Rapid Molecular Analysis of the STAT3 Gene in Job Syndrome of Hyper-IgE and Recurrent Infectious Diseases
2010; Elsevier BV; Volume: 12; Issue: 2 Linguagem: Inglês
10.2353/jmoldx.2010.090080
ISSN1943-7811
AutoresAttila Kumánovics, Carl T. Wittwer, Robert J. Pryor, Nancy H. Augustine, Mark Leppert, John C. Carey, Hans D. Ochs, Ralph Wedgwood, Ralph J. Faville, Paul G. Quie, Harry R. Hill,
Tópico(s)T-cell and B-cell Immunology
ResumoWith the recent discovery of mutations in the STAT3 gene in the majority of patients with classic Hyper-IgE syndrome, it is now possible to make a molecular diagnosis in most of these cases. We have developed a PCR-based high-resolution DNA-melting assay to scan selected exons of the STAT3 gene for mutations responsible for Hyper-IgE syndrome, which is then followed by targeted sequencing. We scanned for mutations in 10 unrelated pedigrees, which include 16 patients with classic Hyper-IgE syndrome. These pedigrees include both sporadic and familial cases and their relatives, and we have found STAT3 mutations in all affected individuals. High-resolution melting analysis allows a single day turn-around time for mutation scanning and targeted sequencing of the STAT3 gene, which will greatly facilitate the rapid diagnosis of the Hyper-IgE syndrome, allowing prompt and appropriate therapy, prophylaxis, improved clinical outcome, and accurate genetic counseling. With the recent discovery of mutations in the STAT3 gene in the majority of patients with classic Hyper-IgE syndrome, it is now possible to make a molecular diagnosis in most of these cases. We have developed a PCR-based high-resolution DNA-melting assay to scan selected exons of the STAT3 gene for mutations responsible for Hyper-IgE syndrome, which is then followed by targeted sequencing. We scanned for mutations in 10 unrelated pedigrees, which include 16 patients with classic Hyper-IgE syndrome. These pedigrees include both sporadic and familial cases and their relatives, and we have found STAT3 mutations in all affected individuals. High-resolution melting analysis allows a single day turn-around time for mutation scanning and targeted sequencing of the STAT3 gene, which will greatly facilitate the rapid diagnosis of the Hyper-IgE syndrome, allowing prompt and appropriate therapy, prophylaxis, improved clinical outcome, and accurate genetic counseling. Hyperimmunoglobulin E syndrome (HIES), or Job syndrome (Online Mendelian Inheritance in Man, numbers 147060 and 243700, http://www.ncbi.nlm.nih.gov/omim/, last accessed December 13, 2009), was first described in 1966 by Davis et al1Davis SD Schaller J Wedgwood RJ Job's syndrome: recurrent, "cold," staphylococcal abscesses.Lancet. 1966; 1: 1013-1015Abstract PubMed Google Scholar in two girls with severe eczema, unusual facial features, chronic cutaneous candidiasis, and cold staphylococcal abscesses. The abscesses were described as being "cold" for their lack of the characteristic features of inflammation, such as warmth, redness, tenderness, and fever. In 1972, Buckley et al2Buckley RH Wray BB Belmaker EZ Extreme hyperimmunoglobulinemia E and undue susceptibility to infection.Pediatrics. 1972; 49: 59-70PubMed Google Scholar described similar patients who also were noted to have markedly elevated IgE. Recurrent bacterial and fungal infections are the major cause of morbidity and mortality in HIES.3Freeman AF Kleiner DE Nadiminti H Davis J Quezado M Anderson V Puck JM Holland SM Causes of death in hyper-IgE syndrome.J Allergy Clin Immunol. 2007; 119: 1234-1240Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar The typical patient with HIES also has a history of recurrent pathological fractures, retained primary teeth, hyperextensible joints, deep-set eyes, increased width of the nose, a full lower lip, and thickening of the nose and ears.4Borges WG Hensley T Carey JC Petrak BA Hill HR The face of Job.J Pediatr. 1998; 133: 303-305Abstract Full Text Full Text PDF PubMed Google Scholar Lymphoid malignancies may also be increased in patients with HIES.5Bale Jr, JF Wilson JF Hill HR Fatal histiocytic lymphoma of the brain associated with hyperimmunoglobulinemia-E and recurrent infections.Cancer. 