Etiologic or causal factors of congenital heart defects:  gene-environment interaction hypothesis

By John Dellinger, PhD, professor, College of Health Sciences, University of Wisconsin-Milwaukee; Sarah Antoniewski, BS, teaching assistant, College of Health Sciences, University of Wisconsin-Milwaukee; Nicia Lemoine, BS; and Andrew Pelech, MD, pediatric cardiologist, Children's Hospital of Wisconsin; professor, Medical College of Wisconsin

Congenital cardiac malformations are the most common non-infectious cause of death in infancy and represent a significant financial and emotional burden on both the family and society at large. Congenital heart defects occur in 0.8 percent to 1 percent of live born infants, affecting more than 1 million children within the United States. According to Kathy Hanson-Morris, RN, MS, and Pelech's 2004 Wisconsin Medical Journal report, the causes of these congenital cardiac heart defects are largely unknown. It is expected that 18 percent are due to chromosomal causes like Trisomy 13, 18 and 21, and deletion syndromes. Eight percent are due to single mutant genes such as Holt-Oram, Alport, Noonan, Leopard and William. Approximately 2 percent are due to other known causes including viruses, drugs and toxics. The vast majority (>73 percent) have been attributed to multifactorial causes, including gene-gene or gene-environmental interactions.

Although the etiologies of the majority of congenital heart defects remain largely unknown there is evidence to suggest that the environment may play a significant role. Recent Wisconsin Pediatric Cardiac Registry studies show that the prevalence rates for left heart hypoplasia in southeastern Wisconsin may correspond with high levels of critical pollutants in the United States' areas of concern (as depicted in Figure 1). We hypothesize because of the geographic high rates that environmental factors may be associated with many of these findings. Further, it is apparent that cardiac malformations cluster in families and that genetic factors influence susceptibility of families for developing congenital cardiac malformations. Studies of CHD, genetics and environmental exposure have yielded variable and inconsistent results, mostly because the elements have been studied one at a time while total toxicant body burden is not assessed. The WPCR has initiated preliminary studies to study gene to environment influences and their conglomerate effect on congenital cardiac defects.

Background and significance
The Centers for Disease Control and Prevention Agency for Toxic Substances and Disease Registry reports that xenobiotics such as environmental toxics and pharmaceuticals have been established as potential reproductive toxicants that can result in altered embryonic development and fetal death. The impacts of reproductive toxicants on congenital anomalies have been of particular interest along the shores of Lake Michigan in areas of concern near Green Bay, Sheboygan and Milwaukee, Wis. because these areas have disproportionately more cases of certain CHDs. This suggests that regionally elevated pollution may be a causal factor. The pollutants of most concern are organochlorine pesticides, polychlorinated biphenyls, dioxins, polybrominated diphenyl ethers and heavy metals, including mercury. One of the major sources of persistent organic pollutant exposure among the general population is food. Food contamination begins with contaminated soil, plants, or water and becomes a health problem as these chemicals migrate up the food chain into fish or animals that people may eat. Environmental factors are most likely acting in an additive adverse manner with many other risk factors.

Dietary and other environmental sources lead to increased body burdens of POPs and mercury in vulnerable populations such as pregnant women. If combined with genetic risk factors for birth defects, then it is logical that geographical information systems studies can illustrate "hot spots." Our earlier, preliminary GIS investigations seemed to confirm this hypothesis.

Genetic abnormalities in association with environmental pollutants appear to be the greatest risk factor in adverse birth outcomes. The cardiovascular system is the first functioning organ in the embryo, and specific genes trigger each stage of the system's development. Some success has been achieved in identifying single gene mutations. The identification of GATA4 mutations is associated with the autosomal dominant forms of CHD, but this is just one example and many more genes associated with CHD need to be studied.

Figure 2: Persistent organic pollutant serology of eight mater-nal-infant pairs with variant forms of congenital heartabnormalities, demonstrating correlation and variablepatterns of toxics distribution.

