Mol. Hum. Reprod. Advance Access published online on August 24, 2007
Molecular Human Reproduction, doi:10.1093/molehr/gam053
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A simple screening method for detection of Klinefelter syndrome and other X-chromosome aneuploidies based on copy number of the androgen receptor gene
Department of Growth and Reproduction, Juliane Marie Centre, Section GR-5064, The National University Hospital of Copenhagen, DK-2100 Copenhagen, Denmark
1 Correspondence address. Tel: +45-35-45-43-30; Fax: +45-35-45-60-54; E-mail: anne.marie.ottesen{at}rh.regionh.dk
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Abstract |
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Due to the high prevalence and variable phenotype of patients with Klinefelter syndrome, there is a need for a robust and rapid screening method allowing early diagnosis. Here, we report on the development and detailed clinical validation of a quantitative real-time PCR (qPCR)-based method of the copy number assessment of the androgen receptor (AR) gene, located to Xq11.2–q12. We analysed samples from 50 individuals, including a healthy male and female controls and patients with Klinefelter syndrome (47,XXY; 48,XXXY) (n = 28), mosaicisms (46,XX/47,XXY/48XXYY; 45,X/46,XY) (n = 3), other sex chromosome abnormalities (46,XX males; 47,XYY)(n = 4) and normal karyotypes (46,XY) (n = 13). The reference range for the AR-copy number was established as 0.8–1.2 for one copy and 1.7–2.3 for two copies. The qPCR results were within the reference range in 17/18 samples (94%) or 30/31 (97%) samples with one or two copies of the AR gene, respectively. None of the Klinefelter patients were misdiagnosed as having a karyotype with only one X-chromosome, and in none of the 46,XY males were two copies demonstrated. We systematically compared qPCR results with those obtained with another PCR-based method, the XIST-gene expression. The XIST-expression based assay was correct in only 29/36 samples (81%). Our findings demonstrated that the AR-qPCR technique is a simple and reliable screening method for diagnosis of patients with Klinefelter syndrome or other chromosomal disorders involving an aberrant number of X-chromosomes.
Key Words: Klinefelter syndrome/qPCR technique/screening/X-chromosome
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Klinefelter syndrome is the most frequent sex chromosomal disorder in males, and in the vast majority of patients, the aberrant karyotype is 47,XXY, with more seldom occurrence of additional X-chromosomes or mosaicisms. The incidence of 47,XXY is estimated as 1 to 667 males, but only about 25% of the expected cases are diagnosed (Bojesen et al., 2003; Bojesen and Gravholt, 2007). Because of the variable severity of symptoms, a large number of patients are never diagnosed. Among the symptoms, tall stature, eunuchoid body proportions and/or cognitive and behavioural disabilities most often lead to diagnosis in childhood (Linden et al., 1995; Lanfranco et al., 2004; Bojesen and Gravholt, 2007). Not infrequently, however, individuals with Klinefelter syndrome are first diagnosed in fertility clinics, as a progressive degradation of the germinal epithelium accelerating at puberty is a hallmark of the Klinefelter phenotype (Skakkebæk, 1969; Aksglaede et al., 2006). Despite ejaculatory azoospermia, a large proportion of Klinefelter patients have focal residual spermatogenesis and may be treated by assisted reproduction techniques (Tournay et al., 1996; Ulug et al., 2003; Lanfranco et al., 2004). These patients could potentially benefit from having their diagnosis established early in life, thus providing a possibility for early androgen substitution, and theoretically, for cryopreservation of semen in late puberty before complete azoospermia occurs. Furthermore, Klinefelter syndrome is associated with an increased mortality (Bojesen et al., 2004) and an elevated risk of diabetes and metabolic syndrome (Bojesen et al., 2006). Other studies have shown that the patients are also at increased risk of developing mediastinal germ cell tumours (Völki et al., 2006), as well as breast cancer (Swerdlow et al., 2005), suggesting a closer follow-up of these patients.
