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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Objective

Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease. Although heterozygous germline gain-of-function NLRP3 mutations are a known cause of this disease, conventional genetic analyses fail to detect disease-causing mutations in ∼40% of patients. Since somatic NLRP3 mosaicism has been detected in several mutation-negative NOMID/CINCA syndrome patients, we undertook this study to determine the precise contribution of somatic NLRP3 mosaicism to the etiology of NOMID/CINCA syndrome.

Methods

An international case–control study was performed to detect somatic NLRP3 mosaicism in NOMID/CINCA syndrome patients who had shown no mutation during conventional sequencing. Subcloning and sequencing of NLRP3 was performed in these mutation-negative NOMID/CINCA syndrome patients and their healthy relatives. Clinical features were analyzed to identify potential genotype–phenotype associations.

Results

Somatic NLRP3 mosaicism was identified in 18 of the 26 patients (69.2%). Estimates of the level of mosaicism ranged from 4.2% to 35.8% (mean ± SD 12.1 ± 7.9%). Mosaicism was not detected in any of the 19 healthy relatives (18 of 26 patients versus 0 of 19 relatives; P < 0.0001). In vitro functional assays indicated that the detected somatic NLRP3 mutations had disease-causing functional effects. No differences in NLRP3 mosaicism were detected between different cell lineages. Among nondescript clinical features, a lower incidence of mental retardation was noted in patients with somatic mosaicism. Genotype-matched comparison confirmed that patients with somatic NLRP3 mosaicism presented with milder neurologic symptoms.

Conclusion

Somatic NLRP3 mutations were identified in 69.2% of patients with mutation-negative NOMID/CINCA syndrome. This indicates that somatic NLRP3 mosaicism is a major cause of NOMID/CINCA syndrome.

Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (MIM no. #607715), also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly-inherited autoinflammatory disease that is characterized by neonatal onset and the triad of urticarial-like skin rash, neurologic manifestations, and arthritis/arthropathy. Patients often experience recurrent fever and systemic inflammation. NOMID/CINCA syndrome is the most severe clinical phenotype of the cryopyrin-associated periodic syndromes (CAPS) that also include the 2 less severe but phenotypically similar syndromes familial cold autoinflammatory syndrome (FCAS; MIM no. #120100) and Muckle-Wells syndrome (MIM no. #191900). CAPS are caused by mutations in the NLRP3 gene, which is a member of the nucleotide-binding oligomerization domain–like receptor (NLR) family of the innate immune system (1, 2).

NLRP3 is an intracellular “sensor” of danger signals arising from cellular insults, such as infection, tissue damage, and metabolic deregulation, and it has been highly conserved throughout evolution. NLRP3 associates with ASC and procaspase 1 to constitute a large multiprotein complex termed the NLRP3 inflammasome. When activated, the NLRP3 inflammasome converts the biologically inactive procaspase 1 into active caspase 1. Caspase 1 produces the cytokines interleukin-1β (IL-1β) and IL-18, which are mainly involved in the inflammatory response (3). Available research suggests that mutated NLRP3 induces autoactivation of the NLRP3 inflammasome in CAPS patients, resulting in an uncontrolled overproduction of IL-1β.

Most CAPS patients carry heterozygous germline missense mutations in the NLRP3 coding region (“mutation-positive” patients) (4, 5). More than 80 different disease-causing mutations have been reported to date (6). However, ∼40% of clinically diagnosed NOMID/CINCA syndrome patients show no heterozygous germline NLRP3 mutation during conventional Sanger-sequencing-based genetic analyses (“mutation-negative” patients). Comparisons of NOMID/CINCA syndrome patients with and without heterozygous germline NLRP3 mutations have revealed no differences in clinical features or response to treatment (4, 7).

In a previous study, we identified a high incidence of somatic NLRP3 mosaicism in “mutation-negative” NOMID/CINCA syndrome patients in Japan (8). We therefore hypothesized that somatic NLRP3 mosaicism may be implicated in the etiology of the disorder, although its precise contribution remains unclear. The aim of the present study was to evaluate both the frequency of NLRP3 somatic mosaicism in NOMID/CINCA syndrome patients and the association between somatic mosaicism and clinical phenotype using an international cohort of mutation-negative NOMID/CINCA syndrome patients.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Study design and participants.

