Y染色体微缺失检测

Genomic analysis using high-resolution

single-nucleotide polymorphism arrays reveals novel microdeletions associated with premature ovarian failure

Megan M.McGuire,B.S.,a Wayne Bowden,B.S.,b Natalie J.Engel,B.S.,a Hyo Won Ahn,M.S.,a

Ertug Kovanci,M.D.,b and Aleksandar Rajkovic,M.D.,Ph.D.a

a Department of Obstetrics,Gynecology,and Reproductive Sciences,Magee-Womens Research Institute,University of Pittsburgh,Pittsburgh,Pennsylvania;and

b Department of Obstetrics and Gynecology,Baylor College of Medicine,Houston, Texas

Objective:To analyze DNA from women with premature ovarian failure(POF)for genome-wide copy-number variations(CNVs),focusing on novel autosomal microdeletions.

Design:Case-control genetic association study.

Setting:Department of Obstetrics and Gynecology,Baylor College of Medicine,Houston,Texas.

Patient(s):Of89POF patients,eight experienced primary amenorrhea and81exhibited secondary amenorrhea before age40years.

Intervention(s):Genomic DNA from peripheral blood samples was analyzed for CNVs using high-resolution single-nucleotide polymorphism(SNP)arrays.

Main Outcome Measure(s):Identi?cation of novel CNVs in89POF cases,using the Database of Genomic Variants as a control population.

Result(s):A total of198autosomal CNVs were detected by SNP arrays,ranging in size from0.1Mb to3.4Mb. These CNVs(>0.1Mb)included17novel microduplications and seven novel microdeletions,six of which contained the coding regions8q24.13,10p15-p14,10q23.31,10q26.3,15q25.2,and18q21.32.Most of the novel CNVs were derived from autosomes rather than the X chromosome.

Conclusion(s):The present pilot study revealed novel microdeletions/microduplications in women with POF.Two novel microdeletions caused haploinsuf?ciency for SYCE1and CPEB1,genes known to cause ovarian failure in knockout mouse models.Chromosomal microarrays may be a useful adjunct to conventional karyotyping when evaluating genomic imbalances in women with POF.(Fertil Sterilò2011;95:1595–600.ó2011by American Society for Reproductive Medicine.)

Key Words:Premature ovarian failure,POF,copy number,CNV,microdeletion and microduplication,SNP microarray,POI,array CGH,chromosomal microarray

Menopause,or the permanent cessation of menses,naturally occurs in most American women between46and54years of age(1). Approximately1%–5%of women in the world experience cessation of their menstrual cycle before age40(2),a condition known as premature ovarian failure(POF)or premature ovarian insuf?ciency. POF encompasses a broad spectrum of individuals,ranging from women with primary amenorrhea to women who have reproduced but experience loss of menstrual cycles before age40,coupled with menopausal serum FSH levels(3,4).Unlike menopause, however,this condition does not always result in permanent loss of ovarian function,as approximately5%–10%of women are able to conceive after the onset of POF(5).

Most POF cases are classi?ed as idiopathic,and these cases are further divided into sporadic and familial forms.Genetics is known to contribute signi?cantly to idiopathic POF cases(6),and $10%–15%of diagnosed women have an affected?rst-degree rel-ative(5).Initial genetic interest has focused on the X chromosome owing to Turner syndrome as well as published associations of POF cases with X chromosome deletions and translocations (7–10).Genes speci?cally expressed from the X chromosome that have been implicated in POF include FMR1(premutation,fragile X syndrome),BMP15,and POF1B(11–14).

In addition to X chromosome abnormalities,POF has been asso-ciated with autosomal gene aberrations.FSHR mutations are associ-ated with POF in Finnish women,although such mutations are rare outside of Finland(15).Other autosomal genes that have been more widely associated with POF include GDF9,FIGLA,FOXO1a, FOXO3a,and NOBOX(13,16–18).

Current genetic guidelines in the evaluation of POF cases recom-mend karyotyping and FMR1premutation carrier screening.Con-ventional karyotyping has been a mainstay to determine genomic imbalances in POF individuals;however,it cannot detect genomic imbalances<3–5Mb in size(microdeletions and microduplica-tions,also known as copy-number variations[CNVs])owing to

Received September9,2010;revised November12,2010;accepted

December22,2010;published online January22,2011.

M.M.M.has nothing to disclose.W.B.has nothing to disclose.N.J.E.has

nothing to disclose.H.W.A.has nothing to disclose.E.K.has nothing to

disclose.A.R.has nothing to disclose.

