Associations of properdin factor B with melanoma

Associations of Properdin Factor B with Melanoma Bruce Budowle, Bruce O. Barger, Charles M. Balch, Rodney C. P. Go, Jeffrey M. Roseman, and Ronald T. Acton

ABSTRACT: We investigated 98 melanoma patients and 135 normal controls for differences in phenotype and genatype frequencies at the properdin factor B locus. A significant negative association with the Bf-F allele and melanoma was found, resulting in an estimated relative risk of 0.5. The estimated relative risk for developing melanoma among people with the BfFF genotype is 0.07. The Bf-S phenotype was significantly increased among the melanoma sample, with an estimated risk of 6.5. The data suggest association of the Bf locus with a melanoma protection and~or susceptibility gene(s). INTRODUCTION The major histocompatibility complex (MHC) system in man is unique compared to other gone systems in that several HLA polymorphisms coded by genes in this complex have significant population associations with a variety of diseases. However, the majority of the disease associations most likely are due to linkage disequilibrium between genes coding for HLA products and a disease susceptibility gone(s) and/or immune response gene(s) [1]. It is possible that genes coding for other known products within this region, such as C2, C4, and properdin factor B (Bf), may be directly involved in the pathogenesis of some diseases. Several of the HLA-associated diseases, such as insulindependent diabetes mellitus (IDDM) [2-5], multiple sclerosis [6], and idiopathic membranous nephropathy [7], have also been shown to be associated with one of the alleles of the Bf system. In certain studies of IDDM [4] and idiopathic membranous nophropathy [7] the Bf-F1 allele has been reported to be more strongly associated with these diseases than the strongest association with any reported HLA specificity. A recent investigation of our population from the southeastern United States by Acton et al. [8] found an increased association between HLA-DR4 and melanoma compared to healthy controls, while Bw38, Bw54, and DR2 were decreased in the patient population. In addition, Pellegris et al. [9] found in an Italian population of melanoma patients that B40 was increased, and Bw35 was decreased signifiFrom the Departments of Microbiology, Public Health, and Surgery, and Diabetes Research and Training Center, University of Alabama in Birmingham, Birmingham, Alabama.

Address requests for reprints to Bruce Budowle, Ph.D., Diabetes Research & Training Center, 1808 7th A v e n u e South, Room 817, Birmingham, AL 35294. Received June 2, 1981; accepted July 20, 1981.

247 © Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 1 0 0 1 7

Cancer Genetics and Cytogenetics5, 247-251 (1982) 0165-4608/82/03024705502.75

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B. Budowle et al. cantly. These aforementioned populations were relatively homogeneous [10]. The possibility exists that malignant melanoma is another disease that may be associated with one of the alleles of Bf. Therefore, the investigation herein reported sought to examine this issue. It is important to add that our population is ideal for such a study on melanoma due to it being relatively nonmigratory and homogeneous with regard to various racial and ethnic groups.

MATERIALS AND METHODS

Serum and/or plasma samples from 98 Caucasian melanoma patients at Stages I, II, and/or III, who presented to the Melanoma Clinic at the University of Alabama, and 135 Caucasian controls from the southeastern United States without a family history of an HLA associated disease were typed for properdin factor B. The controis and patients were all at least third-generation American. The clinical and pathological characteristics of the entire patient series have been published [1113]. In the present study, 77 patients had localized melanoma (Stage I), while 21 patients had evidence of metastatic disease (Stages II and III). The samples were subjected to agarose gel electrophoresis for 11/2 hr, according to the method of Teisberg [14]. The electrophoresed samples were subsequently immunofixed with Bf antisera (Atlantic Antibodies), according to the method of Alper and Johnson [15]. The phenotype relative risks were estimated, using Woolf's odds ratio method [16]. The confounding effects of Bf-F and Bf-S were examined by determining the relative risks of the Bf-FF and Bf-SS genotypes, as suggested by Curie-Cohen [17]. The significance of the Bf alleles was determined by use of chi-square of FisherIrwin exact test where applicable. RESULTS

Our control population is in Hardy-Weinberg equilibrium (×2 = 7.97; df = 4). The control Bf gene frequencies are similar to those reported by Alper et al. (×2 = 7.95; df = 5) [18]. The gene frequencies of the Bf alleles for the melanoma population and controls are presented in Table 1 and the Bf genotypes in Table 2. The Bf-F allele is carried by 35.7% of the melanoma patients, compared with 52.6% of the controls (Table 2). This significant decrease (p < 0.010) results in a relative risk of 0.5 for individuals carrying this phenotype (Table 1). In addition, only 1.0% of the patients are homozygous for Bf-F, compared with 14.8% of the controls (Table 2). The relative risk for people with the Bf-FF genotype developing melanoma is 0.07 (Table 2). There is a significant increase (p < 0.005) of individuals carrying a Bf-S allele among melanoma patients compared to controls, 96.9% vs. 83.0% (Table 2), giving a relative risk of 6.5 (Table 1). Bf-S homozygotes comprise 61.2% of our

Table 1

Bf gene frequencies in melanoma patients and controls Gene frequencies

Gene frequencies

(melanoma)

(controls)

Phenotypic ~

N = 98

N = 135

relative risk

Chi-square

p Value

F

0.184

0.337

0.50

6.52

<0.010

S

0.791

0.633

6.50

11.19

<0.005

F1

0.026

0.015

--

--

--

$1

0.000

0.015

--

--

--

° P h e n o t y p i c r e l a t i v e risks were e s t i m a t e d a c c o r d i n g to W o o l f [16].

