The following is a slightly modified excerpt from a recently submitted proposal for funding.

Fetal growth restriction (FGR) is a serious health risk factor. Infants in the lowest 7.5 percent of birth weight account for two-thirds of infant deaths. Among the FGR infants that live, there is an increased frequency of hypoglycemia, hypothermia, polycythemia, neurodevelopmental deficits, and cerebral palsy. Later in life the risk of hypertension, cardiovascular disease, and non-insulin dependent diabetes is elevated. Therefore, FGR infants account for a substantial proportion of early death and morbidity and chronic illness. The ability to diagnose and to treat FGR early in gestation has enormous potential to reduce childhood death and suffering and to save considerable money devoted to treating its outcomes.

It is well-known that pre-eclampsia, fetal infection, malnutrition, placental damage, and smoking can produce growth restriction. However, most cases of FGR are of unknown cause. Detection occurs as late as the third trimester, delaying clinical support of the pregnancy. When the underlying cause is unknown, care may be restricted to careful monitoring, reactive treatment, and early induction of labor. There is an urgent need to understand the underlying bases for idiopathic FGR in order to provide early diagnosis and to suggest prophylactic measures that can be taken to restore the normal growth trajectory of the fetus. The goal of this research is to elucidate the underlying genetic cause of idiopathic FGR.

FGR has a strong genetic component. Siblings of FGR infants have below-average birth weights, and there is non-random aggregation of low birth weight in families. IGF-I levels are predominantly (93% male, 77% female) controlled by genetic factors, insulin predominantly by the environment (80%), and IGF-II and IGFBP-1 almost equally by genetic and environmental factors. Most of the fetal gain in weight and height occurs during the last trimester of pregnancy and is highly reliant upon the supply of nutrition and of growth factors, such as the insulin-like growth factors I and II. Therefore, a search for genetic influences on fetal growth should focus on those molecules that affect the fetal supply of nutrients and growth factors. The genes of the human GH locus are expressed in both the pituitary and placenta and are ideal candidate genes for idiopathic fetal growth restriction.

Structure of the human growth hormone locus

There are five growth hormone-related genes. GH-N is expressed mainly in the pituitary. Growth hormone variant (GH-V) and three chorionic somatomammotropins (CS-L, CS-A and CS-B) are expressed by the syncytiotrophoblast of the placenta and thus are derived from the fetal genotype. The 5 genes span 46,000 nucleotides in the arrangement: 5' - GH-N - CS-L - CS-A - GH-V - CS-B - 3'. GH-N and GH-V proteins differ at 13 residues. The mature forms of CS-A and CS-B are identical. CS-L appears to be a pseudogene, and a CS-L protein has never been detected. Each gene is in the same transcriptional orientation and is composed of five exons spanning of 1500 nucleotides. Within the GH locus, there are three key regulatory elements that control the expression of the genes: a negative regulatory element called P (GH-V and CS genes), an enhancer (CS genes), and a promoter.

Function of the human growth hormone locus during gestation

GH-N (pituitary). During pregnancy, pituitary GH steadily decreases in concentration in the maternal circulation until it reaches undetectable levels around 24-25 weeks gestation. The GH-N found in the fetal circulation is entirely of fetal origin. Surprisingly, GH-N is not essential for fetal growth because anencephalic fetuses or those with a congenital lack of a pituitary achieve nearly normal length.

GH-V (placental). GH-V is not secreted into the fetal circulation. In the maternal circulation GH-V can be detected at 8 weeks gestation and continually rises until about the 35th week of gestation to its plateau level of 27.5 mU/L. During gestation IGF-I levels increase in parallel with GH-V levels, suggesting that maternal IGF-I production is under the control of placental GH. Via IGF-I, GH-V also plays a role in the growth of the placenta during gestation. Subnormal levels of GH-V may indirectly produce FGR by retarding the growth of the placenta. Alternatively, GH-V may affect fetal growth through its influence on nutrient availability to the fetus. GH-V stimulates gluconeogensis, lipolysis, and anabolism, all of which increase the concentration of nutrients in the blood that are accessible to the fetus. Defects in the secretion of GH-V may restrict fetal growth through nutrient deprivation. GH-V levels are reduced by half (14.9 vs. 26.5 mU/L) in cases of idiopathic intrauterine growth restriction. Similarly, maternal IGF-I levels are reduced (156 vs. 285 m g/L) in FGR.