Supported by National Institutes of Health grant no.R21HD058125.

Reprint requests:Aleksandar Rajkovic,M.D.,Ph.D.,Magee-Womens

Research Institute,204Craft Avenue,Pittsburgh,PA15213(E-mail:

rajkovic@https://www.wendangku.net/doc/5b12423019.html,).

0015-0282/$36.00Fertility and SterilityaVol.95,No.5,April20111595 doi:10.1016/j.fertnstert.2010.12.052Copyrighta2011American Society for Reproductive Medicine,Published by Elsevier Inc.

banding resolution.Recent studies using oligonucleotide and single-nucleotide polymorphism(SNP)microarray technologies have shown that genomic imbalances involving as few as1,000base pairs can be detected(19).It is possible that some of the idiopathic POF cases are due to CNVs.Therefore,we used a SNP based array to determine if women with POF exhibit novel CNVs. MATERIALS AND METHODS

Participant Population,Blood Collection,and Genomic DNA Isolation

The study was composed of89women with POF:eight women who ex-hibited primary amenorrhea and81who exhibited secondary amenorrhea for>6months before the age of40years,with FSH serum levels>40IU/L. Women with a history of pelvic surgery,cancer,radiation exposure,smoking, and genetic syndromes were excluded from the study.Peripheral blood sam-ples were collected from all of the women,and genomic DNA(gDNA)was extracted from5mL whole blood(Puregene protocol;Qiagen,Valencia, CA),as previously described(20).One of the eight women with primary amenorrhea failed genotyping quality control and was excluded from analy-sis.Therefore,a total of88women were available for analysis.This study was approved by the Institutional Review Board at Baylor College of Medicine.

Single-Nucleotide Polymorphism Arrays and Data Analysis Illumina’s(San Diego,CA)HumanCNV370-Duo DNA Analysis BeadChip with>318,000tag SNP markers and52,000markers targeting additional CNV regions(5.5kb median marker spacing)was used to examine CNVs in gDNA for38individuals on the BeadArray500GX.Illumina’s Human660W-Quad v1DNA Analysis BeadChip with550,000tag SNPs and an additional100,000markers targeting common CNV regions(2.5kb median marker spacing)was used for51individuals on the iScan System. The array data were analyzed using Illumina’s GenomeStudio Genotyping Module software.A call rate of>99%was accepted as the validity cutoff for each sample on both chips,which indicates that>99%of the probes success-fully identi?ed a https://www.wendangku.net/doc/5b12423019.html,ing the GenomeStudio software,the log of the signal intensity(log R ratio)highlighted CNVs as an increase or decrease from the baseline intensity which was standardized to zero.The B-allele frequency,standardized at0.5,was coupled with the log R ratio values to examine areas of DNA loss/gain.The concurrent use of these two tools enabled CNVs to be differentiated from regions of copy-neutral loss of heterozygosity.

All CNVs were screened against polymorphisms noted in the Database of Genomic Variants(DGV;http://projects.tcag.ca/variation);those already listed with a high population frequency in the DGV were regarded as benign polymorphisms and subsequently excluded from further analyses.Quantitative Real-Time Polymerase Chain Reaction Quantitative real-time polymerase chain reaction(qPCR)was used to corrob-orate SNP array?ndings(20).Emphasis was placed on unique microdele-tions that were not present in the DGV database.Where possible, a relevant protein-coding gene within each deleted region was chosen as a tar-get,and oligonucleotides were designed for qPCR analysis(Table1).Primers were custom-designed through Primer3Plus(https://www.wendangku.net/doc/5b12423019.html,/ primer3).

Quantitative real-time PCR was conducted by using100ng gDNA,0.4 m mol/L forward and reverse primers,1?iQTM SYBR Green Supermix (Bio-Rad,Hercules,CA),and nuclease-free water,with a total volume of 20m L per well.Five replicates were run for each POF individual for the tar-geted gene within each respective deleted region.A pooled human gDNA sample from?ve women was used as a control(cat.no.G1521;Promega). Threshold cycle(Ct)values were calculated and averaged for each set of ?ve replicates;these values were then used to calculate the standard error of the mean.The2àDD Ct method of quanti?cation was used to analyze fold changes in copy number between control(2n)and deleted regions (1n or absent).

