Damage to the heart after a heart attack typically results in cardiac tissue scarring (fibrosis), which causes strain on the heart and can eventually lead to heart failure. One factor that can lead to fibrosis is increased expression of the connective tissue growth factor gene, CTGF. Regions of CTGF that show little variation across species suggest presence of a crucial function that has been conserved over time (negative selection). Greater variation among CTGF regions suggests potential selective pressure to alter that region of the protein (positive selection). We used the program Molecular Evolutionary Genetics Analysis (MEGA7) to align the CTGF sequences from 19 species. To predict selective pressures on the gene, we used Phylogenetic Analysis by Maximum Likelihood (PAMLX) software. We detected negative selection in CTGF across these species. We will use these same software programs to determine whether the selective pressure varies among individual protein coding regions of the gene. Detection of conserved regions of CTGF can help to identify crucial functional regions of this gene. Patients who have variants in these conserved regions may have an increased risk of fibrosis and identification of these patients may allow for individualized treatment based on this increased risk.

1. Acknowledgements
We would like to thank Plymouth State University, the PSU Research Advisory Council,
the PSU Student Research Advisory Council, and the New Hampshire Idea Network of
Biological Research Excellence for funding support. We would like to thank the
University of New Hampshire Hubbard Center for Genome Studies and Dartmouth
College Molecular Biology Shared Resources Lab for sequencing. We would also like to
thank Jon Bairam, Kevin Chesmore, Joel Dufour, Hailey Gentile, Evyn Grimes, Ethan
Johnson, Kathryn Kahrhoff, Lauren Oakes, Stacy Peterson, Ellen Rounds, Harlie Shaul,
Lorna Smith, Kate-Lyn Skribiski, Zachary Stevens, Amed Torres, Zoe White, and Alycia
Wiggins.
Conclusions
Departments of Biological Sciences and Computer Science at Plymouth State University, Plymouth NH
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• Examine CTGF selection in other species to further define
regions of conservation
• Examine human selection among more ethnically diverse
populations
• Throughout evolution, mutations that alter CTGF have been
selected against, suggesting conservation
• Human CTGF variants that alter amino acid sequence could
have a meaningful impact on the protein’s structure and
function
• Individuals with CTGF variants may have altered susceptibility
to fibrosis
• Current sequence data suggests selection is not occurring in
the PSU sample
Identifying Evolutionary Pressures in the Connective Tissue Growth Factor Gene
Kimberly Amerson, Ashley Kennedy, Kimberly Jesseman, Heather E Doherty, PhD
Future Directions
Negative Selection is Detected in Pairwise Comparisons Between Vertebrates
Human Orngtn Rabbit Pika GuinPg Mouse Rat Elephnt Pig Bovine Opossm TsmDvl AnlLzd Platyps Chcken ZbrFnch Flyctch ChTrtl
Human
Orangutan 0.1786
Rabbit 0.2277 0.2197
Pika 0.1790 0.1620 0.2764
GuinPig 0.2209 0.2301 0.2564 0.2612
Mouse 0.0956 0.0877 0.1273 0.1559 0.1639
Rat 0.0995 0.0913 0.1371 0.1489 0.1601 0.0678
Elephant 0.1628 0.1534 0.1783 0.1733 0.1444 0.0791 0.0810
Pig 0.0994 0.0961 0.1570 0.1654 0.1767 0.0760 0.0686 0.1128
Bovine 0.0974 0.0978 0.1581 0.1447 0.1788 0.0828 0.0803 0.1265 0.0760
Opossum 0.0598 0.0647 0.0702 0.0930 0.0738 0.0635 0.0762 0.0824 0.0618 0.0588
TasmDvl 0.0675 0.0681 0.0714 0.0830 0.0804 0.0639 0.0740 0.0755 0.0684 0.0653 0.0259
AnoleLzd 0.0778 0.0764 0.1106 0.1155 0.0996 0.0844 0.0773 0.0959 0.0839 0.0879 0.0682 0.0675
Platypus 0.1066 0.1091 0.1193 0.1622 0.1442 0.1016 0.0920 0.0986 0.1104 0.1132 0.0535 0.0648 0.1031
Chicken 0.0986 0.1014 0.1285 0.1390 0.1068 0.0757 0.0815 0.1011 0.0888 0.1046 0.0436 0.0594 0.0803 0.1037
ZbrFnch 0.0876 0.0886 0.0951 0.0975 0.0980 0.0654 0.0675 0.0851 0.0663 0.0987 0.0563 0.0721 0.0849 0.0817 0.0599
Flyctchr 0.1063 0.1068 0.1139 0.1238 0.1125 0.0842 0.0917 0.0896 0.0917 0.1234 0.0564 0.0778 0.0919 0.1117 0.0690 0.0965
ChTrtl 0.1051 0.1156 0.0850 0.1174 0.1145 0.1210 0.1085 0.1248 0.1179 0.1229 0.1020 0.1256 0.1550 0.1206 0.1381 0.1770 0.1743
Coelacnth -1.000 -1.000 0.0367 0.0650 -1.000 0.0570 0.0556 0.0795 0.0530 -1.000 0.0523 0.0658 0.0630 0.0696 0.0785 0.0728 0.0922 0.1216
Exon Selection LRT value p-value
1 & 2 < 1; negative 41.379 < 0.0001
3 < 1; negative 85.770 < 0.0001
4 < 1; negative 58.471 < 0.0001
5 < 1; negative 58.471 < 0.0001
Location
Chromosome
Location
Base
Change
A.A.
