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The DAZ genes and impaired spermatogenesis de Vries, J.W.A.

Publication date 2002

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Citation for published version (APA): de Vries, J. W. A. (2002). The DAZ genes and impaired spermatogenesis.

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Download date:29 Sep 2021 Chapterr 4

REDUCEDD COPY NUMBER OF DAZ GENES IN SUBFERTILE AND INFERTILE

MEN. .

|ann W.A. de Vriesa, Mariëtte J.V. Hofferdb, Sjoerd Repping^, Jan M. N, Hoovers5,, Nico J. Leschotb and Fulco van der Veend.

'Centerr for Reproductive Medicine, Academic Medical Center, Amsterdam, The Netherlands s

bb Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands s

Fertilityy and Sterility 2002;77: 68-75 Chapterr 4

Abstract t

Objective(s):: To determine the copy number and identity of the DAZ genes on the YY chromosomes of infertile patients. Design:: Prospective study. Setting:: University medical center. Patient(s):: One hundred and thirty-nine patients with male factor infertility. Intervention(s):: The separate genes were detected by polymerase chain reaction (PCR)) digestion assays of sequence family variants in leukocyte DNA and by fluorescencee in situ hybridization of interphase nuclei and chromatin fibers. Mainn Outcome Measure(s): Number of DAZ genes present. Result(s):: One hundred twenty-nine patients had four genes, 6 patients had two genes,, and 4 patients had none. Three patients had a deletion of the two proximal DAZDAZ genes, and three were missing both distal genes. Semen analysis showed a lesss severe phenotype in patients with only two DAZ genes compared with patientss missing all four genes. Conclusion(s):: In six patients, two different partial deletions were found that were nott detected by PCR with conventional markers. One patient with an AZFb deletionn appeared to also have a partial AZFc deletion that was not detected by routinee PCR. Phenotypic differences between patients with different deletions suggestt a dose effect of the DAZ genes Keyy Words: DAZ genes, partial deletions, AZFc, Y chromosome, male subfertility. Reducedd copy number

INTRODUCTION N

Too date, 10 gene families located on the non-recombining region of the Y chromosomee that are specifically expressed in the testis have been described (1, 2).. The number of copies per gene family varies. RBMY, for instance, has many copiess that are dispersed over the entire length of the Y chromosome, of which onlyy one to four seem to be needed for spermatogenesis (3-5). The DAZ gene familyy consists of four copies located relatively close together in deletion interval 66 on chromosome band Yq11, designated as AZFc. As we demonstrated using fluorescencee in situ hybridization (FISH), the four copies are located in two clusters,, each comprising an inverted pair of DAZ genes (6). At least three different DAZDAZ complementary DNAs have been identified, indicating that at least three DAZDAZ genes are transcribed in the testis during spermatogenesis (6, 7).

Becausee DAZ genes have multiple copies, sequence tagged site markers locatedd in the DAZ genes tend to also have multiple copies. Polymerase chain reactionn (PCR) analysis with conventional markers, the method used in all publishedd deletion studies thus far, does not detect individual gene deletions withinn the DAZ gene family. Therefore, subfertile or infertile men may carry a deletionn of fewer than four of the DAZ genes that goes undetected by conventionall PCR techniques.

Thee number of DAZ genes present can be determined by using FISH and gene-specificc PCR digestion assays. Fluorescence in situ hybridization on interphasee nuclei allows detection of the separate gene clusters, and FISH on extendedd DNA fibers (also called "fiber FISH") can be used to detect the separate geness within the clusters. A gene-specific PCR digestion assay that identifies the fourr different DAZ genes by their sequence family variants was recently developed (6).. It makes use of single nucleotide variants in three different sequence tagged sitess in the DAZ genes. These variants can be identified by using PCR digestion assays,, producing a specific band pattern for each DAZ gene (the so-called DAZ

67 7 Chapterr 4 genee signature) that identifies the four different DAZ genes (6). Deletions of all fourr DAZ genes have been described and are associated with azoospermia or severee oligozoospermia (8, 9). AA partial deletion of the DAZ genes was recently described in a subfertile patientt (10). It is not known how frequently this partial deletion is detected in subfertilee or infertile men. We therefore examined the number of DAZ genes in patientss with variable semen characteristics, all of whom had diagnosed male factorr subfertility or infertility, by using gene-specific sequence family variant PCR andd FISH. To determine whether different phenotypes in these patients are associatedd with the number of DAZ genes present, we related the number of copiess found to semen characteristics.

