Article Polymorphic variants on chromosomes probably play a significant role in infertility

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1 RBMOnline - Vol 11. No Reproductive BioMedicine Online; on web 29 September 2005 Article Polymorphic variants on chromosomes probably play a significant role in infertility Dr Prochi Madon is an Honorary Geneticist at Jaslok Hospital and Research Centre, Mumbai, India, where she set up the Genetics and PGD Laboratory in the Department of Assisted Reproduction in She specializes in cytogenetics and was instrumental in introducing FISH as a rapid, easily available diagnostic test in India, especially for prenatal samples and common haematological malignancies. She obtained her PhD in Applied Biology from the University of Bombay in She is the recipient of the Young Scientist Award in cytogenetics and the Lady Tata Memorial Trust senior research scholarship. She lectures extensively on topics related to cytogenetics. Dr Prochi Madon Prochi F Madon 1, Arundhati S Athalye, Firuza R Parikh Department of Assisted Reproduction and Genetics, Jaslok Hospital and Research Centre, Mumbai , India 1 Correspondence: prochi_madon@yahoo.com; prochimadon@hotmail.com Abstract Polymorphic variants on chromosomes are considered normal, as heterochromatin has no coding potential and nucleolar organizing regions (NOR) contain genes coding for rrna. Variants have been reported in infertility and recurrent abortions. With refined molecular techniques, genes for fertility and viability are now thought to reside in heterochromatin. DNA sequence analysis of human chromosome 9 has shown that it is highly structurally polymorphic, with many intrachromosomal and interchromosomal duplications, and contains the largest autosomal block of heterochromatin. Transcriptional activation of constitutive heterochromatic domains of the human genome in response to environmental stress was reported recently. Heat shock triggers the assembly of nuclear stress bodies on the pericentromeric heterochromatin of human chromosomes including chromosome 9. These are characterized by an epigenetic status typical of euchromatic regions. On acrocentric chromosomes, NOR-associated protein count and morphology was reported to separate benign and malignant melanocytic lesions. Hence all variants may not be normal. The present study of karyotyping 842 individuals attending an IVF clinic with primary infertility or repeated miscarriages, showed polymorphic variants in 28.82% of males and 17.19% of females, which was quite high. It is suggested that variants should not be ignored by cytogeneticists. Screening prospective gamete donors for chromosome variants may help enhance the success of IVF. Keywords: acrocentric stalks and satellites, activation of constitutive heterochromatin, chromosome polymorphic variants, gamete donors, heat shock transcription factors, heterochromatic block on chromosome Introduction Recent progress in the study of the cell nucleus has increased the interest in euchromatin and heterochromatin, the two functionally different parts of the genome, which can be visualized on chromosomes (Kalz and Schwanitz, 2004). Euchromatin is the most active portion of the genome within the cell nucleus (Frenster, 1974) and is gene-rich. Heterochromatin is of two types: constitutive, a stable form present in polymorphic variants; and facultative, a reversible form as seen in the inactive X chromosome or Barr body. Constitutive heterochromatin is formed by tandemly organized highly repeated sequences of satellite DNA that do not encode proteins (Evans et al., 1974b). It does not contain any unique sequences and has no apparent coding potential (Rooney, 2001). The precise role of heterochromatin in the human genome long remained a mystery, as its frequent polymorphisms did not appear to have any functional or phenotypic effect (Mattei and Luciani, 2003). These variants are presently considered normal and are often transmitted from one of the parents, though they could arise de novo. It is now known that heterochromatin is not inert, and is essential for cell and organismal viability in multicellular eukaryotes. Genes required for viability and fertility, are thought to reside in heterochromatin, as are elements required for normal chromosomal inheritance. Heterochromatin plays an essential role in spindle attachment and chromosome movement, meiotic pairing, and sister chromatid cohesion (Karpen and Endow, 1998). Polymorphic variants on non-acrocentric chromosomes usually occur in the paracentric heterochromatin on the long arms of chromosomes 1, 9, 16 and distal heterochromatin of the Y chromosome. Increase in length of the heterochromatic regions

2 on the long arms of these chromosomes, are designated as 1qh+, 9qh+, 16qh+ and Yqh+. Increase in length of the short arm satellites and stalks of the acrocentric D and G group chromosomes (13, 14, 15, 21 and 22) are designated, for example, as 14ps+ and 13pstk+ while increase in length of the short arms themselves are designated as p+ (e.g. 15p+) (Mitelman, 1995; Borgaonkar, 1997). The short arms and satellites of acrocentric chromosomes also contain heterochromatin (Gosden et al., 1981), while the stalks contain repeats of genes coding for 18S and 28S rrna and ribosomal proteins that coalesce to form the nucleolus and are known as the nucleolar organizing regions (NOR) (Makalowski, 2001). Human beings are highly polymorphic with respect to a number of such polycistronic genes (Henderson et al., 1972; Evans et al., 1974a). Therefore, polymorphisms observed in these regions may not essentially be of neutral selective value, and some of the reports suggesting positive association with clinical anomalies could be the result of fluctuations in these regions beyond the limits of tolerance. Review of literature and changing views on heterochromatin There are many reports with conflicting views on the clinical effect of chromosome variants. Some have reported the presence of variants in different clinical conditions such as reproductive failure (Fryns et al., 1985; Sasagawa et al., 1998; Lissitsina et al., 2003; Parikh et al., 2004; Madon et al., 2005), couples with recurrent spontaneous abortions (Patel et al., 1996; Thomas, 1999) and even psychiatric disorders (McConville et al., 1983; Di Gennaro et al., 2004). Reduced heterochromatin content has been found to be associated with disorders such as Noonan syndrome (Podugol nikova and Solonichenko, 1994). Earlier cytogenetic studies (Patil and Lubs, 1977; Nielsen, 1978) on over 10,000 individuals suggested that long Y chromosomes were associated with fetal loss. These were supported by other studies (Genest, 1979; Westlake et al., 1983), though some showed no association between chromosome polymorphisms and recurrent spontaneous abortions (Hemming and Burns, 1979; Blumberg et al., 1982). Others reported that variants of the Y chromosome had no influence on the sperm count and fertility of men (Kalantari et al., 2003). Cases of very large satellites in acrocentric chromosomes of infertile men have also been reported (Pena Videa et al., 2001). Higher frequencies of satellite variants have been observed in adults with reproductive failures (Tsenghi et al., 1976; Rosenmann et al., 1977) child psychiatric populations (Crandall et al., 1972, Christensen and Nielsen, 1974; Say et al., 1977; Funderburk et al., 1978), and in children referred for cytogenetic examination (Krag-Oslen et al., 1980) when compared with the normal population. In a study of spontaneous abortions, Holbek et al. (1974) reported a statistically significant difference for the ph+ and s+ variants that are more frequent in the parents of clinically abnormal and normal abortuses. An exhaustive review of human population cytogenetics (Bhasin, 2005) covers many more studies. Variations in chromosome 9 Among the non-acrocentric human chromosomes, chromosome 9 presents with the highest degree of morphological variations. The mechanisms of origin of inversion 9 (inv 9) are highly complex (Verma, 1999). It was suggested that inversion 9 might have some interchromosomal effect leading to a higher incidence of mitotic disturbances and it is known to be associated with aneuploidies such as trisomy 21 (Murthy and Prabhakara, 1990). Since the introduction of fluorescence in-situ hybridization (FISH) techniques, a few systematic studies have been performed to classify chromosome 9 heteromorphisms in more detail. A human chromosome 9-specific alphoid DNA repeat spatially resolvable from satellite III DNA by FISH was isolated (Rocchi et al., 1991). In one study, four different types of inversion and duplication in the 9qh+ region were reported (Macera et al., 1995) and in all, seven different types of rearrangements leading to inversions on chromosome 9 were described (Ramesh and Verma, 1996; Samonte et al., 1996). However, neither the origin nor the clinical significance of these heteromorphisms was well understood (Teo et al., 1995). Later investigations revealed the fact that constitutive heterochromatin was important for gene regulation, thereby modifying gene inactivity in various ontogenetic phases and specific tissues (Brown et al., 1997). The centromeric regions of human chromosomes are poorly assembled by current mapping and sequencing efforts, as they are poor in euchromatin but repeat-rich, and organized in a highly complex fashion (Horvath et al., 2001). Using FISH with three differentially labelled chromosome 9 specific probes, 12 heteromorphic patterns were distinguished in addition to the most frequent pattern defined as normal (Starke et al., 2002). Although polymorphisms of heterochromatic regions are considered as normal variants in humans, a high frequency of