# Patent

## Title
Human PEN11B-related gene variant associated with lung cancers

## Abstract

The invention relates to a novel human PEN11B-related gene variant, and to the use of the nucleic acid of the gene variant in diagnosing diseases, in particular, lung cancer, e.g. small cell lung cancer.

## Background
FIELD OF THE INVENTION
The invention relates to the nucleic acid of a novel human PEN11B-related gene variant, and the uses of the same in diagnosing diseases associated with the deficiency of PEN11B gene, in particular, lung cancers, e.g. small cell lung cancer (SCLC).
BACKGROUND OF THE INVENTION
Lung cancer is one of the major causers of cancer-related deaths in the world. There are two primary types of lung cancers: small cell lung cancer and non-small cell lung cancer (NSCLC) (Carney, (1992a) Curr. Opin. Oncol. 4:292–8). Small cell lung cancer accounts for approximately 25% of lung cancer and spreads aggressively (Smyth et al. (1986) Q J Med. 61: 969–76; Carney, (1992b) Lancet 339: 843–6). Non-small cell lung cancer represents the majority (about 75%) of lung cancer and is further divided into three main subtypes: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma (Ihde and Minna, (1991) Cancer 15: 105–54). In recent years, much progress has been made toward understanding the molecular and cellular biology of lung cancers. Many important contributions have been made by the identification of several key genetic factors associated with lung cancers. However, the treatments of lung cancers still mainly depend on surgery, chemotherapy, and radiotherapy. This is because the molecular mechanisms underlying the pathogenesis of lung cancers remain largely unclear.
A recent hypothesis suggested that lung cancer is caused by genetic mutations of at least 10 to 20 genes (Sethi, (1997) BMJ. 314: 652–655). Therefore, future strategies for the prevention and treatment of lung cancers will be focused on the elucidation of these genetic substrates, in particular, the genes localized on chromosome 11p15.5, a region shown to be associated with the development of lung cancer (Kondo et al. (1996) Oncogene 12:1365–8; O'Briant and Bepler, (1997) Genes Chromosomes Cancer 18:111–4; Sanchez-Cespedes et al. (1997) Clin Cancer Res 3:1229–35; Bepler et al. (1998) Cancer Detect Prev 22:14–9; Pitterle et al. (1999) Mamm Genome 10:916–22; Xu et al. (2001) Cancer Res 61:7943–9). A human PEN11B gene was mapped on this region (GenBank Accession No. AF020089), suggesting that PEN11B gene may have a role in the tumorigenic process of lung cancer. Therefore, the discovery of gene variants of PEN11B may be important targets for diagnostic markers of lung cancers.
SUMMARY OF THE INVENTION
The present invention provides a PEN11B-related gene variant and the fragments thereof. The nucleotide sequence of the gene variant can be used for the diagnosis of diseases associated with the deficiency of PEN11B gene, in particular, lung cancers, e.g. SCLC.
The invention also provides methods for diagnosing diseases associated with the deficiency of PEN11B gene, in particular, lung cancers, e.g. small cell lung cancer.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A; 1B and 1C show the nucleic acid sequence (SEQ ID NO: 1) and the encoded amino acid sequence (SEQ ID NO: 2) of PEN11BV.
FIGS. 2A–2J show the nucleotide sequence alignment between the human PEN11B gene (SEQ ID NO: 5) and PEN11BV (SEQ ID NO: 1).



DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, all technical and scientific terms used have the same meanings as commonly understood by persons skilled in the art.
The term “base pair (bp)” used herein denotes nucleotides composed of a purine on one strand of DNA which can be hydrogen bonded to a pyrimidine on the other strand. Thymine (or uracil) and adenine residues are linked by two hydrogen bonds. Cytosine and guanine residues are linked by three hydrogen bonds.
The term “Basic Local Alignment Search Tool (BLAST; Altschul et al., (1997) Nucleic Acids Res. 25: 3389–3402)” used herein denotes programs for evaluation of homologies between a query sequence (amino or nucleic acid) and a test sequence as described by Altschul et al. (Nucleic Acids Res. 25: 3389–3402, 1997). Specific BLAST programs are described as follows:
(1) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;
(2) BLASTP compares an amino acid query sequence against a protein sequence database;
(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence against a protein sequence database;
(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames; and
(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
The term “cDNA” used herein denotes nucleic acids that synthesized from a mRNA template using reverse transcriptase.
The term “cDNA library” used herein denotes a library composed of complementary DNAs which are reverse-transcribed from mRNAs.
The term “complement” used herein denotes a polynucleotide sequence capable of forming base pairing with another polynucleotide sequence. For example, the sequence 5′-ATGGACTTACT-3′ SEQ ID NO: 3 binds to the complementary sequence 5′-AGTAAGTCCAT-3′ SEQ ID NO: 4.
The term “deletion” used herein denotes a removal of a portion of one or more amino acid residues/nucleotides from a gene.
The term “expressed sequence tags (ESTs)” used herein denotes short (200 to 500 base pairs) nucleotide sequence that derives from either 5′ or 3′ end of a cDNA.
The term “in silico” used herein denotes a process of using computational methods (e.g., BLAST) to analyze DNA sequences.
The term “polymerase chain reaction (PCR)” used herein denotes a method which increases the copy number of a nucleic acid sequence using a DNA polymerase and a set of primers (about 20 bp oligonucleotides complementary to each strand of DNA) under suitable conditions (successive rounds of primer annealing, strand elongation, and dissociation).
The term “nucleic acid sequence” or “polynucleotide” used herein denotes a sequence of nucleotide (guanine, cytosine, thymine or adenine) in a specific order that can be a natural or synthesized fragment of DNA or RNA. It may be single-stranded or double-stranded.
The term “reverse transcriptase-polymerase chain reaction (RT-PCR)” used herein denotes a process which transcribes mRNA to complementary DNA strand using reverse transcriptase followed by polymerase chain reaction to amplify the specific fragment of DNA sequences.
The term “transformation” used herein denotes a process describing the uptake, incorporation, and expression of exogenous DNA by prokaryotic host cells.
The term “transfection” used herein a process describing the uptake, incorporation, and expression of exogenous DNA by eukaryotic host cells.
The term “variant” used herein denotes a fragment of sequence (nucleotide or amino acid) inserted or deleted by one or more nucleotides/amino acids.
The present invention in the first aspect provides a novel human PEN11B-related gene variant and the fragments thereof.
According to the present invention, human PEN11B cDNA sequence was used to query the human lung EST databases (a normal lung, a large cell lung cancer, a squamous cell lung cancer and a small cell lung cancer) using BLAST program to search for PEN11B-related gene variants. One human cDNA partial sequence (i.e., EST) showing similarity to PEN11B was identified from the ESTs deposited in the SCLC database. The cDNA clone, named PEN11BV (PEN11B variant), was then isolated from the SCLC cDNA library and sequenced. FIGS. 1A–1C show the nucleic acid sequence (SEQ ID NO:1) and the encoded amino acid sequence of PEN11BV.
The full-length of the PEN11BV cDNA is a 2173 bp clone containing a 447 bp open reading frame (ORF) extending from 128 bp to 574 bp, which corresponds to an encoded protein of 149 amino acid residues with a predicted molecular mass of 16.6 kDa. The sequence around the initiation ATG codon of PEN11BV (located at nucleotide 128 to 130 bp) was similar to the Kozak consensus sequence (A/GCCATGG) (Kozak, (1987) Nucleic Acids Res. 15: 8125–48; Kozak, (1991) J Cell Biol. 115: 887–903.).
To determine the variation in sequence of PEN11BV cDNA clone, an alignment of PEN11B nucleotide sequence with PEN11BV was performed (FIGS. 1A–2J). One major genetic deletion was found in the aligned sequences, showing that PEN11BV is a 160 bp deletion in the sequence of PEN11B from 856–1015 bp. This deletion occurs on the 3′-untranslated region (3′UTR). Thus, no change can be observed on the predicted amino acid sequence of PEN11BV as compared with that of PEN11B.
