1Institute of Molecular Genetics-CNR, 27100 Pavia, 2Dibit-San Raffaele Scientific Institute, 20132 Milano, Departments of 3 Biology and Genetics for Medical Sciences and 4Obstetrics and Gynecology, University of Milano, 20133 Milano, 5San Gerardo Hospital, University of Milano Bicocca, 20126 Milano, Departments of 6 Obstetrics and Gynecology and 9 Pathology and Medical Genetics, University of Pavia, 27100 Pavia, 7 Cytogenetics and Genetics Unit, Azienda Ospedaliera Careggi, 50139 Firenze and 8 Department of Gynecology and Human Reproduction, University of Firenze, Firenze, Italy
10 To whom correspondence should be addressed at: Dibit-San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy. Email: toniolo.daniela{at}hsr.it
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Abstract |
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Key words: DACH2/POF1B/premature ovarian failure/susceptibility gene
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Introduction |
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Rare mutations in a few genes responsible for autosomal recessive (Aittomaki et al., 1995) autosomal dominant (Di Pasquale et al., 2004
) or syndromic forms (Crisponi et al., 2001
; Fogli et al., 2003
) of the disease were identified. Recently, two further genes have been implicated in the POF phenotype. One is the premutation allele of the FMR1 gene at the FRAXA locus in Xq27, shown to act as a risk factor for POF with an estimated relative risk of
20 (Murray, 2000
). The other is the G769A mutation in the inhibin
(INHA) gene, which was found to be significantly associated with POF in two independent studies (Shelling et al., 2000
; Marozzi et al., 2002
). Taken together these data indicate that POF may be rarely inherited as a mendelian disorder, and more often is due to mutations in susceptibility genes contributing to POF in a multifactorial fashion.
The involvement of the X chromosome in POF arose from the frequent observation of sex chromosome anomalies in POF patients. Turner syndrome (TS), partial X chromosome monosomies and X;autosome balanced translocations have been observed in association with POF, and their description led to the cytogenetic definition of a critical region for normal ovarian function on the long arm of the X chromosome, corresponding to the Xq13.3-q26/27 interval (Therman et al., 1990). Molecular definition of the critical region by fine mapping of X;autosome balanced translocations confirmed their distribution along a large genomic region, suggesting the presence of several loci involved in the disorder (Sala et al., 1997
; Prueitt et al., 2000
; Mumm et al., 2001
). The finding of chromosomal rearrangements in normal women interspersed with those associated with POF showed that the rearrangements per se could not be responsible for the disorder, but alternative explanations, such as interruption of genes in the critical region, position effects on the expression of X-linked or autosomal genes, or increased apoptosis due to meiotic mispairing of the X chromosomes during oocyte maturation, could not be excluded.
Transcriptional characterization of breakpoint regions led to the identification of three genes interrupted by translocations, the DACH2 gene in Xq21.3 (Prueitt et al., 2002), the DIAPH2 gene in proximal Xq22 (Bione et al., 1998
) and the XPNPEP2 gene in Xq25 (Prueitt et al., 2000
). Most of the other breakpoints described in POF patients were mapped to genomic regions free of transcribed sequences (Mumm et al., 2001
; Prueitt et al., 2002
), suggesting that different genetic mechanisms may be responsible for X-linked POF. However, the role of the interrupted genes was never clarified.
Here we report the mutation analysis of two genes, DACH2 (Mumm et al., 2001; Prueitt et al., 2002
) and POF1B, a fourth gene found to be interrupted by an X;1 balanced translocation in a patient presenting secondary amenorrhea at the age of 17 years (Riva et al., 1996
). The two genes are localized 700 kb apart in Xq21, a gene-poor region where several balanced translocation breakpoints were mapped (Sala et al., 1997
; Prueitt et al., 2000
; Mumm et al., 2001
). Searching for mutations in a large cohort of >200 Italian POF patients with normal karyotype revealed rare coding variants in both genes. Analysis of the mutations found in patients in a panel of >900 selected female controls failed to show association of POF1B, but suggested that rare variants in DACH2 might be related to the POF phenotype.
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Materials and methods |
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Statistical analysis
Casecontrol association was calculated for each coding single nucleotide polymorphisms (cSNP) using a two-tailed Fisher's exact test. Differences in allele frequencies were considered significant at P<0.05
Fluorescence in-situ hybridization mapping
Fluorescence in-situ hybridization (FISH) was performed on metaphase preparations, with PAC or BAC DNA labelled by nick-translation, with biotin-16 dUTP (Roche Diagnostics Gmbh, Mannheim, Germany) as described previously (Rossi et al., 1994). Genomic clones used as probes were obtained by PCR screening of the RPCI1 and RPCI5 human PAC libraries with primer pairs corresponding to Sequence Tag Sites (STSs).
