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Distorted Mendelian transmission as a function of genetic background in Rai1-haploinsufficient mice

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Abstract

The retinoic acid induced 1 gene (RAI1) is the primary causative gene for Smith–Magenis syndrome (SMS). Chromosomal deletion encompassing RAI1 or mutation in RAI1 is responsible for the majority of SMS features. Mouse models with targeted disruption of Rai1 have recapitulated overt SMS phenotypes, including craniofacial abnormalities, obesity, and neurobehavioral anomalies. Penetrance and expressivity of most phenotypes in mice were incomplete due to the mixed genetic background in which they were created. While increased penetrance of craniofacial phenotypes was observed in relatively homogeneous backgrounds, the effect of Rai1 haploinsufficiency on breeding outcome and fitness has not been studied. We analyzed mating results of Rai1+/− mice in a pure C57BL/6J background (≥N10 generations). A significant distortion (P < 0.05) of Mendelian transmission ratio with skewing against Rai1+/− mice was observed. Consequently, a decreased number of Rai1+/− pups and no Rai1−/− pups were obtained from all the breeding pairs. The decreased yield of Rai1+/− pups precluded penetrance studies of other phenotypes in these mice. However, when Rai1+/− alleles were transferred to a slightly variable (∼1% 129/∼99% C57BL/6J) genetic background expected numbers of Rai1+/− pups were obtained. Our results indicate that selection against Rai1-haploinsufficient alleles is governed primarily by modifier genes. Our data show that genetic background or modifier genes also significantly contribute to the severity of the phenotypes in SMS mouse models, mirroring the phenotypic variation observed in humans with Smith–Magenis syndrome and support the need for investigation of modifier loci for both single gene and complex genetic syndromes.

Introduction

Smith–Magenis syndrome (SMS) is a complex mental retardation/congenital anomalies disorder with variable clinical presentation [1]. It is caused by an interstitial deletion of chromosome 17p11.2 that includes the retinoic acid induced 1 (RAI1) gene, or by mutations in RAI1[1]. While haploinsufficiency of RAI1 is responsible for the diagnostic features of SMS, including craniofacial features, neurological and behavioral features, and sleep disturbance, hemizygosity of other genes in 17p11.2 contributes to the systemic features and overall severity of the syndrome [2]. However, striking phenotypic variability is also observed within SMS cases with RAI1 mutations [3], likely due to epistatic interactions and genetic background effects. To characterize the functional effects of Rai1 haploinsufficiency, Rai1-targeted mice were created in a mixed genetic background (C57BL/6J × 129S5/SvEvBrd) [4]. Overt characteristics of the SMS phenotype such as craniofacial anomalies and neurological features including cognition and learning, social paradigms, and sensory and motor behaviors were recapitulated in these mice, although at variable penetrance and expressivity [4], [5]. This is because variability of modifier alleles in the genetic background can either mask or exacerbate specific phenotypes caused due to the targeted gene [6]. The variability of modifier loci decreases with repeated backcrossing to about 10 generations when fewer (0.2% of the genome) polymorphic unlinked loci remain from the original host strain [7]. Notably, Rai1+/− mice at F2 generation (50% 129/50% C57BL/6J) demonstrated only ∼7% penetrance for craniofacial phenotypes [4]. When the Rai1+/− alleles were transferred to relatively homogeneous C57BL/6J backgrounds, the craniofacial penetrance markedly increased, in a graded manner, to 18% (at N2 generation, 25% 129/75% C57BL/6J) [4] and 64% (at N6 generation, ∼2% 129/∼98% C57BL/6J) [5]. However, the effect of haploinsufficiency of Rai1 as a function of genetic background has not been evaluated in relation to the breeding outcome and fitness.

To study the effect of Rai1 haploinsufficiency on breeding outcomes, we analyzed genotypes of >250 pups generated from Rai1+/− mice at ≥N10 generations (∼100% C57BL/6J) and F2 progeny from F1 Rai1+/− hybrids (50% 129/50% C57BL/6J). A significantly distorted Mendelian transmission ratio was observed in all Rai1+/− mice in ∼100% C57BL/6J background due to a reduced number of Rai1+/− pups, precluding other penetrance studies. Surprisingly, as a proof of principle, when Rai1+/− alleles were transferred to a slightly variable genetic background (∼1% 129/∼99% C57BL/6J), the expected number of Rai1+/− pups was rescued. Our analysis confirms the effect of genetic background on the survival of Rai1-haploinsufficient pups and suggests that ∼1% modifier gene variability can greatly alter the fitness constraints on the Rai1+/− alleles. While most studies on SMS is focused on genotyping patients for 17p11.2 deletions or mutations in RAI1, our data on mouse models suggest that phenotypic variability in SMS is largely contributed by genetic modifiers.

Section snippets

Mice breeding and maintenance

Rai1-targeted heterozygous mice (Rai1+/−) were obtained from the Jackson Laboratories (stock # 005981) (www.jax.org). The mice obtained were previously backcrossed to C57BL/6J for about 10 generations creating a ∼100% congenic background. Genotyping was performed as described previously [4]. Breeding experiments were set up for Rai1+/− mice pairing with C57BL/6J mice or Rai1-overexpressing mice (C57BL/6J-Tg(RP23-326M22)1She (line 760), N6–N8 generations, ∼1% 129/∼99% C57BL/6J) [8]. Rai1

Results

Rai1 gene-targeted mice, generated under a pure (∼100% C57BL6/J) background by backcrossing for ∼10 generations, were utilized for these analyses. A breeding scheme was initially set up between Rai1+/− × C57BL/6J, utilizing both male and female mice, and male Rai1+/− × female Rai1+/− mice. Control breeding pairs (C57BL/6J), matched for age, were also set up. The frequency of litters from Rai1+/− mice was significantly decreased with 5 litters/5 Rai1+/− breeder females and 10 litters/8C57BL/6J

Discussion

Evaluation of mouse models for phenotypic consequences of altered gene dosage is of critical importance, particularly when considering the potential consequences of copy number variations in the human genome. Although not all phenotypes in humans are likely to be recapitulated in mice, identification of certain features involving conserved developmental pathways in both species is useful in the study of development. Differences in genetic background have potential effects on penetrance and

Acknowledgments

We thank Heather Wright and Brooke Burns for assistance with mouse husbandry. We thank Drs. Jolene Windle, Michael Grotewiel, Christopher Vlangos, and Matt Dean, and Jeffrey Kidd for critical reading of the manuscript. We also thank anonymous reviewers for constructive suggestions. This research was supported by resources from Virginia Commonwealth University.

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Cited by (5)

  • Smith-magenis syndrome results in disruption of CLOCK gene transcription and reveals an integral role for RAI1 in the maintenance of circadian rhythmicity

    2012, American Journal of Human Genetics
    Citation Excerpt :

    Although some aspects of the circadian feedback loop of expression have been analyzed in SMS subjects,25 these data show a true profile of the core circadian components in SMS tissue and that these circadian alterations are associated with both deletion and mutation of RAI1. Rai1+/− mice, which harbor a targeted mutation of Rai1, are obese and hyperphagic, have decreased sensitivity to pain, and exhibit reduced muscle strength.17,21,26,27 In addition, a chromosomal deletion model of SMS, the Df(11)17 mouse, has the same overall phenotype as the Rai1+/− mouse but also has an altered circadian rhythm, which has not been assessed in the Rai1+/− mouse model.17

1

Present address: Department of Genome Sciences, University of Washington, Seattle, WA, USA.

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