Studies in rats and mice have established that maternal nutrition induces epigenetic modifications, sometimes permanently, that alter gene expression in the fetus, which in turn leads to phenotypic changes. However, limited data is available on the influence of maternal diet on epigenetic modifications and gene expression in sheep. Therefore, the objectives of this study were to investigate the impact of different maternal dietary energy sources on the expression of imprinted genes in fetuses in sheep. Ewes were naturally bred to a single sire and from days 67 ± 3 of gestation until necropsy (days 130 ± 1), they were fed one of three diets of alfalfa haylage (HY; fiber), corn (CN; starch), or dried corn distiller’s grains (DG; fiber plus protein plus fat). A total of 26 fetuses were removed from the dams and longissimus dorsi, semitendinosus, perirenal adipose depot, and subcutaneous adipose depot tissues were collected for expression and DNA methylation analyses. Expression analysis of nine imprinted genes and three DNA methyltransferase (DNMTs) genes showed significant effects of the different maternal diets on the expression of these genes. The methylation levels of CpG islands of both IGF2R and H19 were higher in HY and DG than CN fetuses in both males and females. This result is consistent with the low amino acid content of the CN diet, a source of methyl group donors, compared to HY and DG diets. Thus, results of this study provide evidence of association between maternal nutrition during pregnancy and transcriptomic and epigenomic alterations of imprinted genes and DNMTs in the fetal tissues.

Laron syndrome is an autosomal recessive condition characterized by resistance to growth hormone. We sought to determine the molecular basis of this condition in an Ecuadorean population with a high incidence of affected individuals. Growth hormone receptor gene sequences from an obligate heterozygote were amplified by the polymerase chain reaction and screened for mutations using denaturing gradient gel electrophoresis. Only exon 6 revealed homo- and heteroduplexes on denaturing gradient gels. Sequencing revealed a substitution of guanine for adenine in the third position of codon 180 that did not change the amino acid encoded. Sequencing of the exon 6-exon 7 splice junction from RNA-polymerase chain reaction amplified cellular RNA of an affected individual revealed that the substitution activates a 5' splice site 24 nucleotides upstream from the normal exon 6-intron 6 boundary. Splicing in two probands' lymphoblasts occurred virtually exclusively at the abnormal 5' splice site created by the codon 180 substitution. Exon 6 sequences from 38 patients and 47 relatives were amplified and analyzed by sequencing or dot-blot hybridization with allele-specific oligonucleotides. The substitution was detected in 74 of 76 Laron syndrome patients' GH-receptor alleles. All 26 parents and 12 of 21 unaffected siblings were heterozygous for this mutation. It was absent in 61 unrelated unaffected control individuals. We conclude that the codon 180 nucleotide substitution probably causes Laron syndrome as translation of the observed, abnormally spliced growth hormone receptor transcript would lead to the synthesis of a receptor protein with an 8 amino acid deletion from the extracellular domain.

The identification of genetic loci involved in most forms of congenital heart disease has been hampered by the complex inheritance patterns of these disorders. Atrioventricular canal defects (AVCDs) are most commonly associated with Down syndrome, although non-syndromic cases also occur. Non-syndromic AVCDs have been attributed to multifactorial inheritance. However, the occurrence of a few kindreds with multiple affected individuals has suggested that a major genetic locus can account for the disorder in some families. We have used a combination of DNA pooling and shared segment analysis to perform a high density screen of the entire autosomal human genome in an extended kindred. In so doing, we have identified a genetic locus on chromosome 1 shared by all affected individuals. Our data demonstrate the existence of a congenital heart defect susceptibility gene, inherited as an autosomal dominant with incomplete penetrance, involved in AVCD. Furthermore, our data demonstrate the power of using key isolated kindreds in combination with high density genomic screens to identify loci involved in complex disorders such as congenital heart defects.

Marfan syndrome (MFS) is an autosomal dominantly inherited connective tissue disorder characterized by cardiovascular, ocular and skeletal manifestations. Previously, mutations in the fibrillin-1 gene on chromosome 15 (FBN1) have been reported to cause MFS. We have now screened 44 probands with MFS or related phenotypes for alterations in the entire fibrillin coding sequence (9.3 kb) by single strand conformation analysis. We report four unique mutations in the fibrillin gene of unrelated MFS patients. One is a 17 bp deletion and three are missense mutations, two of which involve 8-cysteine motifs. Another missense mutation was found in two unrelated individuals with annuloaortic ectasia but was also present in unaffected relatives and controls from various ethnic backgrounds. By using allele-specific oligonucleotide hybridization, we screened 65 unrelated MFS patients, 29 patients with related phenotypes and 84 control individuals for these mutations as well as for a previously reported mutation and two polymorphisms. Our results suggest that most MFS families carry unique mutations and that the fibrillin genotype is not the sole determinant of the connective tissue phenotype.