Honghua Li Associate Professor UMDNJ Dept. of Molecular Genetics and Micro The Cancer Institute of New Jersey 195 Little Albany Street New Brunswick. NJ 08903-2681 (732) 235-7330 FAX - 7493 holi@umdnj.edu Visit Dr. Li's Lab!!

Genome-scale understanding the genetic basis of breast cancer. and understanding the mechanisms underlying human immunoglobulin V_H gene complex diversification

Our laboratory is interested in understanding the molecular mechanisms affecting human health in a comprehensive way. Although each of our missions has clearly defined aims. we have been continuously targeting at the biological systems that may have major and comprehensive impact on human health and looking at the life process at different angles.

A.Establishing a Robust Genetic Marker Scoring System for Genome-scale Analysis. The human genetics community desperately needs a highly efficient experimental system for genome-scale analysis with a high marker density. Under the support the National Human Research Institute. we are incorporating 6,000 genetic markers consisting of single nucleotide polymorphisms (SNPs) into a highly efficient and cost effective multiplex genotyping system. Recent progress in our laboratory in using the microarray technique has allowed us to resolve a practically unlimited number of products from multiplex PCR amplification. Our goal is to score the 6,000 SNPs with as few as several tens of assays.

B. Genome-Scale Understanding the Genetic Basis of Breast Cancer. Breast cancer is present as a spectrum of neoplasms. A high degree of heterogeneity has been observed among both invasive carcinomas and their accompanying proliferative lesions. Although extensive histological and cytological studies have been documented. the molecular mechanisms underlying the high degree of heterogeneity remain poorly understood. It is widely believed that breast cancer development is a multi-step process caused by genetic alterations in a number of genes. We hypothesize that this high degree of heterogeneity is a reflection of involvement of different molecular pathways. Because the number of genes involved in epithelial cell proliferation is large. and the occurring sequence of genetic alterations in these genes is presumably random or nearly random during cancer development. the number of pathways underlying breast cancer development could be large. Since each gene may contribute to certain phenotypic effect. the morphology of invasive carcinomas and their accompanying proliferative lesions may vary in a wide range. On the other hand. difference in morphology may represent distinct molecular pathways and may be used for studying these pathways.

To test the above hypothesis. we take the advantage of the fact that loss of heterozygosity (LOH). a common indication of tumor suppressor gene inactivation. is usually associated with cancer development. In most cases. LOH affects relatively large chromosomal regions containing the inactivated genes. Therefore. it can be detected by using genetic markers in these regions. but the markers may or may not be located within the gene. By examining LOH. the correlation between the responsive genes and the corresponding invasive carcinomas or other proliferative lesions may be studied without knowing the genes and their protein products.

In order to study LOH on a gemonic scale. a large number of genetic markers need to be included in the study. Under support of the National Cancer Institute. we have selected 600 makers with a high degree of heterozygosity. These markers are either evenly spaced on the chromosomes or located in the genes known being involved in cancer development. To analyze individual breast carcinomas and accompanying proliferative lesions separately and specifically. we isolate these areas from breast tissue specimens by microdissection. Since the amount of material from microdissection is very small and the number of markers to be included is very large. a highly sensitive and efficient experimental system is needed. A multiplex genotyping system that was developed in this laboratory is suitable for this purpose. With this system. the polymorphic sequences of up to 40 marker loci may be amplified by PCR to a detectable amount simultaneously followed by analyzing the PCR products to determine the genotype of samples. We are incorporating the 600 selected markers into this multiplex genotyping system. We have shown that cells microdissected from each microscopic lesion in a 5-mM paraffin-embedded tissue section are sufficient for the analysis with the genetic markers in several multiplex groups. Because many consecutive tissue sections containing the same set of lesions may be prepared from each breast tissue. it is possible to analyze each lesion with 600 or more markers. Data obtained in this way will allow us to understand the pathways to breast cancer in a comprehensive way. By collaborating with Dr. Helen Feiner. a prominent surgical pathologist at the New York University. this multiplex system has been used to analyze one of the breast cancer form. tubular carcinoma. We are applying this approach to the genetic analysis of other breast carcinomas and their accompanying proliferative lesions. Obviously. this system can be a powerful for analyzing any other cancers and can be used in many other genetic studies.