International collaborators were contacted to identify mutation-negative NOMID/CINCA syndrome cases. A total of 20 DNA samples were received from 4 centers: France (n = 6), The Netherlands (n = 4), Spain (n = 3), and the US (n = 7). DNA samples had been extracted from peripheral blood mononuclear cells or whole blood. All 20 samples had been subjected to conventional sequencing, and no NLRP3 mutations had been identified. In each case, the accuracy of the clinical diagnosis had been confirmed according to the diagnostic criteria (7). The 6 previously reported Japanese cases and 1 Spanish case with NLRP3 somatic mosaicism were also included (8, 9). DNA samples were also collected from 19 healthy relatives of 8 patients (8 from France, 5 from Japan, 2 from Spain, and 4 from the US) to evaluate the causality of somatic NLRP3 mosaicism in a case–control manner, since the clinical features may be modified by genetic and environmental factors.

Written informed consent for NLRP3 gene analysis was obtained from all patients and controls. The study was approved by the Institutional Review Board of the Kyoto University Graduate School of Medicine and was conducted in accordance with the Declaration of Helsinki.

Data collection.

Demographic and clinical data.

The clinicians responsible for each mutation-negative NOMID/CINCA syndrome patient completed a questionnaire to document characteristics such as age, sex, race, symptoms, clinical findings, clinical course, and prognosis. No clinical data were obtained from the healthy controls.

Investigation of NLRP3 gene mosaicism.

Disease-causing mutations in NOMID/CINCA syndrome patients have only been reported in exons 3, 4, and 6 of NLRP3 (6). Thus, the present sequencing was focused on a search for somatic mosaicism of these 3 exons and their flanking intronic regions. After amplifying these genomic regions with the proofreading polymerase chain reaction (PCR) enzyme KOD-Plus polymerase (Toyobo) and dA addition with an LA Taq polymerase (Takara Bio), the amplicons were subcloned into pCR2.1-TOPO vector (Invitrogen). Ninety-six clones were selected at random for each amplicon. The subcloned amplicons were retrieved by PCR with LA Taq polymerase. They were then treated with ExoSAP-IT (USB) and proteinase K (Promega) prior to direct sequencing. The cloned exons were sequenced at the Kazusa DNA Research Institute using a BigDye Terminator kit (version 3.1) and an ABI 3730 DNA sequencer (Life Technologies). Mosaicism was indicated by the detection of >2 subclones carrying the same base variation at the same position in 96 clones.

To purify leukocyte subpopulations, freshly drawn whole blood was separated using sequential dextran and Ficoll-Hypaque density-gradient centrifugation methods. Cell sorting to select T cells, B cells, and monocytes was performed with an AutoMACS Pro Separator (Miltenyi Biotec) or a FACSVantage System (BD Biosciences), as described elsewhere (8, 9). The purity of each cell lineage was >90%. The level of mosaicism was determined by sequencing each source of genomic DNA from 80 clones.

Plasmids and cell lines.

To determine whether the identified NLRP3 mutants cause disease, experiments for assessing 2 pathologic functions were performed as described elsewhere (8). Briefly, ASC-dependent NF-κB activation was performed by a dual-luciferase reporter assay in HEK 293FT cells transfected with NLRP3 mutants. Transfection-induced cell death in the human monocytic cell line THP-1 was performed by transfecting green fluorescent protein–fused mutant NLRP3 into THP-1 cells and then measuring the dead cells with 7-aminoactinomycin D.

Statistical analysis.

The study was designed to detect mosaicism at a 5% allele frequency with >95% possibility. To satisfy this condition, it was necessary to sequence at least 93 clones per patient. The following calculation was used to estimate the number of clones that had to be sequenced: P = 1 − (1 − 0.05)n − n(0.05)(1 − 0.05)n−1 (n = 93, P = 0.956). The study was designed to analyze 96 PCR-fragment clones from each patient. The error rate of the PCR reactions was estimated using a proofreading KOD-Plus enzyme. We analyzed a plasmid vector carrying a normal NLRP3 exon 3, in which 2 distinct errors were detected by sequencing 91 clones. The calculated error rate for this result was 1/87,451 (2/[1,922 bp × 91 clones]). Thus, the probability was negligible that the same errors would be detected more than twice in 96 clones from 1 patient.

To calculate the sample size, we calculated the prevalence of somatic mosaicism among mutation-negative NOMID/CINCA syndrome patients. Eight cases of somatic mosaicism were identified among 15 mutation-negative NOMID/CINCA syndrome patients who were subsequently analyzed by the subcloning method described above. It was assumed that the maximum number of possible somatic mosaicism cases among family controls was 1. On the basis of these data and this assumption, it was calculated that 19 controls were required to ensure a 2-sided alpha level of 0.05 and a power of 0.8.

Continuous variables are presented as the mean ± SD or as the median and interquartile range. Categorical variables are presented as numbers and ratios (with percentages). To compare clinical data between patients with and patients without mosaicism, the Wilcoxon rank sum test was used for continuous variables and Fisher's exact test was used for categorical variables. Fisher's exact test was used to compare the difference in mosaicism ratio between cases and controls. The chi-square test was used to compare the difference in the level of mosaicism between different sources of genomic DNA from each patient.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Somatic NLRP3 mosaicism in mutation-negative NOMID/CINCA syndrome patients.

