Full text of DOI:10.1007/BF03343790

J. Endocrinol. Invest. 23: 659-663, 2000
Characterization of novel genes in AZF regions
,
,
L. Stuppia * **, V. Gatta*, I. Fogh*** , A.R. Gaspari*, R. Grande*, E. Morizio* ****,
,
,
,
D. Fantasia* ****, A. Pizzuti*****, G. Calabrese* ****, and G. Palka* ****
*
Dipartimento di Scienze Biomediche, Sezione di Genetica Medica, Università “G. D’Annunzio”,
*Istituto di Citomorfologia Normale e Patologica CNR, Chieti; ***Istituto di Clinica Neurologica,
*
Università di Milano, Ospedale Policlinico IRCCS, Milano; ****Servizio di Genetica Umana, Ospedale
Civile di Pescara; *****CSS-Mendel IRCCS Roma, Italy
ABSTRACT. The long arm of the Y chromosome
contains genes mapped in loci involved in male
infertility, short stature and gonadoblastoma.
However, many of these genes have not been
fully characterized, and are not currently inves-
tigated in patients affected by such diseases.
We report a study aimed to the genomic char-
acterization of 5 genes mapped within the Y
chromosome: BPY2, PRY, TTY1, TTY2, and VCY.
Exon-intron boundaries were detected for each
gene, and PCR analysis was carried out to in-
vestigate the involvement of these genes in dif-
ferent re-arrangements of the Y chromosome.
It was found that BPY2 and PRY are lost in some
infertile patients with Yq microdeletions, sug-
gesting that they could play a role in male ga-
metogenesis. On the other hand, these patients
retain some copies of TTY1 and TTY2 genes, due
to the presence of copies in Yp, and of VCY,
which has homologous copies on the X chromo-
some. On the basis of their localization, these
genes could be candidate for the gonadoblas-
toma locus (TTY1, TTY2) and for the control
growth locus (VCY).
(J. Endocrinol. Invest. 23: 659-663, 2000)
©
2000, Editrice Kurtis
INTRODUCTION
sent study we investigated the genomic organiza-
tion and the involvement in different rearrange-
ments of the Y chromosome of 5 genes mapped
within the NRY: BPY2 (basic protein Y 2), PRY (PTA-
BL related Y), TTY1 (testis transcript Y 1), TTY2
(testis transcript Y 2) and VCY (variable charge Y,
formerly BPY1).
The human Y chromosome can be divided into two
regions: the recombinant portion (pseudoautoso-
mal region), undergoing meiotic recombination
with the X chromosome, and the non-recombinant
portion (NRY) (1). This latter region represents the
9
5% of the Y chromosome, and contains genes
playing a crucial role in male sex determination and
fertility (1-3). In recent years, several studies have
demonstrated that about 10% of infertile males are
carriers of Yq microdeletions (4-9), and different
genes, such as DAZ, RBMY, USP9Y (formerly DFFRY),
and DBY have been suggested as candidates for
male infertility (10-14). Moreover, the presence on
the Y chromosome of genes involved in gonado-
blastoma and short stature has also been postu-
lated (15, 16). However, few data are so far avail-
able about the number of copies, genomic orga-
nization, localization and functional role of the ma-
jority of the genes mapped within NRY. In the pre-
MATERIALS AND METHODS
Characterization of genomic structure of BPY2
A human Y chromosome YAC library (Research
Genetics, Huntsville, AL) was screened by PCR with
specific primers for BPY2 (1). One of the selected
YAC clones (yOX101) was sub-cloned into a cos-
mid library. Positive clones for BPY2 were submitted
to sequencing, and compared to the cDNA se-
quence of BPY2 (GeneBank no. AF000980) to de-
tect intron/exon junctions. Primers for PCR ampli-
fication of BPY2 were derived for all exons, except
for exons 1-2, amplified by the primers reported by
Lahn and Page (1).
Key-words: Y chromosome, male infertility, gene families.
