Aggressive Periodontitis with Neutropenia Caused by MMD2 Mutation

Aggressive periodontitis causes rapid periodontal tissue destruction and is a disease that occurs at a young age and runs in the patient’s family. Here, we revealed a heterozygous A116V missense mutation in the gene encoding monocyte to macrophage differentiation associated 2 (MMD2) protein in a Japanese family with aggressive periodontitis and neutropenia. Analyses of patients’ peripheral blood revealed a low number of neutrophils but abundant quantity of CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, mutant Mmd2 mice showed severe alveolar bone loss and neutropenia. In patients and mutant Mmd2 mice, differentiation of HSPCs into granulocytes was also impeded, and their granulocytes were functionally impaired. Taken together, A116V mutation in MMD2 gene induced mild neutropenia and slightly limited the immune defense response. Our studies suggested that aggressive periodontitis in association with A116V MMD2 mutation constitutes a new immune system defect that belongs to the same spectrum of severe congenital neutropenia.


Introduction 55
Aggressive periodontitis, formerly called early onset periodontitis or juvenile 56 periodontitis, has an early onset and runs in the patient's family (Nishimura et al., 1990;57 Trevilatto et al., 2002;Llorente, 2006). This disease is characterized by the loss of many teeth 58 due to rapid periodontal tissue destruction, with no evident symptoms in other tissues. The 59 prevalence of this disease is 0.1%-0.2% in Caucasians, 0.4%-1.0% in Asians, and 1.0%-60 3.0% in African Americans (Albandar, 2000). It has been indicated that neutrophil 61 abnormalities lead to the onset of the disease because they allow for bacterial growth, which 62 follows severe periodontal destruction. Especially, abnormalities in neutrophil chemotaxis 63 6 apoptotic cells were higher in number in these patients' setting (data not shown). Interestingly, 99 detailed flow cytometric analysis indicated many CD34 + HSPCs in patients' peripheral blood 100 (CD34 + HSPCs/ CD45 + cells accounted for 3.3 % in patient III-2, 1.7 % in patient III-4, and 101 0.08 % in age-matched healthy subject) (Fig. 2B), which is only transiently seen when 102 peripheral blood is mobilized by granulocyte colony stimulating factor (G-CSF) and 103 high-dose of chemotherapy or plerixafor. However, this condition seen in patients' peripheral 104 blood was persistent rather than transient. Moreover, neutrophil chemotaxis, assessed by 105 stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), was also decreased in the 106 analyzed patients when compared to that of age-matched healthy subjects (Fig. 2C)

Mouse model 137
The exact function of MMD2 has not been elucidated. Therefore, we created a 138 knock-in mouse model (Mmd2 A117V/A117V mice) carrying an amino acid substitution in Mmd2, 139 which corresponded to the A116V mutation observed in the MMD2 human gene. Platinum 140 TALENs were designed to generate a double-stranded break near the targeted nucleotide 141 c.347C>T in exon 4 of Mmd2 gene (Fig. 4A). The target template for homologous 142 recombination was constructed (Fig. 4B). The 25 bp oligonucleotide contained 4 single 143 nucleotide differences, including the nonsynonymous C>T substitution encoding the mouse 144 A117V mutation and a synonymous change that introduced a PstI site (Fig. 4C). The genome 145 editing of the targeted nucleotide mutations resulted in five random deletions and one indel, 146 as the 5 bp, 7 bp, and 11 bp deletions produced frameshift mutations. We used the mice with 147 7 bp deletions in Mmd2 as the knock-out mice (Mmd2 -/mice) ( Fig. 4D and Fig. 4E). 148 Mmd2 mutation causes severe alveolar bone loss 149 Whether severe periodontitis was induced in Mmd2 A117V/A117V and Mmd2 -/mice was 150 investigated using the common ligature-induced periodontitis model (Abe, 2013). Alveolar 151 bone was only markedly absorbed at the time of inflammation in both Mmd2 A117V/A117V and 152 Mmd2 -/mice, compared to wild-type (Mmd2 +/+ ) mice (Fig. 5A). The degree of alveolar bone 153 loss was statistically higher in Mmd2 A117V/A117V , Mmd2 A117V/+ , and Mmd2 -/mice, than in 154 Mmd2 +/+ mice (p = 0.01) (Fig. 5B). 155 Mmd2 mutation causes abnormal differentiation of granulocytes than that in Mmd2 +/+ mice (Mmd2 A117V/A117V mice: p = 0.006 and Mmd2 -/mice: p = 0.03) ( MMD2 mutation was considered to belong to the same spectrum of SCN due to a common 196 mechanism that leads to abnormal number and function of neutrophils. 197 At present, symptomatic treatment is given for aggressive periodontitis; however, it 198 does not fully recover the periodontal tissue. There are reports of SCN patients with absolute 199 neutrophil counts normalized by G-CSF who still have severe periodontitis (Putsep, 2002;200 Carlsson et al., 2006). Therefore, the level of absolute neutrophil count normalized by 201 G-CSF is not enough to maintain normal oral health in these patients. A more detailed 202 examination of the immune response for aggressive periodontitis caused by MMD2 mutation 203 may lead to the development of a new treatment alternative to not only aggressive 204 periodontitis caused by MMD2 mutation but also to periodontitis in general. 205 MMD and MMD2 are two members of the progestin and adipoQ receptor family (Tang 206 et al., 2005). As its name suggests, MMD is involved in macrophage activation and may 207 increase the production of TNF-α and nitric oxide in lipopolysaccharide-stimulated 208 macrophages through ERK1/2 and Akt phosphorylation (Liu et al., 2012). A genome-wide 209 association study in patients with Crohn's disease (CD) identified MMD2 as a CD-related 210 gene (Montero-Melendez, 2013). CD is an inflammatory disease due to abnormal immune 211 reaction to many commensal bacteria in genetically susceptible individuals. Thus, MMD2 212 gene may be involved in the immune response system to chronic bacterial infection. 213 Therefore, in presence of the MMD2 mutation and harmful bacteria, diseases in the intestine 214 and the oral cavity are likely to develop. 215 Additionally, identification of MMD2 may allow to differentiate between chronic and 216 aggressive periodontitis, as until today there is a controversy about whether chronic and 217 aggressive periodontitis should be classified or not as the same type of periodontitis. As a 218 conclusion, our study highlighted the influence of mild immune system defects on the onset 219 of aggressive periodontitis, which should be considered during the diagnosis of the disease. 220 Furthermore, the study of mild neutropenia and related diseases may attract the attention of 221 the medical field other than periodontology and lead to the development of new diagnostic 222 and therapeutic methods. 223

Study family 225
This study was approved by the Human Subjects Committees of Hiroshima University. 226 Written informed consent was obtained from all subjects. All affected individuals were 227 diagnosed with aggressive periodontitis according to periodontal and X-ray examinations. 228 Blood was collected from the four affected and the four unaffected individuals in this family 229 for genetic analyses. Also, blood and bone marrow samples were collected from III-2 and 230 III-4 patients, and FACS analysis, chemotaxis assay, and CT imaging were performed.

Statistical analysis 299
The results are expressed as the mean ± standard deviation. Statistical differences 300 between the mean values of the control and experimental groups were analyzed by using 301 Student's t test. Those p-values ≤ 0.05 were considered statistically significant. 302

Acknowledgments 303
We thank the families involved in this research. We would like to thank Editage (www.     Table S1. Data of complete blood count (Patients