Androgen receptor CAG repeats, non-random X chromosome inactivation, and loss of heterozygosity at Xq25 in relation to breast cancer risk

Human female cells possess two X chromosomes, while male cells have only one. To compensate for X chromosome gene dosage, one X chromosome in each female cell condenses to form a Barr body. Genes on this chromosome are thus shut down in a phenomenon known as X chromosome inactivation (XCI). Which X chromosome undergoes condensation is generally random, therefore theoretically the ratio of paternal inactivation vs. maternal should be 1:1 in a given mass of cells. This phenomenon is known as Lyonization [1, 2].

However, the pattern of XCI in some women is not random (non-random, or skewed Lyonization). Some research suggests that women with non-random XCI are more likely to be affected by X-linked recessive diseases, such as Wiskott-Aldrich syndrome [3], Hunter’s syndrome [4], Haemophilia [5], Duchenne’s muscular dystrophy [6], and Borjeson-Forssman-Lehmann syndrome [7]. Females are carriers of X chromosome mutations and do not usually express the phenotype because the normal allele can compensate for the defect, hence men are more likely to be affected by X-linked recessive diseases. However, non-random XCI in women can mimic the effects of the single X chromosome in men if the mutated X chromosome is active and the normal X chromosome is inactive, leading to expression of X-linked recessive diseases in women.

The androgen receptor (AR) gene has been used to demonstrate the pattern of X chromosome inactivation [8‐10]. The 5′ CpG island of the AR gene is methylated on the inactive X chromosome and hypomethylated on the active X chromosome. Additionally, the AR alleles of paternal and maternal origins may be distinguished by a highly polymorphic CAG triplet repeat within the region of the CpG island. Moreover, the methylation status of a restriction site (GCGC) that is less than 100 bp away from the polymorphic CAG triplet repeat can be recognized by the methylation-sensitive restriction enzymes HpaII and HhaI[11]. Amplification of the CpG island region by polymerase chain reaction (PCR) following methylation-sensitive restriction enzyme digestion is possible only when the AR gene is fully methylated. Therefore, comparing the AR allele ratio with or without digestion of enzyme is a convenient method to determine whether the pattern of XCI is random or non-random.

The AR gene contains a polymorphic CAG trinucleotide repeat that encodes an uninterrupted polyglutamine tract in the N-terminal domain, ranging from 6–39 repeats in healthy individuals [12]. The number of CAG repeats has been linked with hormone-related cancers in both men and women. Several studies have demonstrated that shorter CAG repeat length is associated with increased prostate cancer risk [13]. Multiple studies have also identified associations between CAG repeat length and the risk of ovarian [14], breast [15, 16] and endometrial cancers [17, 18]. In these studies, the allele patterns were defined as the length of the CAG repeat region. For instance, Terry et al. (2005) found that women with of two long AR alleles (≥22 CAG repeats) had an increased risk of ovarian cancer compared with those possessing two short AR alleles (<22 CAG repeats) [19]. However, the results varied with differences in population and sample size [17, 18].

Deletions on the X chromosome often occur in female cancers. Deletions at Xq25 have been reported to be associated with ovarian cancer [20, 21], and a high frequency of loss of heterozygosity (LOH) has been found on chromosome arm Xq. The study of LOH with a series of polymorphic microsatellite markers at Xq25 in breast cancer patients found that LOH of DXS8098 was associated with larger tumour size (>3 cm), higher histological grade, and axillary lymph node metastasis. This suggests that there could be one or more TSGs in this region and that the DXS8098 locus on Xq25 could be used as a prognostic marker for breast cancer.

According to Knudson’s two hit hypothesis, we hypothesized that female breast cancer patients may have a high incidence of LOH at Xq25 and that their tumour tissues would exhibit non-random XCI. The occurrence of breast cancer may result from inactivation of a TSG in this region. When a mutated X chromosome with LOH at Xq25 is active while the normal X chromosome is inactive; cancer may eventuate. Then, we can examine whether there might be a candidate tumour suppressor gene at Xq25.

