Molecular cytogenetic analysis of Xq critical regions in premature ovarian failure

Abstract

Background: One of the frequent reasons for unsuccessful conception is premature ovarian failure/primary ovarian insufficiency (POF/POI) that is defined as the loss of functional follicles below the age of 40 years. Among the genetic causes the most common one involves the X chromosome, as in Turner syndrome, partial X deletion and X-autosome translocations. Here we report a case of a 27-year-old female patient referred to genetic counselling because of premature ovarian failure. The aim of this case study to perform molecular genetic and cytogenetic analyses in order to identify the exact genetic background of the pathogenic phenotype.

Results: For premature ovarian failure disease diagnostics we performed the Fragile mental retardation 1 gene analysis using Southern blot technique and Repeat Primed PCR in order to identify the relationship between the Fragile mental retardation 1 gene premutation status and the premature ovarion failure disease. At this early onset, the premature ovarian failure affected patient we detected one normal allele of Fragile mental retardation 1 gene and we couldn't verify the methylated allele, therefore we performed the cytogenetic analyses using G-banding and fluorescent in situ hybridization methods and a high resolution molecular cytogenetic method, the array comparative genomic hybridization technique. For this patient applying the G-banding, we identified a large deletion on the X chromosome at the critical region (ChrX q21.31-q28) which is associated with the premature ovarian failure phenotype. In order to detect the exact breakpoints, we used a special cytogenetic array ISCA plus CGH array and we verified a 67.355 Mb size loss at the critical region which include total 795 genes.

Conclusions: We conclude for this case study that the karyotyping is definitely helpful in the evaluation of premature ovarian failure patients, to identify the non submicroscopic chromosomal rearrangement, and using the array CGH technique we can contribute to the most efficient detection and mapping of exact deletion breakpoints of the deleted Xq region.

Figure 1

G-banding analysis. The karyotype of the patient with Xq21-q28 deletion of the dominant cell line.

Figure 2

FISH analysis. For FISH analysis using chromosome X centromere specific probe (CEP X) which shows normal female pattern (two green signals) in 90% of cells and X monosomy (one green signal) in 10% of interphase cells (a). The whole painting chromosome X (WC X) identified a normal and a smaller size red colored X chromosome and excluded the possible X chromosome translocation (b).

Figure 3

Picture of Southern blot analysis. EcoRI and EagI double digested DNA samples using radioactive-labelled Stb12.3 probe for Southern blot hybridization. Arrows indicated the 2.8 Kb unmethylated and the 5.2 Kb methylated fragments size. For the case sample we can define the missing 5.2 Kb methylated fragment (circle compassed). POF 283/4, POF 285/4 and POF 287/4 cases indicated those female samples who did not carry FMR1 gene premutation and the background of the POF phenotype should be withstand other genetic deviation. For all these cases we identified two X chromosome normal allels (2,8 kb unmethylated and 5.2 kb methylated).

Figure 4

Picture of repeat primed PCR analysis. Repeat-primed PCR analysis revealed a peak, which corresponds to a 23-CGG with only one AGG interruption.

Figure 5

NimbleGen ISCA plus CGX design profile for X chromosome. a.) The ideogram (below: black, grey and white bars) delineates genomic regions with the cytogenetic bands on the X chromosome. An 67.355 Mb sized loss on chromosome Xq21.3-q28 for the female patient and a black rectangle indicate the length of the loss. b.) Array-CGH workflow. The CGX ISCA plus array showed a 67.355-Mb loss which presented one copy. The 67.355 Mb deleted chromosome segment (GRCh37/hg19; ChrX: 87842016–155255380) is denoted by a bar red line below zero. The blue and yellow dots depict the normalized ration on every probe on the X chromosome. c.) Schematic representation of the chrXq21.3-q28 enlarged region. d.) Next the ideogram listed genes and positions which are affected this patient. This affected region contains 1818 genes and we visualized some of them. We signed with bold font those genes, which can x play a roll to induce the POF/POI phenotype.