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Quantification of κ-deleting recombination excision circles in Guthrie cards for the identification of early B-cell maturation defects

2011; Elsevier BV; Volume: 128; Issue: 1 Linguagem: Inglês

10.1016/j.jaci.2011.01.052

1097-6825

Noriko Nakagawa, Kohsuke Imai, Hirokazu Kanegane, Hiroki Sato, Masafumi Yamada, Kensuke Kondoh, Satoshi Okada, Masao Kobayashi, Kazunaga Agematsu, Hidetoshi Takada, Noriko Mitsuiki, Koichi Oshima, Osamu Ohara, Deepti Suri, Amit Rawat, Surjit Singh, Qiang Pan‐Hammarström, Lennart Hammarström, Janine Reichenbach, Reinhard Seger, Tadashi Ariga, Toshiro Hara, Toshio Miyawaki, Shigeaki Nonoyama,

T-cell and B-cell Immunology

To the Editor: X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by severely decreased numbers of mature peripheral B lymphocytes as a result of a mutation in the BTK gene. Non-XLA is characterized by hypogammaglobulinemia with decreased B-cell counts (less than 2% of mature B cells) in the absence of the BTK gene mutation. Both XLA and non-XLA are caused by an early B-cell maturation defect.1Conley M.E. Broides A. Hernandez-Trujillo V. Howard V. Kanegane H. Miyawaki T. et al.Genetic analysis of patients with defects in early B-cell development.Immunol Rev. 2005; 203: 216-234Crossref PubMed Scopus (159) Google Scholar In patients with XLA and non-XLA, recurrent infections appear between 3 and 18 months of age, whereas the mean age at diagnosis is 3 years.2Kanegane H. Futatani T. Wang Y. Nomura K. Shinozaki K. Matsukura H. et al.Clinical and mutational characteristics of X-linked agammaglobulinemia and its carrier identified by flow cytometric assessment combined with genetic analysis.J Allergy Clin Immunol. 2001; 108: 1012-1020Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar This delayed diagnosis results in frequent hospitalization because of pneumonia, sepsis, meningitis, and other bacterial infections, which frequently require intravenous administration of antibiotics and can be fatal. Frequent pneumonia results in a high incidence of chronic lung diseases.3Plebani A. Soresina A. Rondelli R. Amato G.M. Azzari C. Cardinale F. et al.Clinical, immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study.Clin Immunol. 2002; 104: 221-230Crossref PubMed Scopus (284) Google Scholar Thus, early diagnosis and early treatment, including periodical intravenous immunoglobulin replacement therapy, is essential to improve the prognosis and the quality of life of patients with XLA and non-XLA. In the process of B-cell maturation, immunoglobulin κ-deleting recombination excision circles (KRECs) are produced during κ-deleting recombination allelic exclusion and isotypic exclusion of the λ chain.4Siminovitch K.A. Bakhshi A. Goldman P. Korsmeyer S.J. A uniform deleting element mediates the loss of kappa genes in human B cells.Nature. 1985; 316: 260-262Crossref PubMed Scopus (209) Google Scholar Coding joint (cj) KRECs reside within the chromosome, whereas signal joint (sj) KRECs are excised from genomic DNA. cjKREC levels remain the same after B-cell division, whereas sjKREC levels decrease, because sjKRECs are not replicated during cell division.5van Zelm M.C. Szczepanski T. van der Burg M. van Dongen J.J. Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion.J Exp Med. 2007; 204: 645-655Crossref PubMed Scopus (231) Google Scholar Because the B-cell maturation defects in XLA and non-XLA occur before κ-deleting recombination, KRECs are not supposed to be produced. Therefore, measurements of KRECs have the potential to be applied to the identification of these types of B-cell deficiencies in patients, which consist of around 20% of all B-cell defects.6Eades-Perner A.M. Gathmann B. Knerr V. Guzman D. Veit D. Kindle G. et al.ESID Registry Working Party. The European internet-based patient and research database for primary immunodeficiencies: results 2004-06.Clin Exp Immunol. 