The wooly mutation (wly) on mouse chromosome 11 is associated with a genetic defect in Fam83g
- Legairre A Radden†1,
- Kevin M Child†1,
- Elisabeth B Adkins1,
- Damek V Spacek1,
- Aaron M Feliciano1 and
- Thomas R King1Email author
© Radden et al.; licensee BioMed Central Ltd. 2013
Received: 3 October 2012
Accepted: 7 May 2013
Published: 9 May 2013
Mice homozygous for the spontaneous wooly mutation (abbreviated wly) are recognized as early as 3–4 weeks of age by the rough or matted appearance of their coats. Previous genetic analysis has placed wly in a 5.9 Mb interval on Chromosome 11 that contains over 200 known genes. Assignment of wly to one of these genes is needed in order to provide probes that would ultimately facilitate a complete molecular analysis of that gene’s role in the normal and disrupted development of the mammalian integument.
Here, a large intraspecific backcross family was used to genetically map wly to a smaller (0.8 Mb) span on mouse Chromosome 11 that includes fewer than 20 genes. DNA sequencing of the coding regions in two of these candidates known to be expressed in skin has revealed a 955 bp, wly-specific deletion. This deletion, which lies within the coordinates of both Slc5a10 [for solute carrier family 5 (sodium/glucose cotransporter), member 10] and Fam83g (for family with sequence similarity 83, member G), alters the splicing of mutant Fam83g transcripts only, and is predicted to result in a severely truncated (probably non-functional) protein product.
We suggest that this mutation in Fam83g is the likely basis of the mouse wooly phenotype.
To associate this mutation with a causative molecular defect, wly was fine-mapped to a genetic region where fewer than 20 genes are located—only a few of which are known to be expressed in skin. Direct sequence analysis of the coding regions in two of these candidates has identified a mutant-specific defect in Fam83g (for family with sequence similarity 83, member G) that we propose to be the likely genetic basis of the wly mutation.
Standard inbred strains A/J, C57BL/6J, and NOD/ShiLtJ; and NOD/ShiLtJ-wly/J mutant mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Mice homozygous for the mutant wly allele were reliably identified by the matted appearance of their coats which is first evident by 3–4 weeks of age and persists throughout life. Both male and female wly/wly homozygotes appear to be fully fertile, and we have maintained the NOD/ShiLtJ-wly/J line since 2009 by crossing homozygotes. The treatment and use of all mice in this study were compliant with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Central Connecticut State University (New Britain, CT, USA).
Genomic DNA was isolated from 2–4 mm tail-tip biopsies taken from two-week-old mice, using Nucleospin® kits from BD Biosciences (Palo Alto, CA, USA), as directed. DNA samples from standard inbred and mutant strains that we do not routinely maintain in our colony were purchased from The Jackson Laboratory’s Mouse DNA Resource.
The polymerase chain reaction (PCR) was performed in 13 ul reactions using the Titanium® PCR kit from BD Biosciences, as directed. Oligonucleotide primers for PCR were designed and synthesized by Invitrogen (Carlsbad, CA, USA), based on sequence information available online [3, 4]. In addition to standard microsatellite markers  on Chr 11, six DNA markers based on single-nucleotide-polymorphisms previously reported to differ between strains A/J and NOD/ShiLtJ [3, 4] were also scored. These markers (herein designated SNP1-6) are described in detail in Additional file 1 & Additional file 2. To visualize PCR product sizes, reactions plus 2 ul loading buffer (bromophenol blue in 20% Tris-buffered sucrose) were electrophoresed through 3.5% NuSieve® agarose (Lonza, Rockland, ME, USA) gels. Gels were stained with ethidium bromide (0.5ug/mL) and photographed under ultraviolet light. For sequence analysis, about 1.5 ug of individual PCR amplimers were purified and concentrated into a 30 ul volume using QIAquick® PCR Purification kits (Qiagen, Valencia, CA, USA). Amplimers were shipped to SeqWright, Inc. (Houston, TX, USA) for primer-extension sequencing.
