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Identification of a New Variant in NLRP3 Gene by Whole Exome Sequencing in a Patient with Cryopyrin-Associated Periodic Syndrome

Identification of a New Variant in NLRP3 Gene by Whole Exome Sequencing in a Patient with... Hindawi Case Reports in Immunology Volume 2021, Article ID 2023119, 4 pages https://doi.org/10.1155/2021/2023119 Case Report Identification of a New Variant in NLRP3 Gene by Whole Exome Sequencing in a Patient with Cryopyrin-Associated Periodic Syndrome 1 1,2 1 3 Mahdieh Vahedi, Nima Parvaneh , Saeedeh Vahedi, Mohammad Shahrooei , 1,2,4,5 and Vahid Ziaee Children’s Medical Center, Pediatrics Center of Excellence, Tehran, Iran Department of Pediatrics, Tehran University of Medical Sciences, Tehran, Iran Department of Microbiology and Immunology, Laboratory of Clinical Bacteriology and Mycology, KU Leuven, Leuven, Belgium Pediatric Rheumatology Research Group, Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran Pediatric Rheumatology Society of Iran, Tehran, Iran Correspondence should be addressed to Vahid Ziaee; ziaee@tums.ac.ir Received 14 April 2021; Revised 18 May 2021; Accepted 7 August 2021; Published 17 August 2021 Academic Editor: Elena Bozzola Copyright © 2021 Mahdieh Vahedi et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background.NLRP3geneislocatedinchromosome1andencodesapyrin-likeprotein.Mutationsinthisgeneareassociatedwith an autoinflammatory disease, called cryopyrin-associated periodic syndrome (CAPS). Case Presentation. We report a 1-year-old boywhohadrecurrenturticarialrashsincebirthandjointpainandswelling.Hehadamissensemutationc.G1060T(p.A354S)in exon 5 of the NLRP3 gene which was detected by whole exome sequencing. Conclusion. A novel variant was found in the NLRP3 gene which has not been reported by now. (ereareabout100pathogenicmutationsinNLRP3genein 1. Background patients with CAPS [2–4]. (e relationship between NLRP3 Cryopyrin-associated periodic syndrome (CAPS) is a rare mutations and these diseases was identified in early 2000s. autoinflammatory disorder which inherited as an autosomal We identified a case of CAPS, who had recurrent urti- dominant condition. It includes three clinically overlapping carial rash, high grade fever, and chronic joint involvement. syndromes with a spectrum of severity of symptoms: (1) His symptoms did not response to therapy, and whole familial cold autoinflammatory syndrome (FCAS), (2) the exome sequencing reported new variant in the NLRP3 gene. Muckle–Wells syndrome (MWS), and (3) the chronic in- Whole exome sequencing is a tool for identifying genes fantile neurological, cutaneous, and articular syndrome whichcancauseadisease.Itisalsousefulfordiagnosisinthe (CINCA) [1]. Mutations in NLRP3 which is located on long clinic. arm of chromosome 1 are causal for CAPS. It encodes a protein comprising a pyrin domain, a nucleotide-binding 2. Case Presentation site (NBS) domain, and a leucine-rich repeat (LRR) motif. (e protein is a member of inflammasome complex which A 1-year-old boy was brought to our clinic with joint pain has a major role in innate immunity. (is complex is made and swelling in the left ankle and both knees for more than up of NLRP3, PYCARD, and CASP1. In response to a 14days.Hehadrecurrenturticarialrashsincebirth,andthis pathogen or other damage-associated signals, NLRP3 ini- rash was not triggered by cold or other physical stimulus tiates assembly of this complex. After caspase-1 activation, (Figure 1). His urticarial rash often presented with high proinflammatory cytokines IL-1B and IL-18 are secreted. grade fever. His parents were not blood relatives. On 2 Case Reports in Immunology (a) (b) Figure 1: Urticaria-like rash on the face (a) and trunk (b). examination, neurodevelopmental evaluation was normal, 