Phenotypic Mutation 'seal'

• Allele: seal
• Mutation Type: splice site (2 bp from exon)
• Chromosome: 11
• Coordinate: 94,838,009 bp (GRCm39)
• Base Change: T ⇒ A (forward strand)
• Gene: Col1a1
• Gene Name: collagen, type I, alpha 1
• Synonym(s): Mov-13, Cola1, Cola-1, Col1a-1
• Chromosomal Location: 94,827,050-94,843,868 bp (+) (GRCm39)
• MGI Phenotype: FUNCTION: This gene encodes the alpha-1 subunit of the fibril-forming type I collagen, the most abundant protein of bone, skin and tendon extracellular matrices. The encoded protein, in association with alpha-2 subunit, forms heterotrimeric type I procollagen that undergoes proteolytic processing during fibril formation. Mice lacking the encoded protein die in utero caused by the rupture of a major blood vessel. Transgenic mice expressing significantly lower levels of this gene exhibit morphological and functional defects in mineralized and non-mineralized connective tissue and, progressive loss of hearing. [provided by RefSeq, Nov 2015] ; PHENOTYPE: Mutations in this locus cause variable phenotype, from embryonic lethal to viable/fertile with altered fibrillogenesis. Homozygotes can show impaired bone formation and fragility, osteoporosis, dermal fibrosis, impaired uterine postpartum involution, andaortic dissection. [provided by MGI curators]
• Mapped: Yes
• Institutional Source: Beutler Lab
• Gene Model: not available
• AlphaFold: P11087
• SMART Domains: Protein: ENSMUSP00000001547; Gene: ENSMUSG00000001506

Domain	Start	End	E-Value	Type
signal peptide	1	22	N/A	INTRINSIC
VWC	31	86	1.04e-16	SMART
Pfam:Collagen	97	154	1.1e-9	PFAM
Pfam:Collagen	166	227	7e-10	PFAM
Pfam:Collagen	225	284	2.4e-13	PFAM
Pfam:Collagen	285	344	5.9e-12	PFAM
low complexity region	354	426	N/A	INTRINSIC
internal_repeat_4	427	444	4.93e-7	PROSPERO
low complexity region	447	486	N/A	INTRINSIC
low complexity region	495	516	N/A	INTRINSIC
low complexity region	527	567	N/A	INTRINSIC
internal_repeat_3	570	588	1.25e-9	PROSPERO
low complexity region	590	600	N/A	INTRINSIC
low complexity region	603	627	N/A	INTRINSIC
low complexity region	629	651	N/A	INTRINSIC
internal_repeat_1	652	675	6.29e-11	PROSPERO
internal_repeat_4	658	675	4.93e-7	PROSPERO
low complexity region	678	699	N/A	INTRINSIC
low complexity region	702	717	N/A	INTRINSIC
internal_repeat_2	718	738	2.08e-10	PROSPERO
internal_repeat_1	718	741	6.29e-11	PROSPERO
internal_repeat_3	726	744	1.25e-9	PROSPERO
internal_repeat_5	737	752	9.8e-6	PROSPERO
Pfam:Collagen	768	827	2.8e-12	PFAM
Pfam:Collagen	828	887	6.8e-11	PFAM
internal_repeat_5	944	959	9.8e-6	PROSPERO
internal_repeat_2	952	972	2.08e-10	PROSPERO
Pfam:Collagen	1008	1077	4.8e-8	PFAM
Pfam:Collagen	1068	1127	1.2e-12	PFAM
Pfam:Collagen	1122	1184	2.8e-9	PFAM
PDB:3HR2|C	1185	1205	6e-6	PDB
COLFI	1217	1453	2.04e-162	SMART

• Predicted Effect: probably benign
• Meta Mutation Damage Score: Not available
• Is this an essential gene?: Essential (E-score: 1.000)
• Phenotypic Category: Autosomal Recessive
• Penetrance: 100%
• Mode of Inheritance: Autosomal Recessive
• Local Stock: Embryos
• MMRRC Submission: 030348-UCD
• Last Updated: 2017-05-02 10:56 AM by Katherine Timer
• Record Created: unknown
• Record Posted: 2007-10-15
• Science Writers: Eva Marie Y. Moresco
• Illustrators: Victoria Webster
• Authors: Koichi Tabeta, Xin Du, Bruce Beutler