Total RNA was isolated from tail skin samples taken from 3-month-old mice using the Nucleospin® RNA L kit by Macherey-Nagel (Easton, PA, USA). cDNA was generated using the SMARTer™ RACE cDNA amplification kit (Clontech Laboratories). To amplify Slc5a10-specific cDNA, primers 1F (5’ TGTTCCGGGACCCTTCCACAGGAGACCT 3’), taken from Exon 9, and 1R (5’ ATGACCAGCCGTCCCACCAGCAGCAACT 3’), taken from Exons12 and 13, were used in a “step-down” PCR reaction. The products of this initial reaction were diluted 1:10 in Tricine-KOH buffer (10 mM, pH 8.5) plus 1 mM EDTA, and were amplified again in a standard PCR reaction using a nested primer pair: 2F (5’ AGCGGTCCCTGTCTGCCCGGAACTTGAA 3’), taken from Exon 10, and 2R (5’ TGGGCATCAGCTCCATGACCAGCTTCGGGT 3’), taken from Exon 11. To amplify Fam83g-specific cDNA, primers 3F (5’ ACGGGCAGCCGCACATCAAGGAAGTGGT 3’), taken from Exon 1, and 3R (5’ AGCACAATGGGCTCTGGCTCTGGCTCCT 3’), taken from Exon 4, were used in a standard PCR reaction. The products of this initial reaction were diluted (as above), and were amplified again with a nested primer pair: 4F (5’ TGCGCAAGATGGTCAGCCAGGCGCAGAA 3’), taken from Exon 1, and 4R (5’ ATGGGCTCTGGCTCTGGCTCCTTCTCCA 3’), taken from Exon 4. Final (second-round) amplimers were purified (as described above) and shipped to SeqWright, Inc., for primer-extension sequencing.
Meiotic fine-mapping of wly
Sequence analysis of gene candidates from the wly-critical interval
For some of the genes located in the SNP2 to SNP6 interval (including Slc47a1, Epn2, Grap, Ulk2, Mapk7, and Akap10) loss-of-function alleles have been engineered (by others), and null-allele homozygotes have been reported to display no apparent changes to hair development or coat texture, making them unlikely candidates for the gene responsible for the wly mutation. For several others (including four predicted genes not shown in Figure 3a), available expression data [3, 4] failed to suggest any obvious functional role in skin. By contrast, two other genes in the critical interval, the overlapping Slc5a10 [for solute carrier family 5 (sodium/glucose cotransporter), member 10] and Fam83g (for family with sequence similarity 83, member G) genes, were isolated from cDNA libraries derived from mouse skin . To investigate these “primary” candidates as the possible basis of the mutant wooly phenotype, the coding regions of both genes were sequenced in genomic DNA from A/J, C57BL/6J, NOD/ShiLtJ, and mutant NOD/ShiLtJ-wly/J mice.
While this analysis revealed several sites that are polymorphic among these four strains—some new and some previously reported (as summarized in Additional file 3 and shown in detail in Additional file 4 & Additional file 5)—the only sequence distinction found between the coisogenic NOD/ShiLtJ and NOD/ShiLtJ-wly/J strains was a 955 bp deletion in the mutant strain (see Additional file 6a) that lies in Intron 10–11 of Slc5a10 and removes part of Intron 2–3 and Exon 3 of Fam83g (see Figure 3b). Because none of 17 other inbred strains tested (including nine strains of Swiss origin, and therefore related to or derived from the NOD strain) show this sequence alteration, while wly mutants tested from three distinct colonies do (Additional file 6b), we suggest that this 955 bp deletion is specifically associated with the wly mutation.
A Fam83g deletion is likely to be the molecular basis of the mutant wooly phenotype
Because this deletion lies in Intron 10–11 of the Slc5a10 gene, we amplified sequences between Exons 9 and 13 from cDNA templates based on total RNA isolated from wly/wly or wild type skin to determine if this mutation could affect splicing of Slc5a10 transcripts. Amplimers copied from wly/wly and wild type NOD/ShiLtJ cDNA templates were identical in length, and sequencing verified that Exons 10 and 11 of Slc5a10 are spliced normally in wly/wly mutants (data not shown).
