Skip Navigation


Mol. Hum. Reprod. Advance Access originally published online on April 29, 2008
Molecular Human Reproduction 2008 14(6):371-376; doi:10.1093/molehr/gan022
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
14/6/371    most recent
gan022v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Zhang, J.
Right arrow Articles by He, Y.-s.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, J.
Right arrow Articles by He, Y.-s.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Development of a multiplex quantitative fluorescent PCR assay for identification of rearrangements in the AZFb and AZFc regions

Jun Zhang1,2, Pei-qiong Li1,2, Qi-hong Yu2, Hua-yun Chen2, Juan Li1,2 and Yun-shao He1,2,3

1Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, People’s Republic of China 2 DaAn Gene Diagnostic Center of Sun Yat-sen University, Guangzhou, Guangdong 510080, People’s Republic of China

3 Correspondence address. E-mail: heyunshao2008{at}yahoo.com.cn


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Author Roles
 References
 
The azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions in the human Y chromosome consist of five palindromes constructed from six distinct families of amplicons and are prone to rearrangement. Partial deletion and duplication in the region can cause azoospermia or oligozoospermia and male infertility. The aim of the study was to establish a quantitative fluorescent PCR (QF-PCR) assay to classify AZFb and AZFc rearrangements. A single pair of fluorescent primers was designed to amplify simultaneously the amplicon in AZFc and the length-variant homologous sequences outside of the region as control. Since the copy number of the control sequences is fixed in the human genome, dosage of the target could be easily obtained through comparing the height of the fluorescent peaks between the target and the control after amplification with limited PCR cycles. Most types of rearrangements in AZFb and AZFc regions could be classified with QF-PCR containing four such primer pairs. Eleven types of rearrangement in AZFb and AZFc regions were well discriminated with QF-PCR. In conclusion, QF-PCR is a simple and reliable method to detect rearrangements in AZFb and AZFc.

Key words: AZFb/AZFc/infertility/QF-PCR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Author Roles
 References
 
Five palindromes are included in azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions of the human Y chromosome. Unlike the AZFb region, the structure of AZFc is complicated (Hucklenbroich et al., 2005). Three palindromes constructed from six distinct families of amplicons form the region (Kuroda-Kawaguchi et al., 2001). The first AZFc structure, the prototype structure, was derived from a Y chromosome belonging to the haplogroup R1*(hgrR1*) (‘A’ in Fig. 1). Different types of AZFc rearrangements were reported since then, including whole regional (b2/b4) deletion (B) (Kuroda-Kawaguchi et al., 2001), gr/gr deletion (C) (Fernandes et al., 2002), b2/b4 duplication (D) (Repping et al., 2003), b1/b3 deletion (E) (Repping et al., 2003), gr/gr duplication (F) (Lin et al., 2005), b1/b3 duplication (G) (Lin et al., 2005), b2/b4 duplication (H) (Writzl et al., 2005), b2/b3 inversion (I) (Repping et al., 2004a), g1/g3 deletion (J) (Repping et al., 2004a), g1/g3 duplication (K) (Lin et al., 2005), g1/g3 inversion (L) (Repping et al., 2004a), b2/b3 deletion (M) (Repping et al., 2004a), blue-gray duplication (N) and b2/b3 duplication (O) (Repping et al., 2003). Deletions involving AZFb include AZFb (P5/P1 proximal) deletion (P) (Repping et al., 2002; Simoni et al., 2004) and AZFb + c (P5/P1 distal or P4/P1 distal) whole regional deletion (Q, R) (Vogt, 2005). The internal constructions of the known rearrangement types are illustrated in Fig. 1.


Figure 1
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1: Ideograph of different types of rearrangement at the AZFb and AZFc regions

Ideograph of different types of rearrangement at the AZFb and AZFc regions ‘A’ depicts the haplogroup R1*, from P5 proximal to P1 distal, covering the whole AZFb and AZFc regions except for omitting the fragment ~3.3 M between P4 and b1. Different colors are used for various repeats, according to Kuroda-Kawaguchi et al. (2001). Repeats T1, T2, U3, Gr1, Yel1, Yel2 and Gr2 where our primers designed to amplify as indicators for rearrangements are shown on the top. Different types of deletion, inversion and duplication are depicted from ‘B’ to ‘R’. Positions where duplicated fragments would be inserted are indicated by black arrows. This figure is modified from Lin et al. (2006).

 
Complete deletions of AZFb, AZFc or AZFb + c usually results in azoospermia, whereas partial AZFc deletions yield phenotypes ranging from normospermia to azoospermia (Hopps et al., 2003; Navarro-Costa et al., 2007). Some types of partial AZFc deletions are presumed to be male infertility risks, such as gr/gr deletion (Lynch et al., 2005; Giachini et al., 2007; Imken et al., 2007), b2/b3 deletion (Imken et al., 2007) and b2/b3 duplication (Lin et al., 2007). Moreover, partial AZFc deletions can increase the risk of complete AZFc deletion (Zhang et al., 2007), while opposite results are also reported by other groups (Hucklenbroich et al., 2005; Carvalho et al., 2006; Lin et al., 2007). The typing of rearrangements in AZFb and AZFc regions and their roles in spermatogenesis still need further study.

Sequence tagged sites (STS) analysis is most widely used to detect deletions in AZF regions, while it alone does not allow the detection of AZFc partial duplications. In addition to deletion, duplication events and deletion followed by duplication events have been identified related with male infertility. New methods of screening AZFc rearrangements are needed. Some methods are developed for detecting AZFc rearrangements, such as multiplex PCR with Southern hybridization (Lin et al., 2006), fluorescence in situ hybridization (Repping et al., 2003), PCR assays based on sequence family variants (Machev et al., 2004), plus/minus STSs (Repping et al., 2004b) and DAZ copy number (Writzl et al., 2005). All these methods have advantages and disadvantages to some extent. As illustrated in Fig. 1, partial deletion and duplication cause the lost or gain of specific repeats. In other words, the type of rearrangement can be discriminated by calculating the numerical alterations of each repeats. The repeats U1, U2, U3, Red, Yellow and Gray in the AZFc region all have homologous sequences outside of the region (Kuroda-Kawaguchi et al., 2001). Here we use quantitative fluorescent PCR (QF-PCR) to detect the ratio between the target repeats and the homologous sequences. During QF-PCR process, a pair of primers was designed to amplify the target repeats and the length-variant homologous sequences simultaneously. Since the sequence contents of the amplicons were similar and the PCR was limited in the exponential amplification phase, the ratio between PCR product peaks was the same in the templates. Knowing the dosage of the homologous sequences, we can figure out the dosage of the repeats easily. Combining three such primer pairs with a pair of normal fluorescent primers, we can discriminate most of the rearrangement types in the AZFb and AZFc regions. Different types of AZFb and AZFc rearrangements and their dosages between PCR amplicons are listed in Table I.


View this table:
[in this window]
[in a new window]

  		Table I. Copy numbers of each PCR amplicon in different sample types.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Author Roles
 References
 
Patients and DNA extraction
Sixty samples without rearrangements, 2 samples with AZFb + c deletion (P5/P1 distal or P4/P1 distal), 8 samples with AZFc deletion (b2/b4), 30 samples with Gr1 deletion (including gr/gr deletion and b2/b4 duplication) and 20 samples with U3 deletion (including g1/g3 deletion, b2/b3 deletion and blue-gray duplication) identified with multiplex PCR (Lin et al., 2006), together with 3 47,XYY samples and 2 female samples were used in testing the QF-PCR primers. Patients with oligospermia or azospermia were obtained from the Daan Gene Clinical Diagnostic Center of Sun Yat-sen University. Informed consent was obtained from all patients as well as the approval of Internal Review Boards and Ethics Committee of Sun Yat-Sen University. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions.

STS PCR and quantitative real-time PCR
Multiplex STS PCR was modified from the protocol of Lin et al. (2006). The reaction amplified five AZFc markers, sY1201, 677 bp; sY1291a, 565–580 bp; sY1206a, 412 bp; sY1191a, 368 bp and sY1161, 330 bp, and a control gene pair ZFX/ZFY, 495 bp. The PCR mix contained 20 mM Tris–HCl (pH 8.8 at 25°C), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 1 M betaine, 0.8 µg/µl BSA, 0.1 mM each of the dNTPs, 50 ng of genomic DNA, 1 U of Taq DNA polymerase (Takara, Japan), 1 µM each for ZFX/ZFY, sY1161, sY1191 and sY1206, 2 µM for sY1291a and 10 µM for sY1201 primers. PCRs were carried out under the following conditions: preheated at 95°C for 15 min, followed by 35 cycles of 94°C for 30 s; 63°C for 90 s; 72°C for 60 s and a final extension at 72°C for 10 min. The products were analyzed on 2% agarose gels to observe whether all the amplicons were amplified.

Quantitative real-time PCR (Q-PCR) was used to determine the copy number of DAZ gene which lies in the Amplicon Red as described by Roze et al. (2007). In brief, totally four-copied STS sY586 (G63907 [GenBank] ) located in intron 6 of DAZ gene was chosen for quantification, with the single copy STS sY1064 (G64723 [GenBank] ) located in the AZFa region as a reference. The primer sequences are the same as Roze et al. (2007). Reactions were analyzed on an ABI 7500 Real Time PCR system. Each Q-PCR (25 µl) contained 5 ng of genomic DNA, 0.4 nM primers each and 12.5 µl of the 2x SYBR Green Master Mix (ABI, CA, USA). The initial cycling conditions were 50°C for 2 min and denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s (sY586), 72°C for 30 s and a dissociation step. At least triple tubes of PCR were repeated for each DNA sample. The experiments were performed using the comparative Ct (ddCt) relative quantitation assay method (Livak and Schmittgen, 2001). The data were analyzed using the ddCt method. The relative copy numbers for each STS marker were calculated as follows: the normalized Ct value (dCt) sY586 was calculated by subtracting the average Ct value with that of the sY1064. The ddCt value was calculated by subtracting the dCt value from that of the normal genomic DNA. The relative copy number was finally determined as 2–ddCt.

Multiplex fluorescent PCR
Four pairs of primers were used in QF-PCR. The location and size of the amplicons are listed in Table II. TUT primer pair (DYS26) was picked for amplifying the T1 and T2 regions with the same size 151 bp. U3 primer pair was designed to amplify a 170 bp product in the AZFc region and two products of 165 and 170 bp in Yp11.2 as controls. Primer pair Gray was designed to amplify fragments of 239 bp from Gr1, 243 bp from Gr2 region and 245 bp from the homologous region of chromosome 1 as control. Primer pair Yellow was designed to amplify fragments ~223 bp in Yel1 and 227 bp in Yel2 region. Primers were synthesized by Sangon Technologies (Shanghai, China). Primer sequences are as follow: tUT-F: cAATCTGGTGATGGGTGGAG; TUT-R: gCTGTGCTCCAATCTGAAGC; U3-F: aTTTGGAATCCCCTTTAGGC; U3-R: cTGTCCTGAGCATCTTGATTTC; Gray-F: gCTGTACACATGGTTTGGGAAGG; Gray-R: gCTAGGCACTGTGATGGATGATT; Yellow-F: tTCTCATTGCCTCTCCTCTCTC; Yellow-R: tAATGCTAGATGACGAGTTAGTGG. All forward primers were labeled with FAM at the 5' end.


View this table:
[in this window]
[in a new window]

  		Table II. Size and genomic position of the PCR amplicons.

 
Quantities of the primers were adjusted in the multiplex PCR mix to meet similar amplification rate, especially for primer pairs Yellow and Gray. The PCR mix contained 20 mM Tris–HCl (pH 8.8 at 25°C), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 1 M betaine, 0.8 µg/µl BSA, 0.1 mM each of the dNTPs, 50 ng of genomic DNA, 1 U of Taq DNA polymerase (Takara, Japan), 0.2 µM each for the TUT primers, 0.4 µM each for the U3 primers, 1.0 µM each for the Gray and Yellow primers in a final volume of 25 µl. PCR amplification was carried out in an ABI 9700 thermocycler (ABI) with an initial heating at 94°C for 5 min, followed by 25 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min and a final incubation at 60°C for 45 min.