RESULTS

We analyzed the genomic DNA of89women with POF by using high-resolution SNP arrays to determine the prevalence of novel CNVs(microdeletions/microduplications)which would not be de-tected by conventional karyotype methods.Eighty-eight samples were successfully arrayed.Microarray data revealed a total of50 microduplications and148microdeletions on autosomal chromo-somes,ranging in size from0.1Mb to3.4Mb.These CNVs occurred in72out of88genotyped POF individuals,and they spanned every autosome.To distinguish novel CNVs from seemingly benign non-pathogenic imbalances,we compared our data to the Database of Genomic Variants.The DGV is a compilation of common CNVs de-tected in more than10,000healthy individuals,and it has been used as a control population in several studies(21–23).We found33of the50microduplications and141of the148microdeletions in the control population of the DGV.Therefore,24autosomal CNVs in a total of19women were considered to be novel.There were seven novel autosomal microdeletions(Table2),one in each of seven different women,and17novel autosomal microduplications (Table3),one in each of11women and two in each of three women.

We speci?cally focused on microdeletions because genomic los-ses are more likely to result in a clinical phenotype than genomic gains(24).Seven women with POF exhibited novel hemizygous mi-crodeletions>0.1Mb in size across the autosomes.Six of the novel autosomal microdeletions contained coding regions,and?ve of the

1596McGuire et al.Genomic imbalances in ovarian failure Vol.95,No.5,April2011

deletions were subsequently veri?ed by qPCR analysis:8q24.13, 10p15-p14,10q26.3,15q25.2,and18q21.32(Table2).Control am-pli?cations for regions of no change in copy number did not show detectable Ct shifts.A2àDD Ct(fold change)quanti?cation analysis was also performed(Fig.1),with the standard error of the mean Ct difference for each group of replicates given as the error bars.The fold change was plotted against the mean intensity shift(log R ratio) as measured on the array.All plotted microdeletions showed considerable fold reductions,as indicated by the top bars that lie below the shaded region(2n?diploid).SYCE1and CPEB1were genes included in the veri?ed microdeletions.These genes are known to cause ovarian failure in knockout mouse models(25,26).

Interestingly,microdeletions were enriched in women with pri-mary amenorrhea;two of the seven genotyped women with primary amenorrhea(29%)had novel microdeletions,compared with6of the81women with secondary amenorrhea(7%).Although our study

Fertility and Sterilitya1597

focused on autosomes,we also detected one novel interstitial micro-deletion on the X chromosome in a woman with secondary amenor-rhea,which mapped to Xp11.23–p11.22,where the BMP15gene is located(Table2).

DISCUSSION

Animal models indicate that the majority of candidate genes for ovarian failure are located on autosomes(13,16–18).Although initial genetic screening studies for POF focused on the X chromosome,recent genome-wide studies in humans have found as-sociations with autosomal chromosomes,as well(21,27–29).These ?ndings offer further motivation to continue looking more closely across the entire genome for genomic imbalances that could contribute to the phenotypic spectrum of POF.

Earlier studies have highlighted the possible role of CNVs in the pathogenesis of several common and rare diseases(30–32). Haploinsuf?ciency encountered in microdeletions is a well known cause of human genetic disorders;these microdeletions can occur sporadically or be inherited.Sporadic cases likely arise from nonallelic homologous recombination or unequal crossing over between large highly identical segmental duplications.It has also become clear that the severity of clinical presentations varies widely,with identical microdeletions causing no apparent symptoms in carrier parents yet severe clinical presentation in the offspring(33).The causes of such variable expression are unclear but likely include predisposing polymorphisms in the affected indi-viduals.The objective of the present study was to use a genome-wide approach to detect microdeletions and microduplications in a cohort of89women diagnosed with POF.We hypothesized that novel CNVs associate with POF.

We identi?ed seven novel autosomal microdeletions among88 successfully arrayed POF women.Quantitative real-time PCR was used to corroborate?ve of the six novel autosomal microdeletions that contained known coding regions.In the case of POF-66,the de-letion(0.24Mb)was in a genomic region devoid of known genes, miRNAs,or other noncoding RNAs.Although it is easy to dismiss genomic regions that do not contain known transcriptional units,re-cent data shows that transcriptionally inactive regions can account for an abnormal phenotype when disrupted(34).