C. Understanding the Mechanisms Underlying Human Immunoglobulin V_H Gene Complex Diversification. Human immunoglobulin V_H gene segments are found on three chromosomes. i.e.. 14q32. 15q11 and 16p11. Only the segments on chromosome 14 are found to be expressed. A very recent publication by Matsuda et al. (J. Exp. Med. 2151-2162. 1998) indicates that the V_H region on chromosome 14q32 contains 123 gene segments. V_H gene segments are subdivided into seven families (V_H - V_H7) based on degree of sequence identity. Gene segments in each family share >80% identity. The V_H region is highly diversified in gene segment number and composition. It is believed that no individuals in the human population contain the same set of V_H gene segments. By using family-specific PCR primers. we were able to amplify the V_H sequences in each family from single sperm. The amplified products from a V_H family are then separated by the denaturing gradient gel electrophoresis. capable of resolving DNA sequences differing by as little as 1 bp. In this way. the gene segment number and composition in the haplotypes of a sperm donor can be determined. Based on the analysis of 26 haplotypes. we were able to discriminate the V_H sequences differing allelically or intergenically. and to assign the V_H sequences to the corresponding loci. The data also revealed some of the loci containing null alleles that are mostly present in a heterozygous state and undetectable if diploid cells are used. We are learning the effect of homozygosity for these null alleles on the development of the immune system. To determine the haplotype organization of the V_H gene segments. each single sperm lysate was aliquoted into 12 to 16 tubes. Because the V_H region is ~1,000 bp long. it is unavoidably sheared into several (3 - 4) fragments upon sperm lysis. Aliquoting the lysate ensured separation of single fragments into different tubes. The V_H gene segments in each tube or fragment were then identified by the PCR-DGGE approach. Because sperm with different parental origins for the V_H region could be distinguished by examining the polymorphic V_H sequences and the break points in the V_H region occur randomly. the V_H gene segment organization in each haplotype could be determined by aligning the overlapping fragments from the sperm of the same haplotype. By comparing our haplotype maps with the composite map constructed by Matsuda et al.. (Nat. Genet. 3:88 - 94. 1993) and by Cook et al. (Nat. Genet. 7:162 - 168. 1994). we showed that the V_H region is highly diversified in certain portions and highly conserved in others. We also showed that some of the V_H gene segments involved in autoantibody development are located at the border between a diversified and a conserved region. To understand the mechanisms responsible for the diversification. we are interested in analyzing the V_H region at the nucleotide sequence level to identify the DNA sequences responsible for the diversification. We are also interested in learning the possible association between the physical structure of the V_H region in haplotypes and the susceptibility to autoimmune diseases.

D. Other Interests. Our laboratory is currently collaborating with Dr. Judy Neubauer's laboratory to dissecting the important component in the central nerve system. hypothalamus. at the molecular level. Our goals are to learn the molecular events in the hypothalamus underlying physiological function of the body and to understand the basic process of comprehension and integration of neuron signals in the hypothalamus.

As completion of the multiplex genotyping system with 6,000 SNPs. the system may be used for many genome-scale analyses. While the genome-scale analysis on breast cancer has been on its way. we have initiated the analysis on prostate cancer. By using the same approach used for breast cancer. we should be able to obtain information for understanding the genetic basis of prostate cancer in a comprehensive way.

Selected Publications

Greenawalt DM. Cui X. Wu Y. Lin Y. Wang HY. Luo M. Tereshchenko IV. Hu G. Li JY. Chu Y. Azaro MA. Decoste CJ. Chimge NO. Gao R. Shen L. Shih WJ. Lange K. Li H. (2006) Strong correlation between meiotic crossovers and haplotype structure in a 2.5-Mb region on the long arm of chromosome 21. Genome Res. 16(2):208-14.

Wang HY. Luo M. Tereshchenko IV. Frikker DM. Cui X. Li JY. Hu G. Chu Y. Azaro MA. Lin Y. Shen L. Yang Q. Kambouris ME. Gao R. Shih W. Li H. (2005) A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome. Genome Res. 15(2):276-83.

Saleem A. Dutta J. Malegaonkar D. Rasheed F. Rasheed Z. Rajendra R. Marshall H. Luo M. Li H. Rubin EH. (2004) The topoisomerase I- and p53-binding protein topors is differentially expressed in normal and malignant human tissues and may function as a tumor suppressor. Oncogene. 23(31):5293-300.

Kovvali G. Shiff S. Telang N. Das K. Kohgo Y. Narayan S. Li H. (2003) Carcinogenesis: The more we seek to know the more we need to know - Challenges in the post Genomic Era. J Carcinog. 2(1):1.

Li H. Cui X. Pramanik S. Chimge NO. (2002) Genetic diversity of the human immunoglobulin heavy chain VH region. Immunol Rev. 190:53-68.

Pramanik S. Li H. (2002) Direct detection of insertion/deletion polymorphisms in an autosomal region by analyzing high-density markers in individual spermatozoa. Am J Hum Genet. 71(6):1342-52.

Cui X. Feiner H. Li H. (2002) Multiplex loss of heterozygosity analysis by using single or very few cells. J Mol Diagn. 4:172-177.

Pramanik and Li. (2002) Direct detection of insertion/deletion polymorphism in an autosomal region by analyzing individual haplotypes in spermatozoa with high-density markers. Am J Hum Gent. 71:1342-52

Li. H.. Cui. X.. Pramanik. S.. and Chimgee. NO. (2002) Genetic Diversity of the Human Immunoglobulin. Immunol Rev 190:53-68.

Cui. X. and Li. H. (2000). Human immunoglobulin V_H4 sequences resolved by population-based analysis after enzymatic amplification and denaturing gradient gel electrophoresis. Eur J Immunogenet. 27: 37-46.

Cui. X.. Feiner H.. Lin Z. and Li H. (2000). Multiplex genotype analysis of invasive carcinoma and accompanying proliferative lesions microdissected from breast tissue. J Mol Diagn 2:29-36.

Xu. Y.. Overton. R. W.. McCoy. J. P.. Marmar. J. L. Jr.. Butler. G. H.. Leonard. J. and Li. H. (1998). Complete replication of human sperm genome with egg extracts from Xenopus laevis. Reprod. Biol. 58:641-647.

Cui. X.. and Li. H. (1998). Haplotype mapping by analyzing single DNA fragments from individual spermatozoa. Proc. Natl. Acad. Sci. USA. 95:10791-10796.