept of epigenetic regulation of genes.

3.   Effectively counsel obstetric patients about the importance of a healthy lifestyle.

4.   Identify technical challenges in the development of accurate cell-free fetal DNA (cffDNA) testing.

5.   Weigh the advantages and disadvantages of cffDNA testing in Rh isoimmunization, sex determination, and de­tection of aneuploidy.

Faculty Disclosure

In adherence to ACCME Standards for Commercial Support, Audio-Digest requires all faculty and members of the plan­ning committee to disclose relevant financial relationships within the past 12 months that might create any personal con­flicts of interest. Any identified conflicts were resolved to ensure that this educational activity promotes quality in health care and not a proprietary business or commercial interest. For this program, the faculty and planning committee reported nothing to disclose.

The Barker Hypothesis: Developmental Origins of Health and Disease

Yao Sun, MD, PhD, Associate Professor of Clinical Pediatrics and Director of Neonatal Clinical Programs, University of California, San Francisco, School of Medicine

Hypothesis: what happens to developing fetus in uterus can directly affect adult disease; early evidence  —  growth re­stricted fetuses have higher risk for heart disease

History: studies by Dr. David Barker and other researchers worldwide revealed similar findings; developmental ori­gins of health and disease  —  what happens during development (including in utero) can affect health as well as dis­ease; Ravelli (1976)  —  studied population of pregnant women who endured famine in Netherlands during World War II; children who experienced famine as young children or infants found to be leaner as adults; children born to women pregnant for 6 mo during famine had high rate of adult obesity

Infant mortality and heart disease (Barker, 1986): found that risk of dying in first month of life (due to poor in utero growth and nutrition) had strongest correlation with risk for heart disease in adulthood; babies strong enough to survive past first month of life but exposed to poor socioeconomic conditions during postneonatal period have in­creased risk for stomach cancer, bronchitis, and rheumatic heart disease as adults; although rate of neonatal mortal­ity eventually decreased, rate of heart disease increased in regions with previously high rates of neonatal mortality (correspondence remained); not true of conditions associated with postneonatal deprivation

Birthweight and heart disease (Barker, 1989): examined records of weights at birth and 1 yr of 5654 male infants born in England between 1911 and 1930; those with both weights below average had highest standardized mortality ratio for ischemic heart disease; Barker attributed relationship to in utero effects

Placental size and hypertension (Barker, 1990): low birth weight and large placental size associated with risk for adult hypertension; the bigger the discordance, the higher the risk

Possible rationales for Barker hypothesis: plasticity  —interaction between genetic variation and adaptation to envi­ronment determine development (not strictly Mendelian genetics); thrifty genotype  —  humans developed genome in which fuel intake maximized; if fetus starved of food in utero, genetic adaptation causes metabolism to be “revved up” after birth to maximize food storage, including fat; does not explain why prevalence of diabetes in­creasing; thrifty phenotype  —  conditions to which baby exposed in utero cause (nongenetic) adaptation in baby’s metabolism; predictive adaptive response  —  not necessarily true that growth-restricted babies at highest risk for de­veloping hypertension, diabetes, and heart disease; babies growth-restricted at birth who experience rapid postnatal growth actually at highest risk; due to thrifty phenotype, metabolism of infant deprived in utero “anticipates” same conditions after birth; animal studies show that growth-restricted pups who get lower level of nutrition postnatally experience normal health as adults; however, those that get abundant postnatal nutrition prone to develop adult met­abolic diseases

Adult diseases affected by fetal environment: metabolic  —obesity; hypertension; type 2 diabetes; cardiovascular disease; nonmetabolic  —  cancer (eg, exposure to diethylstilbestrol [DES]); psychologic disorders (eg, higher rate of schizophrenia in babies subjected to in utero starvation)

Ongoing prospective cohort studies: studying mothers and children to determine fetal growth rate and long-term health outcomes of children; Southampton Women’s Study  —  looking at 12,500 young prospective mothers pre­pregnancy; National Children’s Study  —  »750,000 women in pre- and early pregnancy, with target accrual of 100,000 births across 105 centers in United States

Basic mechanisms: animal models used to look at various ways restricting diet in mothers may affect long-term health outcomes in offspring; variations in organ structure  —  can directly affect development of liver (ie, lipid me­tabolism), kidneys (ie, hypertension), hypothalamic-pituitary-adrenal axis (ie, hormonal control), and heart; other   —  alterations in cell number; clonal selection of cell populations

Epigenetic gene regulation: heritable changes in gene expression without change in DNA sequence; heritable changes defined as those that propagate within animal and pass to succeeding generations; permanent alterations in control of gene expression include DNA methylation, histone modification, and RNA interference

DNA methylation: gene expression can be controlled by degree of methylation of cytosine residue; when methyl­ated, DNA combines with histones in different conformations (can make DNA either easier or more difficult to access and express)

Histone modification: histones can be acetylated or deacetylated; basic DNA wraps around histones to form higher complexes, which eventually form entire chromosome; complex of histones and DNA actually affect gene ex­pression