Current research
In 1997, the WPCR was established following recognition that the prevalence of hypoplastic left heart syndrome was greater than expected in certain areas of Wisconsin. The WPCR began recording detailed diagnostic information, demographics, environmental and lifestyle data, and collecting DNA from families with CHD in January 2000. The WPCR houses a database of more than 3,500 registrants, 1,600 completed questionnaires and more than 1,500 DNA samples. It would be beneficial to collaborate with the WPCR to enhance genetic and environmental etiologic findings.

Preliminary work
POPs serology of eight maternal-infant pairs, 16 samples total, with variant forms of CHD was analyzed in a pilot study in collaboration with the National Center for Environmental Health at the CDC. This study demonstrated correlation and variable patterns of toxics distribution between the mothers and their infants' serum.

The data (as shown in Figure 2) depicts two major trends. First, infants' contaminant profiles clearly reflect their mother's profile providing evidence that monitoring of mothers at mid-gestation may provide excellent exposure information about the postnatal infant. And second, there is considerable variability in body burdens of infants with known cardiac defects demonstrating that focusing narrowly on any one particular contaminant is inappropriate for accurately conducting an exposure assessment of risks for CHD.

Future goals
To date, only a limited number of studies have been done using high-density genotyping methodologies for the identification of genes of polygenetic diseases. It would be beneficial to study which genetic variations lead to the more common forms of CHD.

Exposure assessments for newly emerging POPs and studies of the differences in susceptible Wisconsin mothers and their developing fetus with known CHD are needed. Measuring a wide range of CHD infant serum POPs and correlating the maternal and infant levels will help determine which specific pollutants lead to CHD. We could then compare these findings to regional and national serum concentrations.

Great lakes POPs profiles in dietary fish vary according to the body of water, species consumed and size of the fish. However, we have not explored these differences specifically in mothers of CHD infants. It would be beneficial to compare serum POPs in various forms of CHD and correlate with maternal reports of fish consumption as that is the most likely source for many of these contaminants. However, we must exercise considerable caution since fish also can provide very beneficial nutrients such as omega-3s to young mothers. Therefore, understanding the gene-environment interaction also may help us balance the benefits and risks of fish consumption in young mothers.

Infants with congenital heart disease benefit from developmental-based therapies

By Jenni Goldbaum, OTR/L, occupational therapist, Children's Hospital of Wisconsin

Research is just beginning to identify the long-term developmental outcomes of school-aged children with complex congenital heart disease. Many findings show that children with CHD have more significant delays in the areas of gross and fine motor performance and cognitive skills. In order to understand and identify early indicators that may increase risk of cognitive and motor delays, it is necessary to begin looking at this population early on in their hospitalization.

The Physical/Occupational Therapy Department at Children's Hospital of Wisconsin has specialized clinicians who primarily work with infants with complex congenital heart disease, providing continuous care from the Pediatric Intensive Care Unit through the Herma Heart Center Developmental Follow-up Clinic after discharge. These services include evaluation and treatment of early motor and neurobehavioral skills, development of specific care plans to address areas of concern and ongoing parent education.

Patients may be referred to the Physical/Occupational Therapy Department if they have issues such as:

• Edema.
• Prolonged intubation and bed rest.
• Neurological concerns, such as seizures, head bleeds or abnormal muscle tone.
• Premature birth.
• Increased irritability (difficult to calm at rest or during care).
• Decreased arousal level (difficult to wake/keep awake during oral feeding).
• Limited range of motion (neck, upper/lower extremities, ribcage and trunk).
• Developmental delay (infants already receiving birth-to-three or outpatient services prior to surgery).
  

Patients at risk for motor delays include:

• Infants with delayed chest closure.
• Infants who have been on extracorporeal membrane oxygenation.
• Any infant having open heart surgery within the first month of life.
• Children with a genetic diagnosis of predisposition for developmental delays.
• Infants on prolonged bed rest and narcotics.
  