At present the diagnosis of Klinefelter syndrome is based on karyotyping of in vitro cultured G-banded white blood cells arrested in the metaphase. The method is time consuming and artefacts may appear due to culturing, thus it can only be performed in specialized laboratories of cytogenetics. The aim of this study was to develop and test a quick and simple screening method for Klinefelter syndrome and other karyotypes involving an aberrant number of X-chromosomes based on an evaluation of the copy number of a gene (genes) mapped to this chromosome. We used the quantitative real-time PCR (qPCR) technique for detection of the number of copies of a single copy gene, the androgen receptor (AR), located to Xq11.2-q12. This assay was compared with an alternative method based on the detection of the XIST-gene transcript by PCR (Kleinheinz and Schulze, 1994), as well as with conventional karyotyping in a series of patients and controls. We here report on the results indicative of the possibility of using the AR-qPCR assay for screening on large cohorts of subjects.
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Materials and Methods |
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Patients
Fifty individuals in total were included in this study, which was approved by the local Medical Ethics Committee (KF 01265848). Material for analysis was obtained from patients referred to the clinic of the Department of Growth and Reproduction, Copenhagen University Hospital for diagnosis or treatment. There were 10 patients who participated with more than 1 blood sample, and 17 samples were analysed more than once. All patients, except two, had previously been karyotyped in the Department of Clinical Genetics at our hospital. The chromosomal constitution of the patients is listed in Table 1 and included 47,XXY (n = 26); 48,XXXY (n = 2); mosaicism of 46,XX/47,XXY/48,XXYY (n = 1), mosaicism of 45,X/46,XY (n = 2); SRY-positive 46,XX-males (n = 2); 47,XYY (n = 2); 46,XY (n = 13). A healthy male and female were included in the study as controls.
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DNA isolation and quality assessment
Genomic DNA was purified from leukocytes isolated from peripheral blood samples of patients using either the semi-automated method on the QuickGene-810 Nucleic Acid Isolation System (Fujifilm Danmark A/S, Trørød, Denmark) with the QuickGene DNA whole blood kit S, or manually, using a DNA isolation kit for mammalian blood from Roche (Basel, Switzerland). In addition, normal human genomic DNA was purchased from Promega. In total, 95 specimens from patients were qPCR-analysed; 31 were derived from manually isolated DNA, and 64 were from semi-automatically isolated. Agarose-gel electrophoresis demonstrated a marked difference of the DNA fragment lengths among specimens, for details see Table 1. The semi-automated method provided high molecular DNA in 38/39 specimens (97%), and the manual method showed optimal conditions in 4/17 cases (23%). As a consequence, we exclusively used the qPCR data from DNA samples prepared with the semi-automated system, for calculations of reference ranges and statistics.
qPCR analysis with primers for the AR and the glyceraldehyde-3-phosphate dehydrogenase genes
Before each experiment, DNA concentrations were measured using a NanoDrop ND-1000 Spectrophotometer (Saveen Werner AB, Malmö, Sweden). DNA and primers were diluted manually, whereas transfers into PCR plates were done automatically by means of the Theonyx Liquid Performer (Mechatronic Systems, Ebersberg, Germany).
Quantitative PCR was performed on the Mx3000P platform from Stratagene (Cedar Creek, Texas). Conditions for amplification were as follows: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 30 s/62°C for 1 min/72°C for 30 s and 1 cycle at 95°C for 1 min/62°C for 30 s/95°C for 30 s. The final cycle was for producing a dissociation curve.
