Short stature and primary ovarian insufficiency possibly due to chromosomal position effect in a balanced X;1 translocation

Abstract

Background: Primary ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea), a decrease in the initial primordial follicle number, high follicle-stimulating hormone (FSH) levels and hypoestrogenism. Although the etiology of a majority of POI cases is not yet identified, several data suggest that POI has a strong genetic component. Conventional cytogenetic and molecular analyses have identified regions of the X chromosome that are associated with ovarian function, as well as POI candidate genes, such as FMR1 and DIAPH2. Here we describe a 10.5-year-old girl presenting with high FSH and luteinizing hormone (LH) levels, pathologic GH stimulation arginine and clonidine tests, short stature, pterygium, ovarian dysgenesis, hirsutism and POI.

Results: Cytogenetic analysis demonstrated a balanced reciprocal translocation between the q arms of chromosomes X and 1, with breakpoints falling in Xq21 and 1q41 bands. Molecular studies did not unravel any chromosome microdeletion/microduplication, and no XIST-mediated inactivation was found on the derivative chromosome 1. Interestingly, through immunofluorescence assays, we found that part of the Xq21q22 trait, translocated to chromosome 1q41, was late replicating and therefore possibly inactivated in 30 % metaphases both in lymphocytes and skin fibroblasts, in addition to a skewed 100 % inactivation of the normal X chromosome. These findings suggest that a dysregulation of gene expression might occur in this region. Two genes mapping to the Xq translocated region, namely DIAPH2 and FMR1, were found overexpressed if compared with controls.

Conclusions: We report a case in which gonadal dysgenesis and POI are associated with over-expression of DIAPH2 gene and of FMR1 gene in wild type form. We hypothesize that this over-expression is possibly due to a phenomenon known as "chromosomal position effect", which accounts for gene expression variations depending on their localization within the nucleus. For the same effect a double mosaic inactivation of genes mapping to the Xq21-q22 region, demonstrated by immunofluorescence assays, may be the cause of a functional Xq partial monosomy leading to most Turner traits of the proband's phenotype.

Keywords: Chromosomal position effect; DIAPH2; FMR1; Primary ovarian insufficiency; Turner syndrome; X chromosome translocation; X;autosome translocation.

Fig. 1

Patient G banded karyotype. Karyotype at 550 band resolution shows a reciprocal translocation between 1 and X chromosomes (blue arrows): t(X;1)(q21;q41)

Fig. 2

Multicolor Banding assay. G-Banding images, high resolution multicolor banding (MCB) and ideograms of the derivative 1 and derivative X chromosomes compared with the normal X chromosome. The region Xq21-qter translocated downstream the region 1q41 is shown

Fig. 3

Late replication assay. Fluorescent BrdU assay, combined with WCP1 probe FISH, shows that the normal X chromosome was late replicating (yellow arrow). The late replication also interested the region Xq21-q22 translocated to der(1) (white arrow). Red arrow points to der(X)

Fig. 4

FISH analysis of XIST. FISH analysis using XIST probe shows the localization of the XIST gene on normal X chromosome (red arrow) and on the derivative X chromosome (yellow arrow)

Fig. 5

Fluorescent 5-methylcytosine (5-Mc) assay. 5-Mc antibody, combined with WCP1 probe FISH, shows that the X region involved in translocation was heterocromatized (white arrow)

Fig. 6

Nuclear positioning assay. 6A. Interphase Multicolor banding shows the normal X chromosome (yellow arrow) and the Xq21-q22 region on der(1) (white arrow) located peripherally in the observed nucleus, whereas der(X) is located centrally in the nucleus (blue arrow). 6B. Dual FISH using whole chromosome painting probes for chromosome 1 (green) and for chromosome X (red) shows the normal X chromosome (yellow arrow) and the Xq21-q22 region on der(1) (white arrow) located peripherally in the nucleus, whereas der(X) is located centrally in the nucleus

Fig. 7

Gene expression analysis. qRT-PCR in proband versus control blood shows upregulation of DIAPH2 gene (2,05) and of FMR1 gene (1,62). Values represent the average determination ± SEM for 2 experiments carried out in triplicate. A pool of RNA from 3 healthy girls, matching for age was used as calibrator