A Novel Deletion Mutation and Structural Abnormality in the Bruton's ...

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Background: X-linked agammaglobulinemia (XLA) is a heritable primary immune deficiency disorder caused by mutation of Bruton's tyrosine kinase (BTK) gene.
Clin. Lab. 2014;60:859-862 ©Copyright

CASE REPORT

A Novel Deletion Mutation and Structural Abnormality in the Bruton’s Tyrosine Kinase Gene Identified in a Chinese Patient with X-linked Agammaglobulinemia SHIFU WANG 1, *, YANQIN LU 2, 3, *, HU LI 2, 3, ZHAOXIA WANG 4, XINKAI MO 2, 3, ZHENGBIN CHAI 2, 3, JINXIANG HAN 2, 3 * both authors contributed equally to this work Department of Laboratory, Qilu Children's Hospital of Shandong University, Jinan 250022, China Key Laboratory for Biotech-Drugs Ministry of Health, Key Laboratory for Modern Medicine and Technology of Shandong Province, Key Laboratory for Rare & Uncommon Diseases of Shandong Province, Key Laboratory for Virology of Shandong Province, Shandong Medicinal Biotechnology Centre, Shandong Academy of Medical Sciences, Jinan 250062, China 3 School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences. Jinan 250200, China 4 Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, China 1

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SUMMARY Background: X-linked agammaglobulinemia (XLA) is a heritable primary immune deficiency disorder caused by mutation of Bruton’s tyrosine kinase (BTK) gene. The main clinical characteristics of XLA are recurrent respiratory tract infections and profoundly low serum immunoglobulin levels and B cells. Methods: The clinical characteristics of a five-year-old Chinese boy with XLA were described. Mutations of BTK genes were identified by traditional DNA sequencing based on PCR. A three-dimensional model of the truncated BTK protein was constructed. Results: Molecular analysis showed a point deletion of an adenine nucleotide at position 1427 (p.Tyr476Ser), which would cause a frameshift and premature termination at codon 484. Three-dimensional analysis showed that the truncated protein had lost the functional region for both ATP and substrate binding such that tyrosine kinase activity would be affected. Conclusions: The study identified a novel BTK mutation of one Chinese XLA patient. The truncated BTK model identified the loss of a functional domain. (Clin. Lab. 2014;60:859-862. DOI: 10.7754/Clin.Lab.2013.130631) XLA is an autosomal recessive genetic disorder. Mutations in the Bruton's tyrosine kinase (BTK) gene were identified as the cause of XLA in 1993 [2]. BTK is a member of the Tec family of non-receptor protein tyrosine kinases, which are involved in many signal transduction pathways concerning the survival, proliferation, differentiation, and regulation of B cells [3]. BTK is composed of five distinct domains: N-terminal pleckstrin homology and Tec homology domains, a Src homology 3 (SH3) domain, and an SH2 and kinase domain (TK) at the C-terminus [2]. About 85% of cases of the disorder are due to BTK gene mutation [4]. Thus far, 1100 unrelated XLA families with 681 unique mutations and 1252 analyzed alleles have been reported. ___________________________________________

KEY WORDS X-linked agammaglobulinemia, Bruton’s tyrosine kinase, gene mutation, TK domain, three-dimensional structure INTRODUCTION X-linked agammaglobulinemia (XLA, MIM:300755), which is also called Bruton’s agammaglobulinemia or Bruton’s syndrome, is a rare inherited disorder characterized by early recurrent bacterial infections, then remarkable reduction in the serum levels of all immunoglobulin isotypes and an extremely low B lymphocyte count [1]. Clin. Lab. 5/2014

Case Report accepted July 24, 2013

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Mutations in BTK comprise deletion, insertion, splice site, missense, and nonsense mutations. Mutations are found throughout all five domains but mutations in the TK domain are most prevalent, accounting for 46.8% of the total [5]. Although there are no data on the prevalence of XLA in China, BTK mutations in Chinese XLA patients have been reported [6]. Here, we describe a Chinese XLA patient with a novel deletion mutation in the TK domain-encoding region of BTK.