Nucleotide sequences of AR- and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-primers were checked by BLAST-searches and for SNPs by means of the Ensemble- and UCSC-browsers, and although genomic variations of the AR gene are known from the literature (e.g. point mutations in the AR-exon 5), they appear to be rare. Primers for AR, mapped to exon 5, (Forward (Fw) 5'-CGA CCA GAT GGC TGT CAT TC-3' and Reverse (Rev) 5'-CTG GAG TTG ACA TTG GTG AAG G- 3') and for GAPDH (Fw 5'-CTC CCC ACA CAC ATG CAC TTA-3' and Rev 5'-TTG CCA AGT TGC CTG TCC TT- 3') (DNA Technology A/S, Aarhus, Denmark) were diluted initially to 5 and 1 µM, respectively. Mixtures of forward and reverse primers were denatured for 3 min at 95°C and incubated on ice until use. Reaction tubes contained 11.25 ng genomic DNA, 15 µl Brilliant SYBR Green QPCR Master Mix (Stratagene), 7.0 µl primer-mixture of AR (final conc.: Fw 100 nM/Rev 300 nM) or GAPDH (final conc.: Fw 50 nM/Rev 50 nM) and dH2O added for a total volume of 30 µl. For a non-template reaction DNA was substituted with H2O.
Quantification was done by estimation of the copy number of the AR gene using the Mx3000P-software (Stratagene). In principle, the raw data from analysis of the AR gene in a patient were normalized by comparing to data from analysis of the somatic single copy gene, GAPDH of the same patient and this ratio was calibrated to the ratio of a normal male reference DNA following this equation:
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For calculation by this algorithm equal efficiency of the amplification reactions for AR and GAPDH is mandatory, illustrated by parallel standard curves established by analysis of a series of dilutions of normal DNA using the two primer sets. For statistical analysis of data, mean ratios, standard deviation (SD), variance, 95% confidence intervals (CI) and reference ranges of mean ratio ± 2SD were calculated (see Table 2).
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XIST-transcript analysis
The XIST-transcript analysis was performed using a modified protocol described previously by Kleinheinz and Schulze (1994). Total RNA was isolated from 2–5 ml peripheral blood using QIAamp RNA blood mini kit (QIAgen, Valencia, CA, USA). Extracted RNA was dissolved in diethyl pyrocarbonate-treated (DEPC) H2O, concentration measured by means of NanoDrop ND-1000 Spectrophotometer and stored at –20°C.
In each RT-PCR experiment, RNA from a normal female and male constituted a positive and negative control, respectively. For an internal RNA quality control, transcripts were amplified from the house-keeping gene Actin, and a non-template (DEPC-H2O) was also included in each experiment. Complementary DNA (cDNA) synthesis was carried out in a reaction mixture containing RNA (0.5 µg), 1 µl XIST cDNA primer (0.5 µg/µl) (5'-CAG TAT ATT TTA TTT TAC-3'), 1 µl Actin-primer (dT20, 0.1 µg/µl, DNA Technology, Denmark) and DEPC-H2O ad 12.0 µl. Following incubation at 65°C for 1 min and 42°C for 1 min, 8 µl cDNA Synthesis Mixture (4 µl 5x cDNA synthesis buffer; 0.5 µl 100x dNTP-mixture (25 nM of each dNTP in ddH2O) (GE Healthcare, UK); 5.5 µl DEPC-H2O; 0.3 µl reverse transcriptase (AMV)) was added and the final reaction mixture incubated at 42°C for 1 h. The reverse transcription was performed on a Thermomixer compact (Eppendorf, Hamburg, Germany). cDNA products were amplified using a DNA Thermal Cycler PE9700 (Perkin Elmer, Waltham, USA) with the conditions for amplification as follows: 1 cycle of 96°C for 5 min; 10 cycles of 96°C for 30 s/68°C for 40 s/72°C for 1 min 45 s; 30 cycles of 96°C for 30 s/54°C for 40 s/72°C for 1 min 45 s; 1 cycle of 72°C for 5 min and 4°C over-night. Amplification was carried out in a 30–31 µl reaction mixture containing 