A heterozygous germline NLRP3 mutation was detected in 1 of the 27 samples, and this was therefore excluded from the analyses. For each patient, 96 clones were selected at random for each amplicon. These were then sequenced. NLRP3 mosaicism was detected in 18 of 26 patients (69.2%), and the level of allelic mosaicism ranged from 4.2% to 35.8% (mean ± SD 12.1 ± 7.9%; median 10.2%) (Table 1). Seven of the detected NLRP3 mutations were novel (p.G307S, p.K355N, p.M406V, p.T433I, p.F566L, p.E567K, and p.K568N). The remaining mutations have been reported previously in NOMID/CINCA syndrome patients as disease-causing heterozygous germline mutations (p.L264F, p.D303H, p.G307V, p.A439P, p.Y570C, and p.G755R). Each of the 3 NLRP3 mutations, p.F566L, p.E567K, and p.G755R, was detected in 2 unrelated patients. NLRP3 mutation p.D303H was detected in 3 unrelated patients.

Table 1. Somatic mosaicism among mutation-negative NOMID/CINCA syndrome patients*
Country, patientSequence variantProtein variantMosaicism, %
  • *

    NLRP3 mosaicism was detected in 18 of 26 patients (69.2%) with neonatal-onset multisystem inflammatory disease (NOMID)/chronic infantile neurologic, cutaneous, articular syndrome (CINCA syndrome). When samples from 19 healthy relatives of these patients were investigated, no somatic mosaicism was detected. The P value from the comparison of the cases and the controls (18 of 26 versus 0 of 19) was statistically significant (P < 0.0001).

France   
 F11298C>TT433I5.2
 F2907G>CD303H4.2
 F31315G>CA439P21.9
 F41216A>GM406V9.2
 F51698C>AF566L11.5
 F6None
Japan   
 J11709A>GY570C12.2
 J2790C>TL264F4.3
 J3919G>AG307S10.7
 J41699G>AE567K6.5
 J5907G>CD303H11.9
 J6None
Spain   
 S1920G>TG307V9.6
 S2907G>CD303H19.1
 S3None
 S4None
US   
 A11065A>TK355N18.8
 A21698C>AF566L14.6
 A31704G>CK568N9.4
 A42263G>AG755R35.8
 A5None
 A6None
The Netherlands   
 N11699G>AE567K6.3
 N22263G>AG755R6.3
 N3None
 N4None

Analyses in family controls.

To validate the clinical relevance of the NLRP3 mosaicism identified in mutation-negative NOMID/CINCA syndrome patients, samples from 19 healthy relatives were investigated. No somatic mosaicism was detected in any of these samples. The P value from the comparison of cases and controls (18 of 26 versus 0 of 19) was statistically significant (P < 0.0001).

Functional effects of the identified somatic NLRP3 mutations.

Since disease-causing heterozygous germline mutations in NLRP3 have been implicated in necrosis-like programmed cell death and ASC-dependent NF-κB activation (8), experiments were performed to determine whether the mutations identified in patients with somatic mosaicism showed the same effects. All of the identified mutations induced both THP-1 cell death (Figure 1A) and ASC-dependent NF-κB activation (Figure 1B). The in vitro effects of these novel mutations were similar to or even more pronounced than those of previously reported NLRP3 mutations. This strongly suggests that all mutations showing somatic mosaicism have pathogenic effects, including the novel mutations identified in the present study.

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Figure 1. In vitro functional assessment of the identified NLRP3 mosaicism mutations. A, Necrotic cell death of THP-1 cells induced by the identified somatic NLRP3 mosaicism mutations. Green fluorescent protein (GFP)–fused mutant NLRP3 was transfected into THP-1 cells. The percentage of dead cells (7-aminoactinomycin D positive) among GFP-positive cells is shown. Values are the mean ± SD of triplicate experiments, and data are representative of 2 independent experiments. None = nothing transfected; mock = vector without NLRP3; WT = wild-type NLRP3; R260W = NLRP3 with p.R260W (frequent mutations in patients with cryopyrin-associated periodic syndromes). B, ASC-dependent NF-κB activation induced by the identified somatic NLRP3 mosaicism mutations. HEK 293FT cells were cotransfected with WT or mutant NLRP3 in the presence or absence of ASC. The induction of NF-κB is shown as the fold change compared with cells that were transfected with a control vector without ASC (set at 1). Values are the mean ± SD of triplicate experiments, and data are representative of 2 independent experiments.

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Mutation frequency of NLRP3 among various cell lineages and 1 tissue type.