MATERIALSS AND METHODS

PATIENTSS AND SEMEN DONORS.

Fromm October 1997 to December 1998, we enrolled 139 consecutive patientss attending our clinic in whom male factor subfertility or infertility had beenn diagnosed. Semen analysis was performed according to World Health Organizationn criteria (11). To be included, patients had to have no history of alcoholl abuse, orchitis, surgery on the vasa deferentia, bilateral orchidectomy, chemotherapy,, radiotherapy, or obstructive azoospermia (confirmed by testis biopsy).. An andrologic history was taken, testicular volume was measured, and bloodd was collected for karyotyping and DNA isolation. Blood from semen donors withh proven fertility attending our semen bank was used as an internal control. Thee study was approved by the institutional review board, and all patients and semenn donors gave informed consent before participating.

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CONVENTIONALL PCR ASSAYS.

Isolatedd DNA from leukocytes obtained by venipuncture was examined by usingg routine PCR amplification to detect deletions of the following loci: sY81, sY84,, SY182, sY94, sY102, sYl 1 7, sY143, sY147 sYl 52, sY1 53, and sYl 57.

SEQUENCEE FAMILY VARIANTS.

Sequencee family variants that distinguish among DAZ genes were detected byy PCR amplification of the three sequence tagged site markers sY581 (intron 3), sY5866 (intron 6), and sY587 {intron 10). This was followed by enzyme digestion, ass described elsewhere (6). Figure 1 shows the nucleotide variation between these markerss and their association with the different DAZ genes. In brief, digestion of sY5877 with Dra1 produces four fragments of 122 base pairs, 73 base pairs, 49 basee pairs, and 26 base pairs in DAZ1 and DAZ2, which both contain the T variantt of this sequence tagged site. In DAZ3 and DAZ4, which contain the C variantt of sY587, three fragments of 195 base pairs, 49 base pairs, and 26 base pairss are detected. Similarly, digestion of sY581 with 5au3A produces a 189-base pairr fragment and a 63- base pair fragment in DAZ1 and DAZ4 and three fragmentss of 130 base pairs, 59 base pairs, and 63 base pairs in DAZ2 and DAZ3. Finally,, digestion of sY586 with Taq1 produces two fragments of 184 base pairs andd 117 base pairs in DAZ1, DAZ3, and DAZ4, whereas Y586 of DAZ2 remains undigested,, resulting in one 301-base pair fragment.

Sequencee family variant PCR in sY587 distinguished cluster 1 and cluster 2, andd sequence family variant PCR in sY581 distinguished between left or right geness in both clusters. Fragment sizes of the PCR products after digestion with restrictionn enzymes are shown in Table 1. If incomplete DAZ signatures were detected,, tests were repeated.

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FISH H Wee used three sequenced DAZ cosmids as probes for FISH (6). Cosmid 18E88 (Cenbank AC010089) encompasses the 5' end portions of two neighboring DAZDAZ genes (Fig. 1). Cosmid 63C9 (Genbank AC000021) contains exons 2 through 111 —almost an entire DAI gene. Cosmid 46A6 (Genbank AC000022) derives from thee 3' end portion of DAZ; it contains exons 8 through 11 and 35 kb downstream off the gene. Single-colorr FISH was performed on interphase nuclei from lymphocytes accordingg to standard procedures (1 2). Cosmid 1 8E8 was used to detect the presencee of the two DAZ clusters. Because the DAZ genes are located in two clusters,, each containing two DAZ genes in a head-to-head orientation, cosmid 18E88 shows predominantly one signal per cluster. Because superimposition of the twoo signals can lead to misinterpretation, at least 200 interphase nuclei were examinedd before the presence of one or two signals was accepted. Ann average of 20 patients was tested in every FISH procedure. Lymphocytes fromm a fertile donor were included in every FISH procedure as internal control on thee validity of the FISH procedure. The technician performing the counting of the signalss was not aware of the outcome of the PCR experiments. Extendedd chromatin fibers from lymphocytes were prepared by using SDS / ethylenediaminee tetraacetic acid extraction (1 3). Fiber FISH was performed by usingg a combination of probes 18E8 with 46A6 to detect the 5'- and 3' portions of thee DAZ genes and a combination of probes 18E8 with 63C9 to detect the 5' portionn and the body of the DAZ genes in single clusters. Probess were labeled with biotin or digoxigenin, hybridized to target DNA, and detectedd by avidin or antidigoxigenin antibodies conjugated to fluorochromes Cy3 (red)) or fluorescein (green).