In the PICU, therapists help ensure a calm, awake state of arousal for greater success with early interactions and prior to oral feeding. We provide edema massage, and range of motion and soft tissue mobilization when appropriate. We continuously educate and demonstrate appropriate positioning options to promote symmetrical motor development. Once the infant is transferred to the Intermediate Intensive Care Unit, we continue to provide graded strengthening opportunities for progression of developmental skills. We also will fabricate and fit orthotic devices or splints that may be needed. Our team is careful to consider any precautions or contraindications that may be pertinent with post-surgical patients, such as when to safely start tummy time, and how to lift and carry a baby who has had open heart surgery.

Prior to discharge, families are provided with detailed handouts and information regarding appropriate home-based activities and exercises to continue developmental progression and strengthening. Therapists then recommend a birth-to-three program referral for any infant that exhibits developmental concerns or delays and confirm that these patients also have been referred to the Developmental Follow-up Clinic.

As families manage various admissions, surgeries and outpatient clinic visits, therapists provide developmental-based support. By introducing the role of therapies from the beginning, parents are more likely to understand the importance of promoting typical developmental skills and consistently follow through with home programs and therapy appointments.

For more information, contact the Physical/Occupational Therapy Department at Children's Hospital of Wisconsin at (414) 266-2858.

Herma Heart Center Developmental Follow-up Clinic

By Ann Chin, RN, program coordinator, Herma Heart Center Developmental Follow-up Clinic, Children's Hospital of Wisconsin; and Laurel M. Bear, MD, director, Herma Heart Center Developmental Follow-up Clinic, Children's Hospital of Wisconsin; assistant
professor, Pediatrics, Medical College of Wisconsin

Through the Herma Heart Center Developmental Follow-up Clinic, staff provide targeted follow up and planning for intervention to help reduce developmental delays and provide optimal care for children with congenital heart disease. The goal of the program is to partner with primary care providers, medical specialists and families to optimize not only the health of the patient but their development outcomes as well.

The clinic is modeled after a Wisconsin Association for Perinatal Care follow-up program designed for premature infants and Neonatal Intensive Care Unit graduates. A multidisciplinary approach is used to assess each child and is staffed with nurses and physical, occupational and speech therapists who specialize in high-risk follow up.

Each assessment includes a review of developmental milestones and standardized scoring of the child's motor and mental skills, feeding concerns and the building blocks for the development of language. Attention is given to general growth and nutrition, and all patients undergo a hearing evaluation at 6 months of age. Indicators of family stressors also are explored. If delays are identified, the team makes recommendations and referrals for appropriate interventions.

The clinic team uses the Bayley-III Scales of Infant Development as a developmental tool. This assessment tool incorporates a flexible administration format into a standardized procedure. The norms were derived from a national, stratified random sample representative of the U.S. population for children from 1 through 43 months of age. The scores of this testing instrument demonstrate a high degree of stability over time and across age groups. There is a high level of reliability and confidence in the scores a child obtains on the test, as well as concurrent validity.
Results of this developmental tool are reported using five domains of development:

• Cognitive.
• Fine motor.
• Gross motor.
• Expressive language.
• Receptive language.
  

Each child receives a raw score that reflects the number of items receiving credit. This value is translated into the scaled score that has a range of one to 19 with a mean of 10 and standard deviation of three, and a composite score that has a range of 40 to 160 with a mean of 100 and a standard deviation of 15. These values are examined and compared at each of the patient's visits and will allow for interpatient review as well.

Initially, all neonates younger than 30 days old undergoing open heart surgery, all neonates with cyanotic heart disease and any other infant with congenital heart disease considered to be at high risk for developmental delay are referred to the clinic. Children are seen every six months through 3 years of age. Follow-up care with Cheryl Brosig, PhD, a developmental specialist for children older than 3, is arranged if needed.

Using bone marrow cells to treat heart disease
 

By Jos Domen, PhD, assistant professor, Surgery (Cardiothoracic), Medical College of Wisconsin; and Kimberly Gandy, MD, PhD, pediatric cardiothoracic surgeon, Children's Hospital of Wisconsin; associate professor, Surgery (Cardiothoracic), Medical College of Wisconsin

Our research focuses on the use of stem cells and other hematopoietic cells in translational medicine in two specific fields important to heart disease: tolerance induction for organ transplantation and myocardial rescue of congenital cardiomyopathy.

