Absence of testicular DAZ gene expression in idiopathic severe testiculopathies

A. Ferlin1, E. Moro1, M. Onisto2, E. Toscano1, A. Bettella1 and C. Foresta1,3

1 Clinica Medica 3, Department of Medical and Surgical Sciences and 2 Institute of Histology and Embryology, University of Padova, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Deletions of the DAZ (deleted in azoospermia) gene family are frequently responsible for male infertility and are generally assessed by analyses of genomic DNA extracted from peripheral leukocytes. The multicopy nature of this gene prevents the distinction of intragenic deletions or deletions not involving the whole DAZ gene cluster. Thus it is still unclear whether each DAZ copy is effectively expressed in the testis. We analysed, by reverse transcription–polymerase chain reaction (RT–PCR), the expression of DAZ, RBM and SRY genes, in testicular cells from infertile men affected by idiopathic severe hypospermatogenesis, obstructive azoospermia and Sertoli cell-only syndrome. Normal mRNA for DAZ, RBM and SRY were observed in obstructive azoospermia, whereas only SRY transcripts were detected when only Sertoli cells were present. Nine out of 10 patients affected by idiopathic severe hypospermatogenesis had normal expression of SRY, RBM and DAZ, while in one patient no DAZ transcript was detected, suggesting that his testiculopathy was related to the absence of DAZ expression. The lack of DAZ mRNA in testicular cells with an apparently normal DAZ gene constitution on DNA extracted from leukocytes may be explained by different hypotheses: (i) not all the copies of the DAZ gene cluster are transcribed in the germ cells and the reported patient had a small deletion involving only the active ones; (ii) the patient may be mosaic for the DAZ gene having a normal constitution in leukocytes and be deleted for DAZ gene in the testis; (iii) abnormalities of DAZ transcription may exist. These findings highlight the intrinsic interpretative difficulties of normal PCR analysis for DAZ and RBM on leukocytes and suggest caution in the use of germ cells for assisted reproductive techniques in these cases to avoid transmission of genetic abnormalities to male offspring.

Key words: DAZexpression/DAZ gene/RT–PCR/testiculopathy/Y chromosome


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Deletions on the Y chromosome long arm (Yq) in male infertility most frequently involve the DAZ (deleted in azoospermia) gene family and at present 10–15% of idiopathic severe testiculopathies are thought to be caused by the absence of this gene (Reijo et al., 1995Go, 1996Go; Najmabadi et al., 1996Go; Qureshi et al., 1996Go; Stuppia et al., 1996Go; Vogt et al., 1996Go; Foresta et al., 1997Go; Girardi et al., 1997Go; Pryor et al., 1997Go; Simoni et al, 1997Go; Foresta et al., 1998Go; Grimaldi et al., 1998Go; Liow et al., 1998Go; McLachlan et al., 1998Go; Silber et al., 1998Go; Vogt, 1998Go; Kleiman et al., 1999Go; Ferlin et al., 1999Go). Although DAZ is not the only gene present in distal Yq interval 6 (Lahn et al., 1997; Gläser et al., 1998Go; Yen, 1998Go), its high prevalence of deletions in infertile men has suggested that it represents the AZFc (azoospermia factor c) candidate (Vogt et al., 1996Go, 1997Go). This possibility is further strengthened by the high homology of DAZ with a Drosophila male infertility gene, boule (Eberhart et al., 1996Go), which mutation causes spermatogenic arrest. Supporting a major role for DAZ in regulating human spermatogenesis, this gene is testis-specific with transcription limited to germ cells (Menke et al., 1996; Habermann et al., 1998Go; Lee et al., 1998Go); however, its function remains unclear since it is only known to encode an RNA binding protein postulated to be involved in RNA processing, and as yet the RNA binding property of this gene has not been demonstrated (Reijo et al., 1995Go; Saxena et al., 1996Go; Cooke and Elliott, 1997Go; Yen et al., 1997Go). Furthermore, DAZ deletions may be associated with non-unique testicular phenotypes, as they could be detected in azoospermic men with a testicular picture of Sertoli cell-only syndrome as well as in severely oligozoospermic men with different spermatogenic alterations (Reijo et al., 1995Go, 1996Go; Najmabadi et al., 1996Go; Qureshi et al., 1996Go; Stuppia et al., 1996Go; Vogt et al., 1996Go; Foresta et al., 1997Go; Girardi et al., 1997Go; Pryor et al., 1997Go; Simoni et al., 1997Go; Foresta et al., 1998Go; Grimaldi et al., 1998Go; Liow et al., 1998Go; McLachlan et al., 1998Go; Silber et al., 1998Go; Vogt, 1998Go; Ferlin et al., 1999Go). Probably most difficulties in such genotype–phenotype relations arise from the multicopy nature of this gene, present in at least three copies, but probably more, in distal Yq interval 6 (Saxena et al., 1996Go; Gläser et al., 1997Go, 1998Go; Yen et al., 1997Go, 1998). In fact, deletions of DAZ are generally assessed by polymerase chain reaction (PCR) on genomic DNA extracted from peripheral leukocytes, a method that does not allow the distinction of intragenic deletions or deletions not involving all the DAZ copies. Therefore, deletions of this gene reported in infertile men seem to remove the whole of the DAZ gene cluster, while smaller deletions may not be found using this method (Vereb et al., 1997Go; Yen et al., 1997Go). Moreover, due to these methodological problems it is not known whether each DAZ copy is effectively expressed and active in the testis. Theoretically, if each DAZ copy has a role in male germ cell development it could be argued that different spermatogenic alterations might be associated with specific DAZ copy deletions. Alternatively, only one DAZ gene may be really active and responsible for the testicular phenotype, but deletions of this copy may not be seen by PCR due to amplification from the retained DAZ genes.