STATISTICALL ANALYSIS. Thee t-test was used for all statistical analyses.

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FIGUREE 1

Schematicc drawing of the two DAZ gene clusters.

Ü Ü o o Va V a () ) -- /s S CO OO)' O)' CO O ~~CO O— — coo co V V 10 0 >-->->-m m>U 3-- 3 w w

D4Z1 1 D/AZ2 2 cosmids s 63C9 9 13E8 8 Clusterr 1

Ü Ü O O ÜÜ Ü O O T-- CD co o SFVS S COO CO m m mm in >-- ^0^0 0 >-- >-- >- U)) to

DAZ3 DAZ3 DAZDAZ A Clusterr 2 cosmids s 46A6 6

Sequencee family variants (SFVs) between the four different genes. sY587 distinguishess between clusters 1 and 2, sY581 distinguishes between the geness within the clusters, and sY586 distinguishes between DAZ2 and the otherr three genes. The location of the three cosmids used for fluorescence in situu hybridization is also shown.

RESULTS S

SEMENN CHARACTERISTICS.

Twenty-fivee patients had azoospermia, and 84 patients had oligozoospermia;; of these 84 patients, 57 had severe oligozoospermia (<5 million spermm cells/mL). Thirty patients had asthenozoospermia or teratozoospermia with normall sperm counts. Sperm count varied from 0 to 200 x 10'' cells/mL. A

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testicularr volume less than 15 mL in both testes was detected in 71 patients, of whomm 1 5 had a normal sperm count and asthenozoospermia or teratozoospermia andd 45 had oligozoospermia (counts < 20 x 10(J cells/mL); of these 45 azoospermicc patients, 34 had a sperm count less than 10 x 106cells/mL and 11 hadd azoospermia.

Tablee 1 1 1 Fragmentt sizes after restriction digestion.

Sequencee tagged Enzymee DAZ1 DAZ2 DAZ2 DAZ3 DAZ3 DAZ4 DAZ4

sY5811 intron 3 Sau3A 189,63 130,59,63 130,59,63 189,63

sY586intron66 Taq\ 184,117 301 184,117 184,117

SY5877 intron 10 Drat 122,73,49,26 122,73,49,26 195,49,26 195,49,26

Note:: In cluster 1 (DAZ1 and DAZ2), sY587 has the T variant, producing an extraa restriction site for Dra1. In cluster 2 {DAZ3 and DAZ4), sY587 shows the CC variant. In DAZ2 and DAZ3, sY581 has the T variant, producing an extra restrictionn site for Sau3A. In DAZ1 and DAZ4, sY581 shows the C variant. In DAZ2,DAZ2, sY586 is in the T variant, causing loss of a restriction site for Taq1.

KARYOTYPES S

Sixx of the 139 patients had an aberrant karyotype. Three patients had a 47,XXYY karyotype and azoospermia, one patient had a 47,XYY karyotype and oligozoospermiaa (3 x10() cells/mL), one patient had a translocation 46,XY, der (22)t(y;22)(q12;p13)) and oligozoospermia (7x 10(>cells/ mL), and one patient had 47,XY,, mar ish der(15) and azoospermia. Reducedd copy number

MlCRODELETIONS S

Usingg the standard panel of sequence tagged site markers for the Y chromosome,, we found five patients with deletions (Table 2). Deletions of the AZFcAZFc region {sY1 57, sY1 52, sYl 53, and sY1 47) were detected in four patients. Onee patient retained the markers for the AZFc region but was missing sY1 1 7 and sY143,, corresponding to an AZFb deletion.