1977; 39: 2386-2390Crossref PubMed Scopus (47) Google Scholar, 6Leonard GD Posadas E Herrmann PC Anderson VL Jaffe ES Holland SM Wilson WH Non-Hodgkin's lymphoma in Job's syndrome: a case report and literature review.Leuk Lymphoma. 2004; 45: 2521-2525Crossref PubMed Scopus (72) Google Scholar A systematic study of 30 patients and their families led to the development of a scoring system to aid in the diagnosis of HIES.7Grimbacher B Holland SM Gallin JI Greenberg F Hill SC Malech HL Miller JA O'Connell AC Puck JM Hyper-IgE syndrome with recurrent infections: an autosomal dominant multisystem disorder.N Engl J Med. 1999; 340: 692-702Crossref PubMed Scopus (641) Google Scholar Such a scoring system was felt to be necessary because HIES not only lacks pathognomonic markers, but the characteristic manifestations often develop over years, delaying diagnosis. Furthermore, the symptoms and signs often found in HIES are also features of other conditions. For example, elevated serum IgE and severe eczema are found in allergic disorders, as well as other primary immunodeficiency disorders, such as the Wiskott-Aldrich syndrome.8Ochs HD Thrasher AJ The Wiskott-Aldrich syndrome.J Allergy Clin Immunol. 2006; 117: 725-738; quiz 739Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar There is no correlation between the clinical phenotype, NIH score, and the serum IgE concentrations, which can rise and fall during the lifetime of patients with HIES, and some do not even have marked elevations of this immunoglobulin, making the name Job syndrome, perhaps, a more appropriate term than the Hyper-IgE syndrome. Both familial and sporadic cases of HIES have been described.9Grimbacher B Holland SM Puck JM Hyper-IgE syndromes.Immunol Rev. 2005; 203: 244-250Crossref PubMed Scopus (207) Google Scholar, 10Van Scoy RE Hill HR Ritts RE Quie PG Familial neutrophil chemotaxis defect, recurrent bacterial infections, mucocutaneous candidiasis, and hyperimmunoglobulinemia E.Ann Intern Med. 1975; 82: 766-771Crossref PubMed Scopus (121) Google Scholar The familial cases show mostly autosomal dominant inheritance, but autosomal recessive cases have also been reported.11Renner ED Puck JM Holland SM Schmitt M Weiss M Frosch M Bergmann M Davis J Belohradsky BH Grimbacher B Autosomal recessive hyperimmunoglobulin E syndrome: a distinct disease entity.J Pediatr. 2004; 144: 93-99Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar The autosomal recessive cases seem to be an entirely different set of disorders.12Minegishi Y Saito M Morio T Watanabe K Agematsu K Tsuchiya S Takada H Hara T Kawamura N Ariga T Kaneko H Kondo N Tsuge I Yachie A Sakiyama Y Iwata T Bessho F Ohishi T Joh K Imai K Kogawa K Shinohara M Fujieda M Wakiguchi H Pasic S Abinun M Ochs HD Renner ED Jansson A Belohradsky BH Metin A Shimizu N Mizutani S Miyawaki T Nonoyama S Karasuyama H Human tyrosine kinase 2 deficiency reveals its requisite roles in multiple cytokine signals involved in innate and acquired immunity.Immunity. 2006; 25: 745-755Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar13Woellner C Schaffer AA Puck JM Renner ED Knebel C Holland SM Plebani A Grimbacher B The hyper IgE syndrome and mutations in TYK2.Immunity. 2007; 26: 535-; author reply 536Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar14Freeman AF Holland SM The Hyper-IgE Syndromes.Immunol Allergy Clin North Am. 2008; 28: 277-291Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar Mutations in STAT3 (signal transducer and activator of transcription 3) have recently been identified by four research groups in both sporadic and familial patients with HIES.15Holland SM DeLeo FR Elloumi HZ Hsu AP Uzel G Brodsky N Freeman AF Demidowich A Davis J Turner ML Anderson VL Darnell DN Welch PA Kuhns DB Frucht DM Malech HL Gallin JI Kobayashi SD Whitney AR Voyich JM Musser JM Woellner C Schaffer AA Puck JM Grimbacher B STAT3 mutations in the hyper-IgE syndrome.N Engl J Med. 2007; 357: 1608-1619Crossref PubMed Scopus (927) Google Scholar16Minegishi Y Saito M Tsuchiya S Tsuge I Takada H Hara T Kawamura N Ariga T Pasic S Stojkovic O Metin A Karasuyama H Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome.Nature. 2007; 448: 1058-1062Crossref PubMed Scopus (798) Google Scholar17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar18Jiao H Toth B Erdos M Fransson I Rakoczi E Balogh I Magyarics Z Derfalvi B Csorba G Szaflarska A Megarbane A Akatcherian C Dbaibo G Rajnavolgyi E Hammarstrom L Kere J Lefranc G Marodi L Novel and recurrent STAT3 mutations in hyper-IgE syndrome patients from different ethnic groups.Mol Immunol. 2008; 46: 202-206Crossref PubMed Scopus (68) Google Scholar All patients with dominant HIES described to date are heterozygotes, and the STAT3 mutations identified are hypomorphic and localized in the DNA-binding, SH2, and transactivation domains. To date, there have been no clear phenotype-genotype correlations described in patients with HIES with various mutations in the STAT3 gene. All of the specific immunological and somatic defects resulting from the STAT3 mutations leading to HIES are not known, but deficiency in Th17 cells is part of the problem.17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 19Ma CS Chew GY Simpson N Priyadarshi A Wong M Grimbacher B Fulcher DA Tangye SG Cook MC Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3.J Exp Med. 2008; 205: 1551-1557Crossref PubMed Scopus (551) Google Scholar, 20Milner JD Brenchley JM Laurence A Freeman AF Hill BJ Elias KM Kanno Y Spalding C Elloumi HZ Paulson ML Davis J Hsu A Asher AI O'Shea J Holland SM Paul WE Douek DC Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome.Nature. 2008; 452: 773-776Crossref PubMed Scopus (925) Google Scholar Since many common disorders present with elevated serum IgE and recurrent infections, which are also characteristic manifestations of Job syndrome, we have developed a rapid molecular assay to diagnose the disorder. Although mutations can be identified by comprehensive sequencing, the time and cost required are substantial. High-resolution DNA-melting analysis is a mutation scanning technique that monitors the change in fluorescence caused by the release of double-stranded DNA-binding dye from DNA as it is denatured by increasing the temperature.21Montgomery J Wittwer CT Palais R Zhou L Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis.Nat Protoc. 2007; 2: 59-66Crossref PubMed Scopus (242) Google Scholar, 22Wittwer CT Reed GH Gundry CN Vandersteen JG Pryor RJ High-resolution genotyping by amplicon melting analysis using LCGreen.Clin Chem. 2003; 49: 853-860Crossref PubMed Scopus (1017) Google Scholar Both heterozygous and homozygous variants can be identified. When the change in DNA is heterozygous (as in Job syndrome), two heteroduplexes and two homoduplexes are formed following PCR amplification. Each duplex has a different thermal stability, leading to subtle melting curve differences between wild-type and variant sequences.23Reed GH Kent JO Wittwer CT High-resolution DNA melting analysis for simple and efficient molecular diagnostics.Pharmacogenomics. 2007; 8: 597-608Crossref PubMed Scopus (524) Google Scholar, 24Ririe KM Rasmussen RP Wittwer CT Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.Anal Biochem. 1997; 245: 154-160Crossref PubMed Scopus (1267) Google Scholar We have applied this rapid molecular technique to defining the mutations in STAT3 genes in 10 unrelated HIES pedigrees with 16 patients with classic HIES to facilitate rapid diagnosis, therapy, and genetic counseling of families with the disorder. Primers were designed by using LightScanner primer design software (Idaho Technology, Salt Lake City, UT) and synthesized by the University of Utah DNA core facility. The primers were placed at least 15 nucleotides away from the ends of each exon to include most residues influencing splicing. Twelve exons of the STAT3 gene encoding the DNA-binding, SH2, and transactivation domains, where the disease-causing mutations have been described,15Holland SM DeLeo FR Elloumi HZ Hsu AP Uzel G Brodsky N Freeman AF Demidowich A Davis J Turner ML Anderson VL Darnell DN Welch PA Kuhns DB Frucht DM Malech HL Gallin JI Kobayashi SD Whitney AR Voyich JM Musser JM Woellner C Schaffer AA Puck JM Grimbacher B STAT3 mutations in the hyper-IgE syndrome.N Engl J Med. 2007; 357: 1608-1619Crossref PubMed Scopus (927) Google Scholar16Minegishi Y Saito M Tsuchiya S Tsuge I Takada H Hara T Kawamura N Ariga T Pasic S Stojkovic O Metin A Karasuyama H Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome.Nature. 