Following our previous study on Yq PCR screening of infertile men (Ferlin et al., 1999Go), we have analysed the expression of DAZ and, for comparison, of RBM (RNA binding motif) and SRY (sex determining region) genes in testicular cells from infertile men carrying an apparently normal DAZ gene constitution as assessed by PCR on genomic DNA, in order to investigate these aspects and in general to better clarify the role of this gene in idiopathic testiculopathies.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
Expression of Y-linked genes was evaluated in a selected group of patients affected by idiopathic azoospermia (n = 4) or severe oligozoospermia (sperm count <5x106/ml, n = 6) in whom bilateral testicular fine needle aspiration cytology (FNAC) showed severe hypospermatogenesis. Selected subjects presented a normal PCR analysis of Yq, as assessed using 39 sequence-tagged sites (STS) on DNA extracted from peripheral leukocytes, as previously described (Ferlin et al., 1999Go). Details of patient selection, semen analysis, follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone plasma concentrations determination and STS–PCR analysis have been previously given (Ferlin et al., 1999Go), as well as details of FNAC technique and analysis (Foresta and Varotto, 1992Go; Foresta et al., 1992Go, 1995Go; Ferlin et al., 1999Go).

RNA extraction from testicular cells
The presence of DAZ, RBM and SRY mRNA was analysed by means of reverse transcription (RT)–PCR in testicular cells retrieved by FNA. Part of the material retrieved from both testes by FNA for diagnostic purpose was immediately frozen at –20°C and used for RNA extraction. Total RNA was extracted from testicular samples using the RNeasy Method (Qiagen, Germany). Briefly, the cells were recovered by means of centrifugation at 1000 g for 5 min, rapidly homogenized and lysed in the presence of a highly denaturing guanidinium isothiocyanate-containing buffer that immediately inactivates RNases to ensure isolation of intact RNA. Ethanol was added to provide appropriate binding conditions and the sample was then applied to an RNeasy mini spin column where the total RNA binds to the membrane and contaminants are efficiently washed away. Total RNA was then eluted in 30 µl of DEPC-treated water and concentrated to 5 µl by means of Centricon 3 (Amicon, Beverly, MA, USA).

cDNA synthesis
RT of total RNA was performed using 0.2 µg of oligo-dT primer for 1 h at 42°C utilizing 25 U of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim, Germany) in Tris 50 mmol/l (pH 8.3), KCl 75 mmol/l, MgCl2 3 mmol/l, dithiothreitol (DTT) 10 mmol/l, RNasin 1 U (Promega, USA) and 1 mmol/l of dATP, dGTP, dCTP and dTTP in a total volume of 20 µl.