2007; 147: 306-312Crossref PubMed Scopus (109) Google Scholar In addition, some types of combined immunodeficiencies show an arrest in B-cell maturation and can also be identified by this method. The success of newborn screening for T-cell deficiencies by measuring T-cell–receptor excision circles7Morinishi Y. Imai K. Nakagawa N. Sato H. Horiuchi K. Ohtsuka Y. et al.Identification of severe combined immunodeficiency by T-cell receptor excision circles quantification using neonatal Guthrie cards.J Pediatr. 2009; 155: 829-833Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar prompted us to develop a newborn screening method for XLA and non-XLA by measuring KRECs derived from neonatal Guthrie cards. The study protocol was approved by the National Defense Medical College institutional review board, and written informed consent was obtained from the parents of normal controls, the affected children, and adult patients, in accordance with the Declaration of Helsinki. First, we determined the sensitivity of detection levels of cjKRECs and sjKRECs in Guthrie cards using real-time quantitative PCR.5van Zelm M.C. Szczepanski T. van der Burg M. van Dongen J.J. Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion.J Exp Med. 2007; 204: 645-655Crossref PubMed Scopus (231) Google Scholar Normal B cells from a healthy adult were isolated from peripheral blood (PB; mean purity, 88.5%). PB was also obtained from 1 patient with XLA (P20) whose B-cell number was 0.09 in 1 μL whole blood and who was negative for sjKRECs ( 1.0 × 102 copies/μg DNA) of cjKRECs and sjKRECs increased constantly as the B-cell concentrations increased (Fig 1). None of the samples were positive for sjKRECs when the B-cell numbers were less than 20/μL, but cjKRECs were often positive. It has been reported that 90% of patients with XLA have less than 0.2% B cells in the PB at diagnosis.1Conley M.E. Broides A. Hernandez-Trujillo V. Howard V. Kanegane H. Miyawaki T. et al.Genetic analysis of patients with defects in early B-cell development.Immunol Rev. 2005; 203: 216-234Crossref PubMed Scopus (159) Google Scholar Because peripheral lymphocyte numbers in neonates range from 1200 to 9800/μL,8Ozyurek E. Cetintas S. Ceylan T. Ogus E. Haberal A. Gurakan B. et al.Complete blood count parameters for healthy, small-for-gestational-age, full-term newborns.Clin Lab Haematol. 2006; 28: 97-104Crossref PubMed Scopus (41) Google Scholar the absolute B-cell numbers of 90% of patients with XLA are estimated to be 2.4 to 19.6/μL at the time of blood collection for Guthrie cards, although exact B-cell numbers of XLA in neonatal periods are not known at this moment. Because neonates are known to have fewer B cells than infants,9Comans-Bitter W.M. de Groot R. van den Beemd R. Neijens H.J. Hop W.C. Groeneveld K. et al.Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations.J Pediatr. 1997; 130: 388-393Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar and we observed that B-cell numbers are constantly low in patients with XLA throughout infancy (Nakagawa, unpublished data, June 2010), which is consistent with the fact that BTK plays an essential role in B-cell maturation. It is likely that neonates with XLA also have severely decreased B cells. On the other hand, all samples obtained from 400 B cells/μL were positive for both cjKRECs and sjKRECs. We also observed that all healthy infants (1-11 months old; n = 15) were sjKREC-positive (Nakagawa, unpublished data, June 2010) and might have at least 600 B cells/μL whole blood.9Comans-Bitter W.M. de Groot R. van den Beemd R. Neijens H.J. Hop W.C. Groeneveld K. et al.Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations.J Pediatr. 1997; 130: 388-393Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar From these data, it is assumed that at least 90% of patients with XLA are sjKREC-negative, and healthy neonates are positive for sjKRECs on neonatal Guthrie cards. Next, we measured cjKRECs and sjKRECs in dried blood spots in filter papers or Guthrie cards from 30 patients with XLA and 5 patients with non-XLA and from 133 neonates born at the National Defense Medical College Hospital during this study period (August 2008 to October 2009) and 138 healthy subjects of various ages (1 month to 35 years old) to investigate the validity of this method. The levels of B cells of the patients ranged from 0.0% to 1.1% of total lymphocytes and 0.0 to 35.78/μL. IgG levels were 10 to 462 mg/dL (see this article’s Table E1, Table E2 in the Online Repository at www.jacionline.org). Patients with leaky phenotypes1Conley M.E. Broides A. Hernandez-Trujillo V. Howard V. Kanegane H. Miyawaki T. et al.Genetic analysis of patients with defects in early B-cell development.Immunol Rev. 2005; 203: 216-234Crossref PubMed Scopus (159) Google Scholar, 10Kaneko H. Kawamoto N. Asano T. Mabuchi Y. Horikoshi H. Teramoto T. et al.Leaky phenotype of X-linked agammaglobulinaemia in a Japanese family.Clin Exp Immunol. 2005; 140: 520-523Crossref PubMed Scopus (21) Google Scholar were included; 1 patient (P30) had more than 1% B cells and 34.22/μL total B cells, and 4 patients had more than 300 mg/dL serum IgG (P12, P30, P31, P33). All of the normal neonatal Guthrie cards were positive for both cjKRECs and sjKRECs (7.2 ± 0.7 × 103 and 4.8 ± 0.6 × 103 copies/μg DNA, respectively). All healthy subjects of various ages were also positive for both cjKRECs and sjKRECs (Nakagawa, unpublished data, June 2010). In contrast, specimens from all 35 B-cell–deficient patients were sjKREC-negative (<1.0 × 102 copies/μg DNA; Fig 2). All 5 patients with leaky phenotypes were also sjKREC-negative, which might be explained by the hypothesis that leaky B cells of patients with XLA are long-lived B cells that divided several times and have fewer sjKRECs than naive B cells. One patient (P27) was positive for cjKRECs, but other patients were negative for it. RPPH1 (internal control) was detectable at the same level as in normal controls in all samples. These results indicate that sjKRECs are undetectable in XLA and non-XLA and suggest that measurement of sjKRECs in neonatal Guthrie cards has the potential for the use of newborn mass screening to identify neonates with early B-cell maturation defects. Greater numbers of neonatal Guthrie cards should be examined to confirm this potential, and the data obtained from dried blood spots on filter papers must be examined to prove that they truly reflect the data obtained from neonatal Guthrie cards. We should also examine whether screening can reduce the cost of treatment of the bacterial infections and chronic lung diseases in patients with XLA and non-XLA and increase the benefits for these patients. An anticipated pilot study using a large cohort of newborns must address these problems. We also found that T-cell–receptor excision circles and sjKRECs can be measured simultaneously on the same plate. Thus, a pilot study of neonatal screening for both T-cell and B-cell deficiencies could be performed simultaneously. We thank the patients and their families who participated in this study. We also thank Ms Makiko Tanaka and Ms Kimiko Gasa for their skillful technical assistance and members of the Department of Obstetrics and Gynecology at the National Defense Medical College for collecting umbilical cord blood samples