In this case-control study, NF1 patients were associated with periodontal disease, decreased salivary flow rate, compromised amylase activity, and higher prevalence of caries, all in an age-dependent manner. Significantly higher PI scores and DMFT scores, lower unstimulated salivary flow rates and amylase activity, and higher salivary pH values, were observed among NF1 patients aged over 20. For those aged below 20, the above-mentioned parameters were not significantly different between NF1 patients and NF1- controls. Moreover, the difference in the means was the highest for those aged over 40, followed by those aged between 20 and 40, finally those aged below 20. With RNA-seq data from peripheral blood in NF1 patients, gingival tissues from patients in periodontitis, and parotid glands in patients with sicca syndrome, IPA analysis predicted that several chronic inflammatory pathways were involved for the association. These pathways included the fibrosis pathway, which may initiate periodontitis and sicca-like symptoms through the upregulation of TNFSF13B, PML and STAT1.
Although a case of gingival neurofibroma in the attached gingiva has been reported [14, 15], so far there has been a lack of evidence regarding whether NF1 patients may be associated with the periodontal disease without tumors that directly involved the oral cavity. In our study, as all NF1 patients did not have neurofibromas in the maxilla and mandible, our findings for the first time demonstrated that NF1 patients presented with a higher incidence of periodontitis. As periodontitis is an inflammatory disease triggered by dysbiosis in the oral cavity , our findings on periodontitis inpatient with NF1 were in accordance with previous studies pinpointed that NF1 patients presented with low-grade chronic inflammation . On the other hand, our findings on salivary gland dysfunction and dental caries were in phase with a previous case-control study in which the prevalence of hyposalivation in NF1 patients was 4-fold higher than their respective NF1- controls .
Apart from the above-mentioned theory that low-grade chronic inflammation in NF1 patients may initiate or exacerbate periodontal disease , a previous cross-sectional study conducted on 150 individuals demonstrated salivary pH to be more alkaline among individuals with periodontitis, which was similar to our study , in that higher salivary pH values were simultaneously noted in our enrolled NF1 patients. Alteration to alkaline pH values could lead to a decrease in the activity of various salivary anti-microbial proteins and enzymes, which could be evidenced by salivary amylase activity in starch-iodine-saliva experiments. The basic environment could also increase the proteolytic activity of the organism, which promotes the deposition of calcium phosphate, thereby placing NF1 patients at greatest risk for dental caries and periodontal destruction as a feedback loop .
Other factors causing periodontitis and caries in NF1 patients may include fused tooth, misregulation of amelogenesis leading to enamel hypoplasia, microdontia , low levels of serum 25-hydroxy vitamin D3 , and increased osteoclastic activity. Specifically, lower serum 25-hydroxy vitamin D3 among NF1 patients result from increased pigmentation, cutaneous neurofibromas-associated impaired synthesis or accelerated catabolism and its severity increases with age, which may make NF1 patients susceptible to periodontitis in an age-dependent pattern .
Several studies have proposed mechanisms that may explain salivary gland dysfunction in NF1 patients or models. For instance, animal studies have suggested that the NF1 gene plays an important role in the organogenesis of salivary glands . Likewise, a study based on salivary gland tissues from NF1 patients pointed out that the neurofibromin gene is expressed in major and minor salivary acinar, ductal epithelium irrespective of age and sex, which indicates the importance of the NF1 gene in the normal function of the salivary gland . Overall, mechanisms that might contribute to hyposalivation in NF1 patients include defective organogenesis of the salivary gland [22, 23], parasympathetic ganglion supplying the gland , and neurovascular bundles that determine the quality and quantity of saliva that enter the salivary glands .
Moreover, NF1 mutation has been reported to bring about complications that result from collagen loss or inability to produce collagen fibres, which the underlying fibrosis pathways may account for the reported pulmonary complications including interstitial lung disease and pre-capillary pulmonary hypertension [24–26], lack of skin elasticity , and the susceptibility to osteoporosis and osteomalacia  in NF1 patients. Thus, similar to how the fibrosis of alveolar epithelium and connective tissue of lungs result in pulmonary complications of NF1 [24–26], the effect of NF1 mutation on observed gingival tissue loss/ periodontal destruction and sicca-like symptoms may also be driven by inadequate collagen amount in the connective tissue of the periodontium and salivary glands in NF1 patients . As salivary glands function through squeezing the saliva out of these exocrine glands  that are full of type I collagen , inadequate collagen production or defective collagen structure [27–29] in NF1 patients may lead to adverse salivary changes, including both abnormal salivary flow rates and salivary pH values.
The major strengths of this study included matching between NF1 patients and NF1- controls, which we considered confounding factors including age, gender, region, socio-economic status, medical history, personal and deleterious habits that may affect periodontal health and salivary flow. However, limitations of this study include the inability to provide causal inference in case-control studies. Moreover, due to the high mutation spectrum of NF1-associated genes, for which genotype-phenotype correlation has been widely investigated for various phenotypic expression of NF1 [32–36], the lack of gene sequencing data for specific NF1-mutated subtype in this study limited the ultimate scope to provide specific mutated regions that were associated with the observed intraoral complications in this study. Future studies incorporating radiographic findings and genetic analysis are warranted to elucidate the underlying mechanisms of these presenting orofacial complications of NF1.
In conclusion, this study for the first time demonstrated age-dependent periodontal destruction and functional salivary changes correlating with carious lesions in NF1 patients, which were evidenced by intraoral examinations and functional salivary tests. These intraoral complications instead of resulting from direct compression by neurofibromas may develop following systemically upregulated chronic inflammatory pathways.