Change
Published
Frequency
Observed
Frequency
Exon 2 6:131950889 G/A C56Y unpublished 0.057
Exon 2 6:131950812 C/G H83D >0.99 0.989
Exon 2 6:131950778 T/C V94A unpublished 0.057
Exon 3 6:131950313 G/A V174M <0.01 0.006
Human Exon L1 L0 LRT p-value
1 & 2 -337.099 -337.411 0.622 0.430
3 -268.287 -268.509 0.443 0.416
4 -70.051 -70.051 4.00*10-6 ~1
5 -237.590 -237.590 1.15*10-4 ~1
Figure 2: A phylogenetic tree with dN/dS values for each branch
comparing two or more species. The phylogenetic tree was
generated using a bootstrap method and dN/dS values were
calculated using PAMLX. LRT values were calculated to determine
statistical significance. LRT values were compared to a Chi-square
distribution to determine p-values, where p<0.00625 was
considered statistically significant. Branches marked with a *
indicate p-values <0.001. Branches marked with a ** indicate p-
values <0.0001.
Results for Figure 2: dN/dS values calculated for all branches of the
phylogenetic tree were less than one, suggesting negative selection
for the CTGF gene at multiple evolutionary branches. These data
indicate that, throughout vertebrate evolution, there has been
selective pressure to conserve CTGF.
Table 1: A) Nonsynonymous SNPs detected in the CTGF gene in our PSU
population. SNP list includes information about location, base change, amino
acid change, frequency in our population, and published frequency (if
applicable). B) Summary of selective pressures detected in exons of the
CTGF gene within the Plymouth State population. dN/dS values for all exons
were computed for each pairwise comparison using PAMLX. LRT values were
calculated and compared to a Chi-square distribution to determine p-values,
where p<0.05 was considered statistically significant.
Results for Table 1: A) Four protein coding variants have been detected in
the CTGF gene in our PSU population. Although this gene is highly conserved
across a long evolutionary period, multiple genetic variations that alter the
amino acid sequence have been detected in a relatively homogenous
population. Variants in our PSU sample are found in evolutionarily conserved
regions and, therefore, are more likely to impact protein structure and
function. B) The p-values for exonic regions were not significant, suggesting
that within our human population, we observed neutral selection.
Figure 1: CTGF sequences were obtained from Ensembl and values were computed using PAMLX. dN/dS values <1 indicate negative selection, dN/dS values >1
indicate positive selection, and a dN/dS value = -1.0000 indicates that after adjustment for confounding factors, no synonymous variants were present. LRT values
were calculated and compared to a Chi-square distribution to determine p-values. p<0.05 was considered statistically significant. A) Pairwise dN/dS values between
19 vertebrate species for the whole CTGF gene. B) Summary of pairwise dN/dS values between 19 vertebrate species for individual exons of CTGF.
Results for Figure 1: A) All dN/dS values for the full CTGF gene are less than one, indicating negative selection is occurring in this gene. The average LRT value across
all species is 92.97 (L0 = -7959, L1 = -7913) with p-value <0.001 (p<0.05 is significant). The detected negative selection suggests that, as a whole, the CTGF gene has
been conserved throughout its evolutionary history. Strong conservation suggests mutations resulting in alteration to the CTGF protein were selected against,
indicating the importance of the protein’s amino acid sequence to its function. B) Negative selection was seen in all exons, suggesting strong conservation is not
just observed in CTGF as a whole, but also in individual exons of this gene.