Tablee 2 ]|

Microdeletionss detected by routine polymerase chain reaction.

AZFaAZFa AZFb AZFc Patientt sY81 sY86 sY182 sY94 sY102 sY117 sY143 sY152 sY153 sY147 sY157 21488 + + + + + + + - 74822 + + ++ + + + ---- 49711 + + ++ + + + ---- 59522 + + ++ + + + ---- 49377 + + ++ + - - + + + +

Note:: Table shows presence ( + ) or absence (-) of sequence tagged sites detected inn polymerase chain reaction in the three deletion intervals of the Y chromosome. .

Inn two patients, DAZ1 and DAZ2 were detected but DAZ3 and DAZ4 were absent.. In two patients, only DAZ3 and DAZ4 were detected. In four patients, only DAZ3DAZ3 was detected; one of these patients also had an AZFb deletion. In one patient,, only DAZ1 was detected. In the four patients with AZFc deletions, all sequencee family variants were absent, as expected. Althoughh PCR for sY581 and sY587 showed a normal sequence family variantt pattern in 126 patients, 32 of these patients did not have the 301-base pair fragmentt for sY586, which is specific for DAZ2. This fragment was also missing in onee of the two patients who had both DAZ1 and DAZ2 according to PCR for sY5811 and sY587.

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Conversely,, the 301-base pair band was present in one of the two patients inn whom only DAZ3 and DAZ4 were detected on PCR for sY581 and sY587. Tablee 3 1 1 Combinationss of DAZ signatures in 139 patients and the presence of one or moree clusters on fluorescence in situ hybridization. DAZ2 DAZ2 detected d byy PCR Sequencee Family of f variant3 3 sY586b b FISH H Fiberr FISH No.. of Patient (%) DAZ1 DAZ2 DAZ3 DAZ4 Yess No 22 clusters 11 cluster normal clusters 1266 (90.6) ++ + + + 944 32c 126 6 Yes s 2(1.4) ) ++ + - 11 lc -- 22 Yes 22 (1.4) ++ + 1cc 1 2 2 Yes s 44 (2.9) + + 4 4 1d d 33 Yes 11 (0.7) ++ - - 1 1 11 Yes 44 (2.9) Not t determined d

aa Columns show presence (+) or absence (-) of the DAZ genes as detected by polymerasee chain reaction digestion assay of sY581 and sY587. bb Data are expressed as the number of patients in whom the 301-base pair band,, DAZ2 gene, was found or was absent. (( This result conflicts with that of the sY587 and sY581 assay. dd Patient with a 47,XYY karyotype had two clusters per Y chromosome.

FISH H

Fluorescencee in situ hybridization with probe 18E8 on interphase nuclei showedd two clusters in 1 29 patients. In six patients, however, the mean number of signalss per nucleus was significantly lower than in control samples from semen donors.. The mean ( SD) number of signals in these six patients ranged from 1.08 0.28 to 1.26 , compared with 1.84 0.54 to 1.95 0.56 in the control sampless (P < .001). On fiber FISH, all six patients were shown to have retained onee complete DAZ cluster with two genes.

74 4 Reducedd copy number

Off the 129 patients with two signals on FISH, tour genes were also detected byy sequence family variant analysis in 126, including the 32 patients who were missingg the 301- base pair band representing DAZ2 (Table 3). Fiber FISH again showedd only normal cluster images (comprising two genes each) in all these patients.. Six of the nine patients with incomplete sequence family variants had predominantlyy one cluster per cell, and FISH clearly showed two clusters with two geness each in the remaining three patients. Interphase FISH and sequence family variantt PCR were concordant in the 2 patients in whom only DAZI and DAZI weree detected, in the one patient in whom only DAZI was detected, a\-)d in three off the four patients in whom only DAZ3 was detected. In contrast, both patients in whomm only DAZ3 and DAZ4 were detected and one patient in whom only DAZ3 wass detected clearly had two normal clusters with two genes each on FISH.