Accession Number

NCBI RefSeq: NM_007742; MGI: 88467

Alleles Listed at MGI

All alleles(14) : Targeted, knock-out(3) Targeted, other(6) Gene trapped(2) Transgenic(1) Chemically induced(2)

Phenotypic Description

The seal phenotype was detected in two ENU-induced mutant G3 littermates as a defect in hindlimb movement resulting in an abnormal gait. The hindlegs are paralyzed such that seal mice appear to “waddle” like seals on land. Initially, transmissibility appeared sporadic. However, it became clear that homozygotes only demonstrate hind leg paralysis after being picked up for examination by grasping the loose skin over the nape of the neck. Otherwise, seal mice display normal locomotor activity (i.e. walking ability and cage activity). When it occurs, paralysis persists for approximately 8 days before hindlegs regain near-normal movement; most animals retain a mild “seal-like” gait. The abnormal gait of seal mice is associated with physical damage to the spine (visible hemorrhage results from grasping). Seal mice have thin bones and fragile tissues.

Nature of Mutation

The seal mutation mapped to Chromosome 11, and corresponds to a T to A transversion in the donor splice site of intron 36 (GTAAGT -> GAAAGT) in the Col1a1 gene (position 10914 in Genbank genomic region NC_000077 for linear genomic DNA sequence of Col1a1). Col1a1 cDNA from seal mice has not been sequenced. The mutation may result in skipping of the 108-nucleotide exon 36 (depicted below); this deletion would not destroy the reading frame of the encoded type I procollagen, α1 chain [α1(I)]. Col1a1 contains 51 total exons.

     <--exon 35 <--exon 36 intron 36-->  exon 37--> <--exon 51 
10551 GGCCCCCCT……GGCCCCATT GTAAGTATC……………GGTAACGTT………TTCGTGTAA 15350
796   -G--P--P-……-G--P--I-               -G--N--V-………-F--V--*  1417
       correct    deleted                      correct

The donor splice site of intron 36, which is destroyed by the seal mutation, is indicated in blue lettering; the mutated nucleotide is indicated in red lettering.

Protein Prediction

Figure 1.  Domain structure of the mouse Col1a1 protein.  Col1a1 encodes the 1453 amino acid type I procollagen, α1 chain [α1(I)].  The seal mutation is an A to T transversion occurring in the donor splice site of intron 36, indicated by the red asterisk.  The mutation also results in the possible deletion of exon 36 (residues 807-841).  The triple helical region is flanked by N- and C-terminal propeptides.  The N propeptide contains a von Willebrand Factor type C domain (VWFC), shown in teal.  The C-terminal propeptide contains a fibrillar cartilage NC1 domain (FC NC1), indicated in dark blue.

Col1a1 encodes the 1453-amino acid type I procollagen, α1 chain [α1(I)] (Figure 1). α1(I) forms heterotrimers with the type I procollagen, α2 chain [α2(I)] with stoichiometry 2 α1(I): 1 α2(I), forming the extracellular matrix protein type I collagen (Figure 2). [All collagens consist of 3 α chains; each α chain is identified by an arabic numeral followed by a roman numeral in parentheses representing the collagen type it is part of (1).] The protein structure and folding of collagen has been extensively studied [see (2-4) and references therein]. It is described here briefly: Both α1(I) and α2(I) encode left-handed polyproline II-like chains, which supercoil together in a right-handed manner around a common axis to form a triple helix. The bulk of each chain consists of a triple helical domain encoded by 43 exons and 1014 amino acids in the uninterrupted repeating sequence Gly-X-Y. Flanking the triple helical domain are N- and C-terminal propeptides. Regions of the C-terminal propeptide nucleate the triple helix by mediating the association of the chains and tethering them together. The repeating Gly-X-Y sequence undergoes a series of required enzymatic modifications which allow the triple helix to form. These include hydroxylation of a subset of proline and lysine residues in the Y position, and subsequent glycosylation of specific hydroxylated lysines. Chain formation propagates in a C- to N-terminal direction, and depends critically on a glycine residue at every third position of the chain. Glycine is the only residue small enough to fit in the sterically constrained inner aspect of the helix, and mutation to any other residue disrupts helix folding.

 

Figure 2. Crystal structure of a collagen-like peptide. This protein triple helix represents a heteromic triple helix structure consistent with fiber diffraction studies on collagen supercoiling. Based on PDB ID: 1CAG, from Bella J, et. al., Crystal and molecular structure of a collagen-like peptide a 1.9 Angstrom resolution. Science, 1994. Model created with Pymol, Schrodinger, LTD.