Characterization of genomic structure of PRY,
TTY1, TTY2 and VCY
The cDNA sequences for PRY (AF000988), TTY1
(AF000990), TTY2 (AF000991) and VCY (AF000979)
Correspondence: Prof. Giandomenico Palka, Dipartimento di Scienze
Biomediche, Sezione di Genetica Medica, Università “G. D’Annunzio”,
Chieti, Italy.
E-mail: gdpalka@unich.it
659

L. Stuppia , V. Gatta, I. Fogh, et al.
A)
0
5K
10K
15K
20K
25K
PRY (genomic)
clone NH0066M18
BAC clone
RP11-492C2
PRY (cDNA)
Ex.1 Ex.2
Ex.3
Ex.4
Ex.5
B)
Fig. 1 - A) Alignment of cDNA and genomic sequences of PRY with clones NH0427G18 and RP11-492C2; B) Coamplification of ex-
ons 1-2 and 3 of PRY in a fertile male (left) and in patient no. 3 (right), showing deletion of exon 3.
were used for a database screening using the
BLAST program. Genomic sequences with high ho-
mology were detected and aligned with the cDNA
sequences of the five genes. Exon-intron junctions
were detected by the identification of the AG-GT
splice sites in the matching sequences. Specific
primers for the putative exons of each gene were
derived, in order to verify their sequence and size.
tervals 6D-6E, with loss of the DAZ gene (patients 6-
9). PCR reactions were performed with 35 rounds of
the following cycle: 94 C 1 min, 58 C 1 min, 72 C 1
min, except for exon 5 of TTY2 (annealing at 62 C).
RESULTS
Genomic structure of BPY2
Screening of the YAC library with primers for BPY2
showed the presence of a PCR product of about 350
bp in clones yOX101, yOX21, yOX103, yOX190 and
yOX120. YAC yOX101 was sub-cloned in cosmids,
and the cosmid clone 26 identified as containing the
entire genomic sequence of BPY2, which spans 21
Kb and consist of 9 exons. Comparison of BPY2 ge-
nomic sequence with sequences present in Gene-
Bank showed a complete homology with clone
NH0086G22 (AC 006366.2), and a partial homology
for exons 1-2 and 4 with clone NH0109F19.
PCR analysis
PCR was performed on 20 normal males, 5 females,
9
infertile patients with previously detected Yq dele-
tions (patients 1-9) and one female patient with a
6,X,i(Yq) karyotype (patients 10). Four infertile pa-
4
tients had azoospermia (patients 1-4) and 5 oligo-
zoospermia (patients 5-9). Five patients had terminal
deletions, with breakpoint within sub-interval 5Q (pa-
tient 1), 6A (patients 2-4), and 6E (patient 5). The re-
maining 4 cases had interstitial deletions of sub-in-
660

Novel genes in Y chromosome
Genomic structure of PRY, TTY1, TTY2, VCY
sequencing. No band was present in female con-
trols, except for VCY exons. In the patient with an
i(Yq), PCR produced amplification of BPY2, PRY,
VCY and exons 1, 3 and 7 of TTY2, while TTY1 and
exons 2, 4 and 5-6 of TTY2 were not detectable.
All patients with Yq deletions showed normal am-
plification of TTY1, TTY2 and VCY. On the other
hand, patients 1-4 and 6-9 showed absence of
BPY2, although a pale band for exons 1-2 and 4
was sometime observed. All exons of BPY2 were
present in patient 5. Finally, patients 1-4 showed
absence of exons 3, 4 and 5 of PRY (Fig. 1B), while
all exons were present in patients 5-9.
Comparison of cDNA sequences of PRY, TTY1, TTY2
and VCY with genomic sequences present in Gene-
Bank evidenced high homology with clones NH00-
6
6M18 (AC007359.2) and NH047G18 (AC008175.2)
for PRY, the BAC clone RP11-492C2 (AC006335) for
TTY1 and TTY2, and the BAC clone RP11-292P9
(
AC006370) for VCY. Partial homologies were de-
tected also between TTY1 and TTY2 cDNA and clone
NH0066H18, and between PRY cDNA and the BAC
clone RP11-492C2 (Fig. 1A). Alignment among these
sequences allowed to identify the putative genomic
structure of each gene. PRY spans 25 kb and is com-
posed by 5 exons, with the ATG codon within exon
3
. TTY1 spans 21 kb, and is composed by 5 exons.