chromosomal variants in infertile men could support the opinion that the large heterochromatic blocks may destabilize the pairing of chromosomes and cause meiotic arrest, resulting in infertility (Lissitsina et al., 2003). DNA sequence and analysis of human chromosome 9 recently revealed that it is highly structurally polymorphic and contains the largest autosomal block of heterochromatin, which is heteromorphic in 6 8% of humans. Analysis of the sequence also revealed many intra- and interchromosomal duplications including segmental duplications, adjacent to both the centromere and the large heterochromatic block (Humphray et al., 2004). A brief review here of recent reports which have changed earlier views on heterochromatin will help in stimulating renewed interest in chromosome variants, and could lead to the clinical application of basic research. Transcriptional activation of constitutive heterochromatin due to stress stimuli The first report of transcriptional activation of a constitutive heterochromatic portion of the human genome in response to stress stimuli such as heat shock (Rizzi et al., 2004) showed that heterochromatic domains including those present on chromosome 9 are characterized by an epigenetic status typical of euchromatic regions. Stress proteins are a group of proteins that are present in all cells in all life forms. They are induced when a cell undergoes various types of environmental stress such as heat, cold and oxygen deprivation. Exposure of cells to stressful conditions results in the rapid synthesis of a subset of specialized proteins termed heat shock proteins (HSP) which function in protecting the cell against damage. Heat shock proteins are also present in cells under perfectly normal conditions. They act like chaperones, making sure that the cell s proteins are in the right shape and in the right place at the right time. For example, HSP help new or distorted proteins fold into shape, which is essential for their function. They also 727

3 728 shuttle proteins from one compartment to another inside the cell, and transport old proteins to garbage disposals inside the cell. HSP are also believed to play a role in the presentation of peptides on the cell surface to help the immune system recognize diseased cells. The stress-induced activation of HSP genes is controlled by the heat shock transcription factor 1 (HSF1). At the cellular level, one of the most striking effects of stress is the rapid and reversible redistribution of HSF1 into a few nuclear structures termed nuclear stress granules, which form primarily on the 9q12 locus (heterochromatic portion) in humans and also contain a subset of RNA processing factors. Within these structures, HSF1 binds to satellite III repeated elements and drives the RNA polymerase II-dependent transcription of these sequences into stable RNAs, which remain associated with the 9q12 region for a certain time after synthesis, even throughout mitosis. Other proteins, in particular, splicing factors, are also shown to relocalize to the granules upon stress (Metz et al., 2004). Evidence for transcriptional activity within a locus considered so far as heterochromatic and silent and the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure has also been revealed (Jolly et al., 2004). These intriguing findings will certainly give research on these structures a new twist (Sandqvist and Sistonen, 2004). The central role for satellite III transcripts in the targeting and/ or retention of splicing factors into the granules upon stress has been highlighted (Metz et al., 2004). Heat stress has also been shown to induce RNA polymerase III-mediated transcription of repeat sequences in both human and mice. Together, the results suggest that transcription of many heterochromatic regions might be revealed in future, given the right stress conditions (LeBrasseur, 2004). Heat shock transcription factor on the Y chromosome (HSFY) Recently HSFY, which is similar to the HSF family has been mapped on the human Y chromosome in the euchromatic AZFb region as multicopies. Deletion of this region results in severe male infertility. HSFY belongs to the heat shock factor family, which has been shown to be implicated in spermatogenesis both in animals and humans. The characterization of the genomic structure, the number of copies on the Y chromosome and the expression of the gene was recently reported (Tessari et al., 2004). Expression analysis revealed that the three HSFY transcripts are differentially expressed, transcript 1 being present in many tissues including testis and ejaculated spermatozoa, and transcripts 2 and 3 being testis-specific. These data suggest that HSFY could have an important role in human spermatogenesis. Newly available sequence data show that only the HSFY gene located on Yq has a long open reading frame containing a HSFtype DNA-binding