Polymerase chain reaction
PCR was performed using the following primer pairs: sY254 for the DAZ gene (Reijo et al., 1995Go), which amplify the region 1409–1789 (from exon 2 to exon 3) of the genomic DNA of the DAZ gene (clone 63C9); sY14 for the SRY gene (Vollrath et al., 1992Go), which amplify the region 479–948 of the SRY gene; F19/E355 for the RBM gene (Ma et al., 1993Go; Kobayashi et al., 1995Go), which amplify the region 1292–1733 of the RBM-I coding sequence, corresponding to the distal part of exon 11 and the most part of exon 12. PCR was carried out for each gene in 20 µl of reaction volume containing: 6 µl of testicular cDNA diluted 1:20 from total cDNA obtained, Taq polymerase (0.8 U), dNTP (0.2 mM dTTP, dCTP, dGTP, dATP), oligonucleotide primers (10 pmol each) made up in a final concentration of 1x PCR reaction buffer (Tris–HCl 10 mmol/l pH 8.3, MgCl2 1.5 mmol/l, KCl 50 mmol/l). All reagents were obtained from Pharmacia (Milan, Italy). Amplification was performed for 35 sequential cycles, each of them including 1 min of denaturation at 94°C, 1 min of primer annealing at 60°C and 1 min of extension at 72°C; before the first cycle, all samples were incubated for 10 min at 94°C. PCR reaction products were eventually stored at 4°C and then separated on 2% agarose gel by electrophoresis in TAE (Tris–acetic acid–EDTA) buffer at room temperature using a voltage gradient of 8 V/cm for 30–60 min. Experiments were also performed on mRNA samples without AMV–RT to amplify the full length (unspliced) products of SRY, RBM and DAZ. Co-amplification of genomic DNA was also observed due to a contamination that naturally occurs in these circumstances. Furthermore, RT–PCR of glyceraldehyde-6-phosphate dehydrogenase (GAPDH) was used as internal control. In order to obtain a good quality image for Figure 1Go, the RT–PCR fragments from agarose gel were purified and run separately.



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Figure 1. Reverse transcription–polymerase chain reaction (RT–PCR) for DAZ, RBM and SRY genes from testicular cells retrieved by FNA. Lanes A: products of SRY, RBM and DAZ without avian myeloblastosis virus–reverse transcriptase (amplification of testicular DNA, control); lanes B: products of SRY, RBM and DAZ with AMV–RT (amplification of testicular RNA); lanes C: amplification of GAPDH RNA (control). 1: patient with normal spermatogenesis: each gene normally amplified; 2: patient with severe hypospermatogenesis and absent DAZ expression: only RBM and SRY amplified from testicular RNA; 3: patient with Sertoli cell-only syndrome: only SRY is found.

 
RT–PCR from testicular cells was also performed for the same genes in 10 patients affected by obstructive azoospermia, thus representing normal spermatogenesis, as positive controls, and in 10 patients affected by Sertoli cell-only syndrome with normal Yq PCR analysis as well as two patients affected by severe hypospermatogenesis with previously diagnosed DAZ gene deletions, as negative controls. In addition, we studied two patients with a similar severe hypospermatogenesis of known origin (post-mumps orchitis and previous chemo-radiotherapy for non-Hodgkin disease) with normal Yq analysis.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Testicular fine needle aspiration (FNA) allowed us to retrieve a sufficient number of testicular cells to perform RNA extraction with subsequent RT–PCR for the DAZ, RBM and SRY genes, other than to precisely diagnose the testicular alteration related to the seminal pattern, as previously described (Foresta and Varotto, 1992Go; Foresta et al., 1992Go, 1995Go). With this technique, in normal subjects we retrieve Sertoli cells and the different spermatogenic cells, including spermatogonia, primary and secondary spermatocytes, early and late spermatids and mature spermatozoa. Using FNA we have selected a group of patients affected by idiopathic severe hypospermatogenesis, that was characterized by a strong quantitative reduction in the absolute number of germ cells with respect to Sertoli cells, but spermatogenic cells at all maturation levels were present. Sertoli cell-only syndrome was diagnosed when extensive analysis of cytological smears from both testes revealed a complete absence of germ cells. In obstructive azoospermic men FNAC showed the presence of a completely normal spermatogenic process with an increased percentage of mature spermatozoa due to intratubular stasis (Foresta and Varotto, 1992Go; Foresta et al., 1992Go, 1995Go; Ferlin et al., 1999Go).

