Molecular cytogenetic analysis of Xq critical regions in premature ovarian failure
© Beke et al.; licensee BioMed Central Ltd. 2013
Received: 15 October 2013
Accepted: 3 December 2013
Published: 20 December 2013
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.
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.
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.
KeywordsSterility Premature premature ovarian failure (POF) Primary ovarian insufficiency (POI) FMR1 gene analysis Array–comparative genomic hybridization (aCGH) X chromosome deletion Repeat primed PCR G-banding Deletion breakpoint Turner syndrome
Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarion follicles befor the age of 40 years, and its aetiology is still unknown in most cases. Coulon et al. examined 1858 patients with premature ovarian failure, and the age-specific incidence was based on the results: under 40 years of age the incidence was 1:100, and under 30 years of age the incidence was 1:1000 . In addition to a reduction in the duration of fertility, there are other important health issues that may be associated with POF such as an increased risk of overall mortality, cardiovascular diseases, osteoporosis and autoimmune disorders such as diabetes or problems with the thyroid or adrenals . In particular, an association between POF and abnormalities of the X chromosome has been reported several times, and another cause, which can be associated with POF, is the balanced X/autosomal translocations despite their generally neutral clinical effect . In fact, the breakpoints of these aberrations are distributed over the whole X chromosome but in many cases they cluster in a critical region between Xq13 and Xq26 [4–7]. Deletion on the X chromosome reduces both fertility and reproductive lifespan and the basis of studies two loci for Xq-linked POF have been postulated: deletion in POF patients have been localised to chromosome Xq21.3-Xq27 (POF1), while balanced X/autosome translocations have been localised to Xq13.3-Xq21.1 (POF2) . The above-mentioned POF disorder has been attributed to various causes including rearrangements or large deletion of the “critical region” in the long arm of the X chromosome. However, it is interesting that there are several genes which have consistently found to be involved in POF on the X chromosome and autosomes but the POF1 region deletions are by far more commonly associated with POF phenotype. Another abundant reason for the POF is the single gene association that can also be involved in the POF phenotype. Out of the single genes group, the FMR1 gene appears to be the most significant cause of POF disease. The association between FMR1 premutation and POF has been previously investigated at a molecular level by analysing FMR1- related factors such as the repeat tract size. Expansion of (CGG) triplet repeats in the FMR1 gene is associated with several disorders, including fragile X syndrome (FRAXA), fragile X-associated tremor/ataxia syndrome (FXTAS), and fragile X-associated primary ovarian insufficiency (POF). Fragile X syndrome is nearly always characterized by moderate mental retardation in affected males, with full mutation and FXTAS occuring in males who have an FMR1 premutation, and is characterized by late onset progressive cerebellar ataxia and intention tremor. FMR1-related (with the premutation alleles, which may have 55 to 200 CGG) POF (age at cessation of menses <40 years) occurs in approximately 20% of females who have an FMR1 premutation . Since full mutation carriers do not have an increased risk for ovarian dysfunction, the molecular mechanism underlying the association between POF and FMR1 gene premutation alleles, although still unravelled, should not be related to the absence or reduction of the fragile mental retardation protein (FMRP) . The known association between POF and premutation alleles in the FMR1 gene  prompted us to initiate fragile X testing. In this case study, we performed a detailed FMR1 gene CGG repeat number assay and cytogenetic analysis such as FISH, G-banding and molecular cytogenetic analysis, which are based on the array CGH technique. The aim of this case study was to perform molecular genetic and cytogenetic analyses in order to identify the exact genetic background of the pathogenic phenotype.
Genetic testing of the 27 year old female patient was carried out due to suspected premature ovarian failure/primary ovarian insufficiency (POF/POI). Her menses ceased at the age of 25. The first menses (menarche) started at the age of 12. There is no pregnancy or assisted reproductive procedure appearing in her history. Other illnesses, genetic disorders, mental retardation have never occurred in the family. The menopause of her mother occurred at the age of 53. The patient’s body type is average, BMI: 19.4 (average: 18.5 to 24.99). During the examination the disease met the criteria for premature ovarian failure/primary ovarian insufficiency (POF/POI): secondary amenorrhea, ovarian failure before the age of 40, levels of FSH > 40 IU/l in two different measurements and low estrogen levels. The patient has never had surgery significantly affecting both ovaries; ovarian toxic medications have never been used (cytostatic treatment). The patient’s mother has normal karyotype and her father was already deceased. For this case study we presented the details of the molecular and cytogenetic analyses at the index patient which were performed by G-banding; FISH, Southern-blotting, Repeat Primed PCR and array-CGH technique.