FIGUREE 2

Fiberr fluorescence in situ hybridization detection of a complete DAZ gene cluster.

Resultss of fiber fluorescence in situ hybridization on DNA from patient 4937, whoo had an AZFb deletion and a normal DAZ gene cluster. Red signal: 18E8; greenn signal:63C9. (Full color figure on page 151) 75 75 Chapterr 4

Thee latter patient had a 47,XYY karyotype, and FISH showed two clusters per Y chromosome.. Of note, one of the patients in whom only DAZ3 was detected on sequencee family variant PCR and one cluster on FISH also had an AZFb deletion (Fig.. 2)All patients with an aberrant number of DAZ genes had normal clusters comprisingg two genes on fiber FISH, even though some patients had sequence familyy variants concordant with only one gene. In the four patients with AZFc deletions,, FISH confirmed absence of the DAZ gene clusters.

SEMENN CHARACTERISTICS IN RELATION TO DAZ GENE CLUSTERS.

Thee number of DAZ gene clusters was determined by FISH, and the cluster wass identified by sequence family variant PCR (Table 4). For example, if one normall cluster was found on interphase and fiber FISH and sequence family variantt PCR indicated the presence of only DAZ3, the cluster was identified as clusterr 2. Tablee 4 J

Semenn characteristics relative to the presence of DAZ gene clusters.

DAZDAZ gene Severe Mild Asthenozoosp. or No.. of patients cluster deleted Azoosp.* oligozoosp.a,b oligozoosp.'a teratozoosp.a 1299 None 21 54 26 28 33 CI uster2 - 1 - 2 33 Clusterl 2C - 1 44 Both 2 2

Note:: The DAZ gene cluster number was determined by FISH and the clusters weree identified by SFVs. dd Data are the number of patients. bb Mild oligozoospermia is a sperm count of 5 x 106 cells/ mL to 20 x 106 cells/ mL; severee oligozoospermia is a sperm count less than 5 x 106 cells/ mL.

Tablee 4 shows the relation of DAZ gene clusters with semen characteristics. Onee of the three patients with a deletion of cluster 2 had severe oligozoospermia.

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Thee other two patients had a normal sperm count (22-46 x 10&cells/mL) with asthenozoospermiaa (8%-22% motility) and teratozoospermia {12 % -13 % normoform).. Of the three patients with a deletion of cluster 1, two had azoospermiaa and one had oligozoospermia (8 x 106cells/mL). Of the four patients withh deletion of both clusters, two had azoospermia and two had severe oligozoospermiaa {<5 x 106cells/mL).

DISCUSSION N

DAZDAZ genes on the Y chromosome arose from transposition of the autosomal DAZLDAZL gene on chromosome 3. Intragenic amplification, exons pruning, and duplicationn produces four almost identical genes on the Y chromosome in two clusters,, each consisting of two genes oriented in a head-to head manner (6). Thesee genes are located in the AZFc deletion interval (9). Yen (14) identified three off the four genes, but observed a gap in their YAC contig at the place where we suspectt the fourth gene is. Our two-cluster concept is in good agreement with the findingss of Glaser et al. (15). However, those investigators concluded that seven DAZDAZ genes are present on the Y chromosome. We suspect that the probes used by thatt group detected the 10.8-kb segment containing exons 2 through 6 of the DAZ gene,, in which the sY581 marker is located. This region represents the RNA recognitionn motif and is present seven times in the DAZ gene clusters: three times inn DAZ1, twice in DAZ4, and once each in DAZ2 and DAZ3 {Fig. 1). Of the 139 patientss in our study, 129 had four DAZ genes in two clusters.

Deletionn events on the Y chromosome and, especially, in the AZFc region aree relatively frequent; rates range from 1 % to 20% in men with severe oligozoospermiaa and azoospermia (9, 16-22). Thus far, all AZFc deletions describedd are deletions of all four DAZ genes, except in the patient described by Moroo et al. (10). In these deletion studies, only complete deletions of the four DAZDAZ genes could be detected because markers that are shared by the DAZ genes weree used.

