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Principal Investigator : Sher Ali Co-principal Investigator Ph D Students Collaborator Genome
analysis enables understanding organization, evolution and expression of genes
and the development of genetic markers useful for conventional and inventional
diagnosis. Our work relates to the analysis of human Y chromosome in normal
subjects and patients with sex chromosome anomalies. The human Y chromosome is
much smaller and harbors fewer genes than the X chromosome. However, almost
entire Yq region (comprising about 45 million sequences) seems to be critical
for normal spermatogenesis. Thus, sequence analysis of Yq region is envisaged
to uncover wealth of information useful for molecular diagnosis as well as for
understanding its more focussed role in spermatogenesis in the overall context
of autosomal gene(s). Marker
chromosome representing aberrant Y with insertion/deletion mutations was
analyzed further for possible origin of the insertional mutations.
Accordingly, subset sequence of DYZ3 locus representing centromeric
heterochromatin was PCR amplified from HK-459, her parents and two normal
males. The resultant 120 base pair fragment cloned and sequenced was found to
be normal in the patient. GenBank database was searched for possible origin of
32 nt (insertional) sequences in the marker chromosome. No significant
homology was found with any of the entries and thus the origin of these
sequences remained unknown. Another
proband (P-20), a XY female with stigmata of Swyer syndrome showed mutation(s)
in both 5’ and 3’ regions of the HMG box in the SRY gene, not reported
earlier. Computer simulation (http://dot.imgen.bcm.tmc.edu:9331/seq-search/struc-predict.html)
indicated that the aforesaid mutations change the configuration of the SRY
peptide altering the secondary structure of the protein. These changes perhaps
alter the spatial organization of the SRY protein rendering it incapable of
acting accurately as the transcription factor. In
addition to the analysis of 17 patients reported last year, 18 more samples
were screened for the mutational analysis of Y-related loci including PABY1,
SRY, DYZ3, DYS1 and DYZ1following the experimental approach mentioned earlier.
In a total of 35 samples screened, PABY1 was found to be affected (60%), SRY
(48.57%), DYZ3 (68.5%), DYS1 (57.14%) and DYZ1 (51.42%). Of the five loci,
DYZ3 seem to be maximally affected. However, this will remain inconclusive
until more number of patients samples are screened. The details of samples
analyzed and the loci affected are summarized in Figure 1. Yq region of the human Y chromosome is known to harbor different types of repetitive sequences, a number of which have been acquired in due course of evolution and faithfully retained. However, their biological function is not yet fully understood. Working on the hypothesis that these regions may even harbor minisatellite(s) or associated sequences, we conducted minisatellite associated sequence amplification (MASA) using consensus repeat of 33.15 locus. MASA uncovered a male specific band of 513 bp in normal males (Figure 2).
Figure
– 2:
Minisatellite associated sequence amplification (MASA) of human genomic DNA
detecting male specific band. MASA reaction was carried out on human male and
female genomic DNA with a 16 nt primer representing the consensus sequence of
33.15 repeat loci. The MASA product was resolved on 40 cm long 3.5%
non-denaturing polyacrylamide gel. fX174 DNA predigested with HaeIII was used
as the molecular size marker (lane M). Note the 513 bp male specific band in
lanes 1-5 () representing Y chromosome related heterochromatic sequences. This
seems to be the first observation where sequences associated with
minisatellite 33.15 are shared by Y chromosome related heterochromatin.
Cloning and sequence analysis of this 513 bp band (Accession No. AF-134482)
showed high level of homology with several human Y chromosome derived clones
(Table-1). Table-1: MASA generated fragment showing sequence
homology with several Y chromosome derived clones.
Based on the GenBank entries on Y chromosome
related clones, using default server of NCBI However, similar MASA analysis with patients DNA,
positive for Y chromosome failed to show such male specific band indicating
that the minisatellite related heterochromatic sequences are perhaps
obliterated or modulated. Additional analysis of such samples will resolve if
the sequence obliteration/modulation is indeed operative. The MASA complements
other experimental approaches for the identification of cryptic Y-chromosome
in patients with varying degrees of mosaicism. Turner
patients often show cell mosaicism involving both X and Y chromosomes or
aberrated sex chromosomes. The homozygous (XX) or heterozygous (XY) sex
chromosome constitution is suggested to be essential for survival of the
conceptuses during early embryogenesis and for normal development. Thus,
patients having 45, X/46, X, +mar or other mosaic conditions survive owing to
the presence of much needed critical sequences on the homologous loci.
However, their ensuing post-natal normal development and eventual fertility
status is seriously affected. Thus, the study on the origin of marker
chromosome is of relevance to assess if the same represents an aberrated Y
chromosome or intact one because patients with Y mosaicism and dysgenic gonads
are at a greater risk of developing gonadoblastoma. The DYZ3 locus
corresponds to the centromeric heterochromatin on Y whose presence is
imperative for persistent propagation of this locus in subsequent cell
division. The other locus DYS1 lies within the euchromatic region on the Yq
harboring azoospermic factor (AZF). In our study on the patient HK-495,
ultrasonography did not detect ovaries or any kind of streak gonads though
patient and her mother both were positive for DYS1 locus and the mother was
normal and fertile. It is therefore inferred that owing to non-disjunction of
the X chromosome during oogenesis in mother, patient inherited normal X
chromosome from her father and marker chromosome from mother. This argument is
supported by the fact that in none of the cells in the patient, father’s
(normal) Y chromosome was detected. Sequence analysis of the DYS1 region in
the patient, her parents and normal males showed polymorphism, which was in
accordance with the earlier reports. Deletion/insertion within the DYS1 region
in the patient, as opposed to the rest of the individuals including her mother
suggests that this locus has undergone additional sequence modulation. The
presence of DYS1 locus harboring AZF sequences in the patient and her mother
seem to be inconsequential probably because either critical genes in this
region are not expressed or expression is not achieved to the desired level
due to haplo-insufficiency. Alternatively, AZF locus remained inactive in the
absence of SRY and other genes (on the Y- derived marker chromosome) involved
with the development of male gonads. Diagnostic potentials of the above
mentioned loci There may be several
still uncharacterized Y linked loci critical for maintenance of its genetic
integrity. However, five loci reported herein enabled us to detect aberrant Y
including marker chromosome in the patients with sex chromosome anomalies. The
present work suggests that gross deletion of both the arms of Y chromosome
including those of SRY and pseudoautosomal region has given rise to marker
chromosome. However, the Y derived marker chromosome has retained the
centromeric and euchromatic sequences. Although marker chromosome in the
patient represented the aberrant Y, in the absence of SRY and other regulatory
sequences, development of male gonads is not envisaged, which in turn, also
exclude the possibility of its neoplastic development. Analysis of more number
of such Y derived marker chromosome(s) would enable to ascertain, if any of
the above-mentioned loci is preferentially affected in Turner cases. The
present study is envisaged to complement routine diagnosis of the aberrant Y
or its derived marker chromosome facilitating management of the Turner cases
and even genetic counseling. Publications Original peer-reviewed articles 1.
Bashamboo A and Ali S (2001) Minisatellite associated sequences
amplification (MASA) of the hypervariable repeat marker 33.15 reveals a male
specific band in humans. Molecular and Cellular Probes (in press). |