Three-dimensional structural analysis We introduced the deletion mutation into a three-dimensional model of the BTK protein (PDB ID 3P08) to predict the effect on protein structure and function using PyMOL (The PyMol Molecular Graphics System, Version 1.3, Schrodinger, LLC). RESULTS AND DISCUSSION Recurrent upper respiratory tract infections and lower respiratory tract infections are the main clinical symptoms of XLA [6]. Recurrent sinopulmonary infections, arthritis, diarrhea, meningitis, and skin infection are also frequently observed [7]. Sputum cultures identified Streptococcus viridans infection in the patient. Serum ELISA showed positive infection with human rhinovirus. There was no Mycoplasma pneumoniae-IgM, Chlamydia pneumoniae-IgM, adenovirus-IgM, respiratory syncytial virus-IgM, or parainfluenza virus-IgM in serum. No tuberculosis was detected by PCR. Endobronchial tuberculosis infection was reported in one Japanese XLA patient [8]. Lower respiratory tract infection was the main symptom in the patient described here, which occurred with hypogammaglobulinemia. There is reportedly a high prevalence of arthritis in Chinese XLA patients [9]; however, this was not observed in our patient. By DNA sequencing, we revealed a point deletion of an adenine nucleotide at position 1427 (exon 15) in the patient (Figure 1A). This would introduce a tyrosine-toserine substitution at codon 476 and result in the addition of eight subsequent missense amino acids after residue 476 before a premature termination. The same deletion, c.1427delA, was found in the patient’s heterozygous mother (Figure 1B), but not in his healthy father (Figure 1C). The mutation in our patient was a deletion at nucleotide 1427 (codon 476), causing a frameshift and substitution of tyrosine to serine. A previous study reported a T-toG substitution at the neighboring position, nucleotide 1426, causing a tyrosine-to-asparagine substitution [10]. Another report identified a substitution of tyrosine to a stop codon at this same codon 476 (Y 476 X(1)) because of a deletion of a cytosine at the position of 1428 [11]. There are two lobes in the TK domain: the N-terminal and C-terminal lobes. The N-terminal lobe is composed of a five-stranded antiparallel β-sheet, which is primarily responsible for ATP binding. The C-terminal lobe consists mostly of seven α-helices and functions in substrate binding. A linker region (residues 475 - 479) joins the two lobes and participates in ATP binding [12]. As a regulatory tyrosine residue, Tyr-551 in the TK domain plays a role in kinase activation with another regulatory tyrosine residue, Tyr-223. Initially, Tyr-551 is transphosphorylated by lyn and spleen tyrosine kinase (SYK), and then phosphorylation of Tyr-551 activates the autophosphorylation of Tyr-223 (within the SH3 do-

CASE REPORT The study was approved by the Ethics Committee of Shandong Medicinal Biotechnology Center and informed consent was signed by the patient’s parents. The patient was a 5-year-old boy who had been hospitalized for a persistent cough that had lasted over a month. The patient had a history of recurrent colds and high fever and pneumonia three to four times every year. He suffered his first episode of pneumonia when he was six months old and underwent an appendectomy at age 2. Pneumonia and left lower lobe atelectasis were identified by chest computed tomography examination. By bronchoscopic examination, mucosal congestion was commonly seen in the tracheal carina and bilateral lungs. Increased opacity within the posterior, anterior, and lateral basal segments of the left lower lobe was obvious. The patient was initially treated for pneumonia and endobronchial inflammation of the left lower lobe because of his obvious lung pathological changes. Alveolar lavage was introduced into the left lobe. Combining his previous disease history with abnormal serum immunoglobulin and lymphocyte levels, the patient was diagnosed as having XLA. His mother was a carrier (Figure 1B); his sister was not available for examination. Intravenous gammaglobulin was administered four times after diagnosis. Clinical detections of pathogens Pathogens were identified in sputum cultures and in serum samples by ELISA. The presence of tuberculosis DNA was tested for by PCR. Serum immunoglobulin levels and lymphocyte counts were detected by flow cytometry. BTK mutation analysis Genomic DNA was extracted from whole blood using an E.Z.N.A.® Blood DNA Kit (Omega Bio-Tek, Norcross, GA, USA). Eighteen overlapping pairs of primers were designed to cover each exon and flanking exonintron boundary of the BTK gene. The products were amplified by PCR, purified, and sequenced (Beijing Genomics Institute, Qingdao, China). Mutations and single nucleotide polymorphisms were analyzed using Mutation Surveyor software 4.0 (SoftGenetics LLC, PA, USA) and the BTK mutation database (http:// bioinf.uta.fi/BTKbase).