In the present invention, a search of ESTs deposited in dbEST (Boguski et al. (1993) Nat Genet. 4: 332–3) at National Center of Biotechnology Information (NCBI) was performed to determine the tissue distribution of PEN11BV in silico. The result of in silico Northern analysis showed that one EST (GenBank Accession Number BE264379) was found to confirm the absence of 160 bp region on PEN11BV nucleotide sequence. This EST was generated from a SCLC cDNA library, suggesting that the absence of 160 bp nucleotide fragment located between nucleotides 855 to 856 of PEN11BV may serve as a useful marker for diagnosing diseases associated with the deficiency of PEN11B gene, in particular, lung cancers, e.g. SCLC. Therefore, any nucleotide fragments comprising nucleotides 855 to 856 of PEN11BV may be used as probes for determining the presence of PEN11BV under highly stringent conditions. An alternative approach is that any set of primers for amplifying the fragment containing nucleotides 855 to 856 of PEN11BV may be used for determining the presence of the variant.
According to the present invention, the fragments of the nucleic acid sequences of the human PEN11BV can be used as primers or probes. Preferably, the purified fragments of the human PEN11BV are used. The fragments may be produced by enzymatic digestion, chemical cleavage of isolated or purified nucleic acid sequences, or chemical synthesis and then may be isolated or purified. Such isolated or purified fragments of the nucleic acid sequences can be directly used as primers or probes.
Many gene variants have been found to be associated with diseases (Stallings-Mann et al., (1996) Proc Natl Acad Sci U S A 93: 12394–9; Liu et al., (1997) Nat Genet 16:328–9; Siffert et al., (1998) Nat Genet 18: 45 to 8; Lukas et al., (2001) Cancer Res 61: 3212 to 9). Since PEN11BV clone was isolated from SCLC cDNA library and its expression in SCLC was confirmed by in silico Northern analysis, it is advisable that PEN11BV may serve as a marker for the diagnosis of diseases associated with the deficiency of PEN11B gene, in particular, lung cancers, e.g. SCLC. Thus, the expression level of PEN11BV relative to PEN11B may be a useful indicator for screening of patients suspected of having such diseases, and the index of relative expression level (mRNA) may confer an increased susceptibility to such diseases.
Accordingly, the subject invention in a further aspect provides methods for diagnosing diseases associated with the deficiency of PEN11B gene in a mammal, in particular, lung cancers, e.g. small cell lung cancer.
The method for diagnosing the diseases associated with the deficiency of PEN11B gene may be performed by detecting the nucleotide sequence of the human PEN11BV of the invention which comprises the steps of: (1) extracting total RNA of cells obtained from a mammal; (2) amplifying the RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with a set of primers to obtain a cDNA comprising the fragments comprising nucleotides 853 to 858 of SEQ ID NO: 1; and (3) detecting whether the cDNA sample is obtained. If necessary, the amount of the obtained cDNA sample may be detected.
In the above embodiment, one of the primers may be designed to have a sequence comprising the nucleotides of SEQ ID NO: 1 containing nucleotides 853 to 858, and the other may be designed to have a sequence complementary to the nucleotides of SEQ ID NO: 1 at any other locations downstream of nucleotide 858. Alternatively, one of the primers may be designed to have a sequence complementary to the nucleotides of SEQ ID NO: 1 containing nucleotides 853 to 858, and the other may be designed to have a sequence comprising the nucleotides of SEQ ID NO: 1 at any other locations upstream of nucleotide 853. In this case, only PEN11BV will be amplified.
Alternatively, one of the primers may be designed to have a sequence comprising the nucleotides of SEQ ID NO: 1 upstream of nucleotide 855 and the other may be designed to have a sequence complementary to the nucleotides of SEQ ID NO: 1 downstream of nucleotide 856. Alternatively, one of the primers may be designed to have a sequence complementary to the nucleotides of SEQ ID NO: 1 upstream of nucleotide 855 and the other may be designed to have a sequence comprising the nucleotides of SEQ ID NO: 1 downstream of nucleotide 856. In this case, both PEN11B and PEN11BV will be amplified. The length of the PCR fragment from PEN11BV will be 160 bp shorter than that from PEN11B.
Preferably, the primer of the invention contains 15 to 30 nucleotides.
Total RNA may be isolated from patient samples by using TRIZOL reagents (Life Technology). Tissue samples (e.g., biopsy samples) are powdered under liquid nitrogen before homogenization. RNA purity and integrity are assessed by absorbance at 260/280 nm and by agarose gel electrophoresis. The set of primers designed to amplify the expected size of specific PCR fragments of PEN11BV can be used. PCR fragments are analyzed on a 1% agarose gel using five microliters (10%) of the amplified products. To determine the expression level of the gene variant, the intensity of the PCR products may be determined by using the Molecular Analyst program (version 1.4.1; Bio-Rad).
