Linkage analysis previously identified ahyperglycemic quantitative trait locus (QTL), Nidd 2/of, on rat Chromosome 14 in crosses utilizing OLETF (Otsuka Long Evans Tokushima Fatty) rat, a model for type 2 diabetes. A separate QTL study mapped an obesity QTL, Obs5, to the same chromosomal region. A congenic strain placing ca. 38cMOLETF-derivedsegments containing both Nidd2/of and Obs5 on the F344 background was shown to possess mild diabetic and obese phenotypes, suggesting the presence of mutations affecting the glucose metabolism and fat accumulation. In order to localize the loci more precisely, we generated a series of deletionsubcongenic strains in which OLETF-segments were shortened from either ends. We found that there are at least two hyperglycemic QTLs within the Nidd2/of locus. We predict that they are localized towards both ends of the Nidd2/of region. In contrast, Obs5QTL was further narrowed down to a single region of ca. 10 cM fragment

Kose, Hiroyuki

Masuda, Aki

Matsumoto, Kozo

Tokushima University Institutional Repository

INTRODUCTIONEven after the completion of human genome project,it is still increasingly challenging to localize susceptibilitygenes that are involved in the expressionof quantitativetraits, such as obesity, hyperglycemia, hypertension(1, 2). This is largely due to the complex nature of inter-actions among those genes and/or gene-environmentassociations. Genetic dissection of complex trait in hu-man is compromised by genetic heterogeneity and nu-merous environmental variations. Therefore, the ad-vantage of alleviating the complexity by utilizing well-defined animal models in constant breeding conditionfor the study of multigene variance is far more signifi-cant than for the study of single-gene trait.Marker-assisted linkage analyses for quantitativetrait loci (QTL) and subsequent congenic strain con-struction have become a usual measure (3, 4). TheNidd 2/of locus mapped to rat chromosome 14 is oneof the fourteen hyperglycemia QTLs we identified inour previous linkage analysis using the OLETF rat,a model for the type 2 diabetes (5, 6). In an independ-ent genetic analysis we also mapped an obesity QTL,Obs 5 , within an overlapping chromosomal segmentwith Nidd2/of (7).With subsequently generated con-genic strain, this 38 cM segmentwasconfirmed to con-tain polymorphisms related to both abnormal glucoseregulation and retroperitoneal fat accumulation (8).Here we constructed a series of subcongenic strains,in which OLETF-derived segment is shortened fromeither ends in order to further narrow down the frag-ment which includes a QTL down to a few cM. Ourresult indicated that there aremultiple loci in this regionand each QTL appears to play a distinctive role.MATERIALS AND METHODSRat strains and animal proceduresThe F.O-Nidd2/of congenic strain, a parental strainORIGINALFinemappingof thehyperglycemicandobesityQTLbycongenicstrains suggests multiple loci on rat chromosome 14Masuda Akhi, Hiroyuki Kose, and Kozo MatsumotoDivisions for Animal Research Resources, Institute of Health Biosciences, The University of TokushimaGraduate School, Tokushima, JapanAbstract : Linkage analysis previously identified a hyperglycemic quantitativetrait locus (QTL),Nidd 2/of, on rat Chromosome 14 in crosses utilizing OLETF (Otsuka Long Evans TokushimaFatty) rat, a model for type 2 diabetes. A separate QTL study mapped an obesity QTL, Obs5,to the same chromosomal region. A congenic strain placing ca. 38 cM OLETF-derived segmentscontaining both Nidd2/of and Obs5 on the F344 background was shown to possess mild diabeticand obese phenotypes, suggesting the presence of mutations affecting the glucose metabolismand fat accumulation. In orderto localizethe loci moreprecisely,wegeneratedaseriesofdeletion-subcongenic strains in which OLETF-segments were shortened from either ends. We foundthat there are at least two hyperglycemic QTLs within the Nidd2/of locus.Wepredict that theyarelocalized towards both ends of the Nidd2/of region. In contrast, Obs5 QTL was further narroweddown to a single region of ca. 10 cM fragment. J. Med. Invest. 