4. Discussion rd but his weight and height were below the 3 percentile for (e symptoms of cryopyrin-associated periodic fever syn- age. He had significant synovial hypertrophy in both knees. dromesarevariable,andtheyarepresentwithabroadrange (e other parts of physical examinations were normal. of clinical manifestations. Inheritance of CAPS is usually Laboratory findings showed leukocytosis, anemia, and autosomal dominant. (e disease has a spectrum of thrombocytosis. Chest X-ray and abdominal ultrasonogra- symptoms in different generations. (e severity of symp- phy were normal. Echocardiography showed normal ven- tomsincreaseswithage.Forexample,infirstgeneration,the tricular function and no pericardial effusion. Due to disease may present as amyloidosis and renal failure, but in prolonged fever, recurrent urticarial rash, arthritis, and younger children, urticaria and fever can be observed [5–8]. increase in acute phase reactants, the diagnosis of auto- NLRP3 gene is located on chromosome 1q44. It has 11 inflammatory disorders, especially CAPS was suggested for exonsandmultiplealternativelysplicedtranscriptvariants. him. (e genetic test was performed, and whole exome (e gene encodes a pyrin-like protein. NLRP3 is a member sequencing identified heterozygous mutation of NLRP3 of the NLRP3 inflammasome complex. (is protein is an gene, and it was de novo mutation. activator of the NF-kappa B signaling pathway, so it has a role in inflammation and the immune response. Mutations in this gene are associated with an autoinflammatory 3. Exome Sequencing disease known as CAPS (cryopyrin-associated periodic Genomic DNA was extracted from peripheral blood using syndrome). Gain of function mutations in CAPS patients the MG Blood Genomic DNA Extraction Miniprep (Can- leads to hyperactive cryopyrin inflammasome, increased cerRop) according to the manufacturer’s instruction, and myeloid cell-derived proinflammatory cytokine release, wholeexomesequencing wasperformed for thepatient. (e and systemic and tissue inflammation leading to disease library was enriched with an Agilent SureSelect Human All symptoms. Most mutations in patients with CAPS are ExonV6kit(AgilentTechnologiesInc.,USA)andsequenced located in exon 3 [9, 10]. with 100× coverage on Novaseq6000 platform (Illumina (e relationship between genetic alterations in the Inc., USA) to generate 150bp paired-end reads. NLRP3 gene and CAPS was reported first in 2001 [11]. After passing quality control, raw data were aligned In this study, we performed whole exome sequencing in against human reference genome (hg19, NCBI Build 37) by apatientwithadiagnosisofCAPS.(eresultsshowedanew BWA (Burrows–Wheeler Aligner) software (http://www. variant in exon 5 of NLRP3 (NM-001079821.3: c.G1060T, bio-bwa.sourceforge.net). Picard was utilized to remark p.A354S). In silico prediction of pathogenicity was per- duplicates. Variant calling was performed using GATK formed with functional prediction programs that included (Genome Analysis Toolkit) (http://www.broadinstitute.org/ BayesDel_addAF, DANN, EIGEN, FATHMM-MKL, gatk/), and variant annotation was performed by ANNO- CADD, andMutationTaster. So,basedon AmericanCollege VAR online software (http://www.openbioinformatics.org/ of Medical Genetics and Genomics guideline for variant annovar/). classification, this variant is pathogenic by using the Var- (e candidate variants identified by whole exome se- SomeandInterVardatabases[12].