Genome-wide methylation profiling in granulosa lutein cells of women with polycystic ovary syndrome (PCOS).

Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder amongst women of reproductive age, whose aetiology remains unclear. To improve our understanding of the molecular mechanisms underlying the disease, we conducted a genome-wide DNA methylation profiling in granulosa lutein cells collected from 16 women suffering from PCOS, in comparison to 16 healthy controls. Samples were collected by follicular aspiration during routine egg collection for IVF treatment. Study groups were matched for age and BMI, did not suffer from other disease and were not taking confounding medication. Comparing women with polycystic versus normal ovarian morphology, after correcting for multiple comparisons, we identified 106 differentially methylated CpG sites with p-values <5.8 × 10-8 that were associated with 88 genes, several of which are known to relate either to PCOS or to ovarian function. Replication and validation of the experiment was done using pyrosequencing to analyse six of the identified differentially methylated sites. Pathway analysis indicated potential disruption in canonical pathways and gene networks that are, amongst other, associated with cancer, cardiogenesis, Hedgehog signalling and immune response. In conclusion, these novel findings indicate that women with PCOS display epigenetic changes in ovarian granulosa cells that may be associated with the heterogeneity of the disorder.

High frequency of TTTY2-like gene-related deletions in patients with idiopathic oligozoospermia and azoospermia.

Genes located on Y chromosome and expressed in testis are likely to be involved in spermatogenesis. TTTY2 is a Y-linked multicopy gene family of unknown function that includes TTTY2L2A and TTTY2L12A at Yq11 and Yp11 loci respectively. Using PCR amplification, we screened for TTTY2L2A- and TTTY2L12A-associated deletions, in 94 Greek men with fertility problems. Patients were divided into three groups as following: group A (n = 28) included men with idiopathic moderate oligozoospermia, group B (n = 34) with idiopathic severe oligozoospermia and azoospermia, and group C (n = 32) with oligo- and azoospermia of various known etiologies. No deletions were detected in group C patients and 50 fertile controls. However, two patients from group A had deletions in TTTY2L2A (7.1%) and six in TTTY2L12A (21.4%), whereas from group B, four patients had deletions in TTTY2L2A (11.8%) and 10 in TTTY2L12A (29.4%). In addition, five patients from both groups A and B (8%) appeared to have deletions in both studied TTTY2 genes, although these are located very far apart. These results indicate that the TTTY2 gene family may play a significant role in spermatogenesis and suggest a possible mechanism of nonhomologous recombinational events that may cause genomic instability and ultimately lead to male infertility.

DNM1DN: a new class of paralogous genomic segments (duplicons) with highly conserved copies on chromosomes Y and 15.

Screening a testis cDNA selection library for Y-linked genes yielded 79 cDNAs. Of these, 9 matched the 3' region of the dynamin 1 gene (DNM1) on chromosome 9q34 with >90% identity. Fluoresence in situ hybridisation and PCR amplification were used to localise a large number of DNM1-like sequences to human chromosomes 15 and Y. PCR amplification of overlapping Y-linked YACs allowed a more accurate mapping of the Y-linked DNM1-like cDNAs to a euchromatic locus in close proximity to heterochromatin at Yq11.23. A search of the genome database identified 64 highly homologous copies of the DNM1 fragment. Most of these copies were localised to chromosomes 15 and Y, but others mapped to chromosomes 5, 8, 10, 12, 19 and 22. These sequences exhibit all the major features of a duplicon and have been designated DNM1DN (DNM1 duplicon). Evolutionary studies using fluorescence in situ hybridisation indicate that transposition of the DNM1DN sequence to chromosome 15 took place earlier in primate evolution than the transposition to the Y chromosome. The translocation to the Y took place at a time following the divergence of a common ancestor from gorilla, approximately 4-7 million years ago.

Canine TCOF1; cloning, chromosome assignment and genetic analysis in dogs with different head types.

We describe the construction of a dog embryonic head/neck cDNA library and the isolation of the dog homolog of the Treacher Collins Syndrome gene, TCOF1. The protein shows a similar three-domain structure to that described for human TCOF1, but the dog gene lacks exon 10 and contains two exons not present in the human sequence. In addition, exon 19 is differentially spliced in the dog. How these structural differences relate to TCOF1 phosphorylation is discussed. Isolation of a genomic clone allowed the exon/intron boundaries to be characterized and the dog TCOF1 gene to be mapped to CF Chr 4q31, a region syntenic to human Chr 5. Genetic analysis of DNA of dogs from 13 different breeds identified nine DNA sequence variants, three of which gave rise to amino acid substitutions. Grouping dogs according to head type showed that a C396T variant, leading to a Pro117Ser substitution, is associated with skull/face shape in our dog panel. The numbers are small, but the association between the T allele and brachycephaly, broad skull/short face, was highly significant (p = 0.000024). The short period of time during which the domestic dog breeds have been established suggests that this mutation has arisen only once in the history of dog domestication.

TTY2: a multicopy Y-linked gene family.

Genes involved in human male sex determination and spermatogenesis are likely to be located on the Y chromosome. In an effort to identify Y-linked, testis-expressed genes, a cDNA selection library was generated by selecting testis cDNA with Y-cosmid clones. Resultant clones containing repetitive or vector material were eliminated, and 79 of the remaining clones were sequenced. Nineteen cDNAs showed homology with the TTY2 gene, and indicated that TTY2 is part of a large gene family. Screening of a panel of Y-linked cosmids revealed that the TTY2 gene family includes at least 26 members organized in 14 subfamilies. Further investigation revealed that TTY2 genes are arranged in tandemly arrayed clusters on both arms of the Y chromosome, and each gene comprises a series of tandemly arranged repeats. RT-PCR studies for two of these genes revealed that they are expressed in adult and fetal testis, as well as in the adult kidney. None of the genes investigated in detail contain an open reading frame. We conclude that the TTY2 gene family is composed of multiple copies, some of which may function as noncoding RNA transcripts and some may be pseudogenes.