2007; 448: 1058-1062Crossref PubMed Scopus (798) Google Scholar17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar18Jiao H Toth B Erdos M Fransson I Rakoczi E Balogh I Magyarics Z Derfalvi B Csorba G Szaflarska A Megarbane A Akatcherian C Dbaibo G Rajnavolgyi E Hammarstrom L Kere J Lefranc G Marodi L Novel and recurrent STAT3 mutations in hyper-IgE syndrome patients from different ethnic groups.Mol Immunol. 2008; 46: 202-206Crossref PubMed Scopus (68) Google Scholar were tested. Exon 21 is amplified in two overlapping amplicons. PCR was performed on MJ Research PTC-200 thermal cyclers (BioRad, Hercules, CA) by using a 96-well plate format. Each 10-μl PCR includes 0.4 U of KlenTaq1, 88 ng of TaqStart antibody, 1x LCGreen Plus (Idaho Technology), 50 mmol/L Tris (pH 8.3), 1 M betaine, 500 mg/L bovine serum albumin, 1.5 mmol/L Mg2+, 0.2 mmol/L of each Deoxyribonucleotide Triphosphate (dNTP), 0.5 μmol/L primers, and 50 ng genomic DNA. Reaction mixtures were overlaid with 10 μl of mineral oil. An annealing temperature gradient PCR was used to determine the optimal annealing temperature (64°C). The extension temperature was set to 72°C. During optimization, the amplicons were examined by melting analysis, agarose gels, and were sequenced to ensure the desired product. The PCR amplification starts at 95°C for 10 seconds, followed by 40 cycles of 94°C for 5 seconds, 64°C for 5 seconds, 72°C for 6 seconds, and finished by denaturing the PCR product at 95°C for 5 seconds. After PCR, the plates were heated to 95°C for 30 seconds and then cooled to 15°C for heteroduplex formation. The plates were then centrifuged (1500 × g for 1 minute) and analyzed in a 96-well LightScanner (Idaho Technology), as previously described.21Montgomery J Wittwer CT Palais R Zhou L Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis.Nat Protoc. 2007; 2: 59-66Crossref PubMed Scopus (242) Google Scholar, 22Wittwer CT Reed GH Gundry CN Vandersteen JG Pryor RJ High-resolution genotyping by amplicon melting analysis using LCGreen.Clin Chem. 2003; 49: 853-860Crossref PubMed Scopus (1017) Google Scholar The 96-well plates were heated at 15°C per second, and fluorescence was measured from 65°C to 98°C. Melting curves were analyzed with LightScanner software. Finally, the melting profiles were clustered. Clustering of melting curves for genotype identification was performed manually, assisted by the LightScanner software on the high sensitivity setting. Amplicons within each cluster were sequenced to determine genotype. High resolution melting analysis was performed on each of 12 exons of 96 random healthy blood donors. Amplification was successful 98.7% of the time; repeated PCR of the failed samples was amplified in all instances. Analysis of the 96 normal samples was performed to determine the number of variants present in a normal population in each of the exons. Typical melting profiles of the exons are shown in Figure 1, A–C. Heterozygous variants can clearly be distinguished from the loose clustering of wild-type samples (Figure 1). There was only one variant (c.1361T>C, p.L454P in exon 15) in our Utah-based, predominantly Caucasian (96%) population (Figure 1C), which is probably benign because it was found in a presumably healthy blood donor. Holland et al15Holland SM DeLeo FR Elloumi HZ Hsu AP Uzel G Brodsky N Freeman AF Demidowich A Davis J Turner ML Anderson VL Darnell DN Welch PA Kuhns DB Frucht DM Malech HL Gallin JI Kobayashi SD Whitney AR Voyich JM Musser JM Woellner C Schaffer AA Puck JM Grimbacher B STAT3 mutations in the hyper-IgE syndrome.N Engl J Med. 2007; 357: 1608-1619Crossref PubMed Scopus (927) Google Scholar tested 158 unaffected control subjects of various ancestral origins (125 white subjects, 15 Hispanic subjects, 4 black subjects, 2 Asian subjects, 1 white Hispanic subject, 1 black Hispanic subject, and 10 persons of unknown ancestry), and Renner et al17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar sequenced 94 unaffected individuals of diverse ethnic background, and both found no variants in STAT3. The STAT3 gene in 16 patients and 75 unaffected family members from 10 kindreds, representing 91 samples enriched for cases of HIES were analyzed by high resolution melting. The samples were blinded to evaluate the