Each of the 43 exons encoding the triple helical domain of α1(I) begins with a codon for glycine and ends with a Y-position codon. Thus, splice site mutants that undergo exon skipping can still yield in-frame transcripts, although use of cryptic splice sites may also result in one or several out-of-frame transcripts or a premature termination codon. The seal mutation is predicted to cause skipping of Col1a1 exon 36, which encodes amino acids near the center of the triple helical domain. The effect of the mutation on Col1a1 transcription or α1(I) translation is unknown, as is the effect on collagen formation. Such a structural mutation could result in an abnormal protein that is poorly secreted, or if secreted into the extracellular matrix in significant amounts, that dominantly interferes with fibrillogenesis, collagen-matrix or collagen-cell interactions, or with bone mineralization (3).

Expression/Localization

Collagens form the structural basis of skin, tendon, bone, cartilage, and other tissues. Some collagens have a restricted tissue expression pattern; for example types II, IX and XI are found almost exclusively in cartilage and type IV is only in basement membranes (1). Type I collagen, of which the α1(I) chain is a constituent, exists in tendon, bone and skin (4). Most collagens form some type of supramolecular structure, such as fibrils in the case of type I collagen. Collagens are secreted into the extracellular matrix.

Background

Figure 3. Type I collagen formation and possible alterations in the structure due to the α chain mutations. The left side of the diagram above illustrates the normal steps in collagen type I formation and the assembly into mature collagen fibers: 1. Propeptide domains are secreted from the rough ER. 2. C-propeptides asssociate and form procollagen triple helices. 3. After secretion into the cytoplasm, propeptides are cleaved at the N- and C-terminus. 4. Triple helix molecules form cross-linkages to form mature fibrils. 5. Fibrils associate to form collagen type I fibers. The right side of the diagram demonstrates possible disruptions in helix and fiber formation that may be seen in OI. Figure adapted from references (2) and ( 4 ) .

Collagens are the most abundant proteins in the human body, making up approximately 30% of its protein mass (1). There are at least 27 collagen types and 42 α chains in vertebrates, in addition to a variety of proteins containing the collagen triple helix motif (1;4). Fibril-forming collagen orthologues have been identified in invertebrates (5), as well as in bacteria and viruses (6).

 

Some 1100 mutations in various collagens leading to heritable human disease have been reported (1;4). One of the best characterized collagen diseases is osteogenesis imperfecta (OI), in which patients have bone fragility due to mutations in the genes encoding the α1(I) or α2(I) chains of type I collagen (3). OI results in fractures upon minimal or absent trauma, dentinogenesis imperfecta (DI; easily worn or broken grey or brown teeth with possible translucence), and hearing loss (7). OI has varying degrees of severity, from perinatal lethality to severe skeletal deformities with impaired mobility to mild predisposition to fractures (7). Fractures may occur in any bone, but most often occur in the extremities. Cases of OI are classified into seven types (I-VII) based on mode of inheritance, clinical presentation and radiographic examination. Most types (I-V) are inherited in an autosomal dominant manner, likely because mutations that deform an α chain can disrupt the formation of collagens containing wild type chains. Type VII is inherited recessively. Mutations in COL1A1 cause OI type I (OMIM #166200), IIA (OMIM #166210), III (OMIM #259420), and IV (OMIM #166220).

 

Two general classes of mutations in type 1 collagen chains result in OI: those that cause the absence of protein (e.g. prematurely terminated alleles, alleles encoding unstable proteins), and those that cause structural defects in the protein (Figure 3) (3). The first class causes a quantitative decrease in the amount of type I collagen produced, usually by half, and generally results in a mild OI type. The second class of structural mutations results in a wide range of OI phenotypes, depending on the type and location of the mutation. The most common of this type of mutation is a single base substitution in a glycine codon, replacing one of the invariant glycine residues of the triple helical domain with one of eight bulkier amino acids and resulting in destabilization of the triple helix (4). How different glycine substitutions lead to different severities of OI remains a subject of study, and no simple rule for understanding genotype-phenotype relationships exists (3). Various models propose the importance of the identity of the substituting residue, the distance of the mutation from the C-terminus, and the sequence surrounding the mutation (8).

