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New Insights Into the Melanophilin (MLPH) Gene Affecting Coat Color Dilution in Rabbits

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New Insights Into the Melanophilin (MLPH) Gene Affecting Coat Color Dilution in Rabbits G C A T T A C G G C A T genes Article New Insights into the Melanophilin (MLPH) Gene Affecting Coat Color Dilution in Rabbits Julie Demars 1,*, Nathalie Iannuccelli 1, Valerio Joe Utzeri 2, Gerard Auvinet 3, Juliette Riquet 1, Luca Fontanesi 2 and Daniel Allain 1 1 GenPhySE, INRA Animal Genetics, Toulouse Veterinary School (ENVT), Université de Toulouse, 31326 Castanet Tolosan, France; [email protected] (N.I.); [email protected] (J.R.); [email protected] (D.A.) 2 Department of Agricultural and Food Sciences (DISTAL), Division of Animal Sciences, University of Bologna, 40127 Bologna, Italy; [email protected] (V.J.U.); [email protected] (L.F.) 3 GenESI, INRA le Magneraud, 17700 Surgères, France; [email protected] * Correspondence: [email protected]; Tel.: +33-5-61-28-51-15 Received: 29 June 2018; Accepted: 13 August 2018; Published: 23 August 2018 Abstract: Coat color dilution corresponds to a specific pigmentation phenotype that leads to a dilution of wild type pigments. It affects both eumelanin and pheomelanin containing melanosomes. The mode of inheritance of the dilution phenotype is autosomal recessive. Candidate gene approaches focused on the melanophilin (MLPH) gene highlighted two variants associated with the dilution phenotype in rabbits: The c.111-5C>A variant that is located in an acceptor splice site or the c.585delG variant, a frameshift mutation. On the transcript level, the skipping of two exons has been reported as the molecular mechanism responsible for the coat color dilution. To clarify, which of the two variants represents the causal variant, (i) we analyzed their allelic segregation by genotyping Castor and Chinchilla populations, and (ii) we evaluated their functional effects on the stability of MLPH transcripts in skin samples of animals with diluted or wild type coat color. Firstly, we showed that the c.585delG variant showed perfect association with the dilution phenotype in contrast to the intronic c.111-5C>A variant. Secondly, we identified three different MLPH isoforms including the wild type isoform, the exon-skipping isoform and a retained intron isoform. Thirdly, we observed a drastic and significant decrease of MLPH transcript levels in rabbits with a coat color dilution (p-values ranging from 10−03 to 10−06). Together, our results bring new insights into the coat color dilution trait. Keywords: coat color dilution; melanophilin; rabbit 1. Introduction Different coat colors in the European rabbit (Oryctolagus cuniculus) have been selected throughout domestication and are nowadays fixed in specific breeds. Among the various phenotypic traits, coat color dilution corresponds to an altered distribution of eumelanin and pheomelanin pigments in skin and hair [1]. A similar coat color dilution phenotype has been observed in other mammals such as mice. Mutations within proteins encoding the melanosome transport complex were described in the myosin VA (Myo5a)[2], Ras-related protein (Rab27a)[3] and melanophilin (Mlph)[4] genes. In humans, patients suffering from Griscelli Syndrome (GS) exhibit a pigment dilution in their hair and skin, which, depending on the specific genetic variant, may or may not be accompanied by other important symptoms. Interestingly, mutations within MYO5A [5], RAB27A [6] and MLPH [7,8] are respectively responsible for GS1 (OMIM #214450), GS2 (OMIM #607624) and GS3 (OMIM #609227). Only GS3 is characterized by hypomelanosis with no immunologic or neurologic manifestations. Genes 2018, 9, 430; doi:10.3390/genes9090430 www.mdpi.com/journal/genes Genes 2018, 9, x FOR PEER REVIEW 2 of 12 #609227). Only GS3 is characterized by hypomelanosis with no immunologic or neurologic Genes 2018, 9, 430 2 of 12 manifestations. Candidate gene approaches focused on these specific genes were performed in several species presentingCandidate a dilution gene‐ approacheslike coat color focused phenotype. on these The specific mode of genes inheritance were performed of the dilution in several phenotype species is presentingautosomal recessive a dilution-like [1]. Mutations coat color within phenotype. the MLPH The mode gene ofwere inheritance identified of in the mice dilution [4], cat phenotype [9] (OMIA is 000206autosomal‐9685), recessive dog [10–12] [1]. Mutations (OMIA 000031 within‐9615), the MLPH chickengene [13] were (OMIA identified 001445 in‐9031), mice [quail4], cat [14] [9] (OMIA 001445000206-9685),‐93934), dog American [10–12 ]mink (OMIA [15] 000031-9615), (OMIA 001438 chicken‐452646) [13 and] (OMIA cattle 001445-9031), [16] (OMIA 001438 quail‐ [452646).14] (OMIA In rabbits,001445-93934), although American variants minkhave also [15] been (OMIA highlighted 001438-452646) within the and MLPH cattle gene, [16] (OMIA two published 001438-452646). studies haveIn rabbits, suggested although different variants variants have also as the been genuine highlighted causal within mutation the MLPH [17,18].gene, An two alternatively published spliced studies MLPHhave suggested transcript different isoform corresponding variants as the to genuine two‐exon causal skipping mutation was suggested [17,18]. An as alternativelythe causal molecular spliced mechanismMLPH transcript for the isoform coat color corresponding phenotype to [17]. two-exon The c.111 skipping‐5C>A was variant, suggested located as within the causal intron molecular 2 in an acceptormechanism site for for the splicing, coat color was phenotypereported as [ 17the]. most The c.111-5C>A likely variant variant, leading located to this within exon skipping intron 2 in [17]. an Thisacceptor skipping site for of splicing,exons 3 and was 4 reported caused a asframeshift the most leading likely variant to a change leading of two to this amino exon acids skipping followed [17]. Thisby a skippingpremature of exonsstop codon 3 and 4[17]. caused A second a frameshift variant, leading c.585delG, to a change corresponding of two amino to a acids 1‐bp followeddeletion byin exona premature 6 of the stop MLPH codon gene [17 has]. A also second been variant, identified c.585delG, in various corresponding breeds with to a a 1-bpdilution deletion of their in exoncoat [17,18].6 of the ThisMLPH variantgene also has alsoled to been a frameshift identified and in various an altered breeds amino with acid a dilution sequence of with their a coat premature [17,18]. Thisstop codon. variant Both also variants led to a were frameshift identified and in an the altered first study amino and acid are associated sequence with coat a premature color dilution stop incodon. several Both breeds variants (Netherland were identified Dwarf, in Loh, the firstLionhead study Dwarf and are and associated Blue Vienna) with coat [17]. color The dilutionauthors suggestedin several a breeds higher (Netherland relevance of Dwarf,the c.111 Loh,‐5C>A Lionhead variant although Dwarf and an effect Blue Vienna)of the c.585delG [17]. The variant authors on thesuggested dilution a phenotype higher relevance was suggested of the c.111-5C>A for individuals variant who although were not an homozygous effect of the c.585delGfor mutated variant allele aton c.111 the dilution‐5C>A [17]. phenotype However, was only suggested the c.585delG for individuals variant was who highlighted were not and homozygous analyzed by for Fontanesi mutated etallele al. [18]. at c.111-5C>A An association [17]. signal However, with onlythe coat the color c.585delG dilution variant was wasobtained highlighted in various and breeds analyzed (n = by7) includingFontanesi etBlue al. Vienna [18]. An and association Castor Rex signal [18]. with the coat color dilution was obtained in various breeds (n = 7)To including better understand Blue Vienna the and dilution Castor phenotype Rex [18]. in rabbits and to likely discriminate, which of the two previouslyTo better understand reported variants the dilution is the phenotype true causal in variant, rabbits we and analyzed to likely discriminate,their segregation which with of the dilutiontwo previously phenotype reported in Castor variants and isChinchilla the true causalbreeds variant,already weknown analyzed to have their the segregation trait of interest with [18]. the Oncedilution highlighting phenotype the in genetic Castor implication and Chinchilla of the breeds c.585delG already variant, known we to evaluated have the its trait impact of interest on MLPH [18]. transcriptsOnce highlighting in order the to genetic understand implication the molecular of the c.585delG mechanisms variant, involved we evaluated in coat its color impact dilution on MLPH in rabbits.transcripts in order to understand the molecular mechanisms involved in coat color dilution in rabbits. 2. Materials and Methods 2.1. Animals 2.1. Animals The coat coat color color dilution dilution phenotype phenotype was was observed observed in Castor in Castor and andChinchilla Chinchilla breeds breeds (Figure (Figure 1). Both1). breedsBoth breeds are selected are selected for fur for production; fur production; each each includes includes two two lines. lines. The The wild wild type type (wt) (wt) Castor Castor line line has has a normala normal black black-brown‐brown coat coat color color with with a a yellow yellow agouti agouti band. band. The The diluted diluted Castor Castor line line was was derived derived from the wt Castor lineline byby selectingselecting thethe blueblue coatcoat color color phenotype. phenotype. Similarly, Similarly, the the wt wt Chinchilla Chinchilla line line carries carries a anormal normal black-brown black‐brown coat coat color color with with the Ch the allele Ch atallele the Cat locus the C and locus the otherand linethe (dilutedother line Chinchilla) (diluted wasChinchilla) derived was from derived wt Chinchilla from wt with Chinchilla the ash coatwith color the ash phenotype. coat color Nowadays, phenotype. the Nowadays, four lines represent the four linesfour distinctrepresent populations four distinct since populations they are selected since they independently. are selected independently. Figure 1. Characterization of the dilution phenotype in Castor and Chinchilla breeds. The rabbits with Figure 1. Characterization of the dilution phenotype in Castor and Chinchilla breeds.
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