52 : 109-113, February, 2005Keywords :QTL, congenic strain, OLETF, type 2 diabetesReceived for publication December 10, 2004 ; accepted Jaunary 5,2005.Address correspondence and reprint requests toKozoMatsumoto,Divisions for Animal Research Resources, Institute of Health Bi-osciences, The University of TokushimaGraduate School, Kuramoto-cho, Tokushima 770-8503, Japan and Fax : +81-88-633-9429.The Journal of Medical Investigation Vol. 52 2005１０９containing Nidd2/of locus (“of”meaning OLETF rat),was intercrossed with Fischer-344 (F344/Crj, SCL,Inc. Hamamatsu Japan)(8). The resultant F1 progenymale was backcrossed with F344 females. By usingmicrosatellite markers BC1 was screened for chro-mosomes inwhich recombination occurredwithin theNidd2/of region. The progeny with desirable recom-binant chromosomes was crossed to F344 rats and theheterozygote offspringswere selectively bred to fix therecombinant chromosome on the F344 background.The established strain was subsequently maintainedby brother-sister mating. Each strain was named afterthe genome structure in the region. For example, F.O-Ni2/of.rat55 indicates that the OLETF-derived seg-ment extends from the proximal end to the positiondefined by D14Rat55 microsatellite marker. Simi-larly, F.O-Ni2/rat55.of exchanges the distal portionstarting from the locus at D14Rat55.All rats were kept under specific pathogen-free con-ditions. The temperature (21±2℃), humidity (55±10%), and air conditioning were all controlled. Rats hadfree access to tap water and standard laboratory chowand were maintained at a 12-h light and dark cycle (7am/7 pm). This studyconformed to theguidelinesfor thecare and use of Laboratory animals of The Universityof Tokushima Graduate School Institute of Health Bi-oscience.GenotypingDNA isolation and polymerase chain reaction (PCR)amplification ofmicrosatellitemarkerswereperformedas described previously (5).The primers for micro-satellite markers were purchased from ResearchGe-netics, Inc. (Hrinlsville, Ala.).PhenotypingOral glucose tolerance test (OGTT)were performedon male rats of 30 weeks of age as described (5). Theanimals were fasted overnight ; bloodglucose levelsweremeasured with the glucose oxidase method (Arkray,Kyoto, Japan) at 0, 30, 60, 90 and 120 min after oral ad-ministration of 2.0 g glucose (in a 2.8 M glucose so-lution) per kilogram of body weight. Animals wereanesthetized with pentobarbital sodium intraperito-neally one week after the OGTT. The abdominal fatpads consisting of mesenteric, retroperitoneal, andepididymal fats were removed and weighted. The adi-posity index was calculated from each fat pad and bodyweight (percentage of fat weight/body weight)(7).Statistical analysisAll values are expressed as means±SE. The statis-tical significance of differences was evaluated usingANOVA for comparing all traits among congenic strainsand control F344 strains.RESULTSGeneration of the new congenic strainsThe extent of the congenic region for newly estab-lished substrains of F.O-Nidd2/of is described inFigure1.Five of these strains have distinctive deletion patternsof OLETF-derived chromosome from the distal sideof the region and the other five from the proximal side.This is the same strategy as those for identifyingbindingsite of a transcription factor within a promoter regionby making a series of deletion mutants. Therefore as-suming that a single QTL is sole determinant for thehyperglycemia or obesity in F.O-Nidd2/of strain, com-mon region for all the substrains showing the identicalphenotypes to that of the parental strain should newlydefine the QTL.OGTT analysis of Nidd2/of substrainsThe substrains were characterized by an oral glu-cose tolerance test (OGTT), the same test used formapping the Nidd2/of (Table 1). None of the strainsshowed significantly increased blood glucose levelsat any time point during OGTT. Since the entire con-genic region in F.O-Nidd2/of is covered by these strains,this result suggested that there aremore than one QTLwithin this segment. It is worth noting that the neitherof the two strains which exchange the shortest segmentat either end, namely F.O-Ni2/of.rat10 and F.O-Ni2/wox1.of, showed equivalent hyperglycemia to that ofthe parental stain, suggesting that there are twoQTLat both ends of the segment. This is indeed an intrigu-ing observation because camel’s hump-likeLODscorecurve in our original linkage analysis in this region ret-rospectively infers the presence of QTL at both ends(Figure 1)(9). The middle portion might also containadditional QTLs, because althoughstatistical significancewas not achieved, strains with shorter fragment tendto show proportionally lower blood glucose levels(Table 1).Adiposity analysis of Nidd2/of substrainsOLETF rat is genetically susceptible to obese pheno-type and obesity is important for the development ofdiabetes (10, 11). In the previous linkage analyses,we found that in the OLETF rat the distinctive set ofloci plays a role forobesity fromthose for hyperglycemiaphenotype. Furthermore each obesity QTL affects onM. Akhi et al. Fine mapping of diabetic and obesity QTL１１０the adiposity in a body-location specific manner (7).Obs5 was identified for increased retroperitonealfat index. In subcongenic strains, only certain strainsshowed increased retroperitoneal fat index (Table 1).The likely location for the QTL is between D14Rat143and D14Rat55, based on the fact that all strains inFigure 1 that contain all or part of this region have sig-nificant effects on adiposity. In contrast, none of theFigure. 1. Genetic map of chromosomal regions exchanged in the congenic substrains. Lod score plots for linkage to glucoselevel at 30 min in OGTT (solid line) and retroperitoneal fat index (dotted line) are shown (7, 9). The known extent of the OLETF-derived portion carried by each congenic strain is designated by the filled bars; the open ends of this bar designate intervalscontaining the recombinant end points. The strain represented by gray bar, F.O-Ni2/of.rat23, is known to possess OLETF-derived segment at D14Rat23 locus. Thus the interval at either side contains one recombinant end point. The markers ap-pearing in this figure are those that are polymorphic between OLETF and F344 rats and were used for genotype check. Geneticdistances represented in centiMorgans were determined by Kosambi map function. Hatched and Stippled bars indicatethe fine mapped loci for Nidd2/of and Obs5 QTL, respectively.The Journal of Medical Investigation Vol. 52 February 2005 １１１substrains examined pronounced any influences onmesenteric fat accumulation, which is consistent withour linkage analysis and with the fact that the parentalF.O-Nidd2/of strain showednodifference from theF344rat. Epididymal fat index and body weight were alsoaffected in some substrains (Table 1). This is unex-pected because the F.O-Nidd2/of shows no significantinfluence on this type of fat tissue.DISCUSSIONAmong the 14 hyperglycemicQTLswe selected theNidd2/of locus for fine mapping analysis as a next steptoward gene finding. This is because our initial char-acterization of the congenic strain revealed that thelocus appears to bemore significant for the expressionof diabetic phenotypes. Firstly, this locus is responsiveto obesity. When obesity was introduced either by highfat diet or leptin receptor deficiency ( fa/fa), glucoselevels were significantly elevated bymore than simpleaddition of the effect from Nidd2/of and the effect fromthe state of obesity (unpublished observation). Giventhe well-established role of obesity for type 2 diabetes,it stands to reason to expect to find important genesor signaling cascades by studying the QTL like Nidd2/of . Secondly, we demonstrated the epistatic interactionbetween the Nidd2/of and Nidd1/of by analyzing adouble congenic rat possessing the both loci (unpub-lished observation).In the past decade, quantitative trait locusmappingusing animal models has identified hundreds of chro-mosomal regions crucial for the expression of athero-sclerosis, diabetes, hypertension, obesity and othercomplex phenotypes (12-16). Indeed in several reportsQTL was further dissected bymethods similar to whatis described in the current study (17, 18). It is rathersurprising that in many cases it was found that therewere multipleQTLwithin a singleQTLregionas initiallymapped (17, 19). Thus, thenumberofgenes for complextrait is likely to increase as more elaborate mappingwork emerges. Indeed in yeast it was found that un-derlying genes for a certain quantitative phenotypeare tightly linked within a small chromosomal region(20, 21).Before we began this mapping, we were more in-clined to believe that perhaps a singleQTL plays a dualrole for both hyperglycemia and obesity, becauseQTLmapping gives only a rough estimation of underlyinggene location. However, this study clearly demonstratedthat loci for these traits are distinctive and there arelikely to be at least twoQTLs forhyperglycemicpheno-type. Apparent disappearance of diabetic phenotypesamong substrainsmaymerely be due to theweak con-tribution from each QTL, therefore more statisticalpower may be required for detecting them. Alterna-tively constructing a double-subcongenic strain intro-gressing short segments at both endsmay achieve theequivalent diabetic effect to that of the parental congenicstrain.It is interesting that at least threeQTLswith specificfunction are clustered and intricate interaction amongthem is crucial for glucosemetabolism. From thepointof understanding how obesity causes diabetes and howeach QTL associates one another, this region shouldprovide a unique opportunity, as there are limitednum-ber ofgenes thus it represents amodelwithsubstantiallyreduced complexity.Table 1. Phenotypic characterization of congenic substrainsstrain n 0 min 30 min 60 min 90 min 120 min AUC BWMesentericfat padRetroperitoneal fat padEpididymalfat padMesentricfat IndexRetroperitoneal fatIndexEpididymalfat IndexF 344 9 79.2±2.8 105.4±3.9 109.4±2.8 106.3±2.6 102.1±2.1 12357±290 388±5.5 10.65±0.45 8.96±0.29 11.66±0.45 2.74±0.10 2.31±0.07 3.00±0.09F.O-Nidd 2/of 8 81.2±3.3 133.0±8.4† 120.6±5.6¶ 119.5±5.9¶ 119.3±5.2† 14201±692¶ 397±6.6 10.31±0.73 10.36±0.73¶ 10.88±0.59 2.59±0.15 2.60±0.15¶ 2.73±0.12F.O-Ni 2/of.rat 10 10 78.5±2.2 111.6±1.4 106.6±1.6 107.5±1.6 102.5±0.8 12408±120 411±5.2† 12.24±0.56 11.38±0.34‡ 13.91±0.50† 2.97±0.12 2.77±0.07† 3.38±0.11¶F.O-Ni 2/of.rat 55 9 82.6±1.5 102.6±2.3 102.1±1.6 102.4±1.9 104.3±1.9 12016±175 404±3.9¶ 12.28±0.46 12.11±0.45‡ 14.81±0.37‡ 3.04±0.09 2.99±0.09‡ 3.66±0.08‡F.O-Ni 2/of.wox 14 8 82.1±1.7 100.4±1.8 105.4±3.1 105.6±2.2 108.0±2.8 12193±255 426±5.3‡ 12.53±0.86 12.54±0.62‡ 15.08±0.91† 2.93±0.18 2.94±0.13‡ 3.53±0.18†F.O-Ni 2/of.rat 5 8 77.1±2.3 101.5±4.5 103.2±3.9 109.5±4.3 103.0±3.4 12126±404 409±7.3¶ 9.81±0.68 10.32±0.47 12.56±0.59 2.38±0.15 2.52±0.11 3.06±0.11F.O-Ni 2/of.rat 23 7 79.6±3.4 96.0±4.8 97.3±5.8 97.4±4.8 94.3±4.8 11329±331 408±7.7¶ 9.70±0.32 10.58±0.42 12.91±0.69 2.38±0.06 2.59±0.08¶ 3.16±0.13F.O-Ni 2/wox 1.of 7 77.3±1.8 111.3±2.4 108.1±3.6 108.0±3.1 105.7±3.2 12567±303 420±6.2† 11.95±0.78 11.84±0.49‡ 14.00±0.70† 2.83±0.15 2.82±0.09† 3.33±0.13¶F.O-Ni 2/rat 143.of 6 75.0±3.0 125.7±10.2 109.3±3.0 109.7±4.5 108.3±3.8 13090±501 428±7.0‡ 12.25±0.90 10.92±0.57¶ 14.36±0.70† 2.85±0.18 2.55±0.11 3.35±0.12¶F.O-Ni 2/wox 14.of 9 74.6±2.6 113.1±7.7 106.1±3.1 105.4±1.3 106.1±1.0 12450±313 409±11.1 11.14±0.59 11.16±0.79¶ 12.44±1.0 2.71±0.12 2.70±0.14¶ 3.01±0.21F.O-Ni 2/rat 55.of 7 80.1±2.7 111.8±3.5 107.7±2.6 103.9±1.5 108.1±1.8 12527±196 392±11.6 9.51±0.80 10.09±0.58 12.35±0.67 2.41±0.18 2.57±0.11 3.14±0.12F.O-Ni 2/rat 8.of 9 81.4±3.0 109.2±3.6 109.3±2.1 107.2±2.3 105.3±2.1 12575±234 388±6.9 10.55±0.47 9.95±0.25 11.92±0.32 2.72±0.12 2.57±0.06 3.07±0.06All values are given as means±SE. 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Fine mapping of the hyperglycemic and obesity QTL by congenic strains suggests multiple loci on rat chromosome 14

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Fine mapping of the hyperglycemic and obesity QTL by congenic strains suggests multiple loci on rat chromosome 14

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