 

There are several mouse mutants of Col1a1, and they display a range of defects similar to those of OI patients (9-13). A knock-in mutant designated Brittle IV (BrtlIV) expresses a mutant α1(I) chain with a Gly 349 to Cys mutation, reproducing a mutation in a type IV OI patient (12). These mice suffer skeletal deformity, fragility, osteoporosis and disorganized trabecular structure, and display a phenotypic variability ranging from perinatal lethality to long term survival, similar to human OI patients (12). Another notable mouse mutant, the exon2Δ mouse lacking the N-terminal propeptide of α1(I), displayed no defects in collagen fibril formation and appeared as wild type, demonstrating that the N-terminal propeptide is not essential for collagen biogenesis (14).

Putative Mechanism

After single base mutations that substitute a bulkier amino acid for glycine, the second most common type of structural mutation in type I collagen α chains is a splice site alteration (3). Most such α1(I) mutations result in a mild OI type. As mentioned above (Protein prediction), splice site mutations can yield in-frame transcripts, but also out-of-frame transcripts and premature termination codons. In addition to the consequences for the integrity of the collagen triple helix, splice site mutations may affect collagen secretion, or interfere with interactions with ligands such as cell adhesion receptors (e.g. integrins) or other extracellular matrix molecules.

 

In a case marginally similar to seal in which a four-exon in-frame deletion of Col1a1 exons 33-36 caused severe type III OI in two patients, no mutant protein could be detected in mutant dermal fibroblasts and osteoblasts (15).The seal mutation is predicted to cause skipping of exon 36 of Col1a1 and yield an in-frame transcript. However, the consequences of the seal mutation for transcripts, α1(I) protein, or collagen formation have not been examined.

Primers

Primers cannot be located by automatic search.

Genotyping

Seal genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.

 

Primers for PCR amplification

Seal(F): 5’-TGCTCCCGTAAGTACAGAAGACCC-3’

Seal(R): 5’-AGATCCTTTCTCACCAGCAGGACC-3’

 

PCR program

1) 94°C             2:00

2) 94°C             0:30

3) 56°C             0:30

4) 72°C             1:00

5) repeat steps (2-4) 29X

6) 72°C             7:00

7) 4°C               ∞

 

Primers for sequencing

Seal_seq(F): 5’- ACAGAAGTGTTACCCTGAGTC-3’

Seal_seq(R): 5’- TCAGAAGTGAACAGTCACCTGTC-3’

 

The following sequence of 1138 nucleotides (from Genbank genomic region NC_000077 for linear DNA sequence of Cola1) is amplified:

 

10352                                   tgctcccgt aagtacagaa gaccctgatc

10381 tctgttcatc ccttctcccc tacctgcttt ctgcccccac cgcaaacccc acccctttac

10441 tctatccgtt cctctccttc cctaatgttg agacatctct ccaaagtcgt ctccttcttc

10501 ttctagggag accgtggtga ggctggtccc cctggtcctg ctggctttgc cggcccccct

10561 gtgagtatca agaccctcct cattttctgt ccctagctga gacacgaggc atgggacctt

10621 ggggtggctg aatgaggaca gaagtgttac cctgagtcag aggagaaggg tggggaggta

10681 ctggtgtctc caagtgtctc tgcatctcca agtccctatc tgtggccctt cctctagccc

10741 agaggccctc tgctctcagg ctgcctcctc cactcctcca ctctccattc tccctcctgc

10801 ctagggtgct gatggccaac ctggtgcgaa aggtgaacct ggtgatactg gtgttaaagg

10861 tgatgctggt cctcctggcc ctgctggtcc tgctggaccc cccggcccca ttgtaagtat

10921 cttgtcttct gcaccataag ctttggatag ccttggactt ggggctagcc tggatctcat

10981 accttgacac tgtcttacag ggtaacgttg gtgctcctgg acccaaaggt cctcgtggtg

11041 ctgctggtcc ccctgtgagt atcatatgca tctctgtcgc gactccccaa aggcagagac

11101 tggagatgag gccaggtgac aggtgactgt tcacttctga ccacccaatg ttctctccta

11161 ccagggtgct actggcttcc ctggtgctgc tggccgtgtc ggtccccctg gtccctctgt

11221 gagtatctgt ggttctggaa tgaggatggg gtgagacatg tattgtcagg acagcaggcc

11281 tggctggggc ttgccactat gatgctttgg aagcctggac tctgacagtc cttcttgtgc

11341 ccatctaggg aaatgctgga ccccctggcc ctcccggtcc cgttggcaaa gaagggggca

11401 aaggtccccg tggtgagact ggccctgctg gacgtcctgg tgaagttggt cccccaggtc

11461 cccccggtcc tgctggtgag aaaggatct

 

PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is shown in red text.

