Omi, a recessive mutation on chromosome 10, is a novel allele of Ostm1
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Large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis has provided many rodent models for human disease. Here we describe the initial characterization and mapping of a recessive mutation that leads to degeneration of the incisors, failure of molars to erupt, a grey coat colour, and mild osteopetrosis. We mapped the omi mutation to chromosome 10 between D10Mit214 and D10Mit194. The Ostm1 gene is a likely candidate gene in this region and the grey-lethal allele, Ostm1gl, and omi mutations fail to complement each other. We show that om/om mice have reduced levels of Ostm1 protein. To date we have not been able to identify the causative mutation. We propose that omi is a novel hypomorphic mutation affecting Ostm1 expression, potentially in a regulatory element.
Large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis has provided many rodent models for human disease, including several forms of deafness, Branchio-Oto-Renal syndrome, and CHARGE syndrome (Hrabe de Angelis et al. 2000; Smits et al. 2005; Bosman et al. 2005, 2009; Calvert et al. 2011; Hilton et al. 2011). Due to the nature of production of these mutant mice, it is likely that additional mutations are present in the mouse lines studied because mutations are generated at random in the genome and are usually noticed only when they lead to a detectable phenotype. Some mutations may be without any obvious consequence, but other mutations may have additional phenotypes of varying degree that may go undetected because they are not screened for or they may appear in later generations if they are recessive. It has been estimated that an average of around 150 mutations occur in each F1 offspring from an ENU-mutagenized male (Hrabe de Angelis et al. 2000). Many of these additional mutations will be lost by dilution in successive generations through crossing to the wild-type background (Hilton et al. 2011) or by selection against the mutant phenotype; however, some will remain.
We report here a new recessive phenotype that was found in a line selected for a completely different phenotype originating from an ENU mutagenesis screen. The phenotype was initially detected due to high lethality just after weaning in some matings within this mouse line. Here we describe the initial characterisation and mapping of this mutation, that we named omi (om). Omi is a recessive mutation that leads to degeneration of the incisors, failure of molars to erupt, a grey coat colour, and mild osteopetrosis. We mapped the omi mutation to chromosome 10 between D10Mit214 and D10Mit194. The Ostm1 gene is a likely candidate gene in this region, and the grey-lethal allele, Ostm1gl, and omi mutations fail to complement each other. We show that om/om mice have reduced levels of Ostm1 protein. To date we have not been able to identify the causative mutation. We propose that omi is a novel hypomorphic mutation affecting Ostm1 expression, potentially in a regulatory element.
Materials and methods
Animal husbandry and experiments were carried out in accordance with UK Home Office regulations. The omi mutation was detected in a line of mice derived from a large-scale mutagenesis screen (Hrabe de Angelis et al. 2000). Male C3HeB/FeJ mice were injected with three doses of 80 mg/kg ENU at weekly intervals, allowed to recover, and mated with uninjected C3HeB/FeJ females. F1 offspring were screened for a variety of dominantly inherited defects, including deafness and vestibular (balance) defects. The recessive omi mutation was discovered in a mouse line initially isolated because of its dominantly inherited mild head-bobbing behaviour (ABE9, also known as Bob). We were unable to map the original feature of mild head bobbing to any chromosome and no obvious malformation of the inner ear could be detected (Bosman and Steel, unpublished results). Offspring from the original ENU-mutagenized male were outcrossed to wild-type C3HeB/FeJ mice (never exposed to ENU) at least five times, diluting out other mutations resulting from the ENU treatment, before the omi phenotype was discovered. The omi phenotype was not linked to the head bobbing, and mice described here showed no sign of any balance defect. Affected omi mice were provided with a Pico-Vac® soft dietary supplement (LabDiet) which enabled them to survive until adulthood. For all experiments control animals were fed normal diet pellets ad libitum. For all phenotypic analyses the mice were studied on their original C3HeB/FeJ genetic background and unaffected wild-type or heterozygous littermates were used as controls. Grey-lethal mice were obtained from Prof. T. Jentsch and were genotyped as described previously (Chalhoub et al. 2003).
Om/om animals on a C3HeB/FeJ genetic background were outcrossed to C57BL/6J mice. +/om offspring were backcrossed to affected (om/om) C3HeB/FeJ animals. Backcross mice were collected around weaning time. The mice were killed by cervical dislocation, the teeth were photographed, and tissues were taken for DNA purification. The DNA from 28 backcross offspring exhibiting tooth defects was used in a genome scan to link the tooth defect with a chromosome. A panel of 69 markers spanning the autosomes was used to detect polymorphisms between C3HeB/FeJ and C57BL/6J mice (Supplementary Table 1). For further fine mapping, another ten polymorphic markers were used (Supplementary Table 2). Single nucleotide polymorphisms (SNPs) between C57BL/6J and C3HeB/FeJ in the region of interest were identified using a SNP database (http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=snpQF). Primers spanning the SNPs were designed using Primer3 (http://frodo.wi.mit.edu/) with standard settings (Supplementary Table 2 for primer sequence). PCR products were then sequenced with the forward and reverse primers to detect the SNP using standard techniques.
Adult mice were killed by cervical dislocation. For histology, organs of 4-week-old male and female mice (n = 10; five om/om and five +/om) were collected and fixed in 10 % neutral buffered formalin. Heads were subsequently decalcified in 10 % EDTA (pH 8.0) for 7 days, followed by paraffin embedding. Embryos (E16.5, n = 13 from heterozygous × homozygous timed matings) were dissected in ice-cold PBS and decapitated, and the heads were fixed in 10 % neutral buffered formalin for 2 days and processed for paraffin embedding. P7 pups (n = 10 from heterozygous × homozygous matings) were decapitated and the heads were fixed in 10 % neutral buffered formalin and decalcified in 10 % EDTA (pH 8.0) for 7 days, followed by paraffin embedding. Serial sections at 7 μm were cut and stained with haematoxylin and eosin by standard procedures. To analyse the skeletons, mice (seven male +/om, four male om/om, seven female +/om, and four female om/om) were weighed and anaesthetised by intraperitoneal injection of either Avertin (1.25 %) or 100 mg/kg ketamine/10 mg/kg xylazine (dosage of 0.1 ml/10 g mouse weight) at 6–8 and at 12 weeks of age. When ketamine/xylazine was used, the anaesthesia was reversed using a solution of 1 mg/kg Antisedan (0.1 ml/10 g mouse weight). Mice were scanned in a MX20 Specimen Radiography System (Faxitron X-Ray Corporation, Lincolnshire, IL, USA) in combination with Faxitron SR v1.2 software. At 12 weeks the density of the tissues was measured using a PIXImus Densitometer in combination with Lunar PIXImus 2 2.1 software. At 16 weeks of age, all nonfasted mice used for the radiographic skeletal analysis were subjected to a terminal retro-orbital bleed under anaesthesia induced by 100 mg/kg ketamine/10 mg/kg xylazine (dosage of 0.1 ml/10 g mouse weight). A complete blood count and clinical chemistry panel were analysed (http://www.sanger.ac.uk/mouseportal/phenotyping/MAHN/plasma-chemistry/). Whole blood was analysed with an automatic haematology analyser which uses spectrophotometry and volume impedance principle to measure white and red blood cell counts, mean corpuscular volume, haemoglobin, erythrocyte indices (haematocrit, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red blood cell distribution width), platelet counts, and mean platelet volume (scilVet Animal Blood Counter, RAB 015 A Ind.E, 22.02.01). Plasma chemistry was analysed using an Olympus AU400 for the following parameters: sodium, potassium, chloride, glucose, triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein (LDL), NEFAC, glycerol, amylase, alanine aminotransferase, alkaline phosphatase, creatine kinase, aspartate aminotransferase, total bilirubin, total protein, albumin, creatinine, urea, calcium, magnesium, iron, phosphate, lactate dehydrogenase, and uric acid.
Adult mice (n = 2 per genotype) were killed by cervical dislocation and adult brains were frozen in liquid nitrogen and stored at −80 °C until further use. Brain tissue (7 ml per 0.379 g tissue) was lysed by homogenization in an ice-cold Dounce homogenizer in ice-cold DOC buffer [1 % sodium deoxycholate in 50 mM Tris-HCl (pH 9.0)] containing Complete protease inhibitors (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions. The lysate was incubated in ice for 1 h, spun at 4,000 rpm for 1 h, and then cleared through a 5-μm filter. Protein concentration was determined and similar quantities were loaded on a 4–12 % acrylamide gel (Bio-Rad, Hercules, CA, USA) and blotted. Western blots were blocked for 4 h at 4 °C in TBS + 0.1 % Tween-20 (TBST) containing 5 % low-fat milk powder, rinsed in TBST, and then incubated in anti-OSTM1 (catalog No. HPA010851 from Atlas Antibodies, Stockholm, Sweden) diluted 1:2,000 in TBST + 3 % low fat milk overnight at 4 °C. Blots were rinsed and washed 3 × 10 min with TBST at room temperature, then incubated with anti-rabbit-HRP (Horse radish peroxidase) diluted 1:50,000 in TBST + 3 % low fat milk overnight at 4 °C. Blots were rinsed and washed 3 × 10 min and 2 × 30 min in TBST. An ECL Advance kit (Amersham GE Healthcare Life Sciences, Piscataway, NJ, USA) was used to detect HRP activity. Blots were then stripped, blocked, and incubated with anti-PSD95 (catalog No. MA1-046 from Affinity Biosciences, Burwood, VIC, Australia) as described above, except for the use of anti-mouse-HRP as a secondary antibody.
Omi is a recessive mutation affecting teeth and coat colour
The omi mutation is a recessive mutation: numbers of normal and affected mice were analysed to determine mode of inheritance
Cross predicted genotypes
Unaffected × unaffected
+/om × +/om
Unaffected × unaffected
+/? × +/+
Affected × unaffected
om/om × +/om
Affected × unaffected
om/om × +/+
Affected × affected
om/om × om/om
n = 110
n = 121
n = 162
n = 44
n = 10
om/om mice have a reduced lifespan
average age (months)
average age (months)
Teeth are formed in om/om mice but are abnormal
Number of control and om/om mice displaying a feature at P14, P21, and P30–32
+/om (n = 13)
Normal size of teeth
Normal number of teeth
om/om (n = 13)
Normal size of teeth
om/om mice have an abnormal bone morphology and increased bone mass
Body mass and bone mineral densities (BMD) from control and om/om male and female mice at 8 and 12 weeks of age
Lean mass (g)
Fat mass (g)
+/om (n = 7)
om/om (n = 4)
+/om (n = 7)
om/om (n = 4)
+/om (n = 7)
om/om (n = 4)
+/om (n = 7)
om/om (n = 4)
We analysed haematoxylin and eosin-stained sagittal sections of the heads from 4-week-old +/om and om/om mice for tooth and bone abnormalities. In control mice molars had erupted, but in om/om mice molars were still covered by tissue (Fig. 3a, b). The jaw bones of control mice consisted of spongy bone and compacted bone areas (Fig. 3c, e). In the compacted bone, no blood vessels were seen and the bone had a regular structure (Fig. 3c). In contrast, the areas normally formed by compact bone were invaded by blood vessels and had a very irregular structure in om/om mice (Fig. 3d). In addition, the spongy bone appeared disorganised (Fig. 3f).