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Althoughh the four DAZ genes are 99.9% identical in coding sequence, they cann be distinguished by detection of single nucleotide variants in three different intronss of the DAZ gene (6). This distinction is based on Bacteria! Artificial Chromosomee (BAC) clones that each contain no more than two of the four DAZ genes.. In genomic DNA, however, four DAZ genes are present; because the specificc band patterns of the four DAZ gene signatures overlap, a single gene deletionn cannot be excluded, even if all band patterns for the sequence family variantss are present. DAZ genes can be detected at the cluster level by using FISH onn interphase nuclei. On fiber FISH, only one complete or incomplete cluster can bee seen in a single microscopic image because of the distance between the clusterss and breaking of the chromatin fibers. Usingg a combination of sequence family variant PCR and FISH techniques, wee found that six patients had a deletion of one of the two DAZ gene clusters. At leastt three different breakpoints must be involved, because either cluster could be absentt independently. These partial deletions of the DAZ genes would have been missedd if we had used only conventional PCR markers. From these partial deletions,, we obtained new information on the relative orientation of the DAZ genee clusters. Because the patient with the AZFb deletion was also missing cluster 11 and the father had no detectable deletion on the Y chromosome (data not shown),, we conclude that cluster 1 is the proximal cluster and cluster 2 is the distall cluster. The orientation of the genes within the clusters, however, remains to bee elucidated. Sequencee family variant PCR is based on three silent nucleotide substitutionss that were found in different BAC clones from one Y chromosome (6). Onn the basis of results of sequence family variant PCR and FISH on the Y chromosomess of 1 39 men, we conclude that polymorphism in these sequence familyy variants exists. In 32 of 126 patients in whom all genes were detected on sequencee family variant PCR for sY581 and sY587 and FISH, the T substitution in sY5866 in DAZ2 was absent, indicating polymorphism for sY586. In three patients

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inn whom only DAZ3 was detected and one patient in whom only DAZ1 was detectedd on sequence family variant PCR for sY581, single but complete clusters weree found on FISH. In two patients in whom only cluster 2 was detected on sequencee family variant PCR for sY587, two normal clusters were found on FISH. Thesee results indicate polymorphism for sY581 and sY587 as well, albeit to a lesserr extent.

Polymorphismm of the restriction site in sY581 has been described in relation too ethnicity (23). However, neither the number of copies of the DAZ genes nor fertilityy status were evaluated. Therefore, it is unknown whether the men with only thee T or C variant for sY581 in that study represent true polymorphisms or deletionss of some of the DAZ genes. In our study, almost all patients with aberrant copyy numbers were of European origin; one patient in whom only cluster 2 was detectedd came from West Africa (data not shown).

Bothh AZFb and AZFc harbor more than one gene family (1). The AZFb candidatee gene RBMY is dispersed over the entire Y chromosome, and fiber FISH hass shown that it also resides in between the DAZ gene clusters (24). AZFb and AZFcAZFc deletions show different breakpoints in different patients (16, 1 7, 19, 25- 27).27). In this study, we describe a patient with an AZFb deletion that extends into AZFc.AZFc. If many breakpoints exist in this area and if the areas appear to overlap, it mightt be better to not distinguish between these two intervals but rather to examinee the entire contiguous interval by using gene-specific probes for all genes knownn to reside in this deletion interval.

Multicopyy genes can be an evolutionary advantage through dose repetition withh identical gene products (28) or variant repetition with related but not identicall gene products (29). Since at least three but probably all four genes are transcribedd in the adult testis, a gene dose effect for DAZ can be expected. Thee predicted products of the four DAZ genes differ in the number of RNA recognitionn motifs; whether these products differ functionally is not yet known (6).

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Wee related the phenotype on the spermogram to the presence or absence off the different clusters. The absence of both clusters does not always lead to completee absence of spermatogenesis (30). Indeed, two of the four patients with ann AZFc deletion had some spermatozoa in their ejaculate. A less severe phenotypee was found if only one cluster was absent, suggesting a gene dose effect off the DAZ genes. Furthermore, absence of cluster 1 seems to lead to a more severee phenotype than does absence of cluster 2, suggesting possible different functionss of the various DAZ genes. To determine the clinical value of these partiall deletions, a larger sample of affected patients and controls must be studied.

