The Case of XPD: Sometimes Two Different Mutant Genes Are Better than One

Rare inherited disorders have long provided a unique window on the genetic basis of disease. Individuals inherit two copies of a gene; one from each parent. In a recessive disorder, if one copy is defective (a mutant allele), the alternate copy is usually sufficient to maintain normal function of the encoded protein. If an individual inherits two defective copies, protein function is disrupted, leading to disease. Often, two different mutant alleles of the same gene are present in one person, a phenomenon called compound heterozygosity. Whether these still cause disease depends on the gene in question. 
 
The potential for recessive genes to interact has rarely been studied in human disease largely because distinguishing the effects of environment and genetic background from “biallelic” effects is very difficult in humans. In a new study, Jaan-Olle Andressoo, James Mitchell, and colleagues circumvent this problem by using a compound heterozygous mouse model of a severe human syndrome called trichothiodystrophy (TTD) that allowed them to link physical traits (or phenotype) to specific combinations of mutant alleles. 
 
TTD belongs to a class of rare, clinically distinct XPD-related recessive disorders. XPD, a DNA-unwinding enzyme, is essential for both gene transcription and DNA repair of sun-induced damage as a component of the transcription/repair factor IIH (TFIIH) complex. In addition to TTD, XPD mutations cause xeroderma pigmentosum (XP) and Cockayne syndrome (CS). XP results in dramatically elevated cancer risk from extreme sun sensitivity—though, surprisingly, sun sensitivity does not necessarily cause skin cancer—and, in severe cases, primary neurodegeneration. Neither CS nor TTD increase cancer risk but lead to accelerated aging, reduced stature, and degeneration of the nerves’ protective myelin sheath. TTD also causes scaly skin and brittle hair. As its name implies, the even rarer XP combined with CS (XPCS) combines cancer predisposition with neurodevelopmental problems. 
 
In the current model, which Andressoo et al. refer to as the “monoallelic” paradigm of XPD-related disease, “causative” mutations are linked to a specific XPD syndrome. Mutations that aren’t linked to a particular disorder are considered biologically inactive, or null. But the appearance of patients with causative mutations and a rare combination of TTD and XP symptoms has revealed the limitations of this paradigm. To explore the potential role of what the researchers call “biallelic effects” in human recessive disorders, Andressoo et al. asked whether different allele combinations of the enzyme XPD influence the diverse phenotypes associated with XPD-related recessive disorders. They discovered that combinations of XPD recessive alleles produced a variety of biallelic effects, from alleviating the severity of various disease symptoms to improving the function of the interacting genes. 
 
In addition to their existing TTD mouse model (with the causative XPDR722W mutation), Andressoo et al. “knocked in” a mutation found in an XPCS “hemizygote” patient (XPDG602D), who had only one copy of the gene, suggesting that mice carrying two copies of this allele (homozygotes) should live. However, no XpdG602D homozygous mutants lived, and the XpdG602D allele was designated Xpd†XPCS or “lethal.” Lethality was likely caused by the reduced expression of the mutant allele rather than by the mutation itself, the researchers concluded, because they found in a separate study that normalization of XPDG602D expression levels leads to viable homozygous animals. Thus, the XPDG602D protein is likely biologically active but its reduced expression in homozygous Xpd†XPCS animals causes lethality. Knocking in an Xpd mutation (encoding XPDR683W) associated with XP was also homozygous lethal (and designated Xpd†XP), probably for the same reason. 
 
To see if these homozygous lethal alleles might interact with a different disease-causing allele, the researchers generated compound heterozygous mice with the Xpd homozygous lethal allele (Xpd†XPCS) and a TTD-causing allele (XpdTTD). Multiple skin, hair, and aging-related features of TTD were far less severe in the compound heterozygous animals than in animals carrying two copies of the TTD-causing allele. Beyond ameliorating these classic TTD symptoms, the homozygous lethal allele alleviated anemia and developmental delay and also extended lifespan in the compound heterozygotes. Similarly, generating compound heterozygotes from the homozygous lethal XP allele (Xpd†XP) and the TTD-causing allele attenuated the TTD-related skin and weight-loss symptoms. The researchers propose that, due to the low expression levels, the lethal alleles, when homozygous, lead to a transcriptional defect that proves fatal. But when either allele is combined with the TTD-causing allele, the latter steps in to perform the transcription task early enough to prevent embryonic lethality. Then, later, as the skin, hair, and blood cells develop, the lethal alleles recover the deficiencies of the TTD allele. 
 