accuracy of the procedure. After PCR amplification and melting analysis, we evaluated representatives of the main cluster (assumed to be wild-type) and all of the outliers, if present, by sequencing. Sequence variants were detected in three exons: 13, 20, and 21. Melting analysis and sequencing of exon 13 revealed three genotypes in addition to the wild-type (Figure 2, A and B). All three variants changed the R382 codon. R382 is located in the DNA-binding region of STAT3. Although all three variants are clearly different from the most common wild-type genotype, they were not differentiated from each other. Matching the genotype of samples to family pedigrees showed that melting analysis correctly identified all affected family members. As an example, one large family is shown in Figure 2, in which a de novo R382Q mutation arose in the proband (II/4) and was inherited by two of the proband's four children (III/3 and III/4). There were three families (three patients) with the R382W (C to T), three families (six patients) with the R382Q (G to A), and one family (two patients) with the R382G (C to G) mutation (Table 1).17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar The codon of R382 is the most common mutational hotspot described previously,15Holland SM DeLeo FR Elloumi HZ Hsu AP Uzel G Brodsky N Freeman AF Demidowich A Davis J Turner ML Anderson VL Darnell DN Welch PA Kuhns DB Frucht DM Malech HL Gallin JI Kobayashi SD Whitney AR Voyich JM Musser JM Woellner C Schaffer AA Puck JM Grimbacher B STAT3 mutations in the hyper-IgE syndrome.N Engl J Med. 2007; 357: 1608-1619Crossref PubMed Scopus (927) Google Scholar16Minegishi Y Saito M Tsuchiya S Tsuge I Takada H Hara T Kawamura N Ariga T Pasic S Stojkovic O Metin A Karasuyama H Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome.Nature. 2007; 448: 1058-1062Crossref PubMed Scopus (798) Google Scholar17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar18Jiao H Toth B Erdos M Fransson I Rakoczi E Balogh I Magyarics Z Derfalvi B Csorba G Szaflarska A Megarbane A Akatcherian C Dbaibo G Rajnavolgyi E Hammarstrom L Kere J Lefranc G Marodi L Novel and recurrent STAT3 mutations in hyper-IgE syndrome patients from different ethnic groups.Mol Immunol. 2008; 46: 202-206Crossref PubMed Scopus (68) Google Scholar as well as in our data set. It represents ∼40% of all patients with STAT3 mutant HIES described to date. R382W is the most common variant in the literature. In contrast, R382G is a newly described mutation.Table 1Patients with Job Syndrome of Hyper-IgE and Recurrent InfectionsIDSexIgE, IU/mlEczemaAbscessPneumoniaCandida infectionAssociated findingsSTAT3 mutationProtein domain (Exon no.)Type1-1*STAT3 gene was completely sequenced from these patients17; reference sequence for mutations is NM_139276.2.Male4000+++++c.1145G>A; p.R382QDNA-binding (Exon 13)de novo1-2Female98,766+++++c.1145G>A; p.R382QDNA-binding (Exon 13)Transmitted1-3Female16,172+++++c.1145G>A; p.R382QDNA-binding (Exon 13)Transmitted2-1*STAT3 gene was completely sequenced from these patients17; reference sequence for mutations is NM_139276.2.Male1451+++++c.1145G>A; p.R382QDNA-binding (Exon 13)?3-1Male3206+++++c.1913A>G; p.E638GSH2 (Exon 21)de novo4-1Female9477++−++c.1863C>G; p.F621LSH2 (Exon 20)?5-1*STAT3 gene was completely sequenced from these patients17; reference sequence for mutations is NM_139276.2.Male8000+++++c.1144C>T; p.R382WDNA-binding (Exon 13)?6-1*STAT3 gene was completely sequenced from these patients17; reference sequence for mutations is NM_139276.2.Female21,700+++++c.1144C>G; p.R382GDNA-binding (Exon 13)de novo6-2Male334+++++c.1144C>G; p.R382GDNA-binding (Exon 13)Transmitted7-1*STAT3 gene was completely sequenced from these patients17; reference sequence for mutations is NM_139276.2.Male8127+++++c.2069del30bp; p.690_P699delTransactivation (Exon 21†)de novo7-2Female1293+++++c.2069del30bp; p.690_P699delTransactivation (Exon 21†)Transmitted7-3Female1909+++++c.2069del30bp; p.690_P699delTransactivation (Exon 21†)Transmitted8-1*STAT3 gene was completely sequenced from these patients17; reference sequence for mutations is NM_139276.2.Male29,899+++++c.1144C>T; p.R382WDNA-binding (Exon 13)?9-1*STAT3 gene was completely sequenced from these patients17; reference sequence