No Selective Pressure is Observed in CTGF Within PSU Sample
Negative Selection is Detected Throughout Vertebrate Evolution
Connective tissue growth factor (CTGF) is a protein vital to numerous processes
in the body including blood vessel development (Shimo et al., 1999), cellular
proliferation (Frazier et al., 1996), and wound healing (Igarashi et al., 1993). CTGF
is also important in bone and connective tissue development, and has only been
observed in vertebrate species (Ivkovic et al., 2003). When the CTGF gene is
overexpressed, development of scar tissue, known as fibrosis, can occur (Colwell
et al., 2005). Differing levels of fibrosis susceptibility and severity have been
correlated with variations among humans in certain regions of the CTGF gene
(Dessein et al, 2009, Fonseca et al., 2007, Kawaguchi et al., 2009). Identifying
regions of the CTGF gene in which variants may be most likely to impact the
protein’s structure and function could help identify those variants more likely to
alter an individual’s risk of developing fibrosis.
To detect these potentially important regions, the evolutionary history of the
CTGF gene was examined. Some regions of the genome, known as conserved
regions, are under pressure to remain the same (Reviewed in Yang & Bielawski,
2000). Very few changes that alter protein structure and function are observed
between species in regions that are conserved. Alternatively, some regions of the
genome are under evolutionary pressure to change, and variations that may alter
protein structure or function are seen more commonly in these areas. Our goal is
to identify regions of CTGF that are conserved among species, because variations
in these regions among humans are more likely to alter regions of the protein
involved in important functions. To detect regions of conservation, the ratio of
variations that alter the protein sequence (nonsynonymous) to variations that do
not alter the protein sequence (synonymous) were calculated (as described by
Miyata & Yasunaga, 1980). Intra-species variations in humans were also examined
using the CTGF sequences from individuals at Plymouth State University. Variations
that we have detected in our human population that are located in these
conserved regions are more likely to alter CTGF structure and function and
therefore may alter fibrosis severity and risk. Identification of patients with
variants likely to increase fibrosis risk could allow for more individualized and
aggressive treatments to improve long-term prognosis.
Sequence Alignments and Phylogenetic Trees for 19 Species
Ensembl genome browser (Flicek et al., 2014) was used to obtain the CTGF
sequences of 19 vertebrate species. The sequences were aligned using a program
called Molecular Evolutionary Genetics Analysis 7 (MEGA7) (Tamura et al., 2015),
and a maximum likelihood phylogenetic tree was created to be used in phylogenetic
analysis. For analysis of individual exons, exons 1 and 2 were aligned together and
exons 3, 4 and 5 were each aligned separately.
Selection Analysis for 19 Species
To identify regions of the CTGF gene that are highly conserved across the 19 species,
Phylogenetic Analysis by Maximum Likelihood (PAMLX) (Xu & Yang, 2013, Yang,
1997, Yang, 2007) software was used. The codon maximum likelihood (codeml)
branch model was used to calculate the dN/dS ratios for all pairwise comparisons
between species for both the whole gene (Figure 1A) and individual exons (Figure
1B). The same method was used to calculate dN/dS values for each branch node on
the phylogenetic tree (Figure 2). A likelihood ratio test (LRT) was performed for each
branch using the equation LRT=2*(L0-L1), where L0 is the null hypothesis of no
selection and L1 is the alternative hypothesis of selection. LRT values were
compared to a Chi-square distribution to determine p-values. A Bonferroni
correction was made for multiple testing, when needed. dN/dS values > 1 suggest
positive selection, values < 1 suggest negative selection, and values close to one
suggest neutral or no selection.
Detection of Nonsynonymous Variants and Human Population Selection Analysis
DNA from cheek cells obtained from Plymouth State volunteers was extracted,
amplified using PCR and sent to Molecular Biology Core Facility at Dartmouth
College or UNH Hubbard Center of Genome Studies for sequencing. Returned
sequences were then analyzed for variants using PolyPhred software, and variants
that altered the protein sequence were noted (Figure 3A). Returned sequences
were also analyzed for selection within human evolutionary history, using similar
methods to those above, examining exons 1 and 2 together, and exons 3, 4, and 5
individually (Figure 3B). L0 and L1 values for pairwise comparisons between human
sequences were averaged and then LRT values were calculated and significance
determined as described above.
*
0.0946
**
0.0001
**
0.2109
**
0.0001 **
0.0470
**
0.0501**
0.1390
**
0.0091
**
0.1917
**
0.0337
**
0.0942
**
0.0408
**
0.4282
**
0.0721
**
0.0623
**
0.0513
**
0.1585
Methods
Introduction
A
B
A
B
Human
Mouse
Orangutan
Rat
Rabbit
Pika
Opossum
Tasmanian Devil
Guinea Pig
Elephant
Bovine
Pig
Anole Lizard
Chicken
Zebrafinch
Flycatcher
Coelacanth
Platypus
Chinese Softshell Turtle