The omi mutation maps to chromosome 10
Number of affected and normal mice in the out- and backcross confirms recessive inheritance
(om/om × C57Bl6 J)
(outcross × om/om)
n = 43
n = 182
Omi and grey-lethal are noncomplementing mutations
The region on chromosome 10 between SNP rs13480578 and D10Mit194 contains approximately 100 genes. Several genes in the region could lead to a severe phenotype as seen in the om/om mice, but one gene was of particular interest. Ostm1 mutations have been identified in mouse and human as causing very severe osteopetrosis. Grey-lethal (gl) mice have a recessive null mutation in Ostm1 that leads to a grey coat colour and early lethality due to severe osteopetrosis (Chalhoub et al. 2003; Pangrazio et al. 2006). Due to the similarity in phenotype between the grey-lethal and omi phenotypes, we tested for noncomplementation. Offspring of crosses between om/om and +/gl mice were collected. Of 28 pups, 10 had a grey appearance and lacked incisors at P21 and the remaining pups were all normal (Fig. 5a, b). These ten abnormal pups were confirmed to be heterozygous for the grey-lethal allele by genotyping (and thus +/gl, +/om). In contrast to gl/gl mice, that are small at P21 and lethal around 3–4 weeks of age, these compound heterozygous mice were viable on a Pico-Vac® soft diet for at least 12 weeks.
om/om mice do not have a mutation in the coding sequence and 500-bp promoter region of Ostm1 but have reduced Ostm1 protein levels
We sequenced the coding region of Ostm1 in om/om, +/om, and control mice from wild-type C3HeB/FeJ colony (+/+) mice. We were unable to detect a mutation within the coding sequence or the 5′ and 3′ UTRs. Work by Meadows et al. (2007) has shown that Ostm1 expression is regulated by the MIcrophthalmia Transcription Factor (MITF). Analysis of the alignment of human and mouse areas upstream (500 bp) of the transcription start site identified one putative MITF binding site (M box) and three weaker binding sites (E boxes). In addition, Ets and Pu.1 binding sites were identified (Meadows et al. 2007). We sequenced this region for control and om/om mice and did not find any mutation in this promoter region.
Here we described a novel allele (omi) of Ostm1 that results in tooth, bone, and coat colour abnormalities. All homozygote mutant (om/om) mice lose incisors in the first month of life and most molars fail to erupt. We have demonstrated that female om/om mice may suffer from progressive osteopetrosis at a young age. Male om/om mice have decreased BMD at 8 weeks of age, but bone mass increases significantly between 8 and 12 weeks of age, even though mice suffer from poor nutritional status (indicated by low triglycerides, LDL, reduced lean and fat mass). In addition, om/om mice have a shortened life span. It is possible that bone mass would increase further with age in om/om males and an osteopetrotic phenotype could underlie the shortened life span, but further detailed analysis of this complex bone phenotype is required.
The omi mutation is likely to have been induced by ENU mutagenesis. A spontaneous mutation rate of 1.1 × 10−8 per base per generation was estimated for the mouse (Drake et al. 1998), resulting in an average of 28.6 mutations per genome per generation. In contrast, the mutation rate induced by ENU can be approximately 10,000 times higher than the natural background mutation rate (Salinger and Justice 2008; http://cshprotocols.cshlp.org/content/2008/4/pdb.prot4985.full), for an average of 3.97 × 107 mutations in the first generation. After five generations of outcrossing with unexposed C3HeB/FeJ, one would expect 93.75 % of the mutations to have been diluted out, leaving around 2.48 × 105 ENU-induced mutations. Therefore, it is likely that the mutation was ENU-induced, but we cannot exclude the possibility that it arose spontaneously.
The phenotype of the om/om mice is similar but not identical to the previously reported grey-lethal mouse. Grey-lethal is a recessive mutation in Ostm1 but has a more severe phenotype than the om/om phenotype across all aspects of the phenotype. Incisors and molars do not erupt in grey-lethal mice. Some teeth do erupt in om/om mice but most are not maintained and are lost just after weaning. In vitro cultures of tooth germs showed that teeth of grey-lethal mice can develop normally, and the failure to erupt in vivo is due to an absence of bone remodelling (Ida-Yonemochi and Saku 2002). Grey-lethal mice are not viable and die around weaning time, even when the mice are on an appropriate soft diet, whereas omi mice are viable and fertile on a soft gel diet. The grey coat colour is another phenotype that is more severely affected in grey-lethal mice than in omi mice. The grey-lethal mutation has been bred onto various genetic backgrounds, including C3H and C57BL/6J. The phenotype of the grey-lethal mouse is less severe on a Mus spretus genetic background, indicating that modifier genes exist (Vacher and Bernard 1999). The omi mutation arose on an inbred C3HeB/FeJ genetic background and backcrossing to C57BL/6J did not alter the phenotype significantly. Grey-lethal was discovered by Grüneberg (1935) in a stock segregating for Tyrc–e. It arose as a spontaneous mutation in a mixed genetic background. Based on mapping data, Vacher and Bernard (1999) suggested that the mutation arose on a 129 or related genetic background. The grey-lethal mutation was identified as a genomic deletion of the 5′ region of the gene. The deletion spans 7.5 kb and includes the promoter, the first exon, and part of the first intron. A 460-bp sequence corresponding to the 3′ UTR of a LINE1 retrotransposon element was inserted at the deletion breakpoints (Chalhoub et al. 2003). RT-PCR showed that this Ostm1 mRNA is not expressed in gl/gl tissues, making it likely that this is a complete null mutation.