Chromosome and fluorescence in situ hybridization analysis
Chromosome analysis was performed on stimulated peripheral blood cultures on metaphase cells with trypsin and Wright Giemsa stain. Fluorescence in Situ Hybridization (FISH) analysis was carried out on methanol/acetic acid-fixed suspensions. Slide preparation for FISH was made according to standard techniques. X, Y centromere specific probes as well as X Painting probe (Cytocell, United Kingdom) were used for evaluation of sex chromosomes and their possible hidden structural abnormalities. Spectrum Green CEP X and Spectrum Red SRY gene specific probe (Abbott, Germany) were used to detect chromosome X copy number and to control the presence of the SRY gene on the chromosome, respectively. Karyotypes and FISH results were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2013).
Genomic DNA from the patient was isolated from peripheral lymphocytes by the simple salting-out procedure. DNA was subjected to restriction enzyme digestion with Eco RI and the methylation sensitive enzyme Eag I followed by Southern blot analysis and hybridization using the DNA probe StB12.3 . In unaffected females, two bands are visible: a 2.8 Kb fragment corresponding to the unmethylated X and a 5.2 kb allele representing the methylated X chromosome.
Repeat primed PCR
The exact number of CGG repeats was determined by Repeat Primed PCR technology (Amplidex, Asuragene). The genomic DNA sample was diluted (20 ng/μl), and then for PCR was used 2 μl. The PCR reaction was carried out in three primers: the FMR1 gene-specific primers (forward and reverse, FAM-labeled) and a CGG repeat specific primer. The PCR products were separated by size, using capillary electrophoresis. The statistical analyzes were performed with FMR1AnalysisMacro_version 2.1.1 software and GeneMapper software.
Array CGH analysis
Array CGH analysis was performed according to the manufacturer’s protocol on genomic DNA ISCA plus design array of Nimblegen Roche containing 1.4 M probes per sub array. This microarray provides a mean average resolution of approximately 15–20 Kb on chromosomes to detect chromosomal imbalances throughout the whole genome. The CGH protocol involves independent labelling of the patient (test DNA) and the reference genomic DNA (Human Genomic DNA, Promega Madison, WI U.S.A.) with Cy3 and Cy5 dyes using a NimbleGen Dual-Color DNA Labelling Kit (Roche NimbleGen Inc.). Cohybridization of these DNAs to a NimbleGen CGH arrays were performed for 72 hrs at 42˚C. Following hybridisation, the array was washed and dried at room temperature using the wash buffer kit (Roche NimbleGen Inc.). Array CGH was scanned on NimbleGen MS 200 microarray scanner and data was extracted and analysed using NimbleScan software and SignalMap and Deva 1.1 software (Roche NimbleGen Inc.). DNA CNVs were mentioned as gain or loss as a linear ration, and the length of the variation was given in megabase (Mb).
POF associated genes at the critical region on chromosome X which are affected at patient
Premature ovarian failure1B
Premature ovarian failure 2B
This gene is expressed at trace levels in mouse prenatal ovary and is barely detectable or absent from adult ovary, in human and in the mouse respectively.
Basic helix-loop-helix domain-containing class B 9
Premature ovarian failure
Other members of this gene family encode proteins which function as transcription factors, either enhancing or inhibiting transcription depending on the activity of the DNA binding proteins.
Premature ovarian failure
This gene is one of two genes which encode a protein similar to the Drosophila protein dachshund, a transcription factor involved in cell fate determination in the eye, limb and genital disc of the fly.
Premature ovarian failure
The product of this gene belongs to the diaphanous subfamily of the formin homology family of proteins. This gene may play a role in the development and normal function of the ovaries.
Centromeric protein 1
Involved in the gonadal tissue response to FSh and assembly of the kinetochore
It has a critical role in chromosome segregation and with deletions candidate for human X-lined disorders of gonadal development and gametogenesis.
