Mutational analysis in neonatal-onset multisystem inflammatory disease: Comment on the articles by Frenkel et al and Saito et al

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Mutational Analysis in Neonatal-onset Multisystem Inflammatory Disease: Comment on the Articles by Frenkel et al and Saito et al

To the Editor:

We would like to comment on 2 recent reports of atypical neonatal-onset multisystem inflammatory disease (NOMID) mutations that were published in Arthritis & Rheumatism. NOMID, also known as chronic infantile neurologic, cutaneous, and articular (CINCA) syndrome, is a severe autoinflammatory disease that is characterized by the onset of systemic inflammation presenting with fever and urticarial skin rash soon after birth (1). NOMID/CINCA syndrome is usually sporadic, and is caused by de novo mutations in the CIAS1 gene, which encodes the cryopyrin protein (2–5). To date 24 mutations have been found to be associated with NOMID/CINCA syndrome (http://fmf.igh.cnrs.fr/infevers). All but 2 of these mutations are clustered in exon 3, which encodes the NACHT domain of cryopyrin. Since many of the mutations have only been seen in single patients, it is necessary to sequence referred cases to obtain a molecular diagnosis. In most laboratories, only exon 3 of CIAS1 is sequenced. However, CIAS1 mutations have been found only in ∼50% of patients with NOMID/CINCA syndrome (4, 5).

In the report by Frenkel et al (6), the first non–exon 3 mutation was described in a patient with NOMID/CINCA syndrome with an atypical phenotype who presented with recurrent episodes of malaise, headaches, vomiting, arthralgia, growth retardation, and progressive sensorineural hearing loss. Interestingly, the patient had reported only 1 episode of a transient erythematous rash, and his disease was noteworthy for absence of fever.

In the second report, Saito et al (7) described a typical NOMID/CINCA syndrome patient with somatic mosaicism for a mutation in exon 3 of CIAS1. The single strand electropherogram of this mutation (Y570C) showed double, but uneven, peaks, demonstrating heterozygosity and suggesting mosaicism. After subcloning the exon 3 of CIAS1 from various cells, the authors found the frequency of the mutant allele to be 16.7% in whole blood, 12.3% in peripheral blood mononuclear cells, and 19.6% in neutrophils. The authors proposed that somatic mosaicism may account for at least some of the undetected mutations in CIAS1 mutation–negative patients. The patient's parents were mutation-negative, indicating that the Y570C mutation occurred early during embryogenesis.

We have carefully evaluated electropherograms of 14 CIAS1 mutation–negative patients with NOMID/CINCA syndrome both for the presence of lower-than-expected heterozygous exon 3 sequencing peaks, and for the presence of non–exon 3 mutations. We found that when we detected a background peak on one strand, it was always resolved by sequence on the reverse strand. In our laboratory the 1,753-bp exon 3 is sequenced in 4 overlapping amplicons, and each of these is sequenced bidirectionally. If necessary, unresolved peaks are sequenced repeatedly until an unambiguous result is obtained. Although we do not exclude the possibility that undetected somatic mosaicism may explain the apparent absence of the CIAS1 mutation in some patients, we believe that it is necessary to do bidirectional sequencing to resolve background peaks before further considering CIAS1 somatic mosaicism.

In our genetic screen, the exon 3 mutation–negative patients are always screened for mutations in the other 8 exons of CIAS1. As a result, we have identified a novel mutation in exon 4 of CIAS1. Whereas the patient described by Frenkel et al had a mutation in exon 6 of CIAS1 and an atypical phenotype, we identified a different mutation in a 9-month-old patient with the typical rash, fever, headaches, elevated cerebrospinal fluid pressure, and bilateral papilledema of NOMID/CINCA syndrome. The exon 4 mutation was a G>C transversion at nucleotide position 2264, encoding a glycine-to-alanine mutation at position 755 (G755A). This mutation was not found in any of 1,476 Caucasian control chromosomes. Matsubayashi et al recently reported a 2263 G>C nucleotide substitution in a typical patient with NOMID/CINCA syndrome, which resulted in G755R mutation (8). Thus, glycine 755 appears to be the first mutational hot spot outside of exon 3, as we report the second nucleotide substitution at the same amino acid residue. Glycine 755 is located between the first and second leucine-rich repeat (LRR) domains of cryopyrin. Interestingly, mutations that are found in 2 homologous domains, NACHT and LRR, of the caspase activation and recruitment domain 15 protein (nucleotide-binding oligomerization domain 2), give rise to 2 distinct diseases, namely Crohn's disease and Blau syndrome. This is not the case with cryopyrin mutations.

We have excluded somatic mosaicism in 14 CIAS1 mutation–negative patients by applying bidirectional sequencing, suggesting that this mechanism of disease is rare. The identification of a third mutation outside of exon 3 underscores the importance of screening for mutations in other exons of the CIAS1 gene. Finally, these new data confirm our initial report (4) that a significant number of NOMID/CINCA syndrome patients are negative for CIAS1 mutations despite typical clinical presentation, an excellent response to anakinra, and careful screening of the CIAS1 gene.

Ivona Aksentijevich MD*, Elaine F. Remmers PhD*, Raphaela Goldbach-Mansky MD*, Andreas Reiff MD†, Daniel L. Kastner MD, PhD‡, * NIH, Bethesda, MD, † Children's Hospital, University of Southern California Los Angeles, CA, ‡ NIH, Bethesda, MD.

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