Genotyping
One microliter of the PCR products was mixed with 13 µl deionized formamide and 1 µl ABI GS-500 marker (ABI). After incubation at 95°C for 5 min and cooling on ice, the mixture was loaded on ABI 3100 Sequencer (ABI) and analyzed with Genemapper 3.2 software (ABI). Product peaks were differentiated according to the fragment sizes. Product peaks amplified by the same primer pair were numbered from left to right, such as U3-1 for the 165 bp product and U3-2 for the 170 bp one. The height of peak ratios between U3-2/U3-1, Y1/Y2 and (G1 + G2)/G3 were calculated. If there was only one product of the Yellow primer pair, ratio between Y/G3 was calculated. If there were only two product peaks of the primer pair Gray, height of the G1 peak was considered zero in calculating the ratio (G1 + G2)/G3. Results of primer pair TUT, positive or negative, were also recorded.

Statistics
Ratios between U3-2/U3-1, Y1/Y2 (2Yellow peaks) or Y/G3 (1Yellow peak) and (G1 + G2)/G3 are different among different rearrangement types. Samples were divided into three U3-2/U3-1 types, one Y1/Y2 type, two Y/G3 types and four (G1 + G2)/G3 types. The height-of-peak ratios of each sample were calculated and pooled to corresponding ratio groups. Data were analyzed in SPSS 11.0 with a value of P < 0.05 for considering statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Author Roles
 References
 
Electrophoretograms of the QF-PCR products were illustrated in Fig. 2. Samples of AZFb + c deletion (Q, R), AZFc deletion (B), fertile male (A), fertile female (S) and 47,XYY (T) were submitted to QF-PCR for testing the primers. Totally eight ratio groups could be divided from the five types of samples used here. Each height-of-peak ratio calculated from QF-PCR results was pooled to the corresponding ratio group. Average, standard deviation and ranges of each ratio group were calculated and listed in Table III. Sample with single yellow amplicon was unavailable here; we used the data Y1/G1 and Y2/G1 from 20 normal samples as substitution for the ratio group Y/G1 = 1:1. Samples losing products in TUT, U3, Yellow and Gray were consistent with the genomic status according to their rearrangement types. The difference between ratio groups U3-2/U3-1 = 2:1 and U3-2/U3-1 = 1:1 has statistical significance (T-test, P < 0.05). The differences among ratio groups (G1 + G2)/G3 = 1:2, (G1 + G2)/G3 = 1:1 and (G1 + G2)/G3 = 2:1 also has statistical significance (Tamhane’s T2, P < 0.05). In the ratio group Y1:Y2 = 1, the difference between Y1 and Y2 has no statistical significance (paired samples T-test, P > 0.05).


Figure 2
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2: Electrophoregrams of the QF-PCR products

Electrophoregrams of the QF-PCR products ‘A’ depict the electrophoregram of the QF-PCR products from a fertile man. Fragment sizes in bp are shown on horizontal axis on the top. Markers TUT, U3-1, U3-2, Y1, Y2, G1, G2 and G3 are identified according to the size and labeled at the bottom. The height-of-peak ratios are about 2:1 for U3-2/U3-1, 1:1 for Y1/Y2 and 1:1:2 for G1/G2/G3 in this type. The electrophoregram of rearrangement types in our samples are shown below as ‘B’ ‘C’ ‘D’ ‘E’ ‘F’ ‘H’ ‘J, M’ ‘N’ ‘P’ ‘Q, R’ ‘S’ and ‘T’ according to Table I.

 

View this table:
[in this window]
[in a new window]

  		Table III. FQ-PCR results among different amplicon ratio groups.

 
We then used the ratio ranges derived from the results above as criteria in screening samples from the Gr1 deletion group and the U3 deletion group. Derived from limited samples, these ratio ranges were not wide enough to accommodate all results from these two groups. Samples with a ratio out of the range would first be evaluated if the rearrangement type could be classified without this ratio. If feasible, the ratio would be used as new border of this ratio group; otherwise the sample would be tested again. Samples not matching judgment criteria in two consecutive tests were abandoned. Three b2/b4 duplication (D) samples and two blue-gray duplication (N) samples were found in them. Height of peak ratios between U3-2/U3-1 were calculated from b2/b4 duplication samples and pooled to U3-2/U3-1 = 3:1 group. Height of peak ratios between (G1 + G2)/G3 were calculated from blue-gray duplication samples and pooled to (G1 + G2)/G3 = 3:2 group. Data processing was the same as before. Results were listed in Table III. The differences among each U3-2/U3-1 ratio groups and (G1 + G2)/G3 ratio groups had statistical significance (Tamhane’s T2, P < 0.05).