To explore the origin of the NLRP3 mosaicism, mutational frequency was evaluated in various cell lineages and 1 tissue type from 4 Japanese patients with NLRP3 somatic mosaicism. In each patient, the same mutations were found in all of the cell lineages investigated (neutrophils, monocytes, T cells, B cells) and in the buccal mucosa tissue, and no significant difference in mutation frequency was observed between these sources (Table 2).

Table 2. Distribution and quantification of NLRP3 mutations among sources of genomic DNA (4 cell lineages and 1 tissue type)*
PatientSequence variantProtein variantMosaicism, %
NeutrophilsMonocytesT cellsB cellsBuccal mucosa
  • *

    No significant differences in the level of mosaicism were observed among the sources of genomic DNA.

J11709A>GY570C12.69.88.09.58.3
J3919G>AG307S9.110.86.910.69.0
J41699G>AE567K3.52.33.73.42.2
J5907G>CD303H14.48.77.78.513.5

Phenotype–genotype analysis.

Given the previously reported genotype–phenotype association between the NLRP3 gene and CAPS, the clinical characteristics of NOMID/CINCA syndrome patients with somatic NLRP3 mutations were compared with those of patients from previous reports who had the same NLRP3 mutations but with heterozygous germline status (1, 4, 10–13) (Figure 2) (further information is available at http://web16.kazusa.or.jp/download/). All of the patients in these 2 groups had an early onset of the disease, fever, and urticarial rash. The presence of arthritis, bony overgrowth, contractures, hearing loss, and seizure varied in each group of patients, and no significant difference was detected. However, whereas most patients with heterozygous germline NLRP3 mutations presented with mental retardation, this was not the case for patients with somatic NLRP3 mosaicism. A comparison was also made between the clinical data from patients with somatic NLRP3 mosaicism and the data from patients with neither germline nor somatic NLRP3 mutations. Again, a lower incidence of mental retardation was observed in patients with somatic NLRP3 mosaicism (P = 0.03). No other significant differences were detected (Table 3) (further information is available at http://web16.kazusa.or.jp/download/).

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Figure 2. Comparison of the clinical profiles of patients carrying somatic NLRP3 mutations and patients carrying the same mutation, but with germline status. Clinical profiles of patients with mosaicism and those of patients with heterozygous germline mutations are compared for each protein variant (L264F, D303H, G307V, Y570C, and G755R). The data on 4 typical clinical symptoms are shown. Total numbers of patients with mosaicism and total numbers of patients with heterozygous mutation examined are shown as a bar for each protein variant. Each bar is stratified according to the presence or absence of the symptom. For the protein variant L264F, no data on mental retardation were available for the patient with a heterozygous germline mutation.

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Table 3. Clinical profiles of patients with somatic NLRP3 mosaicism and patients with neither germline nor somatic NLRP3 mutations*
 Patients with somatic NLRP3 mosaicism (n = 18)Patients with neither germline nor somatic NLRP3 mutations (n = 8)
  • *

    Except where indicated otherwise, values are the number with the feature/the total number of patients assessed. A lower incidence of mental retardation was observed in patients with somatic NLRP3 mosaicism (P = 0.03). No other significant differences were detected between the groups. IQR = interquartile range.

Age, median (IQR) years12 (1–30)10 (3–21)
No. of men/women10/83/5
Age at onset, median (IQR) months0 (0–24)0.5 (0–33)
Fever17/177/7
Urticarial rash14/148/8
Mental retardation4/176/8
Meningitis13/175/8
Seizures2/181/7
Hearing loss10/182/7
Arthritis14/177/8
Bony overgrowth12/176/7
Contractures7/174/7
Walking disability8/183/7
Biologic therapy10/153/8

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

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.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Ohara and Nishikomori had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Saito, Ohara, Nishikomori, Kambe.

Acquisition of data. Tanaka, Izawa, Saito, Oshima, Ohara, Nishikomori, Goldbach-Mansky, Aksentijevich, de Saint Basile, Neven, van Gijn, Frenkel, Aróstegui, Yagüe, Merino, Ibañez, Pontillo, Takada, Imagawa.

Analysis and interpretation of data. Sakuma, Morimoto, Kawai, Yasumi, Nakahata, Heike.

ROLE OF THE STUDY SPONSOR

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Mitsubishi Pharma Research Foundation supported the data collection for this study, approved the contents of the manuscript, and agreed to submit the manuscript for publication.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

We thank all patients and their relatives for participating in the study. We are grateful to Yuki Takaoka at the Department of Pediatrics, Kyoto University Graduate School of Medicine and Seiko Watanabe at the Department of Human Genome Research, Kazusa DNA Research Institute for their technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES