Introduction

This blog post explores the P3 allele of the equine FLNC gene, which encodes filamin-C. Portions of this blog post serve as additional sources of information to supplement the FLNC Gene Page.

The P3 allele of FLNC carries two missense mutations, shown below. The protein model XP_023495410.1 was used to assign amino acid positions.

Figure 1. Alignment of partial FLNC protein sequences from horse, compared to the P3a and P3b variants. The positions of the amino acids affected by the two substitutions are highlighted in red.

We present data to support the hypothesis that the P3 allele of FLNC is damaging.

For the P3a variant (E794K), the substitution of lysine (positively charged) for glutamic acid (negatively charged) is a somewhat conservative substitution. For the P3b variant (A1248T), the substitution of threonine (polar uncharged) for alanine (hydrophobic) is a nonconservative substitution.

Evolutionary conservation provides convincing evidence that the P3 allele of FLNC is damaging. 

We use public data to show that the reference allele of P3a (E794) is highly conserved across 389 species of mammals, birds, reptiles, amphibians, and fish representing 29 unique sequences of the 41-amino acid query sequence centered on the position of the E794K allele, covering about 400 million years of evolutionary history. A single conservative substitution, E794D, has gone to fixation twice, once in reptiles (two species) and once in fish (two species).

A similar analysis shows that the reference allele of P3b (A1248) is highly conserved across 389 species of mammals, birds, reptiles, amphibians, and fish representing 75 unique sequences of the 41-amino acid query sequence centered on the position of the A1248T allele, covering about 400 million years of evolutionary history. A single missense allele, A1248S, has gone to fixation in an amphibian.

Comparing FLNC protein sequences from multiple species

Evolutionary conservation provides evidence on whether the P3 allele of FLNC is damaging. In this approach, predicted FLNC protein sequences are compared among a number of different species. Amino acid substitutions that have gone to fixation in one or more species are assumed to be selectively neutral; mutations in conserved residues that have not gone to fixation in any species are likely damaging.

The 41-amino-acid portions of the equine FLNC gene centered on the P3a and P3b variants, shown above, were used to conduct a blastp search of the protein sequence database at NCBI, retrieving sequences from mammals, birds, reptiles, amphibians, and fish. Identical sequences were clustered. Sequences represented by only a single species were used as query sequence for additional blastp searches. See the Technical Appendix for details.

Evolutionary Conservation of P3a: Mammals

There is remarkably little variation among mammals in the 41-amino-acid segment centered on the position of the E794K allele. Only four unique sequences are found in 185 mammals, as shown in Figure 2 below.

Figure 2. Alignment of partial FLNC protein sequences from mammals, centered on the position of the P3a variant. The horse sequence was used as a blastp query sequence to retrieve FLNC protein sequences from mammals. Sequences that were identical were clustered. Numbers in parentheses indicate the number of species in a cluster. CLUSTAL output summarizes whether a particular position is a single and fully conserved residue (*), has a conservative substitution with strongly similar properties (:), a somewhat conservative substitution (.), or is not conserved ( ). The sequence of the horse P3a allele E794K (highlighted in red) is shown for comparison, but was not included in the CLUSTAL analysis. See the Technical Appendix for details.

The amino acid sequence of FLNC is highly conserved in this region in mammals. Only one of the 41 amino acids is not conserved. Position 41 can be alanine (A), valine (V), or serine (S). Position 34 has an alanine (A) to glycine (G) mutation in a single species (Chinese hamster).

The E794 allele (highlighted in red) is absolutely conserved in 185 species of mammals. The E794K allele is only seen as a minor allele in horse.

Evolutionary Conservation of P3b: Mammals

The 41-amino-acid sequence around the position of the A1248T variant is highly conserved among mammals. Fourteen unique sequences are found in 185 mammals, as shown in Figure 3 below.

Figure 3. Alignment of partial FLNC protein sequences from mammals, centered on the position of the P3b variant. The horse sequence was used as a blastp query sequence to retrieve FLNC protein sequences from mammals. Sequences that were identical were clustered. Numbers in parentheses indicate the number of species in a cluster. CLUSTAL output summarizes whether a particular position is a single and fully conserved residue (*), has a conservative substitution with strongly similar properties (:), a somewhat conservative substitution (.), or is not conserved ( ). The sequence of the horse P3b allele A1248T (highlighted in red) is shown for comparison, but was not included in the CLUSTAL analysis. See the Technical Appendix for details.

The amino acid sequence of FLNC is highly conserved in the P3b region in mammals, but less so than the region surrounding P3a. One position is not conserved, seven allow amino acid substitutions with strongly similar properties, and 34 positions are absolutely conserved.

The A1248 allele is absolutely conserved in 184 species of mammals. The A1248T allele is only seen as a minor allele in horse.

Evolutionary Conservation of P3a: Vertebrates
 
The 41-amino-acid sequence around the position of the E794K variant is highly conserved among mammals. In order to identify additional amino acid positions for which substitutions have gone to fixation, we identified additional FLNC sequences from birds, reptiles, amphibians, and fish.
 
We identified FLNC sequences from 185 mammals (summarized above), 94 birds, 31 reptiles, 9 amphibians, and 70 fish, a total of 389 species. There were 29 unique sequences in this collection, as shown in Figure 4.

Figure 4. Alignment of partial FLNC protein sequences from vertebrates, centered on the position of the P3a variant. The horse sequence was used as a blastp query sequence to retrieve FLNC protein sequences from vertebrates. Sequences that were identical were clustered. Numbers in parentheses indicate the number of species in a cluster. CLUSTAL output summarizes whether a particular position is a single and fully conserved residue (*), has a conservative substitution with strongly similar properties (:), a somewhat conservative substitution (.), or is not conserved ( ). The sequence of the horse P3a allele E794K (highlighted in red) is shown for comparison, but was not included in the CLUSTAL analysis. See the Technical Appendix for details.

The amino acid sequence of FLNC is highly conserved in the P3a region in vertebrates. Seven positions are not conserved, four allow amino acid substitutions with strongly similar properties, one allows an amino acid substitution with somewhat similar properties, and 28 positions are absolutely conserved.

The FLNC-E794K (P3a) allele found as a minor allele in horse has not gone to fixation in any of the 389 species examined. The E794 allele is invariant as the reference or wild-type allele across 185 species of mammals. The conservative substitution E794D (aspartic acid substituted for glutamic acid) is seen in two reptiles (cluster A15, Anolis sagrei and Anolis carolinensis) and two fish (Oncorhynchus tshawytscha and Leuresthes tenuis).

Evolutionary Conservation of P3b: Vertebrates

The 41-amino-acid query sequence around the position of the A1248T variant is also highly conserved among vertebrates. The protein sequences identified in the search among birds, reptiles, amphibians, and fish identified above represent 75 unique sequences, as shown in Figure 5.

Figure 5. Alignment of partial FLNC protein sequences from vertebrates, centered on the position of the P3b variant. The horse sequence was used as a blastp query sequence to retrieve FLNC protein sequences from vertebrates. Sequences that were identical were clustered. Numbers in parentheses indicate the number of species in a cluster. CLUSTAL output summarizes whether a particular position is a single and fully conserved residue (*), has a conservative substitution with strongly similar properties (:), a somewhat conservative substitution (.), or is not conserved ( ). The sequence of the horse P3b allele A1248T (highlighted in red) is shown for comparison, but was not included in the CLUSTAL analysis. See the Technical Appendix for details.

The amino acid sequence of FLNC is somewhat conserved in the P3b region in vertebrates. Fifteen positions are not conserved, 12 allow amino acid substitutions with strongly similar properties, one allows an amino acid substitution with somewhat similar properties, and 13 positions are absolutely conserved.

The FLNC-A1248T (P3b) allele found as a minor allele in horse has not gone to fixation in other species. The A1248 allele is invariant as the reference or wild-type allele across 185 species of mammals. The A1248 allele is strongly conserved in 389 species of vertebrates; a single species of amphibian (Hymenochirus boettgeri) has an A1248S substitution not seen in any other of the 389 species examined.

Summary

Analysis of evolutionary conservation of FLNC protein sequences strongly supports the hypothesis that the missense alleles that make up the P3 genotype are damaging. Both FLNC-E794K (P3a) and FLNC-A1248T (P3b) are substitutions in highly-conserved positions in the protein. While both of these alleles that make up the P3 haplotype are present as minor alleles in horse, neither allele has gone to fixation in any of 389 species of vertebrates. The P3a and P3b alleles are not tolerated over evolutionary time.