1–2 µl cDNA and 29 µl Master mix 1 µl Rev XIST-primer (0.1 pmol/µl) (5'-ATA GCA ACC AAC TCC CCA GTT TG-3'); 1 µl Fw XIST-primer (0.1 pmol/µl) (5'-TTC TGG CAT CCA CTA CCA CTA CTG-3'); 3 µl Rev XIST-primer (10 pmol/µl) (5'-ACA TCT AGA TGG CTT AAG AT-3'); 3 µl Fw XIST-primer (10 pmol/µl) (5'-CTA CCA CTA CTG ATT AAA CA-3'); 1 µl Actin-primer (Exon 4N, 10 pmol/µl) (5'-CCTGACTGACTACCTCATGAA-3'); 1 µl Actin-primer (Exon 6N, 10 pmol/µl) (5'-ATCAAAGTCCTCGGCCACATT-3'); 3 µl 10x DDRT-buffer (100 mM Tris/HCl (pH = 8.3), 500 mM KCl, 18 mM MgCl2, 1% Triton-X 100, 0.05% gelatine); 3 µl 10x dNTPs (2.5 mM of each dNTP); 16 µl H2O; 0.5 µl Amplitaq Polymerase (5 U/µl, GE Healthcare). The amount and fragment sizes of the resulting PCR products were examined by electrophoresis, using a 2% agarose-gel with ethidium bromide and a 100 bp molecular weight marker (GE Healthcare).
Fluorescence in-situ hybridization
Glass slides coated with leucocytes (enriched by buffy coats) were fixed in acetone 10 min at –20°C and washed in PBS-buffer followed by fixation in 2% paraformaldehyde for 2 min at room temperature. After a wash in PBS-buffer, the specimens were dehydrated in a series of alcohols and air-dried. Hybridizations of an 
-satellite probe for the X-chromosome (SG CEP X, Vysis, Downers Grove, IL, USA) were performed according to the manufacturer, except for a prolonged denaturation of specimens for 7 min at 83.5°C on a heating plate. Cells were counterstained with DAPI. Images were captured with a DM RBE fluorescence microscope (Leica, Heerbrugg, Switzerland) equipped with a Cohu non-cooled CCD camera, and analysis performed using the CytoVysion image analysis system (Applied Imaging, Santa Clara, CA, USA). Signals from at least 20 nuclei were scored. A specimen from an individual with a normal karyotype served as a control.
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Results |
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QPCR analysis of the AR-gene copy number
The method was validated by analysis of the AR-gene dosage in samples of genomic DNA in a series of patients and controls with known karyotypes. A representative example of the ratios obtained in patients with different karyotypes is shown in Fig. 1. All results are shown in Table 1, whereas Table 2 summarizes the statistics of data obtained in subjects with different numbers of X-chromosomes exclusively in specimens of semi-automatically isolated DNA.
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The reference ranges for one and two copies of the AR gene were established by repeated analyses of the same DNA from a normal male and female control in different experiments. On the basis of mean ratio ± 2SD, the reference intervals were set as 0.8–1.2 for karyotypes with one X-chromosome (n = 11) and 1.7–2.3 for karyotypes with two X-chromosomes (n = 11). The reference range for three copies was set as 2.5–4.0, based on only two patients with 48,XXXY. The range in the latter was due to an elevated SD of samples from pt. no. 31, one of the two patients in this group. The three reference ranges were not overlapping with each other, thus allowing an assessment of the AR-gene copy number and the assumed number of X-chromosomes in the patients. A very large proportion of the results obtained with semi-automatically isolated DNA samples were inside the reference ranges: 94% from patients with one X-chromosome and 97% with two X-chromosomes. For the two samples with three X-chromosomes, the overall success ratio was 73%, 66% from pt. no. 31 and 100% from pt. no. 32. The lowest values outside ranges for patients with one, two or three X-chromosomes were: none, 1.6 and 2.1, and the highest values outside ranges were: 1.3, none and 4.1, respectively.