We then use the ranges in Table III as criteria to screen fertile males with normal sperm counts and infertile male samples. In the total 40 fertile male samples, no genetic dosage variation was found, while another three types of rearrangements b1/b3 deletion (E), gr/gr duplication (F) or b2/b4 duplication (H) and AZFb deletion (P) were found in infertile male samples. Electrophoretograms are shown in Fig. 2E, F, H and P. Our QF-PCR assay still has the potential to discriminate three other types of rearrangements, namely g1/g3 duplication (K) or b2/b3 duplication (O) with estimated result of one TUT peak, two U3 peaks about 3:1, two Yellow peak about 1:2 and three Gray peak about 1:1:2, and b1/b3 duplication (G) with estimated result of one TUT peak, two U3 peak about 3:1, two Yellow peaks about 1:1 and three Gray peaks about 2:1:2.

All samples with rearrangements and 10 normal fertile samples were subjected for Q-PCR analysis. The copy numbers of DAZ gene were about 4 (3.6–4.8) in normal control, b2/b4 duplication and blue-gray duplication samples; about 2 (1.6–2.2) in the gr/gr deletion, b1/b3 deletion, g1/g3 deletion or b2/b3 deletion and AZFb deletion samples; about 6 (5.2–6.5) in the gr/gr duplication samples; and about 8 (7.9–8.4) in the b2/b4 duplication samples. All the data were consistent with our QF-PCR results.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Author Roles
 References
 
The AZFb and AZFc regions is full of palindromes. Many kinds of rearrangements have been identified by now. Not only deletions but also duplications sometimes affect spermatogenesis (Lin et al., 2007). Several methods have been developed to screen AZFc partial deletions. The most widely used one is PCR assay that detects the specific STS markers around AZFc (Repping et al., 2003). Only a few types of AZFc deletion can be discriminated with this method while other rearrangements, especially the duplications, are missed. Methods like Southern blotting and Q-PCR measuring DAZ dosage have been reported and have partially compensated the limitation of STS PCR (Lin et al., 2006; Roze et al., 2007).

Here we report a simple and rapid QF-PCR method to discriminate different types of AZFb and AZFc rearrangements. QF-PCR is a method based on capillary electrophoresis and widely used in sequencing, forensic medicine (Butler et al., 2004; Alessandrini et al., 2005), prenatal diagnosis (Cirigliano et al., 2004) and other realms (Fimiani et al., 2006). Through incorporation of fluorochromes into the primers, the PCR products of different size could be separated with different fluorescent peaks. Within the exponential phase of PCR amplification, the amount of the PCR products was assumed to be proportional to the quantity of the initial targeted sequences (Nowacka et al., 2004; Sun et al., 2006). Although QF-PCR is a semi-quantitative method, it is reliable to discriminate genetic dosage with only 1-fold difference. We use the homologous sequences as dosage control to the target amplicon. Control sequences were aligned with online sequence database to exclude copy number variations. Since the dosage of the control is stable, it is easy to judge the dosage of the target amplicon. With only one peak, nothing could be compared. The key to the experimental design is finding a pair of primers that amplifies the target repeats in AZFc and the homologous sequences outside with different product sizes. For amplicons appearing more than once in AZFc region, internal comparison can also be used, such as the primer pair Yellow used here. Considering the incidence of homozygocity between the amplicons, choosing a pair of primers with control outside of the AZFc region is more advisable, such as the primer pair U3 and Gray used here. Although the size of PCR products amplified here were approximately stable over the samples, it is possible that in some populations those would be different from ours, thus making interpretation difficult. Solutions to this problem are the same as other QF-PCR applications, adding more primers or using a more informative marker. In fact, combination of primer pair TUT, U3 and Gray is enough for classification of most of the AZFc rearrangement types. We add primer pair Yellow for confirmation. Our QF-PCR protocol is a simple and reliable way to detect AZFb and AZFc rearrangements from a large population, and it still has the potential to discover new rearrangement types especially when more markers are added. Classically, QF-PCR was used to analyze the status between length-variant alleles such as STR markers. We have expanded it to analyze rearrangements among homologous sequences. Non-allelic homologous recombination (NAHR) is common in the human genome. This protocol will provide researchers with a new idea in exploring the mechanism and detection of NAHR.