Figure 6. The amino acid at the position of the equine P3a allele of FLNC (E794K) is a glutamic acid residue conserved throughout evolution. Sequence conservation at this position is absolute across 185 species of mammals; the E794K allele is seen only as a minor allele in horse. The E794K allele has not gone to fixation in birds (94 species), reptiles (31 species), amphibians (9 species), or fish (70 species). The conservative substitution E794D has gone to fixation twice, once in the reptile lineage (2 species), and once in the fish lineage (2 species). Estimated time of divergence is shown as millions of years ago (Mya).

Figure 7. The amino acid at the position of the equine P3b allele of FLNC (A1248T) is an alanine residue conserved throughout evolution. Sequence conservation at this position is absolute across 185 species of mammals; the A1248T allele is seen only as a minor allele in horse. The A1248T allele has not gone to fixation in birds (94 species), reptiles (31 species), amphibians (9 species), or fish (70 species). The nonconservative substitution A1248S has gone to fixation once in the amphibian lineage (1 species). Estimated time of divergence is shown as millions of years ago (Mya).

Technical Appendix

The purpose of this technical appendix is to permit researchers to reproduce and extend these results independently.

The equine P3 allele is a haplotype with a pair of missense alleles. At the DNA level, the alleles are described as the coordinates and base change in EquCab3.0:

Using the amino acid positions from the protein model XP_023495410.1 allows the missense alleles to be described as FLNC-E754K (P3a) and FLNC-A1248T (P3b), shown below.

Click the link to the UCSC Genome Browser to view the horse genome sequence centered on the position of the E794K(P3a) allele.

Click the link to the UCSC Genome Browser to view the horse genome sequence centered on the position of the A1248T (P3b) allele.

Retrieving protein sequences. 

Protein sequences like those shown in the alignments (Figures 2-5) can be retrieved from NCBI using the blastp tool and a query sequence.

The FLNC gene is one of a three-member family of genes: FLNA, FLNB, and FLNC, encoding filamin A, filamin B, and filamin C, respectively. The query sequences used to retrieve FLNC protein sequences for the analysis shown here have high sequence identity to the corresponding sequences in the orthologous proteins. In well-studied species where we expect a high quality assembly and good gene models (e.g. mouse and human), we can see that these query sequences identify other sequences in the paralogs FLNA and FLNB when we use a FLNC query sequence. 

These matches to paralogs can seen by using a human query sequence against the human protein database, as shown below. Go to the BLASTp server at NCBI.

1. For the query sequence, use:

2. Restrict the search to “Homo sapiens” as shown.

3. Click “BLAST”

The same method can be used to search the protein database with any query sequence and any taxonomic restriction (e.g. birds).

Selected results from BLASTp search of human FLNC protein sequences using the human P3a query sequence are shown below.

The human FLNC P3a query sequence retrieves protein sequences from FLNC (100% identity), FLNA (93% identity), and FLNB (82% identity).

The partial human FLNC query sequence used to retrieve sequences around the position of the P3b allele is:

This sequence can be used as a query to identify related sequences using blastp as shown above. Selected results from BLASTp search of human FLNC protein sequences using the human P3b query are shown below.

The human FLNC P3b query sequence retrieves protein sequences from FLNC (100% identity), FLNA (66% identity), and FLNB (63% identity). Each alignment lists the number of matches to the protein sequence. For the FLNC P3b query sequence, these are lower-quality matches to similar repeats in the protein sequence.

All protein sequences used for this analysis were required to match both P3a and P3b query sequences at a level of identity appropriate for the evolutionary distance between the species. In some species with lower quality sequencing and annotation, part of the FLNC coding sequence is missing, and the query sequence will retrieve sequences from paralogs (FLNA and FLNB) or from other part of the FLNC sequence. These sequences were not included in this analysis.

Aligning protein sequences

Multiple protein sequences were aligned using CLUSTAL.

Evolutionary relationships

Information on evolutionary relationships among species is presented graphically as the Tree of Life.

Download data

The data used for alignments in Figures 2-5 are available as a downloadable spreadsheet. The spreadsheet shows every organism used for this analysis, listing both the scientific and a common name. A single FLNC protein sequence from each species is selected; it must match two 41-amino acid queries (centered on P3a and P3b). Identical sequences were clustered. The P3a and P3b clusters to which each sequence was assigned are also given.