Epigenetics and inheritance: epidemiologic study in humans showed that generation-skipping changes can occur due to diet; looked at small farming community in Sweden over last 200 yr; confirmed Barker hypothesis about in utero effects, but also found that periods of either starvation or overeating during grandparents’ adolescence af­fected mortality rate of their grandchildren in sex-specific manner; among paternal grandmothers who had less food, female grandchildren had better survival rate, whereas grandfathers with less food had male grandchildren with better survival rate; conclusion  —  researchers postulate that adolescent diet during slow growth period changed gametes (sperm and eggs) of grandparents, which eventually affected their grandchildren; mouse study  —  expression of coat color (from yellow to almost brown) depends on degree of DNA methylation; can directly look at methylation and predict coat colors; study showed that DNA of maternal mice fed bisphenol-A became demeth­ylated, which resulted in shift in coat color of progeny to one end of color spectrum; if mother fed methyl donor, coat color of progeny shifted to other end of spectrum; provides clear evidence of diet affecting epigenetic control

Implications for clinical care: maternal diet and health  —  not many current studies that give specific recommenda­tions; in general, information should be used as ammunition to promote healthy lifestyle during pregnancy; other implications of Barker hypothesis  —  not only about growth restriction; excessive weight gain and large birth weight also increase risks in child’s adulthood; smoking and control of blood glucose and blood pressure also shown in an­imal models to affect adult diseases; neonatal and pediatric care  —  current recommendations on feeding of small-for-gestational-age (SGA) infants (encouraging rapid catch-up growth) based on old data; cohort studies mentioned above expected to determine appropriate dietary recommendations

Societal implications: prenatal and perinatal public health education needed; actions of parents (and grandparents) affect child through epigenetic effects as well as through environment; improvements in both health and disease prevalence possible with prenatal intervention

Fetal DNA: The Wave of the Future

Mary E. Norton, MD, Professor of Obstetrics and Gynecology, Stanford University School of Medicine, Stan­ford, CA

History of noninvasive prenatal diagnosis (NIPDx): in 1893, Schmorl identified fetal trophoblasts in lungs of women who died from preeclampsia; extraction of fetal cells in maternal circulation not possible until 1980s (cell sorting developed); great enthusiasm generated for NIPDx with advent of polymerase chain reaction (PCR) and flu­orescence in situ hybridization (FISH); prompted National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) trial

Potential sources of fetal genetic material: intact cells (lymphocytes, trophoblasts, and nucleated red blood cells [RBCs]); cell-free fetal DNA (cffDNA); cell-free fetal messenger RNA

Challenges: differentiating between maternal and fetal cells and DNA (fetal DNA clears quickly from maternal cir­culation, but cells can last for years); appropriate timing and indications for testing; performing diagnostic analysis on tiny bits of fetal DNA or few fetal cells

Intact fetal cells: obvious target because nucleus carries entire fetal genome (can culture from nucleus, or perform FISH and obtain complete karyotype); major obstacles  —  only »1 fetal cell per 1 mL of maternal blood; genetic analysis complicated by “allele drop-out”; high rates of cellular apoptosis (source of cffDNA); trophoblasts  —  can be easily sloughed into maternal circulation; detected at as early as 6 wk gestation; many disadvantages for NIPDx (not focus of research); endocervical trophoblasts   —  various harvesting techniques tried, but none highly success­ful; recent study showed inaccuracy in prediction of male sex; lymphocytes  —  standard for karyotype, but persist in maternal circulation; nucleated RBCs  —  abundant in fetal blood and rare in maternal, but do not persist; focus of most early work on NIPDx, but also present many problems

NIFTY study: started study long before technology to extract and analyze cells available; results  —  disappointing; not good at identifying sex; poor detection rate for aneuploidy; discouraged further research on intact fetal cells

cffDNA in maternal plasma: reported by Lo in late 1990s; found Y chromosome sequences in DNA of 24 of 30 women carrying male fetuses and in 0 of 13 women carrying female fetuses; possible with small serum sample; subsequently confirmed by other investigators; originates from placenta, not fetus; characteristics  —  represents 3% to 5% of total cell-free DNA in maternal plasma; fragments shorter than those of maternal DNA; multiple studies reliably detected cffDNA at >7 wk gestation; increases as pregnancy progresses; has short half-life; undetectable ³2 hr postpartum; challenges  —  difficult distinguishing fetal DNA because concentration and total amount still low; variable from person to person; because fetus inherits half of genome from mother, must look for paternal component (ie, father must be available for testing); when testing for, eg, cystic fibrosis, mother and father must have different mutations for test to be useful (more applicable to autosomal disorders present only in father); fur­ther clinical challenges  —  false positives due to contamination, “vanishing” twins, and placental mosaicism