Itt has been proposed that men with sperm concentrations above 5 x 10(' cells/mLL need not be examined for Y chromosome deletions (31). However, in our study,, three of the six patients with only one DAZ gene cluster had sperm concentrationss greater than 5 x 106 cells/mL These deletions will be transmitted too male offspring, and we do not yet know the risk for widening of these deletions inn future generations, as others have proposed (32).

ACKNOWLEDGMENTS: :

Thee authors thank Laura Brown and David Page, Ph.D., for providing probes 18E8, 63C9,, and 46A6; they also thank Saskia van Daalen and Cindy Korver for technicall support.

REFERENCES S

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4.. Elliott DJ, Millar MR, Oghene K, Ross A, Kiesewetter F, PryorJ, et al. Expressionn of RBM in the nuclei of human germ cells is dependent on a criticall region of the Y chromosome long arm. Proc Natl Acad Sci U S A 1997;94:3848-53. . 5.. Chai NN, Zhou H, Hernandez J, Najmabadi H, Bhasin S, Yen PH. Structure andd organization of the RBMY genes on the human Y chromosome: transpositionn and amplification of an ancestral autosomal hnRNPG gene. Genomicss 1998;49:283-9. 6.. Saxena R, de Vries JWA, Repping S, Alagappan RK, Skaletsky H, Brown LC, ett al. Four DAZ genes in two clusters found in the AZFc region on the human YY chromosome. Genomics 2000;67:256-67. 7.. Yen PH, Chai NN, Salido EC. The human DAZ genes, a putative male infertilityy factor on the Y chromosome, are highly polymorphic in the DAZ repeatt regions. Mamm Genome 1997;8:756-9. 8.. Ma K, Sharkey A, Kirsch S, Vogt P, Keil R, Hargreave TB, et al. Towards the molecularr localisation of the AZF locus: mapping of microdeletions in azoospermicc men within 14 subintervals of interval 6 of the human Y chromosome.. Hum Mol Genet 1992;1:29-33. 9.. Reijo R, Lee TY, Salo P, Alagappan R, Brown LG, Rosenberg M, et al. Diverse spermatogenicc defects in humans caused by Y chromosome deletions encompassingg a novel RNA-binding protein gene. Nat Genet 1995;10383- 93. . 10.. Moro E, Ferlin A, Yen PH, Franchi PG, Palka G, Foresta C. Male infertility causedd by a de novo partial deletion of the DAZ cluster on the Y chromosome.. J Clin Endocrinol Metab 2000;85:4069-73. 11.. World Health Organization. WHO laboratory manual for the examination of humann semen and sperm-cervical mucus interaction. 3rd ed. Cambridge (UK):: Cambridge University Press, 1992. 12.. Redeker E, Hoovers JM, Alders M, van Moorsel CJ, Ivens AC, Gregory S, et al.. An integrated physical map of 210 markers assigned to the short arm'of humann chromosome 11. Genomics 1994;21:538-50. 13.. Fidlerova H, Senger G, Kost M, Sanseau P, Sheer D. Two simple procedures forr releasing chromatin from routinely fixed cells for fluorescence in situ hybridization.. Cytogenet Cell Genet 1994;65:203-5. 14.. Yen HY. A long-range restriction map of deletion interval 6 of the human Y chromosome:: a region frequently deleted in azoospermic males. Genomics 1998;54:5-12. . 15.. Glaser B, Yen PH, Schempp W. Fibre-fluorescence in situ hybridization unravelss apparently seven DAZ genes or pseudogenes clustered within a Y- chromosomee region frequently deleted in azoospermic males. Chromosome Ress 1998;6:481-6. 16.. Krausz C, Bussani-Mastellone C, Granchi S, McElreavey K, Scarselli G, Forti G.. Screening for microdeletions of Y chromosome genes in patients