Organ transplantation currently depends on the continued administration of immunosuppressive drugs to prevent rejection of the transplanted organ. Management of immunosuppression is complicated and often leaves patients vulnerable to infection. A percentage of animals have blood systems that are chimeric, a mix of their own blood cells and cells from a donor, and can accept organ grafts from a blood cell donor without the need for immunosuppressants, a phenomenon known as being tolerant of a transplanted organ graft. 

Tolerance is defined as the ability of a recipient to accept and maintain a genetically disparate organ graft without the need for future immunosuppression. Despite decades of research, hematopoietic transplantation has not been successful in clinical tolerance induction like it has been in the treatment of oncological diseases. Recipients of donor specific blood stem cells (hematopoietic cell transplantation or bone marrow transplantation) have a higher likelihood of being able to accept an organ graft if this blood stem cell transplantation results in high levels of donor-specific cells in the recipient's blood system. Unfortunately, methods of blood stem cell or bone marrow administration that result in high levels of engraftment of donor-derived engraftment often are complicated by graft-versus-host disease, a potentially deadly complication in which the donor lymphocytes reject and attack the host, and severe immunoincompetence of the recipient.

As previously described, hematopoietic stem cells represent a small portion of the whole bone marrow or mobilized peripheral blood inoculum that is typically used clinically. The use of purified hematopoietic stem cells alone, as opposed to the aggregate of whole bone marrow, as the cellular infusate can avoid graft versus host disease. However, recipients continue to be plagued by initial severe immunoincompetence.

Due to improvements in clinical protocols, there is renewed hope for the use of hematopoietic cell transplantation beyond the treatment of oncological diseases. We have chosen to pursue the use of hematopoietic cell transplantation for organ tolerance induction. Our research efforts are aimed at further improving the outcome of hematopoietic cell transplantation by addressing the resultant immunoincompetence.

The addition of certain adjuvant cells to the hematopoietic cell inoculum has the potential to reduce the immunoincompetence. One population of interest is called myeloid progenitors, which can result in a rapidly improved resistance to fungal and bacterial pathogens. These cells do not cause graft-versus-host disease. We now are studying the effect of myeloid progenitors on the tolerance induction to donor-derived grafts after hematopoietic stem cell transplantation. We need to verify that donor-derived tolerance induction is not altered by the administration of myeloid progenitors.

Another area of study in the lab involves the use of hematopoietic cells for myocardial rescue in congenital cardiomyopathy. There has been considerable interest in the last decade in the use of hematopoietic cells for rescue of myocardial function in ischemic cardiomyopathy.

We chose to study the effect of hematopoietic cells in congenital cardiomyopathy. Mice that overexpress tropomodulin throughout the myocardium develop a severe cardiomyopathy by 4 weeks of age. We have demonstrated that the administration of whole bone marrow under certain conditions can result in the recovery of cardiac function in these mice. We currently are studying the method by which such myocardial recovery occurs as well as determining whether this phenomenon can be demonstrated in other models of cardiomyopathy.

Clinical uses of stem cells hold great promise

By Jos Domen, PhD, assistant professor, Surgery (Cardiothoracic), Medical College of Wisconsin; and Kimberly Gandy, MD, PhD, pediatric cardiothoracic surgeon, Children's Hospital of Wisconsin; associate professor, Surgery (Cardiothoracic), Medical College of Wisconsin

Stem cells are characterized by the ability to make more stem cells and to differentiate into the different types of mature cells that are necessary for normal body functions in a controlled manner. These properties have generated great interest in the clinical use of stem cells, as they hold the promise for tissue repair beyond current medical capabilities. As such, they are regularly featured in both the scientific and popular press.

This article attempts to provide some background information on the characterization of stem cells and the differences between the types of stem cells.

Types of stem cells
The debate about the use of stem cells is sometimes muddled by the fact that many different types of stem cells exist in the body. The most potent of these cells are the embryonic stem cells, also called ES-cells. These cells have the ability to differentiate into any cell type present in the body. As the name suggests, ES-cells normally are present in early embryos.

Exciting new research suggests that it may be possible to reprogram adult cells to assume the characteristics of ES-cells. Such potential could alleviate much of the tension involved in the ethical questions that surround the use of ES-cells. However, the reprogramming techniques currently used result in cells that are prone to malignant transformation. This would need to be overcome before clinical use can be considered and before one can conclude that adult cells may have the same potential as ES-cells.

Stem cells also are present in adult tissues. They often are referred to as adult stem cells. These cells are more restricted in differentiation capacity than the embryonic cells and typically are limited to producing cells present in one type
of tissue. In some tissues, where new cells need to be produced continuously, stem cells have been known to exist for a long time. These tissues include the blood, gut and skin. These stem cells need to function throughout life. However, other tissues in which new cells are not typically formed, such as brain tissue, recently have been found to contain stem cells in adults. It is less clear how much clinical potential these cells have and how they can be activated and used clinically. Much work remains to be done to answer these questions.

Hematopoietic stem cells
The blood forming stem cell, also called a hematopoietic stem cell, resides in the bone marrow.

Blood consists of many cell types with specialized functions. Blood cells are responsible for the distribution of oxygen from the lungs to the tissues (red blood cells) and the prevention of infection by parasites, bacteria and viruses. Some of the same cells that fight infection also are capable of recognizing and rejecting transplanted organs. Many types of blood cells are short-lived and need to be replenished continuously. The approximately 100 billion new blood cells that a human being requires every day originate from the hematopoietic stem cells in the bone marrow. These stem cells have seen continued and growing clinical use for almost 50 years. Even though these cells have been purified and studied extensively, much remains to be discovered.

Clinical use of stem cells
The clinical use of stem cells holds great promise. Current research suggests that it will be possible to grow and bioengineer tissue, including valves and muscle, outside the body that can be used to replace or aid damaged tissue. But for most classes of adult stem cells, medical applications remain untested or in the very early phases of clinical testing. The use of so-called mesenchymal stem cells to treat cartilage, bone and heart defects is an example of such a technology in its infancy.

Hematopoietic stem cells are the exception. They have been used in transplantation since 1959, albeit almost exclusively in an unpurified form. By 1995, more than 40,000 transplants were performed annually worldwide. Currently, the main indications for bone marrow transplantation are for treatment of hematopoietic malignancies (leukemias and lymphomas) or for hematopoietic reconstitution after high-dose chemotherapy for treatment of non-hematopoietic malignancies (cancers in other organs). Other indications for hematopoietic cell transplantation include treatment of autoimmune diseases and treatment of diseases involving genetic or acquired bone marrow failure, such as aplastic anemia, thalassemia and sickle cell anemia. Recent clinical trials also study the ability of hematopoietic stem cells to contribute to improved heart function following myocardial infarcts.

Herma Heart Center 2007 statistics through Nov. 30

	1/1/2007	2006	Change from 2006
Total cases	678	710	-4.5%
Open heart	362	358	1.1%
Closed heart	316	352	-10.2%
Patients younger than 1 year old	394	455	-13.4%
Total mortality	1.18%	1.55%	-23.84%

Age at operation	2007 Total	Deaths	Open heart	Deaths	Closed heart	Deaths
0-30 days	164	4	49	4	115	0
31-365 days	230	2	132	2	98	0
1-18 years	249	1	157	1	92	0
> 18 years	35	1	24	1	11	0

Clinics and diagnostics	Through 11/30/2007	Through 11/30/2006
Clinic visits, Milwaukee	8,129	6,880
Clinic visits, Fox Valley	1,055	943
Clinic visits, Gurnee	795	659
Echos	7,404	6,234
Stress/tilts	649	607
Cath lab procedures	441	456
EKGs	4,629	5,264