Novel and nondetected human signaling protein polymorphisms

Roy A. Lynch, Lynne Wagoner, Shunan Li, Li Sparks, Jeffery Molkentin and Gerald W. Dorn, II

Departments of Medicine and Pediatrics, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0542


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The frequency of single nucleotide polymorphisms (SNPs) in downstream signaling proteins was determined by combination heteroduplex HPLC and double-stranded sequencing of genomic DNA from 96–144 congestive heart failure (CHF) patients. Analysis of 56 coding exons in 9 signaling genes revealed 17 novel and 8 previously reported synonymous (no change in amino acid) SNPs, as well as one novel nonsynonymous SNP in the Rad small G protein. Because this initial analysis failed to detect numerous SNPs reported in the NCBI and Celera databases, double-strand sequencing of relevant exons from 74–91 CHF patients was used to confirm the absence of 10 previously reported nonsynonymous SNPs. Our results show that synonymous SNPs are frequent in signaling protein genes, whereas nonsynonymous SNPs are rare, suggesting a high degree of evolutionary conservation among these downstream signaling molecules. Comparisons of our results to the NCBI and Celera databases indicates that 56% of their SNP entries are not detected in our cohort. Importantly, while 31% of database SNPs were verified, 69% of SNPs detected in our cohort are not included in these databases. These findings indicate that caution may be warranted in relying exclusively on SNP databases as catalogs for polymorphic signaling protein genes.

heart failure; G protein


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
REPORTS FROM THE HUMAN GENOME projects indicate that there are as many as 2.1 million human single nucleotide polymorphisms (SNPs) in the genome, of which less than 1% encode an amino acid variation for the associated protein (32). This extraordinary prevalence of DNA polymorphisms, and the potential for SNPs to modify disease, identifies a need to unambiguously delineate genetic variations of candidate effector proteins. In this regard, validated polymorphisms in genes relevant to cardiac disease include angiotensin I-converting enzyme (24), angiotensinogen (13), tumor necrosis factor (10), transforming growth factor-ß (2), and {alpha}- and ß-adrenergic receptors (29), supporting the significance of G protein signaling pathways in cardiac disease. A critical deficiency exists, however, in data on genetic variants of G proteins themselves and their downstream transducers, in heart failure. This is especially surprising in the context of abundant experimental data demonstrating pathological roles for these signaling proteins in the heart (reviewed in Ref. 22).

To begin to address this deficiency, the current study was undertaken to identify both unknown and previously reported SNPs of nine key signaling proteins in a heart failure patient population.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study subjects.
Protocols were reviewed and approved by the University of Cincinnati Institutional Review Board (IRB no. 93-7-26-1). Group 1 (96 patients) included 36 white males, 30 white females, 18 African-American males, and 12 African-American females with an average congestive heart failure (CHF) onset age of 49 yr. These 96 patients included 53 cases of dilated cardiomyopathy, 37 cases of ischemic cardiomyopathy, 1 case of diastolic heart failure, and 5 cases of heart failure secondary to other causes. Group 2 consisted of 144 patients including the above 96 patients plus 35 white males and 13 white females representing 21 cases of dilated cardiomyopathy, 24 cases of ischemic cardiomyopathy, and 3 cases of heart failure secondary to other causes. The average age of CHF onset for group 2 was 52 yr.

Genomic DNA was extracted from whole blood (10–20 ml) using the QIAamp Blood DNA Maxi kit (Qiagen) and manufacturer’s protocols. Working stocks of genomic DNA were prepared at 50 ng/µl concentration (2–4 µl DNA/PCR) and aliquoted into 96-well master plates for high-throughput analysis. A single master plate of 96 patients was used for all initial screenings by heteroduplex HPLC. Some genes were further characterized in an expanded cohort of 144 patients, as noted above.

Intron/exon PCR.
Complete coding regions of each exon for the study genes were amplified using oligonucleotide primers designed from flanking intronic or untranslated sequences (Table 1). The genomic sequences for each gene were obtained from either GenBank [National Center for Biotechnology Information (NCBI)] or the Celera Discovery database; accession numbers are given in Table 1. HotStar Taq polymerase (Qiagen, Valencia, CA) was used for all exon PCR reactions, except Erk2 exon 1 and exon 3, G{alpha}s exon 3 and exon 7–9, Rad exon 5, Ras exon 2 and exon 4 (Advantage cDNA polymerase; Clontech Laboratories, Palo Alto, CA), and G{alpha}s exon 13 (Advantage GC polymerase; Clontech Laboratories). Intron/exon PCR products were optimized individually, using various annealing temperatures and buffers, to yield single products that were visualized using agarose electrophoresis (1% agarose in 1x TBE buffer containing 0.1 M Tris base, 0.09 M boric acid, and 1 mM EDTA). For direct sequencing of PCR products, genomic DNA templates were amplified using forward exon primers (coding strand) containing M13 forward sequence (5'-TGTAAAACGACGGCCAGT-3') and reverse exon primers (antisense strand) containing M13 reverse sequence (5'-CAGGAAACAGCTATGACC-3'). All PCR and sequencing primers were synthesized by the University of Cincinnati DNA Core Facility.