Patients were considered normal for the DAZ gene when PCR on genomic DNA normally amplified primer pairs sY277, sY254, sY279, sY283 and sY255; deletions of this gene were considered when all five primers repeatedly failed to amplify. Similarly, RBM and SRY were studied by PCR using primer pairs F19/E355 and sY14 respectively.

The SRY gene is expressed in different testicular cells (Sinclair et al., 1990Go; Clepet et al., 1993Go; Tricoli et al., 1993Go) and therefore its analysis by RT–PCR has been chosen as an internal control for the presence of testicular mRNA. However, sY14 does not span an intron and therefore it cannot distinguish between amplification of DNA and RNA. On the contrary, the RBM gene, like DAZ, is expressed only in germ cells (above all spermatogonia and spermatocytes) (Elliott et al., 1997Go, 1998Go) and the primers used for its detection amplify across regions containing introns, therefore being an internal control for the presence of mRNA from spermatogenic cells. Primer pairs sY14 and F19/E355 utilized for RT–PCR analysis of SRY and RBM genes produced amplification products of 472 bp and 441 bp, respectively, which correspond to the normal lengths based on cDNA sequences (Sinclair et al., 1990Go; Ma et al., 1993Go). As expected, the product size of sY14 in the absence of AMV–RT was again 472 bp, while that of F19/E355 was ~800 bp, as previously reported. Primers sY254 amplify part of exons 2 and 3 of the DAZ gene and its RT–PCR product was of 106 bp, confirming DAZ cDNA sequence (Reijo et al., 1995Go), while the length of the unspliced (testicular DNA) product was 380 bp, corresponding to the genomic DAZ sequence. Table IGo shows the primers used for RT–PCR with their product size. In all samples a normal RT–PCR amplification was obtained for a basic gene, such as GAPDH.


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Table I. Primers used to amplify SRY, RBM and DAZ RNA from testicular cells by reverse transcription–polymerase chain reaction
 
RT–PCR analysis from testicular cells gave normal amplifications for SRY, RBM and DAZ mRNA in 10 obstructive azoospermic men and in two patients with severe hypospermatogenesis of known origin; only SRY amplification was seen in 10 patients affected by Sertoli cell-only syndrome, confirming the complete absence of germ cells in these testicular preparations. In two patients with severe hypospermatogenesis and known DAZ gene deletions, testicular RT–PCR analysis confirmed the PCR results on genomic DNA, showing normal amplification of SRY and RBM mRNA but not of DAZ. These results, together with normal amplification of GAPDH from each samples allowed us to consider the validity of our experimental conditions. Nine of the 10 patients affected by severe hypospermatogenesis with normal PCR analysis of the DAZ gene on genomic DNA had normal RT–PCR results for SRY, RBM and DAZ mRNA, while one patient showed no amplification of sY254 (DAZ) with normal results for SRY and RBM. Table IIGo shows the relationship between cytological analysis, genomic PCR analysis and testicular RT–PCR results, while Figure 1Go shows examples of RT–PCR. Repeated semen collections from the subject with absent DAZ gene expression repeatedly revealed complete azoospermia. Notably, the testicular cytological picture from this patient was not different from the other subjects affected by severe hypospermatogenesis and confirmed a previous testicular open biopsy performed 1 year before that showed diminished seminiferous tubule diameter containing an increased number of Sertoli cells and a very low number of spermatogenic cells, including spermatids and rare spermatozoa.