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undergoingg intracytoplasmic sperm injection. Hum Reprod 1999;14:1 71 7- 21. . 11 7. Najmabadi H, Huang V, Yen P, Subbarao MN, Bhasin D, Banaag L, et al. Substantiall prevalence of microdeletions of the Y-chromosome in infertile menn with idiopathic azoospermia and oligozoospermia detected using a sequence-taggedd site-based mapping strategy. J Clin Endocrinol Metab 1996;81:1347-52. . 18.. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter, et al.. Human Y chromosome azoospermia factors (AZF) mapped to different subregionss in Yq11. Hum Mol Genet 1996;5:933- 43. 11 9. Pryor J L, Kent-First M, Muallem A, Van Bergen AH, Nolten WE, Meisner L, ett al. Microdeletions in the Y chromosome of infertile men. N Engl J Med 1997;336:534-9. . 20.. Simoni M, Gromoll J, Dworniczak B, Rolf C, Abshagen K, Kamischke A, et al. Screeningg for deletions of the Y chromosome involving the DAZ (Deleted in Azoospermia)) gene in azoospermia and severe oligozoospermia. Fertil Steril 1997;67:542-7. . 21.. Kleiman SE, Yogev L, Gamzu R, Hauser R, Botchan A, LessingJB, et al. Geneticc evaluation of infertile men. Hum Reprod 1999;14: 33- 8. 22.. Krausz C, Quintana-Murci L, McElreavey K. Prognostic value of Y deletion analysis:: what is the clinical prognostic value of Y chromosome microdeletionn analysis? Hum Reprod 2000;15:1431-4. 23.. Agulnik Al, Zharkikh A, Boettger-Tong H, Bourgeron T, McElreavey K, Bishop CE.. Evolution of the DAZ gene family suggests that Y-linked DAZ plays little, orr a limited, role in spermatogenesis but underlines a recent African origin forr human populations. Hum Mol Genet 1998;7: 1371-7. 24.. Glaser B, Hieri T, Taylor K, Schiebel K, Zeitler S, Papadopoullos K, et al. High-resolutionn fluorescence in situ hybridization of human Y-linked genes onn released chromatin. Chromosome Res 1997;5: 23-30. 25.. Girardi SK, Mielnik A, Schlegel PN. Submicroscopic deletions in the Y chromosomee of infertile men. Hum Reprod 1 997;1 2:1 635-41. 26.. van der Ven K, Montag M, Peschka B, Leygraaf j, Schwanitz G, Haidl G, et al.. Combined cytogenetic and Y chromosome microdeletion screening in maless undergoing intracytoplasmic sperm injection. Mol Hum Reprod 1997;3:699-704. . 27.. Silber SJ, Alagappan R, Brown LG, Page DC. Y chromosome deletions in azoospermicc and severely oligozoospermic men undergoing intracytoplasmic spermm injection after testicular sperm extraction. Hum Reprod 1998;13:3332-7. . 28.. Finnegan DJ, Rubin GM, Young MW, Hogness DS. Repeated gene families in DrosophilaDrosophila melanogaster. Cold Spring Harb Symp Quant Biol 1978;42 Pt 2:1053-63. . 29.. Long EO, Dawid IB. Repeated genes in eukaryotes. Annu Rev Biochem 1980;49:727-64. .

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30.. Mulhall JP, Reijo R, Alagappan R, Brown L, Page D, Carson R, et al. Azoospermicc men with deletion of the DAZ gene cluster are capable of completingg spermatogenesis: fertilization, normal embryonic development andd pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmicc sperm injection. Hum Reprod 1997;12: 503-8. 31.. Calogero AE, Garofalo MR, D'Agata R. Factors influencing the variable incidencee of Y chromosome microdeletions in infertile patients. Hum Reprod 1999;14:275. . 32.. Stuppia L, Calabrese G, Franchi PG, Mingarelli R, Gatta V, Palka G, et al. Wideningg of a Y-chromosome interval-6 deletion transmitted from a father to hishis infertile son accounts for an oligozoospermia critical region distal to the RBM1RBM1 and DAZ genes. Am J Hum Genet 1996;59: 1393-5.

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