LOH at Xq25 was determined by examining the frequency of heterozygosity 11 markers in normal tissue DNA and in patients with breast cancer (Table 1). The frequency of heterozygosity of these markers should be greater than 80%. However, some microsatellite markers had a low heterozygosity frequency (DXS8067, DXS8059, DXS8078, DXS8071, and DXS8074), indicating that the allele distribution varies amongst populations. Highly polymorphic markers have a high frequency of heterozygosity which is necessary for suitable analysis of LOH in tumour DNA. The ultimate goal of this project was to identify a candidate TSG. Chromosome arm Xq25 is one of the most frequent LOH regions observed in breast and ovarian cancers [20]. Here LOH of at least one microsatellite locus at Xq25 was identified in 70.7% (46/65) breast cancer patients, which is higher than the percentages reported in previous studies. In comparison, LOH of at least one locus on 7q31 was detected in 34% of sporadic prostate tumours, and the candidate tumour suppressor gene TES was found here [26]. LOH of at least one locus on 9q was observed in 56% of transitional cell carcinomas (TCCs) in bladder cancer [27], and LOH of at least one locus at 17q21 (BRCA1 locus) was found in 60.53% of breast cancers [28]. Candidate TSGs in these regions were subsequently identified. The high frequency of LOH at Xq25 identified in Taiwanese breast cancer patients here indicates that one or more novel candidate TSGs exists at Xq25.

It is possible that one hit to a TSG localized on the X chromosome could be sufficient to induce carcinogenesis because the other allele is inactivated, and several lines of evidence support the hypothesis of the existence of a tumour suppressor gene on Xq25. The X chromosome is involved in the establishment of in vitro immortality and may therefore be important for the control of cell proliferation [29]. In 2002, it was announced that a candidate TSG on the X chromosome was identified using chromosome transfer experiments [30]. Additionally, deletions at Xq25 have been reported to be associated with ovarian cancer [20], and Piao et al. found a high frequency of LOH on chromosome arm Xq in breast cancer cells [31]. Piao et al. also identified that LOH of DXS8098 at Xq25 is associated with a larger tumour size (>3 cm), a higher histologic grade, and axillary lymph node metastasis, suggesting that there could be one or more TSGs in this region [21]. The frequency of allele loss at Xq25 varies between cancer types, being more common in breast cancers (70.7%) than in other cancers (40%) in our study. These results demonstrate a significant (p = 0.014) correlation between LOH at Xq25 and breast cancer risk. The DXS1047 marker showed the highest LOH (47.23%) of the markers tested, and we hypothesized that the regions adjacent to DXS1047 contain one or more candidate TSGs with important roles in breast carcinogenesis but not in other kind of cancers (Table 1).

The XCI patterns in the 57 breast and other cancer tissues were found to be of NN, NR or RR types (Figures 2 and 3). XCI patterns in the breast cancer tumour tissues tended to be non-random, and differed from the other kinds of cancer examined (p = 0.047). These results are similar to those of previous studies, in which non-random XCI patterns have been found to be a predisposing factor for the development of invasive ovarian cancer [24].

If a TSG is localized on the X chromosome, a single step, either a mutation or loss of the active allele from the active X chromosome, is sufficient to induce carcinogenesis because the other allele congenitally inactivated. Large deletions at Xq25 were identified in eight of the breast cancer samples with all of the informative loci tested at Xq25 found to have LOH. Additionally, the patterns of XCI of these eight breast cancer tumour tissues tended to be non-random, indicating that the origin of these breast cancer tissues was monoclonal. Thus, carcinogenesis in these patients could be partially explained by two hits to a TSG at Xq25. Here, the first hit is X chromosome condensation and the second is the large deletion at Xq25, which presumably occurs on the active chromosome. In summary, the fact that frequent LOH at Xq25 of the X chromosome was observed in breast cancer tissues suggests that the X chromosome may indeed harbour one or more TSGs, which might play an important role in breast cancer carcinogenesis.

In this study, an increasing number of CAG repeats was found to be associated with elevated breast cancer risk. Multiple studies have previously indicated associations between the length of the CAG repeat and the risk of ovarian [14], breast [15, 16] and endometrial cancers [17, 18], whereas some breast cancer studies have reported no association [16, 32].

We compared the CAG repeat length of the breast cancer patients with those of similarly-aged healthy controls, and found that two long AR alleles (≥21 CAG repeats) was associated with an increased risk of breast cancer, while women with two short AR alleles (<21 CAG repeats) were likely to be normal (p = 0.00069). However, when comparing the breast cancer patients with the young healthy controls, no significant relationship was observed (Table 4). This suggests that the young healthy control individuals with longer AR alleles might be at increased risk of developing breast cancer later in life, though they are currently in good health. These results further suggest that longer AR alleles may contribute to carcinogenesis in women.

This study identifies an extraordinary high frequency of LOH at Xq25 in Taiwanese patients with breast cancer. This strongly implies that there might be one or more TSGs related to the development of breast cancer at Xq25 in this population.