249

Association of Bf with Melanoma

Table 2

Bf genotypes (%) in m e l a n o m a patients and controls

Bf genotypes

Melanoma (N = 98)

Controls ° (N = 135)

Genotypic relative riskb

p Value

43.7 35.6 14.8 3.O 0.7 0 2.2 0

2.3c 0.6d 0.07e

NS~ NS <0.068

I

SS FS FF SS1 FIS FIF1 FF1 FS1

61.2 33.7 1.0 0 2.O 1.0 1.0 0

m

m

~Expected numbers were calculated accordingto the Hardy-Weinberglaw. (~×2 = 7.97; df = 4). bEstimated accordingto Curie-Cohen [17]. °the relative risk for the Bf-SS genotype was estimated relative to those individuals who are Bf-S positive but not Bf-F. dThe relative risk for the Bf-FSgenotypewas estimated relative to those individualswho are Bf-S positivebut not BfF to eliminate confoundingeffects of Bf-S with Bf-F. ~rhe relativerisk of the Bf-FFgenotypewas estimated relativeto those individualswho are Bf-Fpositive but not Bf-S. ~S = not significant.

m e l a n o m a p o p u l a t i o n compared to 43.7% of the controls (Table 2). The relative risk for i n d i v i d u a l s carrying the Bf-SS genotype developing melanoma is 2.3 (Table 2). However, this latter observation did not reach statistical significance. There was no difference in the frequencies of Bf phenotypes and ganotypes of Stage I patients compared with that of Stages II and III patients.

DISCUSSION This is the first k n o w n report of Bf associations with malignant melanoma. However, it is not the first report of a gene within the MHC associated with melanoma, since Acton et al. [8] have reported an increase of DR4 among patients with this disease. Therefore, it was not surprising to observe a change in the frequency of the Bf alleles, since the loci for HLA-DR and Bf are in linkage disequilibrium [19,20]. Bf-S is significantly increased and Bf-F is decreased among m e l a n o m a patients compared with controls. Since Bf is involved i n the alternate c o m p l e m e n t pathway, it is possible that an allelic form could alter or influence the i m m u n e response of an individual. However, despite the increase of Bf-S, it is not likely a single cause of the disease since three patients did not carry the allele. Since 83.0% of the normal sample is also Bf-S-positive, it is difficult to use this marker to predict with any certainty who is at high risk. In contrast, Bf-FF homozygous individuals may be at low risk for melanoma, which may be of some negative predictive value. In addition to a direct gene action model, the data are consistent with three other possible genetic models: (a) the Bf-S allele is in linkage disequilibrium with a m e l a n o m a susceptibility gane(s); (b) the Bf-F allele is in linkage disequilibrium with a m e l a n o m a protection gene(s); or (c) both linkages exist. Due to the low frequency of the Bf-F1 and Bf-S1 alleles in our population, the Bf system is virtually a two-allele system. For a two-allelic system, a change in the frequency of one allele automatically results i n the reciprocal effect of the other allele. Since relative risk according to Woolf [16] is a measure of an association between phenotypes and considers only one antigen at a time [17], it is difficult to discern which allele, Bf-F or Bf-S, is associated with protection or susceptibility, respectively, to mel-

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B. Budowle et al. anoma and which is changing in response to the other. There are two ways to approach this discrimination. One is to investigate a much larger sample size to obtain better estimates of the frequencies of the rarer alleles. The other is to examine whether specific haplotypes of the MHC are in linkage disequilibrium with susceptibility or protection gene(s) or both, that lie within the MHC. This would also provide a comparison to determine whether DR or Bf is more strongly associated with melanoma. We have also observed that 6% of the first- and second-degree relatives of melanoma probands have melanoma. However, 55% of the first-degree relatives have some form of cancer (unpublished data). Therefore, we are also investigating different genetic transmission models for melanoma and the possible linkage of a cancer susceptibility gene(s) with HLA-Bf haplotypes. It is possible that the changes of Bf frequencies may be secondary to ethnic differences. While this does not invalidate the study, it makes for a different interpretation. Epidemiological studies suggest that m e l a n o m a susceptibility is higher in certain ethnic groups [21-23]. Scotch/Irish are reported to be at highest risk, especially w h e n they have migrated to regions of high insolation [22]. By s u r n a m e and heritage declaration and the fact that 41% of the patients have light-colored hair and 62% have blue, gray, or green eyes, it is possible that these observations result from a higher proportion of Scotch/Irish in the population, especially since Bf frequencies can vary among ethnic groups [24,25]. Ethnic origin is difficult to determine. Thus it is important to have a means to identify high-risk individuals, other than their own declaration of ethnicity. Our results may therefore be due either to identifying an ethnic population at increased risk for developing melanoma or to an associated immunological abnormality that fails to contain the malignant cells. We are presently seeking data to discern which of these hypotheses actually exists. This work was supported by grants from the National Cancer Institute, CA-09128, CA-27197, CA-18609, and CA-13148.

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