Genetic Markers for Oncogenes, Growth Factors, and Cystic Fibrosis *
M. Dean 1, C. Stewart 1, A. Perry 1, B. Gerrard 1, T. Beck 2, U. Rapp 2, M. Drumm3, M. Iannuzzi 3, F. Collins 3, S. O'Brien 2    Hämatol. Bluttransf. Vol 32

1 BCDP, Program Resources Inc., NCI-Frederick Cancer Research Facility, Frederick, MD 21701, USA
2 Laboratory of Viral Carcinogenesis, NCI Frederick Cancer Research Facility, Frederick MD 21701, USA
3 Howard Hughes MedicalInstitute, University of Michigan, Ann Arbor, MI, USA
* This project has been funded at least in part with Federal Funds from the Department of Health and Human Services
under contract number N01-CO-74102 with Program Resources, Inc. The content of this publication does not necessarily
reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products,
or organizations imply endorsement by the U.S. Government

A. Introduction

The techniques of molecular biology have had a dramatic effect on the advancement of human genetics. In particular, the development of restriction fragment length polymorphisms (RFLPs) has allowed researchers to generate genetic markers for virtually any region of the human genome. Most RFLPs occur when a mutation creates or deletes a recognition site for a restriction enzyme, generating a DNA fragment of altered size. In the simplest case this will create two alleles. A DNA probe which hybridizes to this fragment will detect the presence of these alleles in the DNA from different individuals. Probes used to detected RFLPs have been derived from both cloned genes and randomly isolated DNA segments. Thus, each RFLP is a genetically inherited marker for a precise location on a chromosome. By analyzing the inheritance of RFLPs in families, linkage analysis techniques can be used to determine the order and genetic distance between different polymorphic probes [1]. When this information is combined with data on the physicallocation of the probes, agenetic map of a chromosome can be constructed [2]. Genetic maps are powerful tools, useful in the detailed molecular analysis of biological problems. By studying the inheritance of polymorphisms in families afflicted with a genetic disease, the approximate chromosomal location of the gene responsible for the disease can be determined. Linkage with RFLP markers has been detected for several human disease genes including Huntington's disease [3], muscular dystrophy [4], and cystic fibrosis [5-7]. Linkage is often a critical step in the eventual isolation of the gene itself [8]. RFLPs have also provided critical data in the analysis of specific chromosomal abnormalities in human tumors. DNA markers can be used to distinguish between the two homologous chromosomes in the normal cells of a patient and, when compared with DNA from a tumor, can detect the loss of a specific chromosome or chromosomal region. Such analysis helped to identify the region containing the gene responsible for retinoblastoma [9] and regions containing putative genes involved in Wilm's tumor and renal cancer [10,11] among others. In this report we present data on several newly detected RFLPs in biologically important genes (Table 1), describe in detail the identification of new RFLPs in three loci, and discuss the potential usefulness of genetic polymorphisms in the analysis of human disease.

B. Results and Discussion

The c-raf:.1 gene is a member of the family of serine protein kinases and is the cellular homologue of the v-raf gene. vraf was identified as the transforming gene of the murine 3611 acute transforming retrovirus [12]. It is believed that part of the function of the c-raf-1 gene is to act as an intracellular signal transmission molecule for certain hormones or growth factors [13]. The human c-raf-1 gene has been localized to chromosome 3 at bands p24-25 [14]. Using cDNA probes from the human c-raf-1 gene, we have searched for the presence of RFLPs in this locus. The pTuc-8 probe detects a polymorphism with the enzyme EcoRI, with alleles of15 and 12 kb (Fig. 1). In an analysis of 71 unrelated Caucasians, we found a frequency of 0.87 for the 15-kb allele and 0.13 for the 12-kb allele (Table 1). In addition, the EcoRI poly

Table 1. Newly described RFLPs

Fig.1. EcoRI RFLP in the c-raf-l gene. DNA from nine individuals was hybridized with the pTUC-8 cDNA probe.
Size of the alleles in kilobases (kb)

