DIABETES, VOL. 50, JANUARY 2001 199 Brief Genetics Report Evidence That Insulin is Imprinted in the Human Yolk Sac Gudrun E. Moore, Sayeda N. Abu-Amero, Gill Bell, Emma L. Wakeling, Amanda Kingsnorth, Philip Stanier, Eric Jauniaux, and Simon T. Bennett Allelic variation in the size of the insulin (INS) variable number tandem repeat (VNTR) correlates with the expression of both INS in the pancreas and thymus and IGF2 (the gene downstream of INS) in the placenta. In addition, the shorter, class I alleles are associated with type 1 diabetes, whereas the longer, class III alleles are associated with type 2 diabetes, polycystic ovary syn- drome (PCOS), and size at birth. Parent-of-origin effects have been reported for type 2 diabetes and PCOS, thus implicating a role for genomic imprinting in these phenotypes. In mice, Ins2 is imprinted and paternally expressed in the yolk sac. In humans, evidence for the imprinting of INS is circumstantial, with occasional mono- allelic expression in the thymus. In the present study, we found evidence for the imprinted paternal expression of INS in the human yolk sac. Two other imprinted genes from the same cluster are also expressed monoallelically in the human yolk sac. IGF2 was expressed solely from the paternal allele, and H19 was expressed solely from the maternal allele. These data suggest not only further func- tional roles for the human yolk sac in early fetal growth, but also evidence for a potential causal link between the control of insulin expression during development and insulin/growth-related diseases in later life. Diabetes 50:199–203, 2001 A llelic variation at the insulin (INS) variable num- ber tandem repeat (VNTR) is known to regulate the level of expression of both INS in the pan- creas and thymus and IGF2 in the placenta (1–5). The shorter, class I alleles correlate with levels of gene expression that are higher for INS in the pancreas, lower for INS in the thymus, and higher for IGF2 in the placenta. Higher levels of INS expression associated with the longer, class III VNTR alleles in the thymus offer an explanation for the protective role of these alleles in type 1 diabetes suscep- tibility by inducing self-tolerance to preproinsulin peptides (1,2). The class I alleles are associated with type 1 diabetes, and the class III alleles are associated with type 2 diabetes, polycystic ovary syndrome (PCOS), and size at birth (5–10). In PCOS and type 2 diabetes, disease susceptibility is con- ferred in a parental sex-specific fashion (6,9), such that in nuclear families, the susceptible allele is transmitted prefer- entially from fathers. This suggests that the etiological effect is occurring when only the paternal allele is active, implica- tive of parental imprinting. Although the imprinting of IGF2 is well established (11), evidence for monoallelic expression of insulin comes only from mice, in which Ins2 (the mouse ortholog of INS) is imprinted in the yolk sac after 14.5 days (12,13). In mammals, the secondary yolk sac is regarded as being the primitive liver, involved in protein synthesis and nutrient transfer and acting as a stem-cell reservoir (14,15). The secondary yolk sac may also play a role as a primitive pan- creas, because in the fourth week of development, the yolk sac forms the primitive gut, the endoderm of which gives rise to the epithelium and glands of the digestive tract. In humans, there is only circumstantial evidence for the imprinting of insulin (e.g., the parent-of-origin effects and 5 of 23 cases of monoallelic expression in the thymus) (1,2). However, INS lies next to the paternally expressed IGF2 gene and the maternally expressed H19 gene in an imprinted cluster on human chromosome 11p15.5 (11,16). Therefore, we set out to test for INS expression in the human yolk sac and investigate whether it is imprinted. We present evidence that, similar to IGF2 and H19, INS is expressed monoallelically and imprinted in the human yolk sac. To investigate gene expression and imprinting status in yolk sacs, a maternal blood sample, a placental sample, and the yolk sac were collected from 38 subjects. The blood and placental samples were used to provide genomic DNA for genotyping, whereas the yolk sacs were used to extract total RNA. Each fetus was genotyped by polymerase chain reaction (PCR) for INS, IGF2, and H19, and the products were digested with the PstI, ApaI, and RsaI restriction enzymes, which are intragenic polymorphisms for INS, IGF2, and H19, respectively (Figs. 1–3) (4,11,16). This revealed 14 fetal samples that were heterozygous for INS, 20 for IGF2, and 14 for H19 (Table 1). Once fetal het- erozygosity was established, the respective maternal DNA From the Molecular Biology Laboratory for Fetal Development (G.E.M., S.N.A.-A., G.B., E.L.W., P.S.), Division of Paediatrics, Obstetrics and Gynae- cology, Queen Charlotte’s and Chelsea Hospital, Imperial College of Science, Technology and Medicine; the Academic Division of Obstetrics and Gynae- cology (E.J.), Royal Free and University College Medical School, University College, London; the Department of Medical Genetics (A.K.), Wellcome Trust Centre of Molecular Mechanisms in Disease, CIMR, University of Cambridge, Cambridge; and the Oxagen Limited (S.T.B.), Abingdon, Oxon, U.K. Address correspondence and reprint requests to Dr. Gudrun Moore, Molecular Biology Laboratory for Fetal Development, Division of Paedi- atrics, Obstetrics and Gynaecology, Queen Charlotte’s and Chelsea Hospi- tal, Imperial College of Science, Technology and Medicine, Goldhawk Rd., London W6 OXG, U.K. E-mail:gemoore@ic.ac.uk. Received for publication 19 April 2000 and accepted in revised form 25 September 2000. PCOS, polycystic ovary syndrome; PCR, polymerase chain reaction; RT, reverse transcriptase; VNTR, variable number tandem repeat.