   Author Roles
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Author Roles
References
 
J.Z., first author, design, doing most of the work, writing and revising the manuscript; P.-q.L: samples preparation, acquisition of data, drafting part of the article; Q.-h.Y.: samples preparation, acquisition of data, drafting part of the article; H.-y.C.: samples preparation, acquisition of data, drafting part of the article; J.L: samples preparation, acquisition of data, drafting part of the article; Y.-s.H.: corresponding author, design, directing experiments, revising the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Author Roles
 References
 
Alessandrini F, Turchi C, Onofri V, Buscemi L, Pesaresi M, Tagliabracci A. Multiplex PCR development of Y-chromosomal biallelic polymorphisms for forensic application. J Forensic Sci (2005) 50:519–525.[Web of Science][Medline]

Butler JM, Buel E, Crivellente F, McCord BR. Forensic DNA typing by capillary electrophoresis using the ABI Prism 310 and 3100 genetic analyzers for STR analysis. Electrophoresis (2004) 25:1397–1412.[CrossRef][Web of Science][Medline]

Carvalho CM, Zuccherato LW, Bastos-Rodrigues L, Santos FR, Pena SD. No association found between gr/gr deletions and infertility in Brazilian males. Mol Hum Reprod (2006) 12:269–273.[Abstract/Free Full Text]

Cirigliano V, Voglino G, Cañadas MP, Marongiu A, Ejarque M, Ordoñez E, Plaja A, Massobrio M, Todros T, Fuster C, et al. Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR. Assessment on 18,000 consecutive clinical samples. Mol Hum Reprod (2004) 11:839–846.

Fernandes S, Huellen K, Goncalves J, Dukal H, Zeisler J, Rajpert De Meyts E, Skakkebaek NE, Habermann B, Krause W, Sousa M, et al. High frequency of DAZ1/DAZ2 gene deletions in patients with severe oligozoospermia. Mol Hum Reprod (2002) 8:286–298.[Abstract/Free Full Text]

Fimiani G, Laperuta C, Falco G, Ventruto V, D’Urso M, Ursini MV, Miano MG. Heterozygosity mapping by quantitative fluorescent PCR reveals an interstitial deletion in Xq26.2-q28 associated with ovarian dysfunction. Hum Reprod (2006) 21:529–535.[Abstract/Free Full Text]

Giachini C, Nuti F, Marinari E, Forti G, Krausz C. Partial AZFc deletions in infertile men with cryptorchidism. Hum Reprod (2007) 22:2398–2403.[Abstract/Free Full Text]

Hopps CV, Mielnik A, Goldstein M, Palermo GD, Rosenwaks Z, Schlegel PN. Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions. Hum Reprod (2003) 18:1660–1665.[Abstract/Free Full Text]

Hucklenbroich K, Gromoll J, Heinrich M, Hohoff C, Nieschlag E, Simoni M. Partial deletions in the AZFc region of the Y chromosome occur in men with impaired as well as normal spermatogenesis. Hum Reprod (2005) 20:191–197.[Abstract/Free Full Text]

Imken L, El Houate B, Chafik A, Nahili H, Boulouiz R, Abidi O, Chadli E, Louanjli N, Elfath A, Hassar M, et al. AZF microdeletions and partial deletions of AZFc region on the Y chromosome in Moroccan men. Asian J Androl (2007) 9:674–678.[CrossRef][Web of Science][Medline]

Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterston RH, Wilson RK, Silber S, Oates R, Rozen S, et al. The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet (2001) 29:279–286.[CrossRef][Web of Science][Medline]

Lin YW, Thi DA, Kuo PL, Hsu CC, Huang BD, Yu YH, Vogt PH, Krause W, Ferlin A, Foresta C, et al. Polymorphisms associated with the DAZ genes on the human Y chromosome. Genomics (2005) 86:431–438.[CrossRef][Web of Science][Medline]

Lin YW, Hsu CL, Yen PH. A two-step protocol for the detection of rearrangements at the AZFc region on the human Y chromosome. Mol Hum Reprod (2006) 12:347–351.[Abstract/Free Full Text]