(evariantwaspredicted quencing were confirmed by Sanger sequencing (Figure 2). to be deleterious (CADD score 37). Case Reports in Immunology 3 Father PRD Father: PID309-F_NLRP3_pA354S Mother PRD Mother: PID309-M_NLRP3_pA354S Patient PRD Mother: PID309-P_NLRP3_pA354S PRD Reference Figure 2: Sequence electropherogram of NLRP3 (c.G1060T). (e parents were homozygous for wild-type allele, but the patient was heterozygote for mutant allele. (e patient was heterozygous for this variant. (e References mutationwasnotpresentineitherparent,indicatingthatthe [1] Z. Ahmadinejad, S. Mansouri, V. Ziaee et al., “A review on mutation was a de novo or germline mutation. rs180177503 clinical,managementandguidelineforIranianpatients—Part is a single nucleotide variant that can cause c.1060G>A II,” Iranian Journal of Pediatrics (Persian edition), vol. 24, missense mutation based on the NCBI database. (e re- no. 3, pp. 229–240, 2014. ported variant in this study is a c.1060G>T mutation and [2] L. M. Booshehri and H. M. Hoffman, “CAPS and NLRP3,” has not reported yet. Given, prenatal diagnosis was rec- Journal of Clinical Immunology, vol. 39, no. 3, pp. 277–286, ommended to his parents for next pregnancies. All of the NLRP3 exons were sequenced by Sanger sequencing for this [3] M. Gattorno, M. Hofer, S. Federici et al., “Classification patient and his parents, and there was no other pathogenic criteria for autoinflammatory recurrent fevers,” Annals of the or likely pathogenic variant in NLRP3. Rheumatic Diseases, vol. 78, no. 8, pp. 1025–1032, 2019. [4] S. Georgin-Lavialle, A. Fayand, F. Rodrigues et al., “Auto- inflammatory diseases: state of the art,” Presse Medicale, 5. Conclusion vol. 48, no. 1 Pt 2, pp. e25–e48, 2019. [5] J. B. Kuemmerle-Deschner, S. Ozen, P. N. Tyrrell et al., Our study identified a novel variant in NLRP3 gene which “Diagnostic criteria for cryopyrin-associated periodic syn- was associated with CAPS and increased the pathogenic drome (CAPS),” Annals of the Rheumatic Diseases, vol. 76, variant spectrum for this gene. Also, more functional no. 6, pp. 942–947, 2017. studies are important to develop new approaches for [6] J. B. Kuemmerle-Deschner, A. Koitschev, K. Ummenhofer treating CAPS. et al., “Hearing loss in Muckle-Wells syndrome,” Arthritis & Rheumatism, vol. 65, no. 3, pp. 824–831, 2013. [7] S. Fingerhutova,´ J. Fra´nov ˇ a,´ E. Hlava´ckov ˇ a´ et al., “Muckle- Data Availability wells syndrome across four generations in one Czech family: natural course of the disease,” Frontiers in Immunology, (e data used to support the findings of this study are vol. 10, p. 802, 2019. available from the corresponding author upon request. [8] T. Imagawa, R. Nishikomori, H. Takada et al., “Safety and efficacy of canakinumab in Japanese patients with phenotypes Consent of cryopyrin-associated periodic syndrome as established in the first open-label, phase-3 pivotal study (24-week results),” Written informed consent was obtained from patients’ Clinical & Experimental Rheumatology, vol. 31, no. 2, parents. pp. 302–309, 2013. [9] E.NazVillalba,E.Gomezdelafuente,D.CaroGutierrezetal., “Muckle-wells syndrome: a case report with anNLRP3T348M Conflicts of Interest mutation,” Pediatric Dermatology, vol. 33, no. 5, pp. e311–e314, 2016. (e authors declare that they have no conflicts of interest. [10] S. Torreggiani, G. Filocamo, and S. Esposito, “Recurrent fever in children,” International Journal of Molecular Sciences, vol. 17, no. 4, p. 448, 2016. Acknowledgments [11] H. M. Hoffman, J. L. Mueller, D. H. Broide, A. A. Wanderer, (e authors would like to thank the parents for giving and R. D. Kolodner, “Mutation of a new gene encoding a permission to publish the report of this case. putative pyrin-like protein causes familial cold 4 Case Reports in Immunology autoinflammatory syndrome and Muckle-Wells syndrome,” Nature Genetics, vol. 29, no. 3, pp. 301–305, 2001. [12] Q. Li and K. Wang, “InterVar: clinical interpretation of ge- netic variants by the 2015 ACMG-AMP guidelines,” 3e American Journal of Human Genetics, vol. 100, no. 2, pp. 267–280, 2017. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Case Reports in Immunology Hindawi Publishing Corporation

Identification of a New Variant in NLRP3 Gene by Whole Exome Sequencing in a Patient with Cryopyrin-Associated Periodic Syndrome

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Copyright © 2021 Mahdieh Vahedi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Hindawi Case Reports in Immunology Volume 2021, Article ID 2023119, 4 pages https://doi.org/10.1155/2021/2023119 Case Report Identification of a New Variant in NLRP3 Gene by Whole Exome Sequencing in a Patient with Cryopyrin-Associated Periodic Syndrome 1 1,2 1 3 Mahdieh Vahedi, Nima Parvaneh , Saeedeh Vahedi, Mohammad Shahrooei , 1,2,4,5 and Vahid Ziaee Children’s Medical Center, Pediatrics Center of Excellence, Tehran, Iran Department of Pediatrics, Tehran University of Medical Sciences, Tehran, Iran Department of Microbiology and Immunology, Laboratory of Clinical Bacteriology and Mycology, KU Leuven, Leuven, Belgium Pediatric Rheumatology Research Group, Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran Pediatric Rheumatology Society of Iran, Tehran, Iran Correspondence should be addressed to Vahid Ziaee; ziaee@tums.ac.ir Received 14 April 2021; Revised 18 May 2021; Accepted 7 August 2021; Published 17 August 2021 Academic Editor: Elena Bozzola Copyright © 2021 Mahdieh Vahedi et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background.NLRP3geneislocatedinchromosome1andencodesapyrin-likeprotein.Mutationsinthisgeneareassociatedwith an autoinflammatory disease, called cryopyrin-associated periodic syndrome (CAPS). Case Presentation. We report a 1-year-old boywhohadrecurrenturticarialrashsincebirthandjointpainandswelling.Hehadamissensemutationc.G1060T(p.A354S)in exon 5 of the NLRP3 gene which was detected by whole exome sequencing. Conclusion. A novel variant was found in the NLRP3 gene which has not been reported by now. (ereareabout100pathogenicmutationsinNLRP3genein 1. Background patients with CAPS [2–4]. (e relationship between NLRP3 Cryopyrin-associated periodic syndrome (CAPS) is a rare mutations and these diseases was identified in early 2000s. autoinflammatory disorder which inherited as an autosomal We identified a case of CAPS, who had recurrent urti- dominant condition. It includes three clinically overlapping carial rash, high grade fever, and chronic joint involvement. syndromes with a spectrum of severity of symptoms: (1) His symptoms did not response to therapy, and whole familial cold autoinflammatory syndrome (FCAS), (2) the exome sequencing reported new variant in the NLRP3 gene. Muckle–Wells syndrome (MWS), and (3) the chronic in- Whole exome sequencing is a tool for identifying genes fantile neurological, cutaneous, and articular syndrome whichcancauseadisease.Itisalsousefulfordiagnosisinthe (CINCA) [1]. Mutations in NLRP3 which is located on long clinic. arm of chromosome 1 are causal for CAPS. It encodes a protein comprising a pyrin domain, a nucleotide-binding 2. Case Presentation site (NBS) domain, and a leucine-rich repeat (LRR) motif. (e protein is a member of inflammasome complex which A 1-year-old boy was brought to our clinic with joint pain has a major role in innate immunity. (is complex is made and swelling in the left ankle and both knees for more than up of NLRP3, PYCARD, and CASP1. In response to a 14days.Hehadrecurrenturticarialrashsincebirth,andthis pathogen or other damage-associated signals, NLRP3 ini- rash was not triggered by cold or other physical stimulus tiates assembly of this complex. After caspase-1 activation, (Figure 1). His urticarial rash often presented with high proinflammatory cytokines IL-1B and IL-18 are secreted. grade fever. His parents were not blood relatives. On 2 Case Reports in Immunology (a) (b) Figure 1: Urticaria-like rash on the face (a) and trunk (b). examination, neurodevelopmental evaluation was normal, 