Polymorphisms in Intron 8 of the HER-2 Gene

EXAMPLE 11

This example illustrates that the sequence of intron 8 is polymorphic. Intron 8 of the human HER-2 gene is alternatively retained in mRNA, and encodes a novel 79-residue domain at the C-terminus of a part of the extracellular domain of p185HER-2. The product, “herstatin,” of the alternative transcript with the retained intron functions as an autoinhibitor of the HER-2 oncogene. The intron 8 encoded domain, alone, was shown to bind with mM affinity to p185HER-2. (Doherty et al., Proc. Natl. Acad. Sci. USA 96:10,869-10,874, 1999).

Additionally, polymorphisms in the nucleotide and deduced amino acid sequence of intron 8 in the HER-2 gene were identified by sequencing genomic DNA from 15 different individuals. FIG. 8 and SEQ ID NO:1 show the most common nucleotide and corresponding amino acid sequences, respectively, of intron 8. This region contains 10 different polymorphisms (marked by the letters W (2×), Y (3×), R, N, M, and S (2×) in SEQ ID NO: 10; or marked by an “X” in FIG. 8) that result in nonconservative amino acid substitutions (see legend to TABLE 1). For example, the polymorphism (G→C) at nucleotide position 161 (FIG. 8; TABLE 1) would result in a substitution of Arginine (R) for Proline (P) at amino acid residue #54 of SEQ ID NO: 1, or residue #394 of SEQ ID NO:2. The N-terminal Glycine (G), designated as position 1 in FIG. 8 or SEQ ID NO:10, corresponds to amino acid residue #341 in the “herstatin” sequence (Doherty et al., Proc. Natl. Acad. Sci. USA 96:10,869-10,874, 1999). The nucleotide sequence shown in FIG. 1(A) is a polymorphic form that differs at amino acid residues #6 and #73 from the most commonly detected sequence shown here in FIG. 8. This result demonstrates that in the human population there are several variations in the intron-8 encoded domain that could lead to altered biochemical and biological properties among ECDIIIa-containing protein variants. Some identified variants are summarized in Table 1.

Table 1. Sequence variants in the intron-8 encoded domain found in the human population (based on 15 different individuals). Sequence variants 1-11 are listed, showing the base changes at a particular “X” positions relative to that found in the most common DNA sequence shown in FIG. 8. The numbers in parenthesis after each X correspond to the nucleotide position in the DNA sequence shown in FIG. 8. The DNA sequence variants listed here correspond to the variable amino acid positions (“Xaa”) of SEQ ID NO: 1 as follows: X(4) to Xaa(2); X(14) to Xaa(5); X(17) to Xaa(6); X(47) to Xaa(16); X(54) to Xaa(18); X(62) to Xaa(21); X(106) to Xaa(36); X(161) to Xaa(54); X(191) to Xaa(64); X(217) to Xaa(73); and to the variable amino acid positions of SEQ ID NO:2 as follows: X(4) to Xaa(342); X(14) to Xaa(345); X(17) to Xaa(346); X(47) to Xaa(356); X(54) to Xaa(358); X(62) to Xaa(361); X(106) to Xaa(376); X(161) to Xaa(394); X(191) to Xaa(404); X(217) to Xaa(413). The specific amino acid changes (relative to the most common DNA sequence of FIG. 8) for the variable amino acid positions in SEQ ID NO: 1 are: Variant 1, Xaa(2)(Thr→Ser); Variant 2, Xaa(5) (Leu→Pro); Variant 3, Xaa(6) (Pro→Leu); Variant 4, Xaa(16) (Leu→Gln); Variant 5, Xaa(18) (Met→Leu); Variant 6, Xaa(21) (Gly→Asp, Alu or Val); Variant 7, Xaa(36) (Leu→Ile); Variant 8, Xaa(54) (Pro→Arg); Variant 9, Xaa(64) (Pro→Leu); Variant 10, Xaa(73) (Asp→Asn), and Variant 11, Xaa(6) (Pro→Leu) and Xaa(73) (Asp→Asn). The same substitutions apply to the corresponding variable amino acid positions in SEQ ID NO:2.