domain. HSFY is similar to LW-1 on the human X chromosome. Comparison of the presumed DNAbinding domains showed that the HSF-like factors, HSFY and LW-1 belong to a different class than conventional HSF. When deletions on the Yq of males suffering from infertility were screened (Shinka et al., 2004), it was found that HSFY was involved in interstitial deletions on the Y chromosomes for two azoospermic males who had DBY, USP9Y, and DAZ but did not have RBMY located on the AZFb. Expression analysis of factors such as HSFY and LW-1 suggested that they are expressed predominantly in testis. Furthermore, immunhistochemistry of HSFY in testis showed that its expression is restricted to both Sertoli cells and spermatogenic cells and that it exhibits a stagedependent translocation from the cytoplasm to the nucleus in spermatogenetic cells during spermatogenesis. These results may suggest that deletion of HSFY is involved in azoospermia or oligozoospermia. Another recent study also reported a deletion of a novel heat shock gene on the Y chromosome associated with azoospermia (Vinci et al., 2005). Materials and methods Karyotyping was carried out by 72-h culture of whole blood on 842 individuals, mainly couples, attending the IVF clinic of the hospital over a period of 6 years. They were divided into two groups according to their indication. Group A consisted of couples with primary infertility, who failed to achieve conception despite trying for more than 2 years. Group B included couples who had two or more miscarriages, mainly in the first trimester. Giemsa banding (GTG) was done in all cases. Quinacrine fluorescence staining was done to confirm inversion Y. At least 20 metaphases were analysed visually and three karyotyped using Metasystems Ikaros software and a Zeiss Axioskop microscope. Large heterochromatic polymorphisms of chromosomes 9 and Y, which are easily visible by G-banding, were scored. Prominent polymorphisms of the acrocentric chromosomes were also recorded. The criteria used to score variants (qh+, ps+, pstk+) on autosomes was that the variant should be at least twice the size of its corresponding region on the other homologue in all the metaphases screened. The length of the Y chromosome was similarly compared with chromosome 22. Occasional metaphases with spreading artefacts were not taken into account. All slides, fixed cell pellets and karyotype images were archived and patients contact details recorded. Only consistent and very prominent polymorphisms were mentioned in the pictorial reports given to patients, with a note that these were normal variants. FISH was done to characterize or confirm some of the structural and numerical aberrations, but was not used to study the variants. Results The 842 individuals studied comprised 458 men and 384 women. Structural and numerical chromosome anomalies were present in 33 (3.92%) and single cell mosaicism in 26 (3.09%) cases, which will be reported elsewhere. Prominent polymorphic variants on chromosomes 9, Y and acrocentrics were present in 198 (23.52%) cases. Of the 458 men studied, variants were present in 132 (28.82%) cases. These mainly included 36 (7.86%) cases of Yqh+, 28 (6.11%) of 9qh+, 9 (1.97%) of inv(y), 3 (0.66%) of inv(9), and 31 (6.77%) of single acrocentric polymorphisms while multiple variants were seen in 18 (3.93%) men. Of the 384 women studied, variants were present in 66 (17.19%) cases including 36 (9.38%) with 9qh+, 4 (1.04%) with inv(9), 15 (3.91%) with single acrocentric polymorphisms and 11 (2.86%) with multiple variants. The higher frequency of variants in males is due to Y chromosome polymorphisms. The percentages of the main polymorphic

4 variants detected in group A and group B are shown in Table 1. The most striking observation was the association of the 9qh+ variant in 13.86% (28/202) women from group B, while this variant was present in only 4.40% (8/182) women from group A. Partial karyotypes illustrating the types of chromosome variants studied are shown in Figure 1. Pooled published data of the general population (Bhasin, 2005) from several worldwide series showed the frequency of 9qh+, Yqh+ and D/G group variants was 2.44, 2.85 and 3.96%, while in the present study group, the frequency of these variants was 7.60, 7.86 and 8.91% respectively, which is statistically significant (P < 0.001) (Table 2). Table 1. Frequency of main polymorphic variants observed in group A and group B. Group a Gender No. Percentage of variants individuals 9qh+ inv(9) Yqh+ inv(y) D/G Multiple group A Male Female B Male Female a Group A = couples with primary infertility; group B = couples who have had two or more miscarriages, mainly in the first trimester. Figure 1. Different types of polymorphic variants studied. The homologue with the variant is on the right of each pair. 729