4. Discussion rd but his weight and height were below the 3 percentile for (e symptoms of cryopyrin-associated periodic fever syn- age. He had significant synovial hypertrophy in both knees. dromesarevariable,andtheyarepresentwithabroadrange (e other parts of physical examinations were normal. of clinical manifestations. Inheritance of CAPS is usually Laboratory findings showed leukocytosis, anemia, and autosomal dominant. (e disease has a spectrum of thrombocytosis. Chest X-ray and abdominal ultrasonogra- symptoms in different generations. (e severity of symp- phy were normal. Echocardiography showed normal ven- tomsincreaseswithage.Forexample,infirstgeneration,the tricular function and no pericardial effusion. Due to disease may present as amyloidosis and renal failure, but in prolonged fever, recurrent urticarial rash, arthritis, and younger children, urticaria and fever can be observed [5–8]. increase in acute phase reactants, the diagnosis of auto- NLRP3 gene is located on chromosome 1q44. It has 11 inflammatory disorders, especially CAPS was suggested for exonsandmultiplealternativelysplicedtranscriptvariants. him. (e genetic test was performed, and whole exome (e gene encodes a pyrin-like protein. NLRP3 is a member sequencing identified heterozygous mutation of NLRP3 of the NLRP3 inflammasome complex. (is protein is an gene, and it was de novo mutation. activator of the NF-kappa B signaling pathway, so it has a role in inflammation and the immune response. Mutations in this gene are associated with an autoinflammatory 3. Exome Sequencing disease known as CAPS (cryopyrin-associated periodic Genomic DNA was extracted from peripheral blood using syndrome). Gain of function mutations in CAPS patients the MG Blood Genomic DNA Extraction Miniprep (Can- leads to hyperactive cryopyrin inflammasome, increased cerRop) according to the manufacturer’s instruction, and myeloid cell-derived proinflammatory cytokine release, wholeexomesequencing wasperformed for thepatient. (e and systemic and tissue inflammation leading to disease library was enriched with an Agilent SureSelect Human All symptoms. Most mutations in patients with CAPS are ExonV6kit(AgilentTechnologiesInc.,USA)andsequenced located in exon 3 [9, 10]. with 100× coverage on Novaseq6000 platform (Illumina (e relationship between genetic alterations in the Inc., USA) to generate 150bp paired-end reads. NLRP3 gene and CAPS was reported first in 2001 [11]. After passing quality control, raw data were aligned In this study, we performed whole exome sequencing in against human reference genome (hg19, NCBI Build 37) by apatientwithadiagnosisofCAPS.(eresultsshowedanew BWA (Burrows–Wheeler Aligner) software (http://www. variant in exon 5 of NLRP3 (NM-001079821.3: c.G1060T, bio-bwa.sourceforge.net). Picard was utilized to remark p.A354S). In silico prediction of pathogenicity was per- duplicates. Variant calling was performed using GATK formed with functional prediction programs that included (Genome Analysis Toolkit) (http://www.broadinstitute.org/ BayesDel_addAF, DANN, EIGEN, FATHMM-MKL, gatk/), and variant annotation was performed by ANNO- CADD, andMutationTaster. So,basedon AmericanCollege VAR online software (http://www.openbioinformatics.org/ of Medical Genetics and Genomics guideline for variant annovar/). classification, this variant is pathogenic by using the Var- (e candidate variants identified by whole exome se- SomeandInterVardatabases[12].