Chorionic somatomammotropin (or, placental lactogen). CS-A and CS-B are found in both the maternal and fetal circulation. CS is detected in the maternal circulation at six weeks gestation and rises in concentration until 30 weeks to a plateau of 5-7 m g/mL. About 1g/day of CS is produced near term. In the fetal circulation CS levels rise from about 5 ng/mL at 20 weeks gestation to 20-30 ng/mL at term. Acute changes in blood glucose concentrations do not seem to affect CS levels, but chronic increases in blood glucose result in a depression of CS levels. Conversely, maternal starvation for 84-90 hours increases CS levels by over 30%. CS functions to maintain long-term nutrient availability to the fetus, one of the major determinants of fetal growth. In the mother CS increases glucose uptake and incorporation into glycogen, glycerol, and fatty acids. Additionally, CS increases insulin production and resistance to insulin, and this contributes to hyperglycemia following meals that increases glucose availability to the fetus. CS also increases serum levels of nonesterfied fatty acids, ketones and glycerol in the maternal circulation. The long-term consequence is to facilitate the utilization of fatty acids by the mother during fasting and to spare glucose for fetal use. In the fetus CS has anabolic effects that promote growth, including stimulation of amino acid uptake, DNA synthesis, IGF-I and IGF-II production, and insulin release. Maternal production of IGF-I is under the control of GH-V, but fetal IGF-I production is regulated by CS. CS levels are depressed in cases of FGR and serve as a potent indicator of neonatal death or distress. Measurements of CS levels in maternal blood below 4 m g/mL on three or more independent occasions correspond to a 71% chance of fetal distress or asphyxia.

Single nucleotide polymorphisms (SNPs) and small lesions in the GH locus

Single nucleotide polymorphisms (SNPs) and small lesions (deletions and insertions) are a significant potential source of variation in the expression patterns and levels of genes if they occur in binding sites for transcriptional factors. GH-N is the only gene of the GH locus that has been surveyed for polymorphism, and it is unusually rich in variation. In the span from -1015 to +282 there are 22 variable sites, a high number of variants for such a short region. Two of these variants are located within binding sites for transcriptional factors (NF1 and Pit-1) and potentially affect the expression of GH-N.

Importantly, the polymorphic sites within the promoter of the GH-N gene are among the few sites that differ in that region among each of the genes of the GH locus. This is due to recurrent gene conversion within the GH locus. Because of this process, I expect to observe levels of polymorphism in each gene of the GH locus as high as those observed in GH-N. Such high levels of polymorphism provide enormous potential for functionally significant changes in the regulation of GH-V and the CS genes that may have consequences for fetal development and growth. The association of individual polymorphisms and of combinations of polymorphisms (haplotypes) with fetal growth restriction will be determined in this study by sequencing the entire coding and regulatory regions of GH-V, CS-A, and CS-B. This will be the most extensive and statistically powerful assessment of the connection between variation in the GH locus and influence on fetal growth to date.

Identification of gross lesions in the growth hormone locus

Gene deletions within the hGH locus are identified by Southern analysis of HindIII and BamHI digestions of placental DNA. This is the first study that will determine the frequency of gene deletions throughout the entire hGH locus. To date, most studies have focussed only on deletions that encompass the GH-N gene. However, it is likely that at least 2% of all people bear a deletion of one or more genes other than GH-N. Digestion with BamHI cuts the hGH locus at many sites and produces a unique fragment for each gene (two for hGH-V). If any gene has been deleted, BamHI digestion provides an immediate identification of the deleted gene and a rough indication of the extent of the deletion. HindIII cuts the hGH locus much less often than does BamHI and will be highly useful for determining the extent of large-scale deletions.

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