Combining one of the homozygous lethal alleles (Xpd†XPCS or Xpd†XP) with a TTD-causing allele also allowed the normally sun-sensitive XPCS and XP cells to better survive ultraviolet light. This finding suggests that interactions between the alleles produce an effect—resistance to sunlight—that neither has on its own, a phenomenon called “interallelic complementation.” The researchers suspect that complementation occurs as different XPD molecules are plugged into the TFIIH complex at the site of DNA damage. 
 
These results suggest that even though presumed-“null” alleles can’t execute their transcription task, they may still influence disease outcome in compound heterozygous patients, as they have in the mouse model. The evidence that both alleles can contribute to disease phenotype, the researchers conclude, also suggests that it’s time to adopt a biallelic paradigm for compound heterozygous patients with XPD-related disorders.

A majority of colorectal carcinomas and other solid tumours are resistant to cytostatic drugs, which is a significant problem in their treatment (de Vries and Pinedo, 1991). The cause of such resistance may be determined not only by specific cellular mechanisms such as the multidrug resistance (MDR) phenotype associated with overexpression of the MDRJ gene, but also by characteristics of the tumour population, such as the proportion of quiescent cells and adequacy of blood supply (Judson, 1992).
The MDR] gene product, a 170 kDa protein known as P-glycoprotein (Pgp), appears to be a bifunctional transmembrane protein that functions both as an energy-dependent drug efflux pump of broad specificity (e.g. for anthracyclines and other natural hydrophobic compounds) and as a chloride channel (Gill et al., 1992). Several studies have shown that MDR can be conferred upon drug-sensitive cells by transfer of genes encoding Pgp (Gros et al., 1986;Ueda et al., 1987). MDR] RNA and Pgp are found at substantial levels in normal colon, small intestine, kidney, liver and adrenal gland (Fojo et al., 1987), suggesting a normal transporter role for Pgp in these tissues. Pgp has also been found in untreated human cancers derived from normal tissues that express Pgp, such as carcinomas of the colon, liver, kidney, pancreas and adrenal gland (Goldstein et al., 1989). However, it has also been shown that expression of low constitutive levels of MDR] mRNA/Pgp may not necessarily result in the functional expression of the MDR phenotype (Hamada et al., 1987;Chambers et al., 1990;Kramer et al., 1993). Chin et al. (1992) have recently shown that one mutant human p53 protein has a specific stimulatory effect upon the MDR] gene promoter in 3T3 cells and that wild-type p53 exerts specific repression. Expression of p53 protein in colorectal carcinomas is associated with a high degree of tumour DNA aneuploidy (Carder et al., 1993;De Angelis et al., 1993) and a high incidence of p53 gene mutation (De Angelis et al., 1993). It could be speculated that highly aneuploid tumours with mutant p53 express excess Pgp as a result of the stimulatory effect of p53 on the MDR] gene. In this study we have measured Pgp expression and p53 expression in 34 colorectal tumours in order to determine whether Pgp expression is likely to confer initial drug resistance and whether mutant p53 stimulates Pgp expression in vivo.

Clinical material
Thirty-four surgically removed and previously untreated colorectal carcinomas and 13 normal colonic mucosas were collected and studied. All tumours were classified according to Dukes' stage (Dukes, 1932) and histological grade (Jass et al., 1986) (Table I, includes tumour site). Normal mucosa samples were selected from macroscopically normal areas of surgical specimens. Fresh tumour (T) and normal mucosa (N) samples were frozen without buffer at -80C immediately after surgery. During tissue sectioning and preparation procedures for DNA ploidy analysis and immunoblotting, the tumours and normal mucosas were kept on dry ice until used.
DNA indices (DIs) were determined for the tumour set using laser flow cytometry as described previously (De Angelis et al., 1993), and are presented in Table I. The DI characterises a tumour as either DNA aneuploid (DI > 1.00) or diploid (DI = 1.00) (Hiddemann et al., 1984;Dressler and Bartow, 1989). Highly DNA aneuploid tumours were defined as those with a DI > 1.30, and moderately DNA aneuploid (hyperdiploid) tumours as those with a DI > 1.00 and <1.30. The DI has been used as a prognostic parameter (with modest impact, Bauer et al., 1993) in addition to Dukes' stage and histological grade classifications for the prediction of prognosis of patients with colorectal tumours.