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DELETION MUTATION OF BTK GENE IN A CHINESE XLA PATIENT

Figure 1. Chromatograms showing a mutation in the BTK gene. (A) Deletion of “A” at position 1427 in the patient, leading to a premature stop codon. (B) Heterozygous c.1427delA deletion in patient’s mother. (C) Wild-type c.1427A sequence in the patient’s healthy father.

Figure 2. Wild-type and mutant-type crystal structure of the BTK kinase domain. The N-terminal lobe is marked in green, the C-terminal lobe is marked in blue, and the linker region is in red. The amino acid at position 476 is marked in yellow. (A) Homozygous deletion of the C-terminal lobe, as in the patient. (B) Heterozygous deletion of the C-terminal lobe. (C) Wild type.

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main) which may create a docking site for an SH2 domain-containing protein [13]. We modeled the three-dimensional structure of the normal and truncated BTK protein (Figure 2). Homozygous and heterozygous deletion of the whole C-terminal lobe was seen in Figure 2A and 2B, respectively. Truncated proteins formed by premature termination may result in accelerated degradation [14]. Truncated proteins can be functional or nonfunctional, according to the proportion truncated as well as conformational changes. We demonstrated that the truncated BTK protein missing 176 residues at the C-terminus would be nonfunctional. We conclude that this mutation underlies the XLA in our patient. Acknowledgement: This study was supported by the grant for National Key Technology R & D Program of China (No. 2013BAI07 B01 and 2013BAI07B02). We thank the patient and his family members who participated in our study.

5.

Vihinen M, Iwata T, Kinnon C, et al. BTKbase, mutation database for X-linked agammaglobulinemia (XLA). Nucleic Acids Research Jan 1 1996;24(1):160-5.

6.

Lee PP, Chen TX, Jiang LP, et al. Clinical characteristics and genotype-phenotype correlation in 62 patients with X-linked agammaglobulinemia. Journal of Clinical Immunology Jan 2010; 30(1):121-31.

7.

Endo LM, Giannobile JV, Dobbs AK, et al. Membranous glomerulopathy in an adult patient with X-linked agammaglobulinemia receiving intravenous gammaglobulin. J Investig Allergol Clin Immunol 2011;21(5):405-9.

8.

Kawakami C, Inoue A, Takitani K, Kanegane H, Miyawaki T, Tamai H. X-linked agammaglobulinemia complicated with endobronchial tuberculosis. Acta Paediatr Mar 2011;100(3):466-8.

9.

Qin X, Jiang LP, Tang XM, Wang M, Liu EM, Zhao XD. Clinical features and mutation analysis of X-linked agammaglobulinemia in 20 Chinese patients. World J Pediatr 2013;Jan 18.

10. Hagemann TL, Rosen FS, Kwan SP. Characterization of germline mutations of the gene encoding Bruton's tyrosine kinase in families with X-linked agammaglobulinemia. Hum Mutat 1995;5(4): 296-302. 11. Vorechovsky I, Luo L, Hertz JM, et al. Mutation pattern in the Bruton's tyrosine kinase gene in 26 unrelated patients with Xlinked agammaglobulinemia. Hum Mutat 1997;9(5):418-25.

Declaration of Interest: No conflict of interest.

12. Hanks SK, Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J May 1995;9(8):576-96.

References: 1.

Bruton OC. Agammaglobulinemia. Pediatrics Jun 1952;9(6):7228.

13. Afar DE, Park H, Howell BW, Rawlings DJ, Cooper J, Witte ON. Regulation of Btk by Src family tyrosine kinases. Mol Cell Biol Jul 1996;16(7):3465-71.

2.

Vetrie D, Vorechovsky I, Sideras P, et al. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature Jan 21 1993;361(6409):226-33.

14. Maquat LE. Defects in RNA splicing and the consequence of shortened translational reading frames. Am J Hum Genet Aug 1996;59(2):279-86.

3.

Maas A, Hendriks RW. Role of Bruton's tyrosine kinase in B cell development. Developmental Immunology 2001;8(3-4):171-81.

4.

Lopez Granados E, Porpiglia AS, Hogan MB, et al. Clinical and molecular analysis of patients with defects in micro heavy chain gene. J Clin Invest Oct 2002;110(7):1029-35.

Correspondence: Professor Jinxiang Han Shandong Medicinal Biotechnology Centre Shandong Academy of Medical Sciences 18877 Jingshi Road 250062 Jinan, China Tel.: + 86-531-82919888 Fax: + 86-531-82951586 Email: [email protected]

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