DISCUSSION
TTY2 spans 17.8 kb and is composed by 7 exons. VCY
spans 734 bp and is composed by two exons only.
The exon-intron organization of the investigated
genes is reported in Table 1.
In this study, we identified the genomic structure
of five genes mapped within the Y chromosome.
Among these, BPY2, PRY and TTY2 have been
mapped within AZFb and AZFc loci, frequently
deleted in infertile males (5). Due to their specific
testis expression, these genes could play a role in
spermatogenesis, but so far their involvement in Yq
PCR analysis
PCR analysis on normal males produced bands of
the expected sizes for all genes, verified by direct
Table 1 - Exon-intron organization of BPY2, PRY, TTY1, TTY2 and VCY genes.
Gene
Exon
Exon size
(bp)
3’ splice site
acceptor
5’ splice site
donor
Intron size
(kb)
BPY2
1
2
3
4
5
6
7
8
9
31
TGATATGgtattcagcc
ACAGAAGgtattttgat
CACCCAGgtaatgtgag
TTCACAGgtactattgt
TCCAAAGgtataacgat
TGTCGAGgtgagttgac
AGATGATgtgagttatc
CACCCAGgtgatgtgag
0.27
3
4.4
2
2.8
0.7
6.5
0.9
123
137
119
118
106
125
125
322
tgtatttcagGGCCCAA
ctggcaatagTGTGCTT
ctgcccacagGTGATAT
tctttcctagGTGCTGC
ctgtccatagTTCGTGT
cattttctagGCCCAGA
tcctgcctagGTCCTGC
ctgcccgcagGTAAAAT
PRY
1
2
3
4
5
88
87
121
116
824
GTCTGAGgtaagagaat
GACTCAGgtatgattgt
CTGCCAGgtactgtcat
TCTGGAGgtgagttgtc
0.1
10
2.5
9.4
cctttcccagAGAGCTT
tctcttttagAGAGACA
tatttttcagGGATGCA
tccattctagATATGGG
TTY1*
TTY2*
1
2
3
4
5
245
160
115
138
258
GAATCGGgtatgatggg
ACCTGAGgtaaattcaa
GGCAATGgtgacattta
AAGATAGgtgagagtgt
3.4
6.8
2.3
7.5
ttatttgcagGTGAAGT
ctctcctagGATCATG
ccttttctagGACTATC
ttttttgcagGTGAAGG
1
2
3
4
5
6
7
1818
87
149
157
79
198
679
GCATTGTgtgagtattt
TTCTCAGgtaagttcct
CGTGGAGgtgagtcagc
CAATAAGgtaataaggt
CTATTGTgtgagtattt
CAATAAGgtaagatgcg
2.4
7.3
0.7
2.8
0.1
1.2
ccttttgcagAGAGGCC
ttttttttagGCTGGCT
tgtctttcagCCAATCC
attggcgtagTTTCCTT
acttctatagAAAAGCT
ttcctttcagGCTGGGT
VCY
1
2
174
368
GAAGAAGgtgagtgacc
0.2
tcccttccagACTACCA
*partial structure, due to the absence of full lenght cDNA of these genes.