In groups with semi-automatically isolated DNA, the SD ranged from 0.18 to 0.21. An exception was the group of 48,XXXY-samples with a SD calculated to 0.57; however, if the highly unstable results from pt. no. 31 were excluded, the SD was 0.32. In Fig. 1, the histograms illustrate the mean ratio values and SD of a series of patients with different karyotypes (only data from samples of DNA isolated by means of the semi-automated method are shown).
Specificity of the QPCR method in comparison to the XIST method
To evaluate the specificity of the qPCR-based test, we compared the results with previously established karyotypes of the patients. In addition, we simultaneously assessed an assay for the presence of XIST transcripts. This method was previously used in our laboratory for a quick detection of the number of X-chromosomes. In five cases, we also performed fluorescence in-situ hybridization (FISH) analysis with centromeric X-probes directly on white blood cells. The evaluation of the specificity of the XIST-expression based assay demonstrated that in 7 of 36 samples (19%), the result did not correspond to the karyotype.
In 96% of good quality DNA samples, the qPCR analysis of the AR-gene copy number was within the established reference range for one and two copies and in agreement with the karyotype, and in all five cases, the copy number agreed with the number of signals in the FISH analysis (Table 1). Notably, none of the qPCR ratio values outside the reference ranges for one or two copies would result in misclassification. In only one patient (no. 31, 48,XXXY) from whom a series of results with a high SD was obtained, the ratio value (2.1) was erroneously in the range established for two X-chromosomes.
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Discussion |
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This study indicates that quantification of the copy number of the AR gene in genomic DNA purified from blood cells by means of qPCR provides a rapid method for the detection of Klinefelter syndrome and other abnormal karyotypes involving an aberrant number of X-chromosomes. The method is highly specific and reliable and may be used for quick genetic screening for patients with symptoms raising suspicion of Klinefelter syndrome e.g. young boys with accelerated growth pattern, gynaecomastia or cryptorchidism, in cases of infertility combined with a certain phenotypic appearance, as well as for patients with a suspicion of other sex-chromosome aneuploidies, such as XX-male syndrome or Turner syndrome (45,X). We propose that this method can be used for screening in newborns, to quickly detect children that could benefit from early treatment, such as androgen substitution in relation to puberty in patients with Klinefelter syndrome in order to prevent the symptoms associated with hypogonadism. This is of special importance since
10% of Klinefelter patients are diagnosed before puberty (Bojesen et al., 2003; Bojesen and Gravholt, 2007). For the correct diagnosis, the microscopic analysis of G-banded chromosomes is the gold standard. This technique, however, is not suitable for screening as it is time consuming, relatively expensive and can only be reliably performed in specialized cytogenetic laboratories. The first attempt to develop a rapid technique for screening of individuals suspected of Klinefelter syndrome was made by Kleinheinz and Schulze (1994) who used the principle of detecting transcripts of the XIST gene, which in somatic cells is only expressed if more than one X-chromosome is present. We have systematically evaluated the XIST method in this study and found that nearly 20% of the samples were in disagreement with the patients' blood cell karyotype, most likely due to the problems with RNA instability. Apart from the fact that misdiagnosis is an unwanted drawback of a technique, the XIST technique has proven too difficult and, therefore, not practical in the clinical setting.
