Some sheep breeds are naturally prolific, and they are very informative for the studies of reproductive genetics and physiology. Major genes increasing litter size (LS) and ovulation rate (OR) were suspected in the French Grivette and the Polish Olkuska sheep populations, respectively. To identify genetic variants responsible for the highly prolific phenotype in these two breeds, genome-wide association studies (GWAS) followed by complementary genetic and functional analyses were performed. Highly prolific ewes (cases) and normal prolific ewes (controls) from each breed were genotyped using the Illumina OvineSNP50 Genotyping Beadchip. In both populations, an X chromosome region, close to the BMP15 gene, harbored clusters of markers with suggestive evidence of association at significance levels between 1E⁻⁰⁵ and 1E⁻⁰⁷. The BMP15 candidate gene was then sequenced, and two novel non-conservative mutations called FecX^Gr and FecX^O were identified in the Grivette and Olkuska breeds, respectively. The two mutations were associated with the highly prolific phenotype (p_FecX^Gr = 5.98E⁻⁰⁶ and p_FecX^O = 2.55E⁻⁰⁸). Homozygous ewes for the mutated allele showed a significantly increased prolificacy (FecX^Gr/FecX^Gr, LS = 2.50±0.65 versus FecX⁺/FecX^Gr, LS = 1.93±0.42, p<1E⁻⁰³ and FecX^O/FecX^O, OR = 3.28±0.85 versus FecX⁺/FecX^O, OR = 2.02±0.47, p<1E⁻⁰³). Both mutations are located in very well conserved motifs of the protein and altered the BMP15 signaling activity in vitro using a BMP-responsive luciferase test in COV434 granulosa cells. Thus, we have identified two novel mutations in the BMP15 gene associated with increased LS and OR. Notably, homozygous FecX^Gr/FecX^Gr Grivette and homozygous FecX^O/FecX^O Olkuska ewes are hyperprolific in striking contrast with the sterility exhibited by all other known homozygous BMP15 mutations. Our results bring new insights into the key role played by the BMP15 protein in ovarian function and could contribute to a better understanding of the pathogenesis of women′s fertility disorders. Although causes altering reproductive function and leading to several fertility syndromes in women are various, a clear association between mutations in some members of the TGFβ family (BMP15 and GDF9) and abnormal ovarian phenotype has established the importance of these factors for normal female fertility. Some sheep breeds are naturally prolific since they carry major genes affecting ovulation rate and litter size. These species are therefore unique informative models for the study of reproductive genetics and physiology. Through a GWAS conducted in two sheep breeds using highly prolific ewes as cases and normal prolific ewes as controls, we identified an X chromosome locus close to the BMP15 gene in both breeds. Two novel non-conservative BMP15 mutations, one in each population, located in very well conserved domains of the protein were associated with the phenotype at heterozygous and homozygous status. Moreover, we showed that both mutations altered the BMP15 signaling activity, suggesting a novel kind of BMP15 variant responsible for an atypical high prolificacy, in contrast to all other BMP15 variants described so far. Our findings suggest an additional role of the BMP15 protein in folliculogenesis and could contribute to a better understanding of the pathogenesis of women′s fertility disorders.

The imprinted expression of the IGF2 and H19 genes is controlled by the imprinting control region 1 (ICR1) located at chromosome 11p15.5. This methylation-sensitive chromatin insulator works by binding the zinc-finger protein CTCF in a parent-specific manner. DNA methylation defects involving the ICR1 H19/IGF2 domain result in two growth disorders with opposite phenotypes: an overgrowth disorder, the Beckwith–Wiedemann syndrome (maternal ICR1 gain of methylation in 10% of BWS cases) and a growth retardation disorder, the Silver–Russell syndrome (paternal ICR1 loss of methylation in 60% of SRS cases). Although a few deletions removing part of ICR1 have been described in some familial BWS cases, little information is available regarding the mechanism of ICR1 DNA methylation defects. We investigated the CTCF gene and the ICR1 domain in 21 BWS patients with ICR1 gain of methylation and 16 SRS patients with ICR1 loss of methylation. We identified four constitutional ICR1 genetic defects in BWS patients, including a familial case. Three of those defects are newly identified imprinting defects consisting of small deletions and a single mutation, which do not involve one of the CTCF binding sites. Moreover, two of those defects affect OCT-binding sequences which are suggested to maintain the unmethylated state of the maternal allele. A single-nucleotide variation was identified in a SRS patient. Our data extends the spectrum of constitutive genetic ICR1 abnormalities and suggests that extensive and accurate analysis of ICR1 is required for appropriate genetic counseling in BWS patients with ICR1 gain of methylation.

The composition and structure of fleece variation observed in mammals is a consequence of a strong selective pressure for fiber production after domestication. In sheep, fleece variation discriminates ancestral species carrying a long and hairy fleece from modern domestic sheep (Ovis aries) owning a short and woolly fleece. Here, we report that the “woolly” allele results from the insertion of an antisense EIF2S2 retrogene (called asEIF2S2) into the 3′ UTR of the IRF2BP2 gene leading to an abnormal IRF2BP2 transcript. We provide evidence that this chimeric IRF2BP2/asEIF2S2 messenger 1) targets the genuine sense EIF2S2 RNA and 2) creates a long endogenous double-stranded RNA which alters the expression of both EIF2S2 and IRF2BP2 mRNA. This represents a unique example of a phenotype arising via a RNA-RNA hybrid, itself generated through a retroposition mechanism. Our results bring new insights on the sheep population history thanks to the identification of the molecular origin of an evolutionary phenotypic variation.

The imprinted 11p15 region is organized in two domains, each of them under the control of its own imprinting control region (ICR1 for the IGF2/H19 domain and ICR2 for the KCNQ1OT1/CDKN1C domain). Disruption of 11p15 imprinting results in two fetal growth disorders with opposite phenotypes: the Beckwith–Wiedemann (BWS) and the Silver–Russell (SRS) syndromes. Various 11p15 genetic and epigenetic defects have been demonstrated in BWS and SRS. Among them, isolated DNA methylation defects account for approximately 60% of patients. To investigate whether cryptic copy number variations (CNVs) involving only part of one of the two imprinted domains account for 11p15 isolated DNA methylation defects, we designed a single nucleotide polymorphism array covering the whole 11p15 imprinted region and genotyped 185 SRS or BWS cases with loss or gain of DNA methylation at either ICR1 or ICR2. We describe herein novel small gain and loss CNVs in six BWS or SRS patients, including maternally inherited cis‐duplications involving only part of one of the two imprinted domains. We also show that ICR2 deletions do not account for BWS with ICR2 loss of methylation and that uniparental isodisomy involving only one of the two imprinted domains is not a mechanism for SRS or BWS. Hum Mutat 32:1171–1182, 2011. ©2011 Wiley‐Liss, Inc.