The RT-PCR experiment may be performed according to the manufacturer's instructions (Boehringer Mannheim). A 50 μl reaction mixture containing 2 μl total RNA (0.1 μg/μl), 1 μl each primer (20 pM), 1 μl each dNTP (10 mM), 2.5 μl DTT solution (100 mM), 10 μl 5× RT-PCR buffer, 1 μl enzyme mixture, and 28.5 μl sterile distilled water may be subjected to the conditions such as reverse transcription at 60° C. for 30 minutes followed by 35 cycles of denaturation at 94° C. for 2 minutes, annealing at 60° C. for 2 minutes, and extension at 68° C. for 2 minutes. The RT-PCR analysis may be repeated twice to ensure reproducibility, for a total of three independent experiments.
Another embodiment for diagnosing the diseases associated with the deficiency of PEN11B gene may be performed by detecting the nucleotide sequences of the human PEN11BV of the invention which comprises the steps of: (1) extracting total RNA from a sample obtained from the mammal; (2) amplifying the RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) to obtain a cDNA sample; (3) bringing the cDNA sample into contact with the nucleic acid of SEQ ID NO: 1 and the fragments thereof; and (4) detecting whether the cDNA sample hybridizes with the nucleic acid of SEQ ID NO: 1 or the fragments thereof. If necessary, the amount of hybridized sample may be detected.
The expression of gene variants can be analyzed using Northern Blot hybridization approach. Specific fragment comprising nucleotides 853 to 858 of the PEN11BV may be amplified by polymerase chain reaction (PCR) using primer set designed for RT-PCR. The amplified PCR fragment may be labeled and serve as a probe to hybridize the membranes containing total RNAs extracted from the samples under the conditions of 55° C. in a suitable hybridization solution for 3 hr. Blots may be washed twice in 2×SSC, 0.1% SDS at room temperature for 15 minutes each, followed by two washes in 0.1×SSC and 0.1% SDS at 65° C. for 20 minutes each. After these washes, blot may be rinsed briefly in suitable washing buffer and incubated in blocking solution for 30 minutes, and then incubated in suitable antibody solution for 30 minutes. Blots may be washed in washing buffer for 30 minutes and equilibrated in suitable detection buffer before detecting the signals. Alternatively, the presence of gene variants (cDNAs or PCR) can be detected using microarray (bio-chip) approach. The cDNAs or PCR products corresponding to the nucleotide sequences of the present invention may be immobilized on a suitable substrate such as a glass slide. Hybridization can be preformed using the labeled mRNAs extracted from samples. After hybridization, nonhybridized mRNAs are removed. The relative abundance of each labeled transcript, hybridizing to a cDNA/PCR product immobilized on the microarray (bio-chip), can be determined by analyzing the scanned images.
The following examples are provided for illustration, but not for limiting the invention.
EXAMPLES
Analysis of Human Lung EST Databases
Expressed sequence tags (ESTs) generated from the large-scale PCR-based sequencing of the 5′-end of human lung (normal, SCLC, squamous cell lung cancer and large cell lung cancer) cDNA clones were compiled and served as EST databases. Sequence comparisons against the nonredundant nucleotide and protein databases were performed using BLASTN and BLASTX programs (Altschul et al., (1997) Nucleic Acids Res. 25:3389–3402; Gish and States, (1993) Nat Genet 3:266–272), at the National Center for Biotechnology Information (NCBI) with a significance cutoff of p<10−10. ESTs representing putative PEN11BV gene were identified during the course of EST generation.
Isolation of cDNA Clones
One cDNA clone exhibiting EST sequence similar to the PEN11B gene was isolated from the SCLC cDNA library and named PEN11BV. The inserts of these clones were subsequently excised in vivo from the λZAP Express vector using the ExAssist/XLOLR helper phage system (Stratagene). Phagemid particles were excised by coinfecting XL 1-BLUE MRF′ cells with ExAssist helper phage. The excised pBluescript phagemids were used to infect E. coli XLOLR cells, which lack the amber suppressor necessary for ExAssist phage replication. Infected XLOLR cells were selected using kanamycin resistance. Resultant colonies contained the double stranded phagemid vector with the cloned cDNA insert. A single colony was grown overnight in LB-kanamycin, and DNA was purified using a Qiagen plasmid purification kit.
Full Length Nucleotide Sequencing and Database Comparisons
Phagemid DNA was sequenced using the Epicentre#SE9101LC SequiTherm EXCELTMII DNA Sequencing Kit for 4200S-2 Global NEW IR2 DNA sequencing system (LI-COR). Using the primer-walking approach, full-length sequence was determined. Nucleotide and protein searches were performed using BLAST against the non-redundant database of NCBI.
In Silico Tissue Distribution (Northern) Analysis
The coding sequence for each cDNA clones was searched against the dbEST sequence database (Boguski et al., (1993) Nat Genet. 4: 332–3) using the BLAST algorithm at the NCBI website. ESTs derived from each tissue were used as a source of information for transcript tissue expression analysis. Tissue distribution for each isolated cDNA clone was determined by ESTs matching to that particular sequence variants (insertions or deletions) with a significance cutoff of p<10−10.
REFERENCES


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## Claims

1. An isolated nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 1.