Among the genes found within the novel autosomal microdele-tions,two have been strongly linked to abnormal reproductive phe-notypes in knockout mouse models:SYCE1(10q26.3)and CPEB1 (15q25.2).SYCE1has been shown to disrupt the repair of DNA double-strand breaks during meiosis,which leads to apoptosis of germ cells and subsequent infertility of female knockout mice (25).Based on this null phenotype in female mice,SYCE1haploin-suf?ciency in humans may have a similar effect,leading to loss of oocytes and POF(35).The woman who presented with a hemizygous deletion of SYCE1had menarche at14and experienced cessation of menstrual cycles at age21.Another gene,CPEB1,is preferentially expressed in the oocyte and encodes a protein that regulates the translation of mRNAs for proteins involved in synaptenomal com-plex formation during oocyte maturation(35).Adult female mice de?cient in CPEB1lack follicles owing to loss of oocytes,and midg-estation female embryos show oocytes arrested at the pachytene stage(36).CPEB1has also been found to regulate mitotic cell progression in the S and M phases,which are especially important during embryonic cell divisions(26).CPEB1haploinsuf?ciency in humans may accelerate germ cell loss during the reproductive lifespan,as is the case in POF.This hypothesis could support the fact that the woman who presented with a hemizygous deletion of CPEB1exhibited ovarian failure at an earlier age;she never experienced menarche(primary amenorrhea).

Other microdeletions identi?ed in our screen harbor genes that are not known to play a role in reproductive biology.POF-119exhibited a deletion that resulted in haploinsuf?ciency of CCBE1and PMAIP1 (18q21.32).We used microarray data generated from wild-type new-born mouse ovaries(37),as well as NCBI EST databases(Unigene and Gene Expression Omnibus),to determine whether CCBE1and PMAIP1are expressed in the ovaries of female mice.Based on these datasets,both CCBE1and PMAIP1are expressed in murine ovaries.Similarly,CTNNA3(POF-85),ANKRD22,and STAMBPL1 (POF-58)transcripts are present in the murine ovarian transcrip-tome.It is possible that such genes may play a yet-to-be-de?ned role in ovarian development.

We also identi?ed17autosomal microduplications which have not been reported in the DGV.The role of duplications in human disorders is less compelling,because corresponding animal models are lacking or dif?cult to interpret.Two duplications on chromosome1were nearly identical in two distinct individuals(POF-21and POF-106), and they included genes for hydroxy-delta-5-steroid dehydrogenase, 3beta-and steroid delta-isomerase2(HSD3B2),and hydroxyacid oxidase2(HAO2).The role of HAO2in ovarian biology is unknown, despite its expression in the mouse ovary.Conversely,HSD3B2is preferentially expressed in the adrenal glands and ovaries of mice,

McGuire.Genomic imbalances in ovarian failure.Fertil Steril2011.

1598McGuire et al.Genomic imbalances in ovarian failure Vol.95,No.5,April2011

according to the Unigene and BioGPS databases(38,39).HSD3B2is essential for steroid hormone production in humans,and de?ciencies cause a severe form of congenital adrenal hyperplasia(40).Further-more,additional studies report increased expression of HSD3B2in theca cells of women with polycystic ovary syndrome,a frequent cause of infertility in women(41).It is possible that duplication of HSD3B2may lead to androgen and estrogen imbalances within the ovary with subsequent disruption of folliculogenesis,but this hypothesis remains to be tested.

We discovered seven novel autosomal microdeletions and17 novel autosomal microduplications;in addition,we identi?ed one novel microdeletion located on the X chromosome.Our results add to the growing body of recent literature supporting our hypoth-esis that CNVs associate with POF.A recent study on French women used a lower-resolution bacterial arti?cial chromosome(BAC) based array(0.7Mb mean spatial resolution)to identify CNVs that statistically associate with POF(21).As with our study,there was an excess of CNVs on the autosomes compared with the X chro-mosome.Eight CNVs showed statistical association with POF in their study(seven autosomal and one X chromosomal),but no novel CNVs were presented.A second study focused only on the X chro-mosome(22).A BAC tiling array for X/Y chromosomes was used,and the authors discovered new discrete X chromosome intervals as-sociated with POF.

It is important to note that there may be additional CNVs that went undetected by our arrays and those from earlier studies,be-cause even the highest-resolution arrays may tag only$50%of CNVs in the genome(42).In addition to technologic limitations, our study was limited by the lack of family members and the small sample size.However,owing to the high variability in the clinical phenotype of carriers and affected family members with microdele-tions(33),the presence of such microdeletions in a fertile parent would not rule out the pathogenic role of such genomic imbalances in ovarian function in the offspring.Future studies with larger populations will be necessary to determine whether recurring geno-mic imbalances are present in women with POF and to discern the utility of molecular karyotyping methods,such as array comparative genomic hybridization(array CGH),in replacing conventional kar-yotyping.Moreover,animal models that mimic novel human dele-tions and/or duplications may be useful in further delineating the importance of such regions in mammalian reproductive biology. Acknowledgments:The authors acknowledge Lisa Marsh,R.N.,for patient recruitment and data collection.

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