The eight members of the Fam83 family are mostly uncharacterized proteins, in both mouse and man. Fam83a, also known as tumor antigen BJ-TSA-9 , is a novel, tumor-specific protein highly expressed in human lung adenocarcinoma cells. Fam83d, also referred to as spindle protein CHICA , is a cell-cycle-regulated spindle component which localizes to the mitotic spindle and is both up regulated and phosphorylated during mitosis. Defects in the gene encoding Fam83h cause autosomal dominant hypocalicified ameliogenesis imperfecta (ADHCAI) [15–19]. Fam83b, c, f, and g are uncharacterized proteins present across vertebrates, while Fam83e is an uncharacterized protein found only in mammals. Some proteins with structural similarity to Fam83g’s N-terminal domain of unknown function (Pfam domain DUF1669, [20, 21]; superfamily domain SSF5624, [22, 23]; conserved domain cd 09119, [24, 25]; see Figure 4) are known to be phospholipases, but this domain in Fam83g shows only trace similarity to the phospholipase D catalytic domain and lacks the functionally-important histidine residue [26, 27], so while Fam83g may share a similar 3-dimensional fold with some phospholipase D-like enzymes, it is unlikely to display phospholipase D-like activity. An additional proline-rich domain has been identified from amino acid 470 to 511 (Prosite profiles, PS5099, [28, 29]; see Figure 4), but again no function has yet been assigned. Whatever their functional significance, both of these conserved protein domains would be partially or entirely removed by the Fam83g mutation in wooly mice (see Figure 4). We predict that this mutant Fam83g protein is, therefore, likely to be non-functional, consistent with our suggestion that the lack of normal Fam83g product in wly/wly homozygotes may be responsible for the mutant wooly phenotype.
More direct evidence for a causal link between this deletion and the wooly phenotype would require, for example, transgenic rescue of mutant homozygotes, or recreation of the wooly phenotype in engineered Fam83g “knock-out” homozygotes. While such single-addition and single-subtraction experiments are beyond our laboratory’s ability to perform, we anticipate testing for complementation between NOD/ShiLtJ- wly/J and a recessive, Fam83g knock-out variant (as soon as one becomes available). Non-complementation (i.e., production of phenotypically mutant wly/Fam83gk.o. offspring) would provide definitive proof that homozygousity for Fam83g defects, alone, is the molecular basis of the mutant wooly phenotype.
No other similar mutations affecting skin or hair have been described in this region on mouse Chr 11 , nor are we aware of any human conditions involving skin or hair in the orthologous region of human Chr 17p11-12 . In man, the term “woolly hair” (WH) is used to describe a group of inherited hair shaft disorders characterized by fine and tightly curled hair , but—based on their distinct phenotypes, chromosomal locations, and, in some cases, known molecular bases [see [32, 33]—none of these described disorders appears related to mouse wooly. We therefore suggest that the NOD/ShiLtJ- wly/J mouse strain may provide a unique animal resource, the study of which will be crucial to any future investigation of Fam83g and its functional role in the normal or disrupted development of the mammalian integument. For example, it will be interesting to learn where Fam83g is expressed in normal and mutant hair follicles (e.g., in the dermal papilla, the epithelial lineage, or other hair-follicle-associated structures), and whether its expression varies during the anagen, catagen and telogen phases of the hair follicle growth cycle. While one previous histological evaluation failed to uncover a microscopic manifestation of the wooly phenotype , we hope—especially with molecular probes made possible by the likely genetic assignment of wly to Fam83g—that in situ-based expression or immunohistological analyses can now be approached to finally reveal the cellular basis of the wooly phenotype.
The 955 bp deletion we describe appears to be specifically associated with the wly mutation; it alters the splicing of mutant Fam83g transcripts; and is predicted to generate a severely truncated, mutant Fam83g protein. We therefore suggest that this defect is likely to be the molecular basis of the mutant wooly phenotype.
TRK is a professor in the Biomolecular Sciences Department at Central Connecticut State University (New Britain, CT). LAR, KMC, EBA, DVS, and AMF were undergraduates majoring in Biomolecular Sciences or Biochemistry when they conducted this research.
The authors thank CCSU undergraduates Nisrine Dagamseh, Michelle Kayser, Andrea Nixon, Andy Morrison, Lori LeBlanc, Randy Taylor, Amarilis Perez, Jose Horak; and all the high-school interns (Edie Tinker, Mariam Hasan, Somaly Chhean, Rick Deschenes, Alicia Davis, Joshua Wrice, Marcelino Thillet, Ananda Thillet, Danerick Peralta, Diego Peralta, Ashley Feliciano, Krystal Garcia, Kayla Garcia, Jodalis Montalvo, Milagros Molina, Brianna Cirinna, and Rebecca Fuentes) for help with DNA isolation and marker typing. We also thank Mary Mantzaris for excellent animal care. This work was supported by NIH AREA Grant 1R15AR059572.
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