(evariantwaspredicted quencing were confirmed by Sanger sequencing (Figure 2). to be deleterious (CADD score 37). Case Reports in Immunology 3 Father PRD Father: PID309-F_NLRP3_pA354S Mother PRD Mother: PID309-M_NLRP3_pA354S Patient PRD Mother: PID309-P_NLRP3_pA354S PRD Reference Figure 2: Sequence electropherogram of NLRP3 (c.G1060T). (e parents were homozygous for wild-type allele, but the patient was heterozygote for mutant allele. (e patient was heterozygous for this variant. (e References mutationwasnotpresentineitherparent,indicatingthatthe [1] Z. Ahmadinejad, S. Mansouri, V. Ziaee et al., “A review on mutation was a de novo or germline mutation. rs180177503 clinical,managementandguidelineforIranianpatients—Part is a single nucleotide variant that can cause c.1060G>A II,” Iranian Journal of Pediatrics (Persian edition), vol. 24, missense mutation based on the NCBI database. (e re- no. 3, pp. 229–240, 2014. ported variant in this study is a c.1060G>T mutation and [2] L. M. Booshehri and H. M. Hoffman, “CAPS and NLRP3,” has not reported yet. Given, prenatal diagnosis was rec- Journal of Clinical Immunology, vol. 39, no. 3, pp. 277–286, ommended to his parents for next pregnancies. All of the NLRP3 exons were sequenced by Sanger sequencing for this [3] M. Gattorno, M. Hofer, S. Federici et al., “Classification patient and his parents, and there was no other pathogenic criteria for autoinflammatory recurrent fevers,” Annals of the or likely pathogenic variant in NLRP3. Rheumatic Diseases, vol. 78, no. 8, pp. 1025–1032, 2019. [4] S. Georgin-Lavialle, A. Fayand, F. Rodrigues et al., “Auto- inflammatory diseases: state of the art,” Presse Medicale, 5. Conclusion vol. 48, no. 1 Pt 2, pp. e25–e48, 2019. [5] J. B. Kuemmerle-Deschner, S. Ozen, P. N. Tyrrell et al., Our study identified a novel variant in NLRP3 gene which “Diagnostic criteria for cryopyrin-associated periodic syn- was associated with CAPS and increased the pathogenic drome (CAPS),” Annals of the Rheumatic Diseases, vol. 76, variant spectrum for this gene. Also, more functional no. 6, pp. 942–947, 2017. studies are important to develop new approaches for [6] J. B. Kuemmerle-Deschner, A. Koitschev, K. Ummenhofer treating CAPS. et al., “Hearing loss in Muckle-Wells syndrome,” Arthritis & Rheumatism, vol. 65, no. 3, pp. 824–831, 2013. [7] S. Fingerhutova,´ J. Fra´nov ˇ a,´ E. Hlava´ckov ˇ a´ et al., “Muckle- Data Availability wells syndrome across four generations in one Czech family: natural course of the disease,” Frontiers in Immunology, (e data used to support the findings of this study are vol. 10, p. 802, 2019. available from the corresponding author upon request. [8] T. Imagawa, R. Nishikomori, H. Takada et al., “Safety and efficacy of canakinumab in Japanese patients with phenotypes Consent of cryopyrin-associated periodic syndrome as established in the first open-label, phase-3 pivotal study (24-week results),” Written informed consent was obtained from patients’ Clinical & Experimental Rheumatology, vol. 31, no. 2, parents. pp. 302–309, 2013. [9] E.NazVillalba,E.Gomezdelafuente,D.CaroGutierrezetal., “Muckle-wells syndrome: a case report with anNLRP3T348M Conflicts of Interest mutation,” Pediatric Dermatology, vol. 33, no. 5, pp. e311–e314, 2016. (e authors declare that they have no conflicts of interest. [10] S. Torreggiani, G. Filocamo, and S. Esposito, “Recurrent fever in children,” International Journal of Molecular Sciences, vol. 17, no. 4, p. 448, 2016. Acknowledgments [11] H. M. Hoffman, J. L. Mueller, D. H. Broide, A. A. Wanderer, (e authors would like to thank the parents for giving and R. D. Kolodner, “Mutation of a new gene encoding a permission to publish the report of this case. putative pyrin-like protein causes familial cold 4 Case Reports in Immunology autoinflammatory syndrome and Muckle-Wells syndrome,” Nature Genetics, vol. 29, no. 3, pp. 301–305, 2001. [12] Q. Li and K. Wang, “InterVar: clinical interpretation of ge- netic variants by the 2015 ACMG-AMP guidelines,” 3e American Journal of Human Genetics, vol. 100, no. 2, pp. 267–280, 2017.

Journal

Case Reports in ImmunologyHindawi Publishing Corporation

Published: Aug 17, 2021

References