Immunoblotting
Slices (approximately 0.5-1.0 mm thick) of tumour and normal mucosa were cut with scalpels and boiled for 3-4 min in sodium dodecyl sulphate (SDS) sample buffer (Laemmli,   differentiated; HD, 265-301 of exon 8. Twenty tumours were also analysed for mutations in codon region 189-215 of exon 6. The optimal conditions for PCR and CDGE have been described elsewhere (Borresen et al., 1991;Smith-Sorensen et al., 1993). DNA sequencing Tumour samples shown to be mutated by CDGE were submitted to direct sequencing in order to determine exactly which codon was affected. A new PCR product was made using primers outside the ones used for CDGE. The PCR products were sequenced directly with standard dideoxysequencing reactions using Dynabeads M280-streptavidin (Dynal AS, Oslo, Norway) as solid support (Hultman et al., 1989). It has previously been shown that CDGE is a more sensitive method of detecting mutants than this type of sequencing (Borresen et al., 1991). Therefore not all mutants found by CDGE were confirmed by sequencing.

Statistical analysis
Fisher's two-tailed 2 x 2 contingency test was used to check for significance of correlations (P-values) between any two parameters. P-values < 0.05 were considered to denote a statistically significant difference.

Results
Pgp was detected in 44% (15/34) of the tumours and in 100% (13/13) of the normal mucosas examined (Figure 1; Table II) (P = 0.0005), with higher levels of expression seen in normal mucosa generally (mean expression 0.260 ± 0.22) as assessed by densitometry, in contrast to a mean expression of 0.061 ± 0.12 for all of the tumours. Table III demonstrates the lower levels of Pgp expression in a subset of the tumour group compared with the levels seen in the corresponding normal mucosas; it was observed in two cases (92-5 and 92-9) 1970) containing protease inhibitors. Representative slices were also cut from the same areas of the tumours as used for immunoblotting, processed for routine histology, and examined by one of us (OPFC) in order to estimate the percentages of tumour and normal mucosal cells in each section. The mean percentage of tumour cells for all sections examined (n = 31) was 90.4% ± 12.6%.
The proteins were first separated by electrophoresis in 8.0% SDS-polyacrylamide gels (Laemmli, 1970). Immunoblotting was done as described previously (De Angelis et al., 1993) using the anti-Pgp monoclonal antibody C219 (Centocor, PA, USA), the anti-p53 monoclonal antibodies PAb 1801 and PAb 421, and the anti-p83 monoclonal antibody 34C1 (courtesy of T Stokke). The amount of p83 (nucleoplasmic protein expressed at equivalent levels in proliferating and non-proliferating cells; T Stokke and S Funderud, unpublished) was determined on the same blots in order to control for gel loading, cell concentration and protein degradation. A biotin-streptavidin alkaline phosphatase staining procedure was used to detect the primary antibodies (Amersham, UK). The amounts of the different proteins were evaluated by densitometry using ImageQuant densitometry software (Molecular Dynamics, USA) to analyse stored gel images generated with an Agfa photo scanner (Agfa, Germany). Levels of protein expression are reported relative to p83 expression.
Mutation analysis within the p53 gene DNA from 28 colorectal tumour samples was subjected to mutation analysis using polymerase chain reaction (PCR) followed by constant denaturant gel electrophoresis (CDGE). We screened for mutations in the conserved domains corresponding to the following codons:  (Tables II and III that the level of Pgp expression in the tumours was higher than in the corresponding normal mucosas. There was significant association neither between Pgp expression and Dukes' stage (A plus B vs C plus D; P = 0.51) nor between Pgp expression and tumour site in the colon (P= 1.00, right colon vs left colon/rectum). Additionally, there was not a significant association between Pgp expression and histological grade (P = 1.00, poorly differentiated vs moderately and highly differentiated tumours).
Fifty per cent (12/24) of the aneuploid tumours were positive for Pgp, compared with 40% (4/10) of the diploid tumours (P = 0.71). The densitometry results demonstrated a trend which showed that the highly aneuploid tumours expressed the lowest amounts of Pgp (mean = 0.034 ± 0.08), the moderately aneuploid tumours more than double that seen in the highly aneuploid tumours (mean = 0.086 ± 0.09), the diploid tumours approximately the same amounts as those seen in the moderately aneuploid tumours (mean = 0.078 ± 0.19) and the normal mucosas the highest amounts of Pgp (mean = 0.260 ± 0.22). However, this trend was not significant since the standard deviations for each sample group were high and overlapped with the other groups, and because each of the sample groups tended to be small in size.
Pgp expression was significantly associated neither with p53 expression (P = 0.73) nor with incidence of p53 gene mutations (P = 0.70). The level of Pgp expression in the p53-positive tumours was determined to be 0.043 ± 0.087, approximately half the level of expression seen for the p53negative tumours (0.081 ± 0.156, although this difference was not significant because of the high and overlapping standard deviations for each group.