5 730 Table 2. Comparison of the percentage of polymorphic variants in the study and control groups. Data from various published population studies (Bhasin, 2005) were pooled as the control group. Group Discussion Percentage of variants (no. cases) 9qh+ Yqh+ D/G group Study 7.60 (842) 7.86 (458) 8.91 (842) Control 2.44 (7995) 2.85 (49,568) 3.96 (73,013) The present cytogenetic study of 842 individuals attending the IVF clinic, showed a high incidence of chromosome variants compared with population surveys (Bhasin, 2005). In the light of recent research, this probably indicates that normal polymorphic variants play a significant, as yet unknown, role in different clinical conditions including infertility. Pooled data from 24 published studies (Bhasin, 2005) on 73,013 individuals worldwide showed the frequency of acrocentric D-group variants to be 2.58% and G-group variants to be 1.38%. The frequency of D and G group variants was therefore 3.96%. The present data on 842 individuals attending the infertility clinic showed a single D or G group variant in 5.46% (46/842) cases, while multiple variants were present in an additional 3.44% (29/842) cases. The frequency of D and G group variants in this study was therefore 8.91% (75/842). Similarly, pooled data on 49,568 males from 51 studies (Bhasin, 2005) showed the presence of a long Y chromosome in 2.85% cases. In the present study, a long Y chromosome was present in 7.86% (36/458) cases. Pooled data on 7995 individuals from 34 series where the frequency of 9qh+ was recorded (Bhasin, 2005) showed that the 9qh+ variant was present in 2.44% cases in the general population. In this series, 9qh+ was present in 7.60% (64/842) cases. Many earlier reports had also shown a high incidence of heterochromatic and acrocentric short arm variants in infertility and spontaneous abortions (Tsenghi et al., 1976; Patil and Lubs, 1977; Rosenman et al., 1977; Nielson, 1978; Genest, 1979; Westlake et al., 1983; Fryns et al., 1985; Patel et al., 1996; Sasagawa et al., 1998; Thomas, 1999; Lissitsina et al., 2003) but were ignored. It is evident from the literature that various authors have utilized different scoring methods (Bhasin, 2005) to study variations in heterochromatin. The present study has accounted for only large polymorphisms which are double or more in size compared with the normal homologue (which acts as an internal standard reference) to avoid inclusion of any disputable cases. The discussion about the significance of constitutive heterochromatin has been controversial for a long time. It was once considered junk DNA (Verma, 1988). The selfish DNA hypothesis (Orgel and Crick, 1980) stated that the frequently repeated DNA sequences associated with heterochromatin were of no value to the organism. It is known that junk DNA is not really junk, and the assumption of its functionality is widespread. Aside from protein coding, DNA sequences may include signals controlling replication. It was suggested (Manuelidis, 1990) that during interphase, chromosomes are localized in specific parts of the nucleus in different cell lines due to a three-dimensional structure imparted to them by folding of junk DNA and may also index different genetic compartments for orderly transcription and replication. Later work (Macera et al., 1995) has shown that non-coding DNA may play a role in the suppression of genes and suggests that some clinical conditions result from changes in non-coding DNA. This enigmatic part of the genome has unusual cytological, molecular and genetic properties, including differential control of replication, condensation throughout the cell cycle, and the ability to silence gene expression (Karpen and Endow, 1998). In both flies and humans, heterochromatin is concentrated in large megabase-sized blocks, predominantly in the centric and subtelomeric regions of all chromosomes. It contains tandemly repeated short sequences of satellite DNAs (e.g. hundreds of kilobases of...aagagaagag...), middle repetitive elements (e.g. transposable elements and ribosomal RNA genes), and some single-copy genes. Genes are only a small part of the genome of the mammal. New epigenetic research is looking for correlations between coding and non-coding DNA and adjacent proteins. Recent investigations on the role of constitutive heterochromatin in the human genome are concentrating on function studies in different ontogenetic stages and might give us new perspectives to the possible importance of its mutations in clinical cytogenetics (Kalz and Schwanitz, 2004). The dynamic characteristics of nuclear domain formation in response to cellular stress have recently been emphasized (Shav-Tal et al., 2005) by demonstrating that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs. Nucleolar segregation is observed under some physiological conditions of transcriptional arrest leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. Contrary to prevailing