Faculty Bibliography

MicroRNAs (miRNAs), a class of small noncoding RNAs that regulate gene expression, have fundamental roles in biological processes, including cell differentiation and proliferation. These small molecules mainly direct either target messenger RNA (mRNA) degradation or translational repression, thereby functioning as gene silencers. Epithelial cells of the uterine lumen and glands undergo cyclic changes under the influence of the sex steroid hormones estradiol-17beta and progesterone. Because the expression of miRNAs in human endometrium has been established, it is important to understand whether miRNAs have a physiological role in modulating the expression of hormonally induced genes. The studies herein establish concomitant differential miRNA and mRNA expression profiles of uterine epithelial cells purified from endometrial biopsy specimens in the late proliferative and midsecretory phases. Bioinformatics analysis of differentially expressed mRNAs revealed cell cycle regulation as the most significantly enriched pathway in the late proliferative-phase endometrial epithelium (P = 5.7 x 10(-15)). In addition, the WNT signaling pathway was enriched in the proliferative phase. The 12 miRNAs (MIR29B, MIR29C, MIR30B, MIR30D, MIR31, MIR193A-3P, MIR203, MIR204, MIR200C, MIR210, MIR582-5P, and MIR345) whose expression was significantly up-regulated in the midsecretory-phase samples were predicted to target many cell cycle genes. Consistent with the role of miRNAs in suppressing their target mRNA expression, the transcript abundance of predicted targets, including cyclins and cyclin-dependent kinases, as well as E2F3 (a known target of MIR210), was decreased. Thus, our findings suggest a role for miRNAs in down-regulating the expression of some cell cycle genes in the secretory-phase endometrial epithelium, thereby suppressing cell proliferation.

The etiology and pathogenesis of endometrial polyps (EPs) are only partially understood. To better understand how sex steroids regulate polyp growth, we investigated the messenger RNA (mRNA) expression of the genes of reproductive steroid hormone receptors (estrogen receptors alpha [ERalpha] and beta [ERbeta], G protein-coupled receptor 30 [GPR30], and progesterone receptor [PR]) in EP tissue and autologous normal appearing endometrium (R) Within each patient, the normal appearing endometrial tissue remote from the site of the endometrial polyp (R) was taken as an internal control. Relative expressions of genes of interest within the endometrial polyp were compared to expressions of respective genes within the internal control tissue (i.e. R). R is the abbreviation for normal appearing endometrium in the later calculation formula. Ten patients diagnosed with EP in a tertiary care center were included in this study. Directed biopsies were obtained under hysteroscopy from the EP and from a normal appearing site remote from EP along the opposite uterine wall in each patient. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used for gene expression profiling in the paired tissue samples. The relative gene expression between EP and normal appearing endometrium in each patient was analyzed with 2(-DeltaDeltaCt) method. We found that ERalpha, ERbeta, GPR30, and PR were expressed in both normal appearing endometrium and EP in each patient. ERalpha, ERbeta, GPR30, and PR showed no difference in relative expression in EP samples compared with paired normal endometrial samples from the same uterine cavity. However, the relative expression of PR correlated with that of GPR30 (r = .70, P = .023), suggesting that the co-expression of PR and GPR30 may be a contributory mechanism in the pathogenesis of EPs at least in a subset of women.

Nucleolar channel systems (NCSs) are membranous organelles appearing transiently in the epithelial cell nuclei of postovulatory human endometrium. Their characterization and use as markers for a healthy receptive endometrium have been limited because they are only identifiable by electron microscopy. Here we describe the light microscopic detection of NCSs using immunofluorescence. Specifically, the monoclonal nuclear pore complex antibody 414 shows that NCSs are present in about half of all human endometrial epithelial cells but not in any other cell type, tissue or species. Most nuclei contain only a single NCS of uniform 1 microm diameter indicating a tightly controlled organelle. The composition of NCSs is as unique as their structure; they contain only a subset each of the proteins of nuclear pore complexes, inner nuclear membrane, nuclear lamina and endoplasmic reticulum. Validation of our robust NCS detection method on 95 endometrial biopsies defines a 6-day window, days 19-24 (+/-1) of an idealized 28 day cycle, wherein NCSs occur. Therefore, NCSs precede and overlap with the implantation window and serve as potential markers of uterine receptivity. The immunodetection assay, combined with the hitherto underappreciated prevalence of NCSs, now enables simple screening and further molecular and functional dissection.

Intercellular adhesion molecule-1 (ICAM-1) is involved in the pathogenesis of multiple sclerosis (MS), whereas sequence variations in the ICAM-1 gene could potentially be responsible for the genetic susceptibility to MS. We studied an association of MS with the 13,848A>G (K469E) polymorphism of the ICAM-1 gene in Finnish and Spanish cases and controls and affected families. An increased risk for the AA (Lys(469)/Lys(469)) genotype was found in both populations. The effect observed was found to be strongest among the HLA-DQB1*0602-positive subjects, which implies genetic heterogeneity of MS. Meta-analysis of all published datasets supports increased risk of MS for the ICAM-1 Lys(469) homozygotes (relative risk = 1.3, p = 0.002).