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Table II. Results of reverse transcription–poymerase chain reaction (RT–PCR) analysis for SRY, RBM and DAZ mRNA from testicular cells observed in this study
 
Testicular volumes were significantly lower and FSH plasma levels significantly higher in patients with Sertoli cell-only syndrome or severe hypospermatogenesis with respect to normal values (10.6 ± 3.9 ml and 17.1 ± 6.4 IU/l, respectively, P < 0.05), without differences in LH and testosterone plasma levels, confirming the primary testiculopathy involving only the spermatogenic system (data not shown). These parameters were not different in the patient with absent DAZ mRNA with respect to the other patients affected by severe hypospermatogenesis.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microdeletion analysis of Yq has become an important tool in the diagnostic process of infertile patients, especially for candidates for assisted reproductive techniques. Therefore, this analysis is routinely performed in most reproductive centres and it is easily done by a simple PCR method on DNA extracted from peripheral leukocytes utilizing a set of Yq-specific STS. These primers are selected to amplify the few genes with suspected functions in spermatogenesis, namely RBM, DAZ and DFFRY (Brown et al., 1998Go; Mazeyrat et al., 1998Go) as well as other Yq loci that may be deleted in infertile men that include AZFa, AZFb and AZFc regions (Reijo et al., 1995Go, 1996Go; Najmabadi et al., 1996Go; Qureshi et al., 1996Go; Vogt et al., 1996Go; Foresta et al., 1997Go, 1998Go; Girardi et al., 1997Go; Pryor et al., 1997Go; Simoni et al., 1997Go; Stuppia et al., 1997Go; Grimaldi et al., 1998Go; Liow et al., 1998Go; McLachlan et al., 1998Go; Silber et al., 1998Go; Vogt, 1998Go; Kleiman et al., 1999Go; Ferlin et al., 1999Go). DAZ is the most frequently deleted gene in azo-oligozoospermic patients (Reijo et al., 1995Go; Najmabadi et al., 1996Go; Reijo et al., 1996Go; Vogt et al., 1996Go, 1997Go; Foresta et al., 1997Go; Girardi et al., 1997Go; Pryor et al., 1997Go; Foresta et al., 1998Go; Grimaldi et al., 1998Go; Liow et al., 1998Go; McLachlan et al., 1998Go; Silber et al., 1998Go; Vogt, 1998Go; Ferlin et al., 1999Go), but the mechanism by which its absence causes a spermatogenic impairment is still unclear, since little is known on the role of this RNA-binding protein in human germ cell development. Most difficulties in studying the DAZ gene and its functions derive from its multicopy nature, that prevents the detection of mutations within the gene or deletions not involving the entire gene cluster (Saxena et al., 1996Go; Vereb et al., 1997Go; Yen et al., 1997Go), therefore not allowing examination of the role of each DAZ copy.

To better understand the functional activity of the DAZ gene and to overcome misleading results obtained by PCR analysis on genomic DNA, in this study we analysed and compared by RT–PCR the expression of DAZ, and for comparison that of RBM and SRY genes, in testicular cells from infertile men affected by obstructive azoospermia, Sertoli cell-only syndrome and severe hypospermatogenesis. This latter testiculopathy was selected, and not maturation arrests, since DAZ deletions have been so far more clearly correlated with a quantitative reduction of germ cells instead of with impairment of the maturation progression. Sertoli cell-only syndrome was selected as control. All subjects presented a normal Yq, as shown by PCR analysis on genomic DNA extracted from peripheral leukocytes. Normal amplification of such genes was observed both when spermatogenesis was completely normal (obstructive forms) and when it was quantitatively reduced, but with presence of all germ cell subtypes (severe hypospermatogenesis), whereas only SRY was detected when only Sertoli cells were present. These findings confirmed previous studies, clearly demonstrating that RBM and DAZ are transcribed in a germ cell-specific manner (Elliott et al., 1997Go, 1998Go; Menke et al., 1997Go; Habermann et al., 1998Go; Lee et al., 1998Go), even if RT–PCR did not enable us to distinguish in which spermatogenic cell subtype these genes are specifically expressed. The expression of SRY cannot be determined in our experiments, since this gene does not contain introns; and therefore RT–PCR analysis could not distinguish between amplification of DNA or RNA; however, since no genomic DNA was amplified with RBM and DAZ primers in these reactions, we can assume RNA has been identified.