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Table 1. Intron/exon PCR primer sets and conditions

 
DNA heteroduplex analysis by HPLC.
Amplified exons were characterized by temperature-dependent denaturing high-performance liquid chromatography (dHPLC) on Transgenomic WAVE apparatuses and eluted using a linear gradient (typically 10% to 20%) of acetonitrile in 0.1 M triethylammonium acetate over 7–10 min. Optimal melting temperature and elution gradient acetonitrile programs for each PCR product were initially estimated using WAVEMaker 4.0 software (Transgenomic). Three exon PCR products that displayed typical (common) elution profiles were directly sequenced to verify PCR specificity and for comparison to published sequence. Exon PCR products that showed similar atypical profiles were grouped together for each gene exon. All exon PCR products from each atypical group were directly sequenced to identify and verify DNA polymorphisms. To determine the accuracy of dHPLC in detecting SNPs, the results of dHPLC and direct sequencing were compared from 96 patients for G{alpha}11 exon 2 (see RESULTS).

DNA sequencing.
For sequencing, exons were amplified from genomic DNA using primers tagged on the 5' ends with M13 sequences, permitting direct sequencing of PCR products using dye primer chemistry (Applied Biosystems, Foster City, CA) and thus minimizing sequencing artifacts while optimizing sequencing reads. Sequencing reactions were analyzed using an Applied Biosystems model 3100 genetic analyzer and ABI Sequence Analysis software according to manufacturer’s protocols (Applied Biosystems). Resulting DNA sequences were aligned with reference sequences using Vector NTI software (InforMax, Bethesda, MD), and DNA polymorphisms were confirmed visually from sequence electropherograms.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
dHPLC for DNA polymorphism discovery.
Because published studies have demonstrated profound functional and structural effects of modulated cardiac heterotrimeric and small G proteins or Erk MAP kinase activity (reviewed in Ref. 22), we screened the coding exons of three heterotrimeric GTP-binding proteins, five small G proteins, and one MAP kinase for DNA polymorphisms in heart failure patients. Genomic sequence published in the NCBI and Celera Discovery databases was used to define intron-exon boundaries and flanking intronic sequences and to design PCR primers that span the coding region of each exon. Of the 59 coding exons targeted for analysis, it was possible through optimization of PCR primers, reaction conditions, and type of polymerase, to achieve single product amplification in 56, or 95% (see Table 1). Amplified exons were analyzed by dHPLC, and elution profiles were classified by visual analysis of chromatograms as typical (i.e., most common) or one or more atypical patterns. For every exon, at least three different typical profile patients and all atypical profile patients were sequenced to identify the DNA polymorphisms detected by HPLC analysis.

To compare the sensitivity and specificity of screening for DNA polymorphisms by dHPLC with direct sequencing, exon 2 of G{alpha}11 was evaluated in 96 patients by both methodologies. Complete information was obtained in all 96 patients. Direct sequencing identified four different DNA polymorphisms: G/A at position 16772 in 26 patients, G/A at position 16825 in 3 patients, T/G at position 17023 in 60 patients, and C/T at position 17035 in 1 patient (Fig. 1A). At the time of analysis, none of these polymorphisms was reported in the electronic databases. As expected, heterozygote combinations of SNPs were more prevalent than homozygous (73% vs. 27%). Blinded comparative dHPLC analysis of the same patient exons revealed atypical profiles for all 66 sequence-verified heterozygote patient DNAs (Fig. 1B). Patient exons containing multiple heterozygote polymorphisms often displayed unique atypical dHPLC profiles (G/A 16772 + T/G 17023 in 14 patients; G/A 16772 + G/A 17035 in 1 patient) (Fig. 1B). In contrast, elution profiles of the 23 patient exons with homozygous SNPs were typically indistinguishable from the wild-type dHPLC profile (data not shown). This requirement for DNA polymorphism heterozygosity represents a known but significant limitation of heteroduplex dHPLC in establishing the true frequency of SNPs (25). However, because homozygous SNPs are unlikely to occur in the absence of heterozygous SNPs in a large sample (zero occurrence in our comparative analysis), the ease and rapidity of dHPLC compared with sequencing made it optimal for high-throughput SNP discovery.



