Alterations in candidate genes PHF2, FANCC, PTCH1 and XPA at chromosomal 9q22.3 region: pathological significance in early- and late-onset breast carcinoma

Abstract

Introduction: Younger women with breast carcinoma (BC) exhibits more aggressive pathologic features compared to older women; young age could be an independent predictor of adverse prognosis. To find any existing differences in the molecular pathogenesis of BC in both younger and older women, alterations at chromosomal (chr.) 9q22.32-22.33 region were studied owing to its association in wide variety of tumors. Present work focuses on comparative analysis of alterations of four candidate genes; PHF2, FANCC, PTCH1 and XPA located within 4.4 Mb region of the afore-said locus in two age groups of BC, as well as the interrelation and prognostic significance of alterations of these genes.

Methods: Deletion analysis of PHF2, FANCC, PTCH1 and XPA were examined in a subset of 47 early-onset (group-A: < or = 40 years) and 59 late-onset (group-B: > 40 years) breast carcinomas using both microsatellite and exonic markers. Methylation Sensitive Restriction analysis (MSRA) was done to check for promoter methylation. Quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemisty (IHC) was done in some genes to see their relative mRNA and protein expressions respectively. Clinico-pathological correlation of different parameters as well as patient survival was calculated using different statistical softwares like EpiInfo 6.04b, SPSS 10.0 etc.

Results: Either age group exhibited high frequency of overall alterations in PHF2, FANCC and PTCH1 compared to XPA. Samples with alteration (deletion/methylation) in these genes showed reduced level of mRNA expression as seen by Q-PCR. Immunohistochemical analysis of FANCC and PTCH1 also supported this observation. Poor patient survival was noted in both age groups having alterations in FANCC. Similar result was also seen with PTCH1 and XPA alterations in group-A and PHF2 alterations in group-B. This reflected their roles as prognostic tools in the respective groups in which they were altered.

Conclusion: Overall alterations of PHF2, FANCC and PTCH1 were comparatively higher than XPA. Differential association of alterations in FANCC and PTCH1 with that of PHF2, XPA and two breast cancer susceptibility genes (BRCA1/BRCA2) in the two age groups suggests differences in their molecular pathogenesis and dysregulation of multiple DNA repair pathways as well as hedgehog dependent stem cell renewal pathway.

Figure 1

(a) Representative autoradiograph showing loss of heterozygosity (LOH) (b) homozygous/hemizygous deletion (HD/HED) analysis in microsatellite markers (c) HD/HED of exonic markers. → indicates loss of corresponding alleles.

Figure 2

Pattern of deletion at different chr.9q22.32-22.33 markers in early- and late-onset BC.

Figure 3

(a) Histogram showing pattern of methylation in the four genes. (b) Representative agarose gels showing methylation in PHF2, FANCC, PTCH1 and XPA done by MSRA. H, HpaII digested DNA; M, MspI digested DNA; U, undigested DNA. Controls: A 445-bp fragment of β3A adaptin gene (K1) and 229-bp fragment of RARβ2 exon-1 (K2).

Figure 4

Patten of overall alterations in 4 candidate genes at chr9q22.32-22.33.

Figure 5

Box plot representing the relative expression level of PHF2, FANCC and PTCH1 genes done by Q-PCR as shown in the y-axis. Each box shows the distribution of expression levels from 25th to 75th percentile. The median is shown as a line across the box, whereas the '+' is the calculated mean expression level for the particular subtype. Overall, the cases consistently had lower expression of the candidate TSGs.

Figure 6

Immunohistochemistry of FANCC and PTCH1 (a-c) FANCC expression pattern (d-f) PTCH1 expression pattern. BC samples showing high (a and d) or moderate (b and e) or low (c and f) expression of FANCC and PTCH1 genes respectively. Arrow (→) indicates the expression pattern of FANCC and PTCH1 in primary BC. All magnifications are at 20×.

Figure 7

Kaplan-Meier 5-year survival probability curves with cumulative survival of BC patients by alteration status of PHF2, FANCC, PTCH1 and XPA in (A.) early-onset and (B.) late-onset BC. Survival time was defined as the time from surgery to the patient's death, known recurrence or the last time the patient was known to be alive. The log rank test was used to assess the differences in the patient survival between cases with plot for the differences in overall survival among cases with any alteration (Deletion +ve/Alteration -ve or Deletion -ve/Methylation +ve or Deletion +ve/Methylation +ve) and no alteration (Deletion -ve/Methylation -ve) of the analyzed genes. N denotes sample size.