notion, a groupof nucleoplasmic proteins, mostly RNA-binding proteins, were shown to relocalize from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. The metaphase nucleolar organizer regions (NOR) contain ribosomal genes associated with proteins such as upstream binding factor(ubf) and RNA polymerase I (RPI). These genes are clustered on the short arm stalks of the human acrocentric chromosomes, which exhibit polymorphic variation. Some NORassociated proteins termed AgNOR proteins,can be specifically stained by silver (Ag). A new aspect of human AgNOR, the nonrandom location of differently shaped structures on the five NOR-carrying chromosomes, was described earlier (Heliot et al., 2000). To assess if the quantity of AgNOR proteins predicts the behaviour of actinic keratosis, a standardized AgNOR analysis was performed (Tuccari et al., 2001). It was suggested that AgNOR quantity may help identify actinic keratosis with high proliferative activity and increased tendency to develop into invasive squamous cell carcinoma. Another study of the AgNOR count and morphometry in benign and malignant melanocytic lesions showed that a combination of the two parameters improved their discriminating ability (Li et al., 2003). This demonstrates that in the near future, polymorphic variants may be used as markers for certain clinical conditions.

6 The first report of transcriptional activation of a constitutive heterochromatic portion of the human genome in response to stress stimuli such as heat shock (Rizzi et al., 2004) may serve as a landmark and further extensive research on clinical cases with known heterochromatic variants may help in furthering understanding of certain conditions such as unexplained repeated spontaneous abortions, infertility, idiopathic mental subnormality, psychiatric disorders, attention deficit hyperactivity disorder with autism in children and a host of other conditions. However, scientists are just beginning to unravel the mysteries of heterochromatin and it will take some time before any clinical correlation with variants is established. Active collaboration between cytogeneticists and cell biologists worldwide, wherein selected samples with heterochromatic variants can be sent for research should be encouraged. Variants should be recorded and also mentioned in the patients reports for later use, without causing alarm. This will avoid a repetition of karyotyping in future, if polymorphic variants are found to play a role in certain clinical conditions. In conclusion, based on the evidence from recent publications on transcriptional activation of constitutive heterochromatin, the role of heat shock transcription factors, nucleolar segregation and capping during transcriptional inhibition, the use of NOR in differentiating malignant and benign tumours and data of a high frequency of polymorphic chromosome variants in couples with infertility, it is postulated that some polymorphic variants in heterochromatic and NOR regions could play a significant role in certain clinical conditions. In the light of recent research by cell biologists, suggesting that heterochromatin may have more important cellular roles than previously thought, it is suggested that chromosome variants should not be ignored by cytogeneticists and clinicians, but should be re-evaluated and recorded for future use, as all polymorphic variants may not be normal. With the current practice of oocyte and sperm donation in IVF, karyotyping all prospective gamete donors and screening out those with variants may take us one step further in the quest to enhance the success and take-home-baby rate of IVF. Randomized controlled trials may help to delineate this point. If at a later date, the success rate with variant-free donors does show an improvement, infertile individuals with variants and repeated IVF failures may stand a better chance with screened donor gametes. Further, development of commercially available FISH probes, which could detect specific variants in the couple, could enhance the selection of embryos by preimplantation genetic diagnosis. Acknowledgements We thank our staff, colleagues and entire team for their dedicated effort, technical and clinical support. This work was partially funded by Research Grants RP293 and RP317 from Jaslok Hospital and Research Centre, Mumbai, India. The authors declare that they have no financial or commercial interests in this paper. References Bhasin M 2005 Human population cytogenetics: a review. International Journal of Human Genetics 5, Blumberg B, Shulkin J, Rotter J et al Minor chromosomal variants and major chromosomal anomalies in couples with recurrent abortions. American Journal of Human Genetics 34, Borgaonkar DS 1997 Chromosomal Variation in Man; a Catalogue of Chromosomal Variants and Anomalies. Wiley-Liss, New York. 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