for mutations is NM_139276.2.Male38,918?????c.1144C>T; p.R382WDNA-binding (Exon 13)?10-1Female15,776+++++c.1145G>A; p.R382QDNA-binding (Exon 13)?10-2Female40,009+++++c.1145G>A; p.R382QDNA-binding (Exon 13)Transmitted†The mutation in family 8 includes the splice acceptor site.17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar* STAT3 gene was completely sequenced from these patients17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar; reference sequence for mutations is NM_139276.2. Open table in a new tab †The mutation in family 8 includes the splice acceptor site.17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar Melting analysis and sequencing of exon 20 detected one heterozygous mutation in the SH2 domain (not shown). F621L is also a newly described mutation. Melting analysis and sequencing of exon 21 (tested by two overlapping amplicons, due to its size) detected two genotypes in addition to the wild-type (Table 1).17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar Exon 21 encodes part of the SH2 domain of STAT3 protein. E638G is a de novo mutation in the proband with 13 unaffected siblings who were tested (Figure 3, A and B). A large, 30-bp deletion was found in exon 21, which encodes the beginning of the transactivation domain, in a family with an affected father and two affected daughters (Figure 4, A and B). The in-frame deletion of 10 amino acids occurred de novo in the proband (II/4), and was inherited by the two daughters (III/3 and III/4), and was detected in all three patients.Figure 4Deletion in the transactivation domain. A: A large, 32-bp deletion occurred in the proband (II/4) and was inherited by two of the proband's children (III/3 and III/4). B: This large heterozygous deletion was detected in all three patients. Family members were tested if available; family members not labeled as WT (wild-type) or HIES were not tested. IgE serum levels are shown in IU/ml if known.View Large Image Figure ViewerDownload Hi-res image Download (PPT) All of the variants in the STAT3 gene matched a clinically-documented patient with HIES in the family pedigrees. We found no HIES mutations or polymorphisms among the unaffected relatives. There were two unrelated cases, however, where the clinical diagnosis and the molecular testing did not match. The family pedigree of the first patient is shown in Figure 5. The proband (II-3) and her son (III-1; patients 6-1 and 6-2 in Table 117Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) were found to have the same missense mutation (R382G) in exon 13 of STAT3. The proband's father possessed some of the signs and symptoms of HIES (numerous abcesses), but not high IgE, thus the clinical diagnosis was uncertain. Genetic testing revealed a wild-type STAT3 sequence in this individual. In the second family, one brother of the proband (Brother A) was given a clinical diagnosis of HIES, and the proband's daughter was given a diagnosis of HIES as well.10Van Scoy RE Hill HR Ritts RE Quie PG Familial neutrophil chemotaxis defect, recurrent bacterial infections, mucocutaneous candidiasis, and hyperimmunoglobulinemia E.Ann Intern Med. 1975; 82: 766-771Crossref PubMed Scopus (121) Google Scholar Mother and daughter (patients 10-1 and 10-2 in Table 117Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) had the same R382Q mutation; however, "Brother A" had a wild-type STAT3 sequence despite the elevated IgE and onychomycosis, highlighting the utility of genetic testing. DNA from the parents and grandparents of the proband were not available for testing.10Van Scoy RE Hill HR Ritts RE Quie PG Familial neutrophil chemotaxis defect, recurrent bacterial infections, mucocutaneous candidiasis, and hyperimmunoglobulinemia E.Ann Intern Med. 1975; 82: 766-771Crossref PubMed Scopus (121) Google Scholar Most cases of classic HIES have unaffected parents, suggesting de novo mutations. In the reported 10 pedigrees, we demonstrated de novo mutations in four families (see Figures 2, 3, and 4), whereas in the other six families we did not have DNA from both parents. In this report, 96 healthy blood donors and 91 DNA samples from HIES families collected over 35 years were scanned for variants by a newly developed melting assay. All of the mutations