References

   1.  Myllyharju, J. and Kivirikko, K. I. (2004) Collagens, modifying enzymes and their mutations in humans, flies and worms, Trends Genet. 20, 33-43.

   2.  Baum, J. and Brodsky, B. (1999) Folding of peptide models of collagen and misfolding in disease, Curr. Opin. Struct. Biol. 9, 122-128.

   3.  Marini, J. C., Forlino, A., Cabral, W. A., Barnes, A. M., San Antonio, J. D., Milgrom, S., Hyland, J. C., Korkko, J., Prockop, D. J., De, P. A., Coucke, P., Symoens, S., Glorieux, F. H., Roughley, P. J., Lund, A. M., Kuurila-Svahn, K., Hartikka, H., Cohn, D. H., Krakow, D., Mottes, M., Schwarze, U., Chen, D., Yang, K., Kuslich, C., Troendle, J., Dalgleish, R., and Byers, P. H. (2007) Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans, Hum. Mutat. 28, 209-221.

   4.  Brodsky, B. and Persikov, A. V. (2005) Molecular structure of the collagen triple helix, Adv. Protein Chem. 70, 301-339.

   5.  Exposito, J. Y., Cluzel, C., Garrone, R., and Lethias, C. (2002) Evolution of collagens, Anat. Rec. 268, 302-316.

   6.  Rasmussen, M., Jacobsson, M., and Bjorck, L. (2003) Genome-based identification and analysis of collagen-related structural motifs in bacterial and viral proteins, J Biol. Chem. 278, 32313-32316.

   7.  Steiner, R. D., Pepin, M. G., and Byers, P. H. (Updated 28 Jan 2005) Osteogenesis Imperfecta. In: GeneReviews at GeneTests: Medical Genetics Information Resource (database online). Copyright, University of Washington, Seattle. 1997-2007. Available at http://www.genetests.org. Accessed 10 Oct 2007.

   8.  Byers, P. H. (2001) Folding defects in fibrillar collagens, Philos. Trans. R. Soc. Lond B Biol. Sci. 356, 151-157.

   9.  Bonadio, J., Saunders, T. L., Tsai, E., Goldstein, S. A., Morris-Wiman, J., Brinkley, L., Dolan, D. F., Altschuler, R. A., Hawkins, J. E., Jr., Bateman, J. F., and . (1990) Transgenic mouse model of the mild dominant form of osteogenesis imperfecta, Proc. Natl. Acad. Sci. U. S. A 87, 7145-7149.

 10.  Harbers, K., Kuehn, M., Delius, H., and Jaenisch, R. (1984) Insertion of retrovirus into the first intron of alpha 1(I) collagen gene to embryonic lethal mutation in mice, Proc. Natl. Acad. Sci. U. S. A 81, 1504-1508.

 11.  Liu, X., Wu, H., Byrne, M., Jeffrey, J., Krane, S., and Jaenisch, R. (1995) A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling, J Cell Biol. 130, 227-237.

 12.  Forlino, A., Porter, F. D., Lee, E. J., Westphal, H., and Marini, J. C. (1999) Use of the Cre/lox recombination system to develop a non-lethal knock-in murine model for osteogenesis imperfecta with an alpha1(I) G349C substitution. Variability in phenotype in BrtlIV mice, J Biol. Chem. 274, 37923-37931.

 13.  Pereira, R., Khillan, J. S., Helminen, H. J., Hume, E. L., and Prockop, D. J. (1993) Transgenic mice expressing a partially deleted gene for type I procollagen (COL1A1). A breeding line with a phenotype of spontaneous fractures and decreased bone collagen and mineral, J Clin. Invest 91, 709-716.

 14.  Bornstein, P., Walsh, V., Tullis, J., Stainbrook, E., Bateman, J. F., and Hormuzdi, S. G. (2002) The globular domain of the proalpha 1(I) N-propeptide is not required for secretion, processing by procollagen N-proteinase, or fibrillogenesis of type I collagen in mice, J Biol. Chem. 277, 2605-2613.

 15.  Wang, Q., Forlino, A., and Marini, J. C. (1996) Alternative splicing in COL1A1 mRNA leads to a partial null allele and two In-frame forms with structural defects in non-lethal osteogenesis imperfecta, J Biol. Chem. 271, 28617-28623.