Lin YW, Hsu LC, Kuo PL, Huang WJ, Chiang HS, Yeh SD, Hsu TY, Yu YH, Hsiao KN, Cantor RM, et al. Partial duplication at AZFc on the Y chromosome is a risk factor for impaired spermatogenesis in Han Chinese in Taiwan. Hum Mutat (2007) 28:486–494.[CrossRef][Web of Science][Medline]

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods (2001) 25:402–408.[CrossRef][Web of Science][Medline]

Lynch M, Cram DS, Reilly A, O’Bryan MK, Baker HW, de Kretser DM, McLachlan RI. The Y chromosome gr/gr subdeletion is associated with male infertility. Mol Hum Reprod (2005) 11:507–512.[Abstract/Free Full Text]

Machev N, Saut N, Longepied G, Terriou P, Navarro A, Levy N, Guichaoua M, Metzler-Guillemain C, Collignon P, Frances AM, et al. Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility. J Med Genet (2004) 41:814–825.[Abstract/Free Full Text]

Navarro-Costa P, Pereira L, Alves C, Gusmao L, Proenca C, Marques-Vidal P, Rocha T, Correia SC, Jorge S, Neves A, et al. Characterizing partial AZFc deletions of the Y chromosome with amplicon-specific sequence markers. BMC Genomics (2007) 8:342.[CrossRef][Medline]

Nowacka J, Helszer Z, Walter Z, Plucienniczak A, Kazewski B. Adaptation of PCR technique for quantitative estimation of genetic material from different regions of chromosome 21 in cases of trisomy 21. Acta Biochim Pol (2004) 51:995–1001.[Web of Science][Medline]

Repping S, Skaletsky H, Lange J, Silber S, Van Der Veen F, Oates RD, Page DC, Rozen S. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet (2002) 71:906–922.[CrossRef][Web of Science][Medline]

Repping S, Skaletsky H, Brown L, van Daalen SK, Korver CM, Pyntikova T, Kuroda-Kawaguchi T, de Vries JW, Oates RD, Silber S, et al. Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet (2003) 35:247–251.[CrossRef][Web of Science][Medline]

Repping S, Korver CM, Oates RD, Silber S, van der Veen F, Page DC, Rozen S. Are sequence family variants useful for identifying deletions in the human Y chromosome? Am J Hum Genet (2004) a 75:514–517. author reply 517–519.[CrossRef][Web of Science][Medline]

Repping S, van Daalen SK, Korver CM, Brown LG, Marszalek JD, Gianotten J, Oates RD, Silber S, van der Veen F, Page DC, et al. A family of human Y chromosomes has dispersed throughout northern Eurasia despite a 1.8-Mb deletion in the azoospermia factor c region. Genomics (2004) b 83:1046–1052.[CrossRef][Web of Science][Medline]

Roze V, Bresson JL, Fellmann F. Quantitative PCR technique for the identification of microrearrangements of the AZFc region. J Assist Reprod Genet (2007) 24:241–248.[CrossRef][Web of Science][Medline]

Simoni M, Bakker E, Krausz C. EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art 2004. Int J Androl (2004) 27:240–249.[CrossRef][Web of Science][Medline]

Sun X, Yan M, Zhang Y, Zhou X, Wang C, Zheng F, Xiong C. Practical application of fluorescent quantitative PCR on Trisomy 21 in Chinese Han population. Mol Biol Rep (2006) 33:167–173.[CrossRef][Web of Science][Medline]

Vogt PH. AZF deletions and Y chromosomal haplogroups: history and update based on sequence. Hum Reprod Update (2005) 11:319–336.[Abstract/Free Full Text]

Writzl K, Zorn B, Peterlin B. Copy number of DAZ genes in infertile men. Fertil Steril (2005) 84:1522–1525.[CrossRef][Web of Science][Medline]

Zhang F, Lu C, Li Z, Xie P, Xia Y, Zhu X, Wu B, Cai X, Wang X, Qian J, et al. Partial deletions are associated with an increased risk of complete deletion in AZFc: a new insight into the role of partial AZFc deletions in male infertility. J Med Genet (2007) 44:437–444.[Abstract/Free Full Text]

Submitted on January 28, 2008; resubmitted on April 21, 2008; accepted on April 25, 2008.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
14/6/371    most recent
gan022v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Zhang, J.
Right arrow Articles by He, Y.-s.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, J.
Right arrow Articles by He, Y.-s.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?