were hypomorphic and heterozygous, and the inheritance was autosomal dominant in all cases where we had sufficient data to make the determination. The melting assay was 100% sensitive for the identification of STAT3 mutations in autosomal dominant HIES and along with targeted sequencing of the abnormal exon it can be performed in a single day. These performance characteristics are in line with other studies in which the authors used melting analysis (for reviews on sensitivities and specificities see van der Stoep et al25van der Stoep N van Paridon CD Janssens T Krenkova P Stambergova A Macek M Matthijs G Bakker E Diagnostic guidelines for high-resolution melting curve (HRM) analysis: an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner.Hum Mutat. 2009; 30: 899-909Crossref PubMed Scopus (114) Google Scholar and Farrar et al26Farrar JS Reed GH Wittwer CT High resolution melting curve analysis for molecular diagnostics.in: Patrinos GP Ansorge WJ Molecular Diagnostics. Elsevier, Oxford2009: 229-245Google Scholar). It is likely that not all STAT3 mutations that cause HIES are known. Because mutation scanning relies on the melting profile of the PCR amplicon, new mutations are easily identified. When a novel melting profile is observed, the targeted exon is sequenced. Both known and previously unknown mutations in the DNA-binding, SH2, and transactivation domains of STAT3 have been detected in our study. Most patients with classic HIES have mutations in the STAT3 gene. Minegishi et al16Minegishi Y Saito M Tsuchiya S Tsuge I Takada H Hara T Kawamura N Ariga T Pasic S Stojkovic O Metin A Karasuyama H Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome.Nature. 2007; 448: 1058-1062Crossref PubMed Scopus (798) Google Scholar identified mutations in STAT3 in 8 of 15 sporadic HIES cases (53.3%). In contrast, Holland et al15Holland SM DeLeo FR Elloumi HZ Hsu AP Uzel G Brodsky N Freeman AF Demidowich A Davis J Turner ML Anderson VL Darnell DN Welch PA Kuhns DB Frucht DM Malech HL Gallin JI Kobayashi SD Whitney AR Voyich JM Musser JM Woellner C Schaffer AA Puck JM Grimbacher B STAT3 mutations in the hyper-IgE syndrome.N Engl J Med. 2007; 357: 1608-1619Crossref PubMed Scopus (927) Google Scholar found mutations in all 50 sporadic and familial patients studied with HIES, and Renner et al,17Renner ED Rylaarsdam S Anover-Sombke S Rack AL Reichenbach J Carey JC Zhu Q Jansson AF Barboza J Schimke LF Leppert MF Getz MM Seger RA Hill HR Belohradsky BH Torgerson TR Ochs HD Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar including our group, failed to find a mutation in only 1 of 38 patients from 35 families. Jiao et al18Jiao H Toth B Erdos M Fransson I Rakoczi E Balogh I Magyarics Z Derfalvi B Csorba G Szaflarska A Megarbane A Akatcherian C Dbaibo G Rajnavolgyi E Hammarstrom L Kere J Lefranc G Marodi L Novel and recurrent STAT3 mutations in hyper-IgE syndrome patients from different ethnic groups.Mol Immunol. 2008; 46: 202-206Crossref PubMed Scopus (68) Google Scholar investigated 12 patients from 9 families and found mutations in STAT3 of all HIES cases. This wide variation in the number of patients with HIES without a mutation in STAT3 (from 0% to 46.7%) suggests differences in the diagnostic criteria used by the clinicians or in the ethnic background. The finding that, at least in some cases of autosomal recessive HIES, the gene-defect may be in Tyrosine Kinase 2 (TYK2),12Minegishi Y Saito M Morio T Watanabe K Agematsu K Tsuchiya S Takada H Hara T Kawamura N Ariga T Kaneko H Kondo N Tsuge I Yachie A Sakiyama Y Iwata T Bessho F Ohishi T Joh K Imai K Kogawa K Shinohara M Fujieda M Wakiguchi H Pasic S Abinun M Ochs HD Renner ED Jansson A Belohradsky BH Metin A Shimizu N Mizutani S Miyawaki T Nonoyama S Karasuyama H Human tyrosine kinase 2 deficiency reveals its requisite roles in multiple cytokine signals involved in innate and acquired immunity.Immunity. 