as well as Drs Wataru and Masuko Hirose. We are also indebted to Prof J. Patrick Barron, Chairman of the Department of International Medical Communications of Tokyo Medical University, for his pro bono linguistic review of this article. Tabled 1Characteristics of patients with XLAPatient no.Unique patient no.Age (y)Serum Ig (mg/dL)CD19+BTK mutationSourceSexIgGIgAIgM% Lymph/μLGenomic DNAcDNAAmino acidGuthrieDry spotP16700M87 T1178-1G>TSplice acceptor defectxP27180M215<10<100.077.0411593_11594 insA144_145insAArg49 frameshiftxP37220M80<11 T763C>TArg255XxP47278M29559570.113.5229269G>T1178-1G>TSplice acceptor defectxP573234M1140∗Trough level during intravenous immunoglobulin therapy. A182T>AIle61AsnxP681124M458∗Trough level during intravenous immunoglobulin therapy.0130.505.3223570T>G426T>GTyr142XxP781318M628∗Trough level during intravenous immunoglobulin therapy.10960.606.8723570T>G426T>GTyr142XxP881419M2600NA0.203.0116180C>T344C>TSer115PhexP981513M600∗Trough level during intravenous immunoglobulin therapy.<10 T142-1G>TSplice acceptor defectxP1081611M12050.000.00150kb deletion of BTK, TIMM8A, TAF7L, DRP2xP1181710M102240.8035.7836288C>T1928C>TThr643IlexP1282413M4626270.4114.4927518C>A895-11C>ASplice acceptor defectxP138345M<237<37430.000.0025715_26210del776+57_839+73delExon 9 deletionxP1483821M<50 C1631+1G>CSplice donor defectxP1583916M604∗Trough level during intravenous immunoglobulin therapy.<1 C1631+1G>CSplice donor defectxP1684711M698∗Trough level during intravenous immunoglobulin therapy.26110.081.8625536delG655delGVal219 frameshiftxP1787714M201980.21NA32357T>C1750+2T>CSplice donor defectxP188805M23339410.06NA10941-?_14592+?del1-?_240+?delExon 1-3 deletionxP198888M<212 A83G>AArg28HisxP2089121M195 G1638C>GCys502TrpxP219580M<50<1090.8027.1431544_31547 delGTTT1580_1583del GTTTCys527 frameshiftxP227012M115 A336C>ATyr112XxP239110M<10<6 C1350-2A>CSplice acceptor defectxP249370M60 T82C>TArg28CysxP259380M<20<4<60.000.0036269-?_36778+?del1909-?_2418+?delExon 19 deletionxP269390M60 T82C>TArg28Cysx P2789012M<237<37 A1909-8G>ASplice acceptor defectxP289446M12 T1921C>TArg641CysxP299485M<237<37 A1909-8G>ASplice acceptor defectxP3010535M38651131.1034.2232259A>C1654A>CThr552ProxAge, Age at analysis of KRECs; CD19+ % Lymph, CD19-positive cell percentage in lymphocytes; CD19+ /μL, CD19-positive cell number in 1 μL whole peripheral blood; M, male; NA, not available; Serum Ig, serum levels of immunoglobulins at diagnosis.BTK mutation’s reference sequences are NCBI NC_000023.9, NM_000061.2, and NP_000052.1.∗ Trough level during intravenous immunoglobulin therapy. Open table in a new tab Age, Age at analysis of KRECs; CD19+ % Lymph, CD19-positive cell percentage in lymphocytes; CD19+ /μL, CD19-positive cell number in 1 μL whole peripheral blood; M, male; NA, not available; Serum Ig, serum levels of immunoglobulins at diagnosis. BTK mutation’s reference sequences are NCBI NC_000023.9, NM_000061.2, and NP_000052.1. Tabled 1Characteristics of patients with non-XLAAge (y)Serum Ig (mg/dL)CD19+SourcePatient no.Unique patient no.SexIgGIgAIgM% Lymph/μLBTK mutationGuthrieDry spotP315964F386<660.4221.27NormalxP327190F<50<5<50.000.00NormalxP338358M311323200.091.88NormalxP349150M<212<37<200.000.00NormalxP359470M<21<37<390.000.00NormalxAge, Age at analysis of KRECs; CD19+ % Lymph, CD19-positive cell percentage in lymphocytes; CD19+ /μL, CD19-positive cell number in 1 μL whole peripheral blood; F, female; M, male; Serum Ig, serum levels of immunoglobulins at diagnosis. Open table in a new tab Age, Age at analysis of KRECs; CD19+ % Lymph, CD19-positive cell percentage in lymphocytes; CD19+ /μL, CD19-positive cell number in 1 μL whole peripheral blood; F, female; M, male; Serum Ig, serum levels of immunoglobulins at diagnosis.