Pulsed-field gel electrophoresis analysis and Southern blot
Genomic DNA from lymphoblastoid cell lines of patient LA1 and of female and male controls was digested with restriction enzymes as suggested by the supplier (Promega, Madison, WI, USA) and fractionated by pulsed-field gel electrophoresis (PFGE) in 1.5% agarose gel, at 170 V for 20 h at 14°C with pulse intervals of 5 s. Southern blots and hybridizations were performed using standard procedures.
Reverse transcriptasePCR
Total RNA from lymphoblastoid and hybrid cell lines was extracted by standard techniques. Reverse transcription was performed from 1 µg of total RNA as described previously (Bione et al., 1998). PCRs were performed in 50 µl reactions with 0.2 mM dNTPs, 0.5 µM of each primer, 1.5 mM Mg2 + and 1.25 U Taq Polymerase (Promega) in the buffer supplied. Primer sequences and amplification condition for XIST and MIC2 (Tribioli et al., 1994
) and for POF1B, CHM and ZNF6 can be found on-line at www.sanraffaele.org/research/toniolo.
DNA methylation analysis
Genomic DNA from male and female peripheral blood leukocytes and ectopic brain samples was extracted by standard procedures and digested with restriction enzymes as per supplier's instructions (Promega). Undigested samples were incubated in the same buffer and conditions but without enzyme. Digested and undigested DNAs were purified by ethanol precipitation and amplified with primers DACH-12F and DACH-20R in the same conditions used for denaturing high-performance liquid chromatography (DHPLC).
DHPLC mutation analysis
PCR fragments corresponding to all exons of the POF1B and DACH2 genes were amplified from primers pairs listed in Supplementary data (www.sanraffaele.org/research/toniolo). All PCR amplifications were carried out from 50 ng of genomic DNA in 50 µl volume with 0.2 mM dNTPs, 0.5 µM of each primer, 1.5 mM Mg2 + and 1.25 U Taq Polymerase (Promega) for 40 cycles. Cycles were for 30 s at 94°C, 30 s at suitable annealing temperature (see Supplementary data) and 30 s at 72°C, with 7 min of final elongation at 72°C. DHPLC analysis was performed on an automated HPLC (WAVE system; Transgenomic, Omaha, NE, USA). PCR products were subjected to denaturation and reannealing from 95°C to 65°C for 30 min prior to DHPLC analysis. Fragments were fractionated on a linear gradient of acetonitrile at a flow rate of 0.9 ml/min (see Supplementary data for temperature and gradient conditions (www.sanraffaele.org/research/toniolo).
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Results |
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The DACH2 gene is inactivated
The pattern of inactivation of the DACH2 gene, localized 700 kb distal from POF1B and interrupted by a POF-associated breakpoint (Prueitt et al., 2002), was also investigated.
Since the DACH2 gene is not expressed in lymphoblastoid cell lines, its inactivation status was established by analysis of the methylation pattern of a CpG island at its 5' end (Figure 2C). Genomic DNA extracted from male and female peripheral blood leukocytes was digested with the restriction enzymes HpaII and BssHII, sensitive to DNA methylation. A fragment of 378 bp surrounding the start codon of the gene and containing two BssHII and five HpaII recognition sites was amplified from digested and non-digested DNAs. A PCR product was detected from undigested as well as from digested female genomic DNA, whereas it was not detectable in male DNA digested with either enzymes (Figure 2C). To verify that methylation of the DACH2 CpG island was not due to its lack of expression in white blood cells, we performed HpaII restriction analysis in DNA extracted from female brain samples in which the gene was expressed (not shown). The presence of a PCR product from amplification of HpaII-digested female DNA confirmed the inactivation status (Figure 2C).
Mutation analysis of the POF1B gene
The contribution of the POF1B to the aetiology of POF was evaluated by mutation analysis. In all patients, we analysed by DHPLC exons and splice junctions aiming at identification of causative mutations in coding regions and in splice junctions. Synonymous mutations and intronic variants of undetermined role were not considered. The 17 exons of the POF1B gene were analysed in 223 POF patients presenting primary amenorrhea (n=16) secondary amenorrhea (n=183) or EM (n=24), and revealed the presence of 30 SNPs. Fifty-two patients of the collection were not analysed as they were not available when the POF1B analysis was carried out, but they could not change the results obtained with the first group of 223. Five variants were found in the coding portion of the gene (cSNPs), and in all cases the nucleotide replacement resulted in a missense mutation of the POF1B protein. The T1191A/L349M substitution found in exon 10 is a common variant, with a frequency of the minor allele of 0.24. The genotypes at this locus were determined in 95 patients and in 95 controls, and did not show differences between cases and controls, excluding its association with the disease (data not shown). The remaining four variants (C239S, R329Q, Q434K and C444Y) were found in a total of six patients with menopause ranging in age from 22 to 40 years (Table I). The presence of these variants was assessed in a panel of 900 controls. The R329Q and the Q434K variants were found three times and twice, respectively, in the control group, whereas the C239S variant was found more frequently, as it appeared 18 times in the 900 control DNAs. The remaining 210 controls were not analysed as they were not enough to change the statistical significance of results. The C444Y variant was found twice among patients and was not found in controls. By itself, it showed a significant association to the disease (P=0.0393), but when corrections for multiple testing were introduced, the association was lost (P=0.157). The remaining three variants were not associated.