Nine out of 10 patients affected by idiopathic severe hypospermatogenesis showed the presence of normal testicular mRNA for RBM and DAZ, while in one patient no DAZ transcript was detected, suggesting that in this case the testiculopathy was likely to be related to the absence of DAZ expression. The lack of DAZ mRNA detection in testicular cells with an apparently normal DAZ gene constitution on DNA extracted from leukocytes may be explained by different hypotheses.

At the moment none of these hypotheses (deletion of the functional DAZ copies, mosaicism, abnormalities of transcription or post-transcription) can be verified, and confirmation of the association between absent DAZ expression and severe testiculopathy is difficult. Northern or Western blot experiments on testicular tissue were not possible since the material retrieved by FNA was not sufficient for these analyses. Furthermore, no experiments were possible on spermatozoa, as the patient was repeatedly found to be azoospermic.

The testicular picture of the reported patient is very similar to that reported in the presence of a specific genomic DAZ deletion (Reijo et al., 1995Go, 1996Go; Najmabadi et al., 1996Go; Vogt et al., 1996Go; Foresta et al., 1997Go, 1998Go; Girardi et al., 1997Go; Pryor et al., 1997Go; McLachlan et al., 1998Go; Silber et al., 1998Go; Vogt, 1998Go; Ferlin et al., 1999Go). Several lines of evidence indicate that the absence of DAZ is more likely to produce a depopulation of germ cells, and both cytological (by FNA) and histological (by open biopsy) examination in the reported patient showed a picture of severe hypospermatogenesis, characterized by a quantitative reduction of spermatogenic cells with conserved relative proportions. These data further suggest that DAZ may induce, by unknown mechanisms, a depopulation of spermatogonia without affecting mitotic and meiotic phases.

Expression of RBM has been observed in all patients affected by idiopathic hypospermatogenesis. Given the small number of patients at risk for Yq microdeletions examined in this study and the low prevalence of RBM deletions in infertile men (Reijo et al., 1995Go, 1996Go; Najmabadi et al., 1996Go; Qureshi et al., 1996Go; Vogt et al., 1996Go; Foresta et al., 1997Go, 1998Go; Girardi et al., 1997Go; Pryor et al., 1997Go; Liow et al., 1998Go; McLachlan et al., 1998Go; Silber et al., 1998Go; Vogt, 1998Go; Ferlin, 1999), this finding is not surprising. However, this gene is in multiple copies both in the short and in the long arm of the Y chromosome, representing active genes as well as pseudogenes (Chai et al., 1997Go, 1998Go; Elliott et al., 1997Go; Gläser et al., 1997Go). Primer pairs used for RT–PCR in this study amplify a region between exon 11 and 12 (outside the SRGY box) and we observed a single amplification product that may originate from a single gene or from different genes with sequence homology in this region. In fact, the RBM transcripts substantially differ only in the number of copies of the SRGY box (Prosser et al., 1996Go; Chai et al., 1997Go), and it is possible that the region examined in the present study may not harbour significant differences among the RBM copies detected by RT–PCR.

Finally, the finding that patients with severe hypospermatogenesis may be infertile because of the lack of DAZ activity despite an apparent normal peripheral constitution of this gene has two other major consequences. It highlights the intrinsic interpretative difficulties of normal PCR analysis for DAZ, as well as for RBM, on leukocytes and it further suggests caution in the use of ejaculated spermatozoa and intratesticular cells for assisted reproductive techniques, since it has been demonstrated that spermatozoa carrying Yq deletions are able to fertilize and to give rise to pregnancies by means of these techniques (Kent-First et al., 1996aGo,bGo; Mulhall et al., 1997Go; Rossato et al., 1998Go; Silber et al., 1998Go).


    Acknowledgments
 
The financial support of Telethon–Italy (grant no. E.C699 E.C0588) is gratefully acknowledged.


    Notes
 
3 To whom correspondence should be addressed at: Clinica Medica 3, University of Padova, Via Ospedale 105, 35128 Padova, Italy Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 1, 1999; accepted on May 24, 1999.