With the advent of quantitative PCR, several research groups have used this method for detection of the number of X-chromosomes. The AR-gene dosage, was in fact, used previously to distinguish males and females in a study of duplications of gene loci in hereditary neuropathies, but because of the different focus of that study the authors analysed only small numbers of subjects and did not intend to use this method for detection of altered sex chromosomes (Poropat and Nicholson, 1998). A few years later, another study (Cirigliano et al., 2002) established a fluorescent qPCR-based method of the X-linked HPRT gene for prenatal diagnosis of sex chromosome aneuploidies in amniotic samples. Detection of sex chromosome disorders, in particular Turner syndrome, was the aim of another recent study, which used a panel of SNP markers that span the X- and Y-chromosome in a quantitative pyrosequencing assay (Meng et al., 2005). The advantage of that method is its broad spectrum of genotyping, however, the methodology is still too complex for screening. On the other hand, the extremely simple qPCR-based assessment of the copy number of the AR gene, which we have developed in this study, was in excellent agreement with the numbers of X-chromosomes observed in karyotyping and can be employed for screening of large cohorts of patients. We decided to use just one locus of the long arm of the X-chromosome for the primary screening, based on the fact that duplication of the q-arm alone in males results in a Klinefelter syndrome phenotype (Mark et al., 1999). In agreement with this, our simple test with only one primer set for the AR gene mapped to Xq11.2–q12 showed a markedly high degree of correspondence with the karyotypical information in 47,XXY-, 46,XX- (SRY-positive) and 46,XY-patients. Thus our observations differed from a study which used qPCR for detection of gene copy numbers and demonstrated that the use of two reference genes were critical for accurate discrimination between one and two copies of a gene (Hoebeeck et al., 2005). If used for general population screening in neonatal wards, the assay must be as simple and cheap as possible, thus we advocate using only one primer pair, which in our study was shown to be reliable and robust. In andrology or fertility clinics, where the majority of male infertility patients are normally virilized, and where a deletion analysis of the Y-chromosome is routinely performed, there is no need for testing additional genes mapped to other regions of the X-chromosome. However, in clinical laboratories, which investigate patients with more complex disorders of sex differentiation (DSD), addition of at least one additional primer set for investigation of an extra locus on the p-arm would be advisable.
In our AR-qPCR assay, the reference ranges for one and two X-chromosomes (0.8–1.2 and 1.7–2.3) did not overlap with each other, and even though the ratio values in two cases were outside the two respective reference ranges, they would not lead to misdiagnosis. The relatively narrow width of the reference ranges for one and two X-chromosomes not only minimizes the risk of misdiagnosis, but also increases the potential for detection of some of the mosaic cases, despite the fact that our method presumably has a limited value for low-percentage mosaicism.
The data obtained from the AR gene is normalized in relation to the GAPDH gene, which is calibrated to equal data derived from a normal male reference. The method of evaluation partially compensates for discrepancies in DNA quality and deviations in measurements of the DNA concentration, providing equal efficiency of the amplification reactions of the two genes. For obtaining stable and reliable qPCR results, the integrity of genomic DNA is essential, and although this was the case in our study for one of the specimens from a patient with the karyotype 48,XXXY, the statistics showed a high SD of repeated analyses. The elevated SD might, however, be ascribed to the fact that we calibrated the analysis to a normal male having one X-chromosome. Using a normal female with two X-chromosomes as calibrator might reduce the overall final error on the relative quantities for specimens with three X-chromosomes because the magnitude of error is dependent of the difference in quantification cycle between test and calibrator (Hellemans et al., 2007). It is important to stress that the qPCR technique is intended as a screening method, and for confirmation of the diagnosis, especially in cases with suspected genotype-phenotype discrepancy, karyotyping using G-banding remains mandatory.
The AR-qPCR technique, which was tested in this study primarily for a screening for Klinefelter syndrome but also for other chromosomal constitutions involving an aberrant number of X-chromosomes, demonstrated the potential for application in the routine laboratory due to the reliability and robustness. Among other benefits of this application is that a result is provided within a day, 20 ng DNA is used for the analysis, and the technique is easy and inexpensive. Thus, the assay can potentially be used for screening nationwide, provided that additional studies will validate our rapid screening test in larger cohorts of patients with sex chromosome aneuploidies and in newborns.
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Acknowledgements |
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We are grateful to Lene Lykke Hansen for the excellent technical assistance with the FISH analysis and to Thomas Rattenborg (AH-diagnostics) and Maria Kirchhoff (Department of Clinical Genetics, National University Hospital of Copenhagen) for the theoretical support in relation to the qPCR analysis. This study was supported by the Danish Cancer Society and the Copenhagen Hospital Cooperation Research Foundation.
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Submitted on July 6, 2007; accepted on July 17, 2007.
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