We have previously found evidence for linkage as well as allelic and haplotype association between the myelin basic protein (MBP) gene and multiple sclerosis (MS). These findings have, however, not been reproduced in other populations. Here, we have analyzed association between MBP and MS in a new set of 349 Finnish triad families. Families with a parent born in the Southern Ostrobothnian region in western Finland (Bothnia families, n=98) were analyzed as a separate group since our previous studies included a high proportion of patients and families from this high-incidence region. Other families (n=251) were collected at five hospitals in southern, eastern, and northern Finland. The MBP short tandem repeat was genotyped, and haplotype patterns were verified by sequencing. In the Bothnia families, the previously detected associations with the 1.27 kb allele and haplotype 1.27-B10 were confirmed (P=0.01 and 0.02, respectively), whereas in the other families there was not even a trend toward association. These results demonstrate a geographic/genealogical restriction in the association between MS and the MBP short tandem repeat, highlight the importance of genealogical information in genetic studies of complex traits, and may provide an explanation why the association has not been found in many other populations.

Genome-wide linkage analyses performed in a Finnish study sample have identified four potential predisposing loci for multiple sclerosis (MS). Here we made an effort to restrict the wide linkage region on chromosome 17 with a dense set of 31 markers using multipoint linkage analyses and monitoring for shared marker alleles in MS chromosomes. We carried out the linkage analyses in 22 Finnish multiplex MS families originating from a regional subisolate that shows an exceptionally high prevalence of MS in order to minimize the genetic and environmental heterogeneity of the study sample. Thirty markers on the 23 cM initial interval gave positive pairwise LOD scores. We monitored for shared haplotypes among affected family members within a family, and identified an approximately 4 cM region flanked by the markers D17S1792 and ATA43A10 in 17 out of the 22 families (77.3%). The multipoint linkage analyses using Genehunter and SIMWALK 2.40 provided further evidence for the same 4 cM region, for example a maximal multipoint NPL score of 5.98 (P<0.0002). We observed nominal evidence for association to MS, with one marker flanking the shared region, and this association was replicated in the additional set of families. Using the combined power of linkage, association and shared haplotype analyses, we were thus able to restrict the MS locus on chromosome 17q from 23 cM to a 4 cM region covering a physical interval of approximately 2.5 Mb. Thus, this study describes the restriction of an MS locus outside the HLA region into a segment approachable by molecular tools.

Several studies have previously provided some albeit weak evidence for linkage or association between chromosome 19q13 and multiple sclerosis (MS) susceptibility. We performed a two-stage association analysis with 19 markers spanning 7 Mb/5.5 cM of 19q13. In stage 1 analysis (135 MS families) allelic and haplotypic associations were found with markers within or close to the ApoE-ApoC subregion. These observations were taken as a hypothesis, which was tested in stage 2 in 125 families. However, none of the initial associations were replicated suggesting that they were most likely due to chance. Linkage analysis was performed in 27 Finnish multiplex families using 10 microsatellites spanning 23 Mb/24 cM of 19q13. DNA was available from 72 MS patients and 150 unaffected relatives. Parametric and non-parametric linkage analyses did not provide evidence for linkage when all families were tested. After stratifying the families according to HLA-DR15 there was weak evidence for linkage to the 19q13.1 subregion in DR15 negative families (LOD(max)=1.8). Taken together these results do not support a major role of chromosome 19q13.2-q13.3 in MS susceptibility among Finnish MS patients, whereas conclusions on the 19q13.1 subregion are less clear and this region requires further study.

We analyzed the HLA class II haplotypes in 249 Finnish nuclear families and compared the frequencies of parental haplotypes transmitted or non-transmitted to multiple sclerosis (MS) patients. The most important predisposing haplotype was DRB1*15-DQB1*0602 (P<10(-6)) as expected and a weak predisposing effect of DRB1*04-DQB1*0302 was revealed after the elimination of DRB1*15-DQB1*0602. HLA-DRB1*01-DQB1*0501 and DRB1*13-DQB1*0603 were negatively associated with MS in transmission disequilibrium test, but only the DRB1*13-DQB1*0603 association remained significant (P=0.008) after the elimination of DRB1*15-DQB1*0602 haplotypes. Based on this study HLA class II haplotypes exhibit both predisposing and protective effects in MS.

Multiple sclerosis (MS) is an oligo- or polygenic disease but no specific susceptibility genes have been identified so far. In the Finnish population we have previously found evidence for linkage between MS and the myelin basic protein gene (here called Golli-MBP gene) suggesting that either Golli-MBP or another gene in its vicinity contributes to MS suceptibility. Here we have screened the Golli-MBP gene for nucleotide variations and carried out multipoint association analyses in a Finnish case-control data-set as well as in an independent data-set composed of 151 MS families from Finland and Sweden. In both data-sets we found association between MS and alleles in the 1.27 kilobase (kb) range at a tetranucleotide repeat element (TGGA)n which is located 1 kb upstream of the MBP exon 1. Haplotype analyses suggested that the MS-associated 1.27 kb alleles can be split into predisposing and non-predisposing variants and provided evidence that the candidate DNA region contributing to MS susceptibility should be located at the Golli-MBP gene within a 20-25 kb segment that was conserved in the predisposing haplotypes. These findings suggest a role for the Golli-MBP locus in MS susceptibility, at least in a subset of patients, and serve as a basis for highly focused attempts to identify predisposing mutation(s).