661

L. Stuppia , V. Gatta, I. Fogh, et al.
deletions has not been tested. The study of these
genes with primers specific for the single exons al-
lowed to obtain interesting data. In our series, BPY2
gene was lost in all patients with deletion of DAZ, in
agreement with another report (17). The presence
of exons 1-2 and 4 of BPY2 in some patients with
AZFc deletion suggests the existence of multiple
copies of these exons only along the Y chromo-
some, as confirmed by the partial homology of
these regions with clone NH0109F19. These par-
tial copies of BPY2 are likely non-functional. If BPY2
is lost in all patients with deletion of DAZ, it would
be of great interest to investigate the contribute of
BPY2 to the phenotype induced by deletions of
DAZ. In addition to BPY2, also PRY appears to be
involved in Yq deletions. In fact, exons 3-5 of this
gene were absent in patients deleted for the entire
interval 6. Since PRY had been mapped in sub-in-
tervals 4A, 6C and 6E (1), it is possible that only ex-
ons 1 and 2 of PRY are present in Yp, as confirmed
by the analysis of clones containing PRY sequences
present in GeneBank. In fact, clone NH0066M18
contains the entire PRY coding sequence, while on-
ly sequences of exons 1 and 2 are present in BAC
clone RP11-492C2. This BAC clone contains also
TTY1 and TTY2 genes, and likely spans a region of
Yp, since TTY1 have been mapped only in Yp (1).
Thus, only partial PRY copies should be present in
Yp, while copies in Yq contain the entire coding re-
gion. According to the refinement of PRY mapping
within interval 6 reported (18), these copies are con-
tained within AZFb, and PRY could be a candidate
for male infertility due to deletions of this locus.
PCR analysis of TTY2 gene showed an amplifica-
tion product for all exons in patients with Yq dele-
tions, while only exons 1, 3 and 7 were present in
the patient with i(Yq). TTY2 had been mapped with-
in sub-intervals 4A and 6C (1), and the localization
of several copies within interval 6 had been con-
firmed by other studies (18). Also in this case, it is
possible that copies in Yp and Yq are not identical
in the genomic organization. In fact, cDNA se-
quence of TTY2 shows complete homology with
BAC clone RP11-492C2, but only partial homolo-
gy with clone NH0066M18, including sequence of
annealing of primers for exons 1, 3 and 7. Se-
quence analysis aimed to the detection of TTY2 se-
quences specific only for Yq copies are in progress,
in order to test the involvement of this gene in Yq
deletions. Studies able to identify which copies of
TTY2 are expressed could help in clarify whether
or not the loss of copies in Yq of this gene could
be of some relevance in male infertility. Moreover,
copies in Yp of TTY2 are mapped in sub-interval
and therefore we cannot exclude a role of this gene
in this tumor.
TTY1 gene was retained in all patients with Yq dele-
tions, but was lost in the patient with i(Yq), con-
firming that all copies of this gene are mapped on-
ly in Yp (sub-intervals 3C and 4A) (1). This would ex-
clude a role for TTY1 in male infertility, but again
indicates this gene as candidate for gonadoblas-
toma. Since available cDNA sequences of TTY1 and
TTY2 are not complete, other studies are in
progress in order to identify the presence of other
putative exons in their genomic sequences. Finally,
PCR analysis of VCY demonstrated the presence of
copies of this gene also in female samples, in
agreement with a recent report describing the pres-
ence of an X-homologous (VCX) (19). The presence
of X-linked copies do not rule out a role for VCY in
spermatogenesis, since other genes with X-homo-
logues, like USP9Y or DBY, are candidate for male
infertility (13, 20). However, due to its localization in
sub-interval 5G, outside the three AZF loci, VCY
could be involved in short stature rather than in
male infertility, since a putative growth control gene
has been mapped in this sub-interval (16). In con-
clusion, the present study provides new informa-
tion about five genes mapped within the human Y
chromosome. All these genes could be involved in
diseases such as male infertility (BPY2, PRY), go-
nadoblastoma (TTY1, TTY2) and short stature (VCY).
The availability of specific primes for all exons will
be of great help in the study of these genes in pa-
tients with the above reported diseases. Moreover,
these data will allow to obtain genomic probes
which could be used in FISH experiments useful to
establish the number of copies and the localization
of these genes.
ACKNOWLEDGMENTS
This work has been supported by the Italian Telethon (grant no.
E.832) and by the Italian MURST.
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