One possibility is that the difference in the type of mutation could cause the difference in phenotype. Omi was identified in a mouse line that came from an ENU mutagenesis program. Therefore, it is likely to be a point mutation rather than a retroviral integration as is the grey-lethal mutation. Omi could be a hypomorphic allele rather than a complete null mutation, which would explain the difference in phenotype. Our data suggest that om/om mice have reduced levels of Ostm1 protein, confirming that it is not a null mutation. Our current hypothesis is that the omi mutation could lie in a regulatory element and lead to a reduction in Ostm1 expression. As enhancer elements have been identified at many kilobases up- or downstream from the transcription start site, further fine mapping of the omi mutation in combination with new sequencing technologies will be required to identify the causative mutation.
Osteopetrosis in humans was described in the early 1900s by Albers-Schönberg, and by the 1940s it was clear that the severity of the phenotype varied greatly. In the most severe forms, deficient bone resorption leads to haematological failure, cranial nerve compression, short stature, and brittle bones. Various inheritance patterns have been described for the different types of osteopetrosis. Autosomal dominant osteopetrosis is variable but usually mild. Various forms of recessive osteopetrosis have been reported, some of which are associated with the most severe phenotypes (including those caused by mutations in the TCIRG1, CLC7, and OSTM1 genes), whereas mutations in other genes (CAII and PLEKHM1) give a milder osteopetrotic phenotype. Recently, long-term survival in infantile malignant autosomal recessive osteopetrosis secondary to homozygous p.Arg526Gln mutation in CLCN7 was described (Kantaputra et al. 2012). Interestingly, the phenotype of omi mice greatly overlaps the description of a case study of recessive mild osteopetrosis (Kahler et al. 1984). As in omi mice, patients presented with oligodontia, striation of the long bones, abnormalities of the zygomatic arch, sclerosis in the skull, and mild anaemia. To date, only four different human OSTM1 mutations have been identified, always leading to recessive malignant osteopetrosis. This does not exclude the possibility that other hypomorphic OSTM1 mutations could lead to a mild osteopetrotic phenotype in humans. The recent identification of a similar mutation in CLC7 (Kantaputra et al. 2012) confirms the idea that many more subtle mutations may be identified in the human population. Therapy for osteopetrosis is presently unsatisfactory and much work needs to done to unravel the gene defects and to identify new treatments to improve symptoms. Recent efforts to identify novel genes involved in bone homeostasis by using ENU mutagenesis screening in mice have identified one novel candidate for osteopenia (Barbaric et al. 2008) and one for osteopetrosis (Ochotny et al. 2011). Although omi mice have a mutation that affects a gene already known to be involved in bone homeostasis, it is likely to be hypomorphic making it a suitable model for validating novel therapeutic treatments.
This study was supported by the Wellcome Trust (grant No. 098051), the MRC, and the EC (BMH4-CT97-2715). The authors thank Heike Brinkmann for collection of backcross mice, Rosalind Lacey for animal care and data collection, Mark Collins for advice on Western blotting and the PSD95 antibody, and Prof. Dr T. Jentsch for the grey-lethal mice. E. A. Bosman thanks W. Skarnes for support and P. Tate for valuable comments.
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