Clinical applications of cffDNA

Rh isoimmunization: recent proposal  —  draw maternal blood sample from Rh negative women and look for RhD gene in fetus; if fetus appears to be Rh-negative, withhold Rho(D) immune globulin (RhoGAM); decreases costs and prevents risks associated with immunization; argument  —  data from recent meta-analysis indicates accuracy of test »95%; in »5% of cases, RhoGAM would be withheld from Rh-negative mother with Rh-positive fetus; de­cision analysis performed by speaker and colleagues found that 13 of 1000 Rh-negative women would become sensitized due to 5% error rate (20 times higher sensitization rate than that with giving RhoGAM to all Rh-nega­tive mothers)

Potential benefits of sex determination: X-linked disorders  —carried by small number of women; congenital adre­nal hyperplasia (CAH)  —  autosomal recessive disorder; 1 in 8 at-risk female fetuses virilized (can be minimized by high doses of glucocorticoids during pregnancy); can perform early test to limit steroid exposure in at-risk pregnant women; applicable to small population; single gene disorders  —  screening of all pregnancies with cffDNA unlikely (parents can be screened first); has greater utility as noninvasive testing for at-risk pregnancies; patients at high risk want definitive information; in women with 25% to 50% risk for single gene disorder, trade-off of 1 in 300 risk for miscarriage acceptable and any chance of false-negative result unacceptable; receptiveness to such testing in at-risk population yet to be seen

Aneuploidy testing: largest area of commercial interest (expensive test applicable to 4 million births per year); more complicated than single gene testing or Y chromosome; detection of trisomy 21  —  can distinguish copies of chromosome 21 using single gene polymorphism testing (genes present in 1 to 1 ratio in disomic mother, and 1 to 2 ratio in trisomic fetus); only miniscule amount of “extra” genetic material present when fetus trisomic (ratio of genes 1 to 1.002; difference difficult to determine accurately)

Conclusions: cffDNA testing likely in near future, but accuracy unlikely to approach 100% (due to, eg, vanishing twins, mosaicism); patients unlikely to make decision about terminating pregnancy based on maternal blood test without 100% accuracy; may simplify screening process, rather than being useful as diagnostic test; unantici­pated downside  —  currently, many patients screened for, eg, trisomy 21, without understanding of reasons for and circumstances of testing; some unhappy with and bewildered by results of tests they did not necessarily want; may be even more problematic if testing simpler and more accurate (ie, more detailed informed consent may be necessary before performance of cffDNA testing)

Ethical issues in cffDNA testing: sex selection  —  ethical implications similar to those of early ultrasonography or in­vasive prenatal diagnosis; possible effect on rates of pregnancy termination  —  improvements in testing do not nec­essarily lead to increases in abortion; many women choose to have testing to prepare for birth of affected fetus; increased availability of noninvasive testing may further encourage such preparation and result in better outcomes for some infants

Direct-to-consumer testing: possible with cffDNA; women could be making decisions about their pregnancy out­side of medical arena; may be of benefit in places in which women cannot have prenatal testing because of cultural or political issues; may allow earlier (ie, safer) medical terminations; downside of earlier testing  —  detection of ab­normalities in fetuses destined to spontaneously abort

Speaker’s concerns about NIPDx: development of testing largely driven by private industry; publicly held compa­nies have been accused of fraud and misrepresentation of data associated with tests under development; difficult to determine true status of technology because much of research not being published in peer-reviewed literature

Acknowledgments

Drs. Sun and Norton spoke at Obstetrics and Gynecology Update: What Does the Evidence Tell Us?, held October 27-29, 2010, in San Francisco, CA, and sponsored by the University of California, San Francisco, School of Medicine, Department of Obstetrics, Gynecology, and Reproductive Sciences. For a list of upcoming CME meetings presented by the University of California, San Francisco, School of Medicine, visit their website at http://www.cme.ucsf.edu/cme. The Audio-Digest Foundation thanks the speakers and the University of California, San Francisco, School of Medicine, Department of Obstet­rics, Gynecology, and Reproductive Sciences for their cooperation in the production of this program.

Suggested Reading

Barker DJ et al: Weight in infancy and death from ischaemic heart disease. Lancet. 1989 Sep 9;2(8663):577-80; Barker DJ et al: Fetal and placental size and risk of hypertension in adult life. BMJ. 1990 Aug 4;301(6746):259-62; Bianchi DW et al: Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn. 2002 Jul;22(7):609-15; Dolinoy DC et al: Ma­ternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development. Proc Natl Acad Sci U S A. 2007 Aug 7;104(32):13056-61; Finning K et al: Effect of high throughput RHD typing of fetal DNA in maternal plasma on use of anti-RhD immunoglobulin in RhD negative pregnant women: prospective feasibility study. BMJ. 2008 Apr 12;336(7648):816-8; Kaati G et al: Transgenerational response to nutrition, early life circumstances and longev­ity. Eur J Hum Genet. 2007 Jul;15(7):784-90; Lo YM et al: Presence of fetal DNA in maternal plasma and serum. Lancet. 1997 Aug 16;350(9076):485-7; Norton ME: First-trimester screening for chromosomal abnormalities: advantages of an in­stant results approach. Clin Lab Med. 2010 Sep;30(3):565-71.