morphism was shown to segregate properly in three large three-generation pedigrees (data not shown). Specific deletions in the short arm of chromosome 3 have been described in patients with small-cell lung cancer (SCLC) and renal cell carcinoma [15,16]. U sing RFLPs within the c-raf-110cus, we have examined DNA from a total of 83 human lung carcinomas (Sidthansen et al., unpublished). In an analysis of ten paired (normal versus tumor) SCLC DNA samples, five informative cases showed loss of heterozygosity at this locus. A BglI polymorphism which shows 50% heterozygosity in a normal population was used to analyze 73 unpaired lung carcinoma DNAs. Fifteen of 31 non-SCLC samples showed heterozygosity; however, none of the 42 SCLC samples were heterozygous. This striking apparent loss of heterozygosity at the c-raf-1 locus in SCLC provides evidence that the c-raf-1 locus is deleted in smallcell lung carcinoma. Recently, Siezinger et al. [17] used RFLPs in the c-raf-l gene to detect linkage between this gene and the gene responsible for Von Hippel-Lindau syndrome, an inherited cancer syndrome. The polymorphism described here can be used to further study families with this disease in order to pinpoint the location of the gene. We used probes from the interleukin-1alpha (IL-1alfa) gene to screen for RFLPs at this locus. IL-1alfa is a secreted protein that is involved in the stimulation of the growth of lymphocytes. While secreted principally from macrophages, the protein is also produced from keratinocytes and lymphocytes and plays a role in the stimulation of lymphocytes and fibroblasts [18]. Using an IL-1alfa cDNA probe we detected a two-allele polymorphism with the enzyme TaqI, and found that these alleles have frequencies of 0.74 and 0.26 (Table 1 ). In addition, we found that a fragment of this gene, detected by several different enzymes, showed a high degree of variability between individuals. This type of variation is characteristic of

Fig. 2. Polymorphisms in the intcrleukin-l-alpha gene. TaqI detects a two-allele site
polymorphism as well as a high polymorphic VNTR

polymorphisms known as VNTRs (variable number of tandem repeats). VNTRs are regions of DNA containing a group of tandem-repeated sequences [19]. The number of repeats at a given locus is often highly variable between different individuals, making these polymorphisms very informative for genetic analyses. Examination of the published genomic sequence of the genomic IL-l alfa gene [20] revealed the presence of a group of tandem repeats in the intervening sequence between the fifth and sixth exons (Fig. 2). The sequence of these repeats is typical of those found in other VNTRs which have been characterized.. The ILlalfa gene has been mapped to chromosome 2q13-21 and the polymorphisms we have described will be useful genetic markers for this region. In addition, these polymorphisms can be used to test whether the IL-l alfa gene is linked to any human genetic disease for which family pedigrees are available.

Fig. 3. Polymorphisms for probes tightly linked to the CF gene. The positions of the chromosome-jumping clones CF 70-32, CF 31, and CF 29 are shown in relation to the pJ3.11 probe. RFLPs detected by these clones are displayed with sizes in kilobases. S, SacI; M, MspI; T, TaqI; P, PvulI; E, EcoRV

Cystic fibrosis (CF) is the most common fatal genetic disease among Caucasians; it is caused by a recessive mutation at a single locus. The gene responsible for cystic fibrosis has been linked to polymorphic markers that map to chromosome 7q31 [21]. Recently, the location of the gene has been narrowed to a 700-kb region between the probes XV2c and pJ3.11 [22]. In order to clone and characterize additional sequences from this region, we have used chromosome jumping [23, 24] to isolate sequences surrounding the pJ3.11 locus. Briefly, chromosome jumping involves the circularization of large DNA fragments and the cloning of the ends of such fragments. By generating a library of chromosome-jumping clones and screening with a probe from the pJ3.11 locus, we have been able to isolate sequences spanning 300-400 kb surrounding pJ3.11 (Fig. 3). Using probes from this region we have searched for RFLPs and have discovered three new polymorphisms to date (Table 1). Two of these RFLPs are detected with the CF29 probe, which is located approximately 150 kb from pJ3.11, away from the CF gene. The third RFLP is detected with CF70-32, a jumping clone that lies closer to the CF gene (Fig. 3; unpublished data). While all three of these RFLPs are fairly informative, in preliminary analysis they all show a high degree of linkage disequilibrium with the MspI RFLP detected by pJ3.11. In other words, all four of these RFLPS reveal very little new information and increase the heterozygosity only from 53% to 58%.

C. Conclusion

Further progress in the molecular analysis of human genetic disease will rely on the development of increasingly detailed chromosome maps. At present, most of the maps of human chromosomes were constructed principally with anonymous DNA markers, not known to be part of genes. We have begun to characterize RFLPs in biologically important genes, many of which have been mapped to specific chromosomallocations by in situ hybridization. The list of genes in which we have detected RFLPs includes the proto-oncogenes and growth factors craf-1, c-jun, c-ovc-2, and IL-l alfa, and cell surface molecules and receptors such as the retinoic acid receptor, the T -cell receptor zeta gene, and the T 4 gene, receptor for HIV. In addition to providing markers for specific chromosomallocations, the RFLPs in these genes will allow them to be tested as candidate genes for human genetic diseases.


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