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Fig. 1. G{alpha}11 exon 2 single nucleotide polymorphism (SNP) analysis by denaturing HPLC (dHPLC) and sequencing. A: schematic of G{alpha}11 exon 2 intron/exon structure with positions of detected SNPs. An illustration of intron/exon boundaries is shown at the top along with intron sizes. G{alpha}11 exon 2 DNA sequence is shown below illustration with intronic sequences indicated by lowercase letters and exonic sequence indicated by bold capital letters. Primer locations are indicated by underscored sequences. Each SNP location is indicated by a bold single letter (polymorphisms) above the common base sequence (in parentheses) along with the corresponding base number (refer to NCBI accession no. ac005262). B: example dHPLC elution profiles and corresponding sequence polymorphisms for G{alpha}11 exon 2. Typical or normal elution profiles (N; blue) are shown along with individual atypical elution profiles (profiles A–C; red) on the left with polymorphic DNA sequence shown on the right, corresponding to the atypical elution profile. Accompanying text on the far right of each profile describes the corresponding polymorphism.

 
To validate dHPLC in screening for a previously identified, pathologically significant polymorphism, ß2-adrenergic receptors were assayed by dHPLC for the Ile164 polymorphism, which is associated with accelerated mortality in heart failure (15). Of note, this polymorphism is not known to occur as a homozygous trait (17), presumably because of early lethality, and so is particularly suited for dHPLC analysis. Forty-two heart failure patients were assayed, and each of their genotypes was correctly identified by dHPLC, indicating 100% sensitivity and 100% specificity (Fig. 2).



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Fig. 2. Detection of the ß2-adrenergic receptor C->T polymorphism (T164I mutation) by dHPLC. Typical or normal elution profiles (N; blue) are shown along with the C/T atypical elution profile (Aß; red) on the left, with corresponding DNA sequence from the T164I patient shown on the right.

 
SNP discovery in heterotrimeric G proteins.
The {alpha}-subunits of G{alpha}q/G{alpha}11 (two different gene products with virtually identical functions; reviewed in Ref. 27) and G{alpha}s have important roles in the normal development and pathogenic responses of the heart (4, 31, 34). Therefore, coding exons of these genes were carefully screened for novel SNPs by dHPLC. Table 2 provides gene names and exon numbers, NCBI or Celera database accession numbers for genomic sequences used, novel and previously reported SNP, and the frequency of detection. Ten novel SNPs were detected in these G{alpha} proteins, each of which was either "conservative" (no change in coding amino acid) or was located in noncoding introns. Certain exons had the majority of polymorphisms (G{alpha}11, exons 2 and 6; G{alpha}s, exons 7–9), suggesting that these may be hypervariable regions of the genome. Four previously reported (NCBI and/or Celera) intronic or conservative SNPs for G{alpha}11 exon 6 and one known conservative SNP for G{alpha}s exon 7 were also detected in our patient cohort (Table 2).


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Table 2. SNPs detected for heterotrimeric G proteins

 
Unexpectedly, a number of candidate polymorphisms reported in the electronic databases were not detected in our patients. In Tables 24, these putative polymorphisms, detected by neither dHPLC analysis or direct sequencing, are designated "not detected" (ND). To determine whether this represented a defect in our data, vs. possible sequencing artifact in the SNP databases, every exon containing putative nonconservative SNPs was amplified and directly sequenced. In no instance did the sequencing reveal that dHPLC had failed to detect a SNP. Rather, database SNPs listed for G{alpha}q exons 2, 6, and 7 were not detected by sequencing of 74, 86, and 91 patients, respectively, and database SNPs for G{alpha}s exons 10/11 and 13 were not present in 87 and 74 patients, respectively (Table 2; shown as a frequency of 0 for total patients sequenced).


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Table 3. SNPs detected for small G proteins

 

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Table 4. SNPs detected for Erk2, Rad1, and H-ras

 
SNP identification in small G proteins.
The Rab small G proteins regulate subcellular protein transport by targeting vesicle-membrane interactions (19). Modified Rab function and expression has been associated with development of experimental heart failure (35). Therefore, the genes for Rabs 3a, 4, and 5c were analyzed in CHF patients for novel or previously reported SNPs. As shown in Table 3, one conservative and one intronic SNP were detected for Rab 3a exon 3, and one conservative SNP was detected for Rab 5c exon 3. Two of these three SNPs were reported in the electronic databases, and one database SNP was not detected in our cohort (accession no. rs237764). Indeed, at the time of analysis, there are no reported nonconservative SNPs for these three Rab proteins.

Whereas the Rab G proteins are not signaling factors per se, Ras and Rad are. Furthermore, several known Ras mutations are associated with malignancy in the heart (14, 12, 7). Therefore, CHF patients were screened for SNPs in the Rad1 and H-ras genes (Table 4). In Rad, a novel SNP resulting in Q66P substitution (T/G at position 2248 in exon 2) was identified in 2 of 96 patients. In addition, four previously unreported conservative or intronic SNPs were detected in Rad exon 2 and H-ras exons 3 and 4. A known conservative SNP for H-ras in exon 1 (T/C; accession no. rs1047212) was identified in 83 of 144 patients (Table 4).

Finally, because of its well established role in myocardial growth and cytoprotection (1, 36), patients were screened for Erk2 polymorphisms (Table 4). Two previously unknown conservative SNPs were identified in Erk2 exons 2 and 7, and two conservative NCBI database SNP candidates in Erk2 exon 5 were not detected by dHPLC analysis (designated ND). Interestingly, except for the Rad SNP reported herein, our analysis did not detect any of the nonconservative SNPs previously reported for Erk2, Rad, or H-ras.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In these studies, we screened a relatively large number of heart failure patients for DNA polymorphisms in eight G proteins and the Erk2 MAP kinase. Our results support two important conclusions. First, the high degree of conservation for these signaling pathway components (with only a single nonsynonymous SNP detected in 9 signaling protein genes) indicates that, in contrast to adrenergic receptors, SNPs of these downstream signal transducers are not likely to be significant modifiers of heart failure. Second, the rather poor concordance between our results and those of Celera and NCBI SNP databases suggests that caution is warranted when relying exclusively upon existing SNP databases as a "catalog" of existing SNPs.

Compared to G protein coupled receptors, where nonconservative (i.e., amino acid changing) polymorphisms are relatively common [2 in ß1-adrenergic receptors (AR), 3 in ß2-AR, and 3 reported to date in {alpha}1-, {alpha}2-, and {alpha}3-AR (29)], the downstream signaling effectors of these receptors, G{alpha}q, G{alpha}11, and G{alpha}s, have remarkably few. Although we found 26 different polymorphisms in a total of 15.3 Mb of primarily exonic genomic DNA [0.17%, within the expected frequency of less than 1% (32)], only one of these polymorphisms encoded an alternate amino acid. Thus there appears to be relatively less tolerance for amino acid variance in encoded proteins for these signaling molecules, compared with their upstream receptors. Indeed, there are far fewer heterotrimeric G{alpha} subunit proteins (~16) (27) than there are G protein-coupled receptors (estimated at more than a thousand) (9). An individual G protein therefore represents a convergence point for many dozens or hundreds of distinct receptors, and it is reasonable that even minor alterations encoded by variant amino acids could be poorly tolerated. The functional significance of the Rad Q66P polymorphism reported herein, the only nonconservative SNP to be reported for this small G protein, is not known at this time.

In all, this study detected 17 novel silent (intronic or conservative exonic) SNPs in the nine genes tested. We confirmed another eight previously reported silent SNPs, but could not detect, by dHPLC or double-stranded sequencing, five silent SNPs reported in the SNP databases. More importantly, from the aspect of structure-function relations in variant signaling proteins, one novel SNP was detected that resulted in an amino acid change in Rad exon 2 (Q66P). However, direct sequencing of between 74 and 91 patients (148 to 182 chromosomes) failed to detect 10 previously reported nonsynonymous SNPs in the SNP databases. Thus, for nonsynonymous SNPs in these 8 genes, the databases were validated 0% of the time, in that 10 reported SNPs were not detected and 1 legitimate SNP was unreported. Overall, of the 26 SNPs detected in the present study, only 8 were previously reported, corresponding to a 31% correlation with the NCBI and Celera database candidate SNPs.

The possible reasons for nonconcordance between our results and the electronic SNP databases are that either our data or the database data are inaccurate. In the current study, SNP discovery was performed using heteroduplex DNA HPLC, which we demonstrated was highly sensitive for detecting single or multiple heterozygous DNA mismatches but was virtually blind to homozygous mismatches. Thus, using our methods, the true allelic frequency of SNPs is not determined, since homozygous variants are not detected. However, in our patient cohort of 96–144 subjects, it is highly unlikely that a SNP would occur as a homozygous and never as heterozygous. Although a possible solution to the problem of dHPLC detection of homozygous polymorphisms would be to promote heteroduplex formation by adding wild-type genomic DNA ("spiking") to each exon PCR reaction, this would necessitate the identification of wild-type DNA templates for each exon tested, prior to high-throughput analysis. On the other hand, in examining our patient cohort for known SNPs, i.e., those reported in the NCBI or Celera databases, we did not rely on dHPLC, but performed complete double-stranded capillary sequencing of each patient’s amplified genomic DNA. In all cases, the sequence chromatograms were individually scrutinized, and in the case of any ambiguity, the samples were reamplified and resequenced. Although our high-throughput approach necessarily precluded us from obtaining every sequence on every patient (due to PCR vagaries), we successfully genotyped 95% of assayed patient exons. Thus every effort was made to assure the integrity and accuracy of our data.

The current approach contrasts with that of the Human Genome Project efforts, which undertook massive genotyping of a relatively few number of subjects [five for Celera (32) and ~9 for NCBI (20)] and which did not focus on specific genes or coding exons as we did. Furthermore, it is unclear that all of the human genome subjects were completely sequenced through both strands, and that suboptimal or ambiguous sequence was manually identified and reanalyzed. These limitations are understandable, and perhaps expected, for an initial screening of the entire human genome. Indeed, Liggett’s laboratory (28) recently reported that nonsynonymous SNPs reported in the dbSNP database for 25 G protein-coupled receptors could be confirmed only 32% of the time in a careful screening of 60 individuals. On the other hand, Marth and coworkers (18), in a detailed analysis of 1,200 candidate SNPs from dbSNP, confirmed more than 80%, which supported the utility of the SNP databases as representative of the general population.

In contrast to the human genome efforts, which analyzed normal individuals, our study used DNA from patients with various stages of CHF. CHF is a leading cause of morbidity and mortality that for untreated patients results in an average lifespan of only 2 yr (5, 11), and even when treated results in only a 50% survival rate over 5 yr (26). In this country, heart failure most frequently presents as a dilated cardiomyopathy resulting from myocardial infarction, chronic myocardial ischemia, or as the sequelum of viral infection (3, 6), and the only "cure" is cardiac transplantation. After decades of intense investigation, the only two classes of drugs demonstrated to prolong lives of heart failure patients are ß-adrenergic receptor antagonists (21, 23) and angiotensin-converting enzyme inhibitors (8, 30), which suggests a critical role for receptor-mediated signaling in the pathogenesis of heart failure. Indeed, recent studies have demonstrated striking effects of variant polymorphic adrenergic receptors as modifiers of CHF progression, suggesting that genetically variant signaling proteins can affect this disease (33, 16, 29). Thus we searched for signaling protein polymorphisms in heart failure patients, but found only one nonsynonymous SNP, Rad Q66P, among the genes tested. The current data therefore do not support a role for SNPs of these G proteins in the pathogenesis of CHF. Although it is formally possible that the lack of concordance of our data with the SNP databases reflects unique characteristics of the CHF population, we believe this to be unlikely.

In summary, we have examined genes coding for a number of signaling proteins to identify the presence and frequency of both novel and previously reported SNPs. A combination of heteroduplex HPLC and double-stranded sequencing of genomic DNA from 56 coding exons in nine signaling genes of 96–144 CHF patients delineated 17 novel and 8 previously reported silent SNPs and identified a novel nonsynonymous SNP in the Rad small G protein (Q66P). Because this initial analysis failed to detect numerous SNPs reported in the NCBI and Celera databases, double-strand sequencing of relevant exons from 74–91 CHF patients was used to confirm the absence of 10 previously reported nonsynonymous SNPs. Our results showed that 56% of database SNP entries were not detected in our study. In addition, although only 31% of database SNPs were verified, 69% of novel SNPs detected in the present study were not included in the databases. In addition to demonstrating that nonsynonymous SNPs are rare in signaling protein genes, while silent SNPs occur frequently, these data caution against exclusive reliance on existing electronic SNP databases for presence or absence of genetic variants in this group of signaling proteins.


    ACKNOWLEDGMENTS
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-52318 and the HuGe IdEA of the University of Cincinnati Cardiovascular Center.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: G. W. Dorn II, Division of Cardiology, Univ. of Cincinnati Medical Center, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0542 (E-mail: dorngw{at}ucmail.uc.edu).

10.1152/physiolgenomics.00030.2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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