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Discussion |
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POF1B, a gene of unknown function, was found interrupted by the breakpoint of a de-novo X;1 balanced translocation in a patient presenting secondary amenorrhea at the age of 17 years (Riva et al., 1996). The POF1B gene is a good candidate for POF and for TS-associated ovarian dysgenesis, as it escapes X inactivation and has no Y homologue. POF1B maps to the long arm of the X chromosome, the region that first diverged between the X and the Y, between 200 and 300 million years ago (Lahn and Page, 1999
): conservation of two active copies of the gene for this long evolutionary time is not common, and strengthens the hypothesis that a double dose of POF1B in females is functionally relevant. The pattern of expression of Pof1b in the ovary was studied in the mouse (not shown), where it was absent from adult ovary but was found between days E16.5 and P5, suggesting that the gene may be critical for one of the steps required for early ovary development leading to the final number of germ cells.
To clarify the contribution of POF1B in the aetiology of the disorder, we performed a casecontrol study on >200 POF patients from Northern and Central Italy and >900 geographically matched controls, selected among women who had entered menopause at the physiological age. In all patients we analysed exons and splice junctions, with the aim of identifying causative mutations in coding regions and in splice junctions. Rare variants affecting amino acid composition were found, but the genetic analysis failed to demonstrate association. Thus, mutations in POF1B are not involved in POF or their contribution cannot be demonstrated using the very heterogeneous group of patients available.
The DACH2 gene was subjected to X inactivation, as were the two genes previously found interrupted in POF patients, DIAPH2 and the XPNPEP2 (Bione et al., 1998; Rizzolio and Toniolo, unpublished results). These genes may be required in double dose throughout the life of the oocytes, when the presence of two active X chromosomes is a rule. Missense mutations were found in DACH2; they were in highly conserved residues and in most cases (P36L, R37L, F316S) they caused non-conservative amino acid substitutions. In Drosophila, dac is a transcriptional cofactor known to participate in a nuclear complex consisting of sine oculis (so), eyes absent (eya) and dachshund (dac) (Chen et al., 1997
; Heanue et al., 1999
). Highly conserved vertebrate homologues have been identified that are coexpressed in multiple organs including eye, inner ear, pituitary gland, muscle and kidney, and seem to act synergically during organogenesis (Davis et al., 2001
; Li et al., 2002
; 2003
). The Drosophila eya gene was reported to have an essential role in fruit fly oogenesis for the correct differentiation of somatic follicle cells into polar cells (Bai and Montell, 2002
). No evidence of the involvement of dac was reported, but the high level of functional conservation lead us to suggest that a complex involving DACH2 and one of the mammalian EYA and SIX genes may be important for development of mammalian gonads, and that alteration of the human DACH2 protein may act as a risk-factor for POF by altering the correct process of ovarian follicle differentiation.
The missense mutations found in patients in the DACH2 gene were more frequent in patients (2.7%) compared with the control group (0.7%). The difference was statistically sign0ificant (two-sided Fisher's exact test: P=0.0125). Replication in a similarly large independent population may definitively confirm our data. Since none of the mutations by itself was significantly enriched in the patients, our results suggest that a number of different private mutations that have arisen recently in DACH2 may be involved in POF. Reduced fitness due to low fertility may well explain this result, as well as the observed incomplete penetrance of some of the mutations found. Both factors could be responsible for the genetic heterogeneity of the disorder. In conclusion, while DACH2 mutations may be too rare to be useful in diagnostic for POF, they should be considered in the analysis with other risk factors such as FRAXA premutation, or the many that are expected but as yet unidentified.
The DACH2 gene is localized 700 kb distal from POF1B, and several X breakpoints in POF patients were mapped between the two genes. It is thus possible that the X chromosome breakpoints outside of the gene may exert a position effect on the expression of DACH2 gene. We did not test directly the CHM gene, but the data available tentatively exclude its involvement in POF (van den Hurk et al., 1997). Mutations in DACH2 were found in patients entering amenorrhea at very variable ages, ranging from 17 to 42 years. The phenotype of the patients carrying balanced translocations was more severe, as most of them presented with primary amenorrhea or with secondary amenorrhea at a very young age and after few and very irregular cycles. Based on this observation, we propose that POF associated with balanced translocation in the Xq critical region may be caused by a position effect of the chromosomal rearrangements on X-linked as well as autosomal genes. They may possibly also alter the expression of POF1B. Further characterization of the genomic region and of the genes involved and the analysis of a larger group of patients may definitively demonstrate this point, but the data presented here begin to shed new light into the very complex role of the genes in the critical region for ovarian failure.
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Acknowledgements |
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