Progesteronereceptor memebrane component-1
Premature ovarian failure
This gene encodes a putative membrane-associated progesterone steroid receptor. The protein is expressed predominatly in the liver and kidney.
BCL6 Corepressor-like 1
BCORL1 interacted with class II histon deacetylases suggesting that they are involved in its function as a corepressor
Deletion of BCORL 1 gene may potentially lead to insufficient repressor of apoptosis resulting in atresia of ovarian follicles.
Angioedema induced by ACE inhibitors, susceptibility to
XPNPEP2, the X-linked gene that encodes membranous aminopeptidase P (APP), has been reported to associate with APP activity. The membrane aminopeptidase P (XPNPEP2) is largely limited in distribution to endothelia and brush border epithelia.
Fragile X mental retardatin 1
Fragile X syndrome Fragile X tremore/ataxia syndrome Premature ovarian failure 1
The protein encoded by this gene binds RNA and is associated with polysomes. The encoded protein may be involved in mRNA trafficking from the nucleus to the cytoplasm.
Fragile X mental retardation 2
Mentar retardation, X-linked, FRAXE type
This gene encodes a putative transcriptional activator that is a member of the AF4\FMR2 gene family.
For the index patient as we mentioned before using the Southern blot assay, we detected a normal and active X chromosome and the absence of the methylated FMR1 allele, according to Lyon hypotheses, should be inactivated in 50% of cells . As scientific studies described, the haploinsuffiency of the genes, which are located in the missing region on one of the chromosome X, could be a promising explanation for the POF disease background, especially when it involves Xq28. A haploinsufficient gene is described as needing both alleles to be functional in order to express the wild type. The lack of expression of those missing genes that normally escape X inactivation may threaten ovarian function . Based on the scientific studies we can conclude that with this POF, the affected patient may be configured by abnormal X chromosome pairing and the epigenetic modification . The epigenetic effects would be supportable if the patient had a POF affected relative carrying the same deletion. In this case we confirmed a “de novo” X-chromosome deletion, so we cannot make any phenotype comparison. We conclude that cytogenetic analysis might be the first step in the investigation of POF/POI, as it might make some subsequent analysis steps unneccessary (i.e. Southern blot and PCR).
Here we present a patient affected with POF disease, where molecular and cytogenetic analyses revealed that she was a carrier of a large deletion spanning from Xq21.31-q28. Sample DNA of the POF phenotype affected women were collected so as to identify the exact molecular genetics background. As the scientific studies suggested, the most frequent gene mutation that can cause the POF phenotype, is the CGG triplet repeat number increasing which denoted that these females are the permutation status carrier. For the index patient we confirmed a large deletion on one of the X chromosomes and we couldn’t detect premutation status at the FMR1 gene. In order to identify the exact breakpoints of the X chromosome deletion, we made the array CGH analyses. As we verified the deletion on the database, we realised that this deletion region consisted of 795 genes, and 10 of them were considered as POF associated genes. Comparing the phenotype and the molecular genetic results, we concluded that for this patient, we established the POF1 disease and offering the prenatal diagnostic at possible future pregnancies is crucial.
Written informed consent was obtained from the patient for publication of this case report and accompanying images.
BCL 6 corepressor-like-1
Basic helix-loop-helix domain-containing class B
Centromeric protein 1
Comparative genomic hybridisation
Copy number variation
Drosophila dashsund homolog 2
Drosophila diaphanous homolog 2
Fluorescens in situ hybridisation
Fragile mental retardation 1 gene
Fragile X mental retardation 2
Fragile mental retardation protein
Fragile X syndrome
Follicle stimulating hormone
Fragile X-associated themor ataxia syndrome
Progesterone receptor membrane component-1
Premature ovarian failure
Premature ovarian failure 1B
Propyl aminopeptidase gene.
The authors wish to thank the patient with POF disease for providing her DNA sample and personal data. Written informed consent was obtained from the patient. The authors are gratefull to Éva Gönczi, Margit Czimbalmos, Eszter Kis, Tóth Zsuzsa, Linda Gyurcsó-Deák, Krisztina Császár, Ágota Szepesi for their valuable technical help.
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