Discussion
Pgp was expressed in 44% of the colorectal tumours and in all of the normal mucosas examined in this study, with the highest levels of expression seen in normal mucosa. The positive tumours generally expressed less Pgp than normal mucosa. Our results are in agreement with those of a previous study (Fujii et al., 1993), which demonstrated that 44% of colon carcinomas were positive for Pgp by immunofluorescence analyses using both flow cytometry and immunohistochemistry in sections, as well as with previous studies demonstrating Pgp expression generally in normal colonic mucosa (Fojo et al., 1987) and colonic tumours (Goldstein et al., 1989). Peters et al. (1992) found positive Pgp expression in all but one tumour; the tumours had significantly higher levels of Pgp than the normal mucosas. In the columns 'Pgp status' and 'p53 status', the amounts of each protein were visually evaluated from the immunoblots and described as absent (-), present in low amounts (+) and present in high amounts (+ +).

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This discrepancy could be explained by their lack of gel loading/concentration standards when quantifying Pgp expression (p83 in our study) and different methods of isolation of cells from tumour and mucosal samples.
More than half of the colorectal tumours examined in this study were negative for Pgp expression, possibly reflecting their clonal origin from a cell which did not originally express Pgp (Noonan et al., 1990). This Pgp-negative cell might be a crypt epithelial cell, since these have been shown to be negative for Pgp expression, whereas the surface epithelial cells lining the lumen (apical brush border cells) are Pgppositive (Cordon-Cardo et al., 1990). This reflects a differentiation-dependent pattern of expression as normal mucosal cells move up the crypt toward the surface. We estimated the percentage of normal mucosal cells in the tumour sections which might influence the number of Pgppositive tumours scored. We are confident that the immunoblotting results reflect the biological characteristics of tumour cells, since very few non-tumour epithelial cells were seen in the sections examined (<10%) from the same areas of the tumours used for immunoblotting. Several tumours in our study which contained some normal cells were not scored as positive, thus contamination is not a significant problem in this study. It may mean, however, that heterogeneous tumours with a low number of Pgp-positive cells may be scored as Pgp-negative. Heterogeneity of Pgp staining and tumour sampling are other factors which may influence the number of positive tumours scored. However, these factors do not influence our main conclusion, which is that colorectal tumours either do not express Pgp at all (lack of differentiated phenotype) or do so to a much lesser extent than normal mucosa.
There was no correlation of Pgp expression with Dukes' stage, histological grade, or tumour site, confirming the results of Pirker et al. (1993). We also found no significant correlation between Pgp expression and DNA aneuploidy, consistent with the results of Sinicrope et al. (1994). However, these results are not in agreement with those of Danova et al. (1992), who demonstrated a correlation between DNA aneuploidy and higher levels of Pgp expression relative to diploid tumours and normal tissue. Chin et al. (1992) found that one mutant human p53 protein (point mutation at codon 175, Arg to His substitution) had a specific stimulatory effect upon the MDR] gene promoter in 3T3 cells, suggesting that colon tumours which express mutant p53 should overexpress Pgp (relative to the levels of Pgp expressed in normal mucosa). However, we found no association between mutant p53 and Pgp expression. Where there was Pgp expression in the p53-positive tumours, it was still lower than that seen in normal mucosa. Additionally, tumour 92-1 in our tumour set expressed the particular codon 175 mutant p53 as studied by Chin et al. (1992), but did not express Pgp. This independence of Pgp and mutant p53 expression is supported by recent studies of B-cell chronic lymphocytic leukaemia (El Rouby et al., 1993), acute myelogenous leukaemia (Zhao et al., 1992) and myelodysplastic syndromes (Preudhomme et al., 1993), which demonstrated that MDRJ gene overexpression is independent of p53 gene mutations/mutant p53 protein.
It was not possible in this study directly to correlate Pgp expression in colon carcinomas with response to cytostatics and overall survival, since only one of the patients received chemotherapy for his tumour. However, the lack of Pgp expression in many colon tumours suggests that resistance mechanisms other than MDR are responsible for the initial resistance to cytostatic drugs often seen in colon tumours. Non-MDR' mechanisms which have been shown to be involved in mediating resistance to cytostatic drugs (other than anthracyclines) in colon tumours are the activity of glutathione S-transferase (Waxman, 1990;Clapper et al., 1991;De Waziers et al., 1991;Moorghen et al., 1991;Peters et al., 1992) and the alteration of DNA-associated topoisomerase II which is involved in post-replication repair (Redmond et al., 1991).