2006; 25: 745-755Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar but that in the majority if not all of autosomal dominant HIES cases the defect is in STAT3, strongly suggests that the TYK2-STAT3 signaling pathway is the key to the pathogenesis of HIES because TYK2 can phosphorylate and activate STAT3. The assay presented here is expected to identify STAT3 mutations in more than 90% of patients who have been given a clinical diagnosis of classic HIES, and can easily be expanded, if necessary, to include new mutations in other STAT3 exons. DNA variant scanning by high-resolution melting is a rapid and cost effective method. The PCR amplification for high-resolution melting requires only standard unlabeled PCR primers and a fluorescent dye that detects heteroduplexes and can be included in the PCR mixture before amplification. It takes about 2 hours after DNA purification to perform PCR amplification and high-resolution melting. High-resolution melting is an in-tube method that requires no opening of the tube or plate after PCR amplification, greatly simplifying the testing process and preventing amplicon contamination with rapid turn-around times. The cost of scanning one sample as described here is estimated to be $29, including disposables, PCR reagents, and the LCGreen Plus dye. The estimated reagent cost of full bi-directional sequencing of the same sample at our facility is $165 (12 PCR products, sequenced in both directions, $6.90 for each sequencing reaction). Therefore, the major time and cost component of the method described here is reflexive sequencing. Bi-directional sequencing of an exon costs $13.80. The amount of sequencing required is determined by the number of variant melting curves detected. As long as there are fewer than nine sequencing reactions required per sample, our assay is cost effective. We encountered four to six variant melting curves caused by wild-type outliers at most (Figure 1) per 96-well plate that translate into less than one potentially avoidable additional sequencing reaction per patient. The ordering pattern of the test is the other determining factor for the amount of reflexive sequencing required. If genetic testing is only ordered for patients with a high NIH score,15Holland SM DeLeo FR Elloumi HZ Hsu AP Uzel G Brodsky N Freeman AF Demidowich A Davis J Turner ML Anderson VL Darnell DN Welch PA Kuhns DB Frucht DM Malech HL Gallin JI Kobayashi SD Whitney AR Voyich JM Musser JM Woellner C Schaffer AA Puck JM Grimbacher B STAT3 mutations in the hyper-IgE syndrome.N Engl J Med. 2007; 357: 1608-1619Crossref PubMed Scopus (927) Google Scholar then most samples will be positive and require reflexive sequencing, thus increasing the cost by two sequencing reactions (bi-directional sequencing of an exon, $13.80) and lengthening the time required. However, if the test is also used for screening and differential diagnosis in addition to confirmation of clinically documented cases, the majority of the samples will be negative and can be returned rapidly without reflexive sequencing in a time and cost-effective manner. Our experience with similar tests supports the second scenario because most cases of HIES are born to unaffected parents, and clinicians have to maintain a high level of suspicion to diagnose primary immunodeficiencies based on repeated or unusual infection. HIES lacks specific symptoms and signs and is difficult to diagnose with currently available screening assays. High resolution melting analysis of the STAT3 gene provides rapid diagnosis of this potentially fatal disorder, and thus, facilitates both prophylaxis and symptomatic therapy to prevent further morbidity and mortality and genetic counseling of these patients and their families. The rapidity of the high-resolution melting analysis followed by targeted sequencing allows detection of STAT3 mutations as a clinically useful diagnostic test for this primary immunodeficiency syndrome characterized by diverse manifestations. Attila Kumánovics, Carl T. Wittwer, and Harry R. Hill designed the experiments and edited the article. Attila Kumánovics wrote the article and performed the laboratory experiments together with Robert J. Pryor and Nancy H. Augustine. Harry R. Hill took care of most of the patients, and he, Mark F. Leppert, and John C. Carey collected the pedigrees and DNA samples. Ralph J. Faville, Jr., Paul G. Quie, Hans D. Ochs, and Ralph J. Wedgwood identified and cared for patients, provided clinical information, enrolled patients, and collected DNA samples for the study.
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