The present international multicenter study investigated 26 NOMID/CINCA syndrome patients who were mutation negative according to conventional sequencing along with 19 family controls to determine whether low-level mosaicism is a disease-causing genetic mechanism. Following our first report of low-level somatic mosaicism in a NOMID/CINCA syndrome patient (14
), we reported a new method of detecting low-level NLRP3
mosaicism, in which lipopolysaccharide (LPS) induced cell death specifically in NLRP3
mutation–positive monocytes (8
). However, this method requires fresh live monocytes, special equipment such as a cell sorter, and experience in its use due to the rapid time course of LPS-induced necrotic monocytic death. For these reasons, application of this method was not feasible in an international collaborative study. We therefore opted to use genomic DNA, since it is easier to handle and can be stored and shipped. Based on our previous study in Japanese patients showing that the frequency of mutant alleles could be <5%, we designed a subcloning and Sanger-sequencing strategy that could detect this very low allelic mutation frequency.
Presuming that the present cohort is representative of the 40% of NOMID/CINCA syndrome patients who are mutation negative according to conventional sequencing, the results suggest that ~28% of all NOMID/CINCA syndrome patients may carry somatic NLRP3 mosaicism. CAPS patients present with a continuous spectrum of symptoms, and a degree of genotypic overlap is observed between disease subtypes. Although the present study focused on the most severe NOMID/CINCA syndrome phenotype, it is possible that somatic NLRP3 mosaicism may also occur in milder forms of CAPS. The presence of somatic mosaicism should also be investigated in patients with other dominantly inherited autoinflammatory diseases caused by gain-of-function mutations and who are mutation negative according to conventional sequencing.
Among the 18 patients with somatic NLRP3 mosaicism, we found 6 mutations that have previously been identified in NOMID/CINCA syndrome patients as heterozygous germline mutations. We also identified 7 novel mutations, which were confirmed as being functionally active and presumably pathogenic. Functional in vitro assays showed that these novel mutations had greater disease-causing capacity than the previously described mutations. This suggests that the novel mutations may be deleterious and unrecognized if inherited as heterozygous germline mutations.
The present study also addressed the important question of how somatic NLRP3 mosaicism modifies clinical presentation. Although no statistically significant differences in age at disease onset, skin symptoms, joint involvement, or response to IL-1 blockade were detected, milder neurologic involvement was observed in patients with somatic mosaicism. Comparisons with NOMID/CINCA syndrome patients carrying the same NLRP3 mutations but with heterozygous germline status made this tendency more prominent. Although the level of somatic mosaicism in blood leukocytes was relatively low, it remains unclear how these low-level mutations influence clinical presentation, including disease severity. One interesting hypothesis is that the difference in the severity of neurologic manifestations is a function of the level of mosaicism. For ethical and technical reasons, it was not possible to evaluate the level of mosaicism in central nervous system (CNS) cells or glial cells in the present study, and this therefore awaits investigation in future studies.
The mechanism through which NLRP3
somatic mosaicism occurs also requires elucidation. The present study demonstrated that similar proportions of neutrophils, T cells, B cells, monocytes, and buccal cells carried the mutated allele. Therefore, the mutation leading to mosaicism must have arisen before the pluripotent stem cells committed to hematopoietic progenitor stem cells or ectoderm-derived nonhematopoietic cells. Several mechanisms for mosaicism have been proposed, including chimerism due to cell fusion with an aborted dizygotic twin and a mutational event during early embryogenesis (15
). The latter mechanism is more likely in the present cohort, since mosaicism at similar frequency was detected in several cell types. To verify the hypothesis of a mutational event during embryogenesis, and to determine the point at which this occurred, it would be helpful to analyze other tissues. However, obtaining such tissues from patients may be ethically problematic.
Approximately 12% of the patients in the present cohort carried neither germline nor somatic NLRP3
mutations and may therefore be considered to be genuinely mutation negative. However, it is possible that these patients have NLRP3
mutations that have been overlooked. A recent report described a mutation in the 5′-untranslated region of NLRP3
in a patient with FCAS (16
), although it remains unclear how this noncoding mutation causes disease. Another possibility is that an extremely low frequency of NLRP3 mosaicism may have been missed. The subcloning and Sanger-sequencing strategy used in this study set the detection limit of mosaicism at 5%. Considering the range of NLRP3
mosaicism detected (4.2–35.8%), the median (10.2%), and the identification of 2 patients with <5% mosaicism, it is indeed likely that patients with an even lower level of NLRP3
mosaicism may have been overlooked. Recent advances in next-generation DNA sequencing technology may resolve this technical problem, although the associated error rate could be problematic. Another possibility is that NLRP3
mutations were present in uninvestigated cell lineages, such as those from CNS tissue, bone tissue, or skin. Future studies of NOMID/CINCA syndrome should investigate these tissues while searching for mutations in other genes.
In conclusion, the present study has clearly demonstrated that a significant proportion of NOMID/CINCA syndrome patients who were mutation negative according to conventional sequencing carried somatic NLRP3 mutations with a variable degree of mosaicism. Clinicians should therefore consider somatic mosaicism as a possible cause of disease in mutation-negative NOMID/CINCA syndrome patients and implement appropriate therapy. The early diagnosis of NOMID/CINCA syndrome and prompt initiation of therapy would improve clinical outcome. Further goals in this research field are the refinement of genetic screening and the verification of the functional consequences of all detected somatic mutations. Systematic screening for somatic mosaicism will provide new insights into the etiology of human disease.