Reference
Complete the bibliographic reference for the article according to AJE format.

Bretherton-Watt D, Given-Wilson R, Mansi JL, Thomas V, Carter N, Colston KW. Vitamin D Receptor Gene Polymorphisms are Associated with Breast Cancer Risk in a UK Caucasian Population. British Journal of Cancer 2001;85(2):171-175

Category of HuGE information
Specify the types of information (from the list below) available in the article:

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

1. Prevalence of gene variant
2. Gene-disease association

Study hypotheses or purpose
The authors study hypotheses or main purpose for conducting the study.

Purpose: To assess whether vitamin D receptor (VDR) polymorphisms in both the 3' and 5' end of the gene are associated with breast cancer risk in a UK Caucasian population.

Gene(s)
Identification of the following:

1. Gene name
2. Chromosome location
3. Gene product/function
4. Alleles
5. OMIM #

1. Gene: vitamin D receptor (VDR)
2. Chromosome location: 12q12-q14
3. Gene product/function: In humans, the product of VDR is the vitamin D receptor, whose ligand is the vitamin D metabolite 1, 25 dihydroxyvitamin D 3 (1, 25-D). The VDR is a member of the steroid-hormone family of nuclear receptors, that are responsible for the regulation of a number of other hormone-responsive genes.
4. Alleles: The VDR gene contains a number of known polymorphisms. A polymorphic start codon (FoKI restriction site) in the 5' end of the gene results in the VDR proteins that differ in length by three amino acids. Polymorphisms in the 3' end of the gene (BSM1, ApaI, TaqI restriction sites) are thought to be tightly linked to the variable length poly A sequence in the 3' untranslated region (3' UTR), which in turn is thought to be a translational control site.
5. OMIM #: 601769

Environmental factor(s)
Identification of the major environmental factors studied (infectious, chemical, physical, nutritional, and behavioral)

Health outcome(s)
Identification of the major health outcome(s) studied

1. Case-control
2. Cohort 
3. Cross-sectional
4. Descriptive or case series
5. Clinical trial
6. Population screening

Case definition
For study designs 2, 3, and 6, the following are defined, where available:

1. Case selection criteria
2. Exclusion criteria
3. Gender
4. Race/ethnicity
5. Age
6. Time period
7. Geographic location
8. Number of participants

1. Case definition: Breast cancer, as confirmed by histopathological records of core biopsy and resection specimens. All women had a surgical procedure (wide local excision or mastectomy) with or without post-operative radiotherapy. All women had had a recent mammogram.
2. Exclusion criteria: Not mentioned if certain cases were excluded.
3. Gender: Female only
4. Race/ethnicity: Caucasian only
5. Age: Ages ranged from 29.0 to 91 years, with an average age of 62.1 years. The median time since diagnosis was 4.3 years.
6. Time period: Not mentioned
7. Geographic location: Patients were recruited through the Combined Breast Clinic at St. George’s Hospital in South-West London.
8. Number of participants: A total of 181 patients were included in this study.

Control definition  
For study design 1, the following are defined, if available.

1. Control selection criteria
2. Matching variables
3. Exclusion criteria 
4. Gender
5. Race/ethnicity
6. Age
7. Time period
8. Geographic location
9. Number of participants

1. Control Selection Criteria: Control subjects were selected after a recent mammogram revealed that breast cancer was not present in the patient. Control subjects were recruited through the UK National Breast Screening Program for South-West London.
2. Matching Variables: Matching criteria was not specifically mentioned, though the control subjects were all women and of an age range similar to that of the case subjects
3. Exclusion Criteria: Not specified.
4. Gender: Female only.
5. Race/ethnicity: Caucasian only.
6. Age: Ages ranged from 50 to 81 years, with an average age of 55.2 years.
7. Time Period: Not mentioned.
8. Geographic location: South-West London.
9. Number of Participants: A total of 241 controls were used in this study.

1. Environmental factor
2. Exposure assessment
3. Exposure definition
4. Number of participants with exposure data (%
   of total eligible)

1. Gene
2. DNA source
3. Methodology
4. Number of participants genotyped (% of total eligible) 

1. Gene: vitamin D-receptor (VDR)
2. DNA source: 10 mL blood sample
3. Methodology : DNA was extracted from blood cells using the QIAamp blood kit. Genomic DNA was amplified by PCR using primers located upstream and downstream from the sequence of interest. Polymorphisms were determined by digesting the PCR products with the BSM1 (to indicate the 3' polymorphism) and the FoKI (to indicate the 5' polymorphism) restriction endonuclease. The fragments were separated by agarose gel electrophoresis. For the poly A analysis, a 425 base pair PCR product was separated on a 6 % PAGE-urea gel. The poly A region, under these conditions, resolves itself into 2 distinct types, long (18-24bp) and short (13-17bp).
4. Number of participants genotyped: all 241 controls were genotyped, as were all 181 cases.

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

Prevalence of gene variants are summarized in Table 1. Note that non-restriction site polymorphism is denoted by the capital letters B and F, and the polymorphism is denoted by the lower case b and f. The intronic BSM1 polymorphism was significantly associated with increased breast cancer risk, as was the long poly A variant. In fact, the BSM1 polymorphism was found to display strong linkage with the variable length poly A genotypes (410/419 samples). No significant association was found between the FoKI polymorphism and breast cancer.

Table 1 : Prevalence of Genotypes/Gene disease association

*Attributable Fraction (bb) = (147/419)(2.32-1)/2.32 = 20%

Further analysis of clinical/pathological characteristics of the breast cancer group was performed to see if any association existed between cancer types and BSM1 polymorphism. Because of the small numbers of BB genotype, data was pooled for BB and Bb genotypes. A significant association existed only between the BSM1 polymorphism and tumor grade. The results are summarized in Table 2.

Table 2: Further analysis of cancer group in relation to BSM1 genotype

The authors conclude that there exists a highly significant association between the risk of breast cancer and the 3' VDR gene polymorphisms BSM1 and variable length poly A microsatellite in a UK Caucasian population. The authors also conclude that a significant association exists between the BSM1 polymorphism and tumor grade. These findings add additional support for the significant association between VDR gene polymorphisms and the risk for breast cancer. The authors acknowledge that these findings have been contradicted in other studies examining different populations, and they discuss some of the problems with equating different polymorphisms between studies.

Comments
Provide additional insight, including methodologic issues and/or concerns about the study

The main finding of the significant association between the BSM1 polymorphism and breast cancer is very interesting, but there are a few aspects of this study that are problematic. Past studies have resulted in conflicting results, such as the finding in Hispanic populations whereby the SS/BB genotype was associated with an increased breast cancer risk rather than a reduced risk. Such a discrepancy among populations indicates that screening for the BSM1 genotype might not be a useful tool for identifying at-risk individuals. Future research of interest would be a general study to determine overall gene prevalence in various populations and studies to elucidate the functional effect of the different genotypes. The high result of the AF calculation is, in part, due to the prevalence of the BSM1 polymorphism in the studied population, and indicates the importance of this genetic characteristic as a marker for increased breast cancer risk.