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From www.bloodjournal.org by guest on June 17, 2017. For personal use only. Two-Exon Skipping Due to a Point Mutation in p67-phox-Deficient Chronic Granulomatous Disease By M. Aoshima, H. Nunoi, M. Shimazu, S. Shimizu, 0. Tatsuzawa, R.T. Kenney, and S.Kanegasaki The cytosolic 67-kD protein in phagocytes (p67-phoxl andB lymphocytes is one of essential components ofthe superoxide-generating systemin these cells, and its defect causes an autosomal recessive type ofchronic granulomatous disease (CGD). We performed mutationanalysis of p67-phox mRNA from a CGD patient who lacks the protein and foundan inframe deletion from nucleotide 694 t o 879, which corresponds t o t h e entire sequence of exons 8 and 9. This sequence encodes one of two Src homology 3 domains and a part of proline-rich domain in p67-phox and lack of these domains seem t o have influenced stability of this protein. To know causative reason for thedeletion, we analyzed genomic DNA for p67-phox using two sets of primersthat covered exons 8 and 9 with adjacent introns. The DNA fragments from the patient were shown t o be same in length as those fromcontrol. However, the single-strand conforma- tion-polymorphism analysis of the fragments showed that a patient’s specimen that included the splice junction of exon 9 exhibited different m o b i l i from thecontrol. By sequencing of the fragment, a homozygous G t o A replace+l of intron9 was foundt o be a sole mutament at position tion, which reduced thematching score of the splicing sequence to theconsensus calculated according t o t h eformula proposed by Shapiro and Senapathy (Nucleic Acids Res 15:7155,1987). The reduced matching score at the splice doner site (5’ splice site] of intron 9 and the original low matching score at theacceptor site (3’splice site) of intron 7 may explain the skipping of exon 8 and 9, and another predicted mechanism is discussed on the basis of Shapiro and Senapathy’s hypothesis. 0 1996 by The American Society of Hematology. (Gly-78 to Glu) was responsible for the protein deficiency HRONIC GRANULOMATOUS disease (CGD) is a in one CGD patient. Skipping of exon 3 due to T to C rare inherited disorder whereby phagocytes cannot replacement in the splice donor site in intron 3 was also generate superoxide anion and its derivatives to other active found recently.’8In this study, we report a new type of oxygen species that may be used for killing infectious agents. p67-phon-deficient CGD, whose mRNA lacks the entire Patients with this disease have recurrent, life-threatening insequence of two exons, namely exons 8 and 9. A single fections with catalase-positive bacteria and fungi.’ The sunucleotide replacement found at position + l of intron 9 peroxide generating system in phagocytes and B lymphogreatly reduces the matching score of the splicing sequence cytes’ consists of a membrane-bound catalytic component, to the consensus29and seems to be responsible for the delecytochrome bSs8,and cytosolic components that seem to be required for activation of the system. A functionally active tion. Two exon skipping due to a point mutation at a splice enzyme complex is formed on stimulation of macrophages site has rarelybeen reported, and this is the first case in or neutrophils by assembly of the necessary components on CGD. the plasma membrane. This complex carries an electron from MATERIALS AND METHODS reduced form of nicotinamide-adenine dinucleotide phosCase. The patient is a 24-year-old man with a history of recurphate to molecular oxygen to form superoxide anion, which rent skin abscess, pneumonia, and cervical lymphadenitis, whose isthen released to the outside of the cells or inside the parents were first cousins. His first episode of cervical lymphadenitis phagosomes.3 and gastroenteritis was at the age of 9. The patient’s neutrophils The catalytic component, cytochrome bSs8,is composed showed normal chemotaxis and phagocytosis, but failed to generate of two subunits, gp91- and p22-phox (phagocyte oxidase). 02-(2.9%of control) on stimulation with FMLP. Cytosolic components include p47- andp67-phoxand a Epstein-Barr virus transformation. B lymphoblast cell lines small guanosine-triphosphate binding protein Ra~-p21.‘“~ were established by immortalization with Epstein-Barr virus (EBV) These five proteins were shown to be sufficient to support as previously de~cribed.’~ Briefly, the collected mononuclear cells superoxide-generation in a cell free ~ y s t e m .Any ~ . ~ defect in from the patient were exposed to EBV obtained from B95-8 culture the four “phox” proteins causes CGD, which occurs either medium for 2 hours, then cultured for 4 weeks. as an X-linked or an autosomal di~order.~ The gene responsible for the former type of CGD codes for gp91-phox and From The Institute of Medical Science, the University of Tokyo, genes responsible for the latter for p22-, p47-, and p67-phox. MitsubishiYuka Bio-Clinical Laboratories Inc, Tokyo, National The mRNAs that code for allphox proteins have been cloned Center of Pediatric Treatment, Setagaya-ku, Tokyo, Japan: and the and sequenced and the genomic organizations r e p ~ r t e d . ~ ” ~National Institute of Allergy and Infectious Disease, National InstiIt is known that p67-phox deficiency is rare in the United tutes of Health, Bethesda, MD. States,” Europe,*’ and Japan (manuscript in preparation). Submitted December 27, 1995; accepted April 25, 1996. Supported in part by the grants in aid from the Ministry of EducaMutations that cause this type of CGD are heterogeneous,’ tion, Science and Culture of Japan. in contrast to the cases in p47-phox deficiency, the majority Address reprint requests to H . Nunoi, MD, The Instituteof Medical of whichseem to be accounted for by a common dinucleotide Science, the University of Tokyo, 4-6-1 Shiroganedai, Minatoku, d e l e t i ~ n , ’although ~ ~ ~ ~ some patients were heterozygous for Tokyo 108, Japan. this deletion and had a second missense rnutaion in the other The publication costs of this article were defrayed in part by page allelle.’33’5We found a homozygous AG dinucleotide insercharge payment. This article must therefore be hereby marked tion in one patient with p67-phox deficiency, which would “advedsement” in accordance with 18 U.S.C. section 1734 solely to bring about a frame shift into mRNA resulting in an early indicate this fact. stop codon.26de Boer et alZ7reported that a mutation re0 1996 by The American Society of Hematology. sulting in a nonconservative amino acid change in p67-phox 0006-4971/96/8805-0016$3.00/0 C Blood, Vol 88,No 5 (September l), 1996:pp 1841-1845 1841 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. AOSHIMAET AL 1842 Table 1. OligonucleotidePrimers Used for Reverse Transcriptase-PCR ~~ ~ No. 1 2 3 4 5 6 7 8 9 10 Sequences Position Direction Sense Antisense Sense Antisense GGGTGTGCCTGAGACAAAAGAA Sense Antisense ATCCTTCATGCTGTCTTCTGAAAG Sense GCTGGAACACACTAAGCTGAGCTA Antisense TTTCTTCAGCTTTGTAGTGTGA Sense CTTAAAGAAGCCTTGATTCAGCTT CGCAACTCAACTGGTTCAAGGTAG Antisense AGGCCTCCTAGTITCTACCTAATC CCTCCTTCTTGGCATACATGAAAG TTCGAGGGAACCAGCTGATAGACT TTGAACATGACCGTGGCCCAGTTA 1-24 407-430 326-349 856-879 772-795 1237-1260 1182-1205 1612-1635 304-327 900-923 Set of primers 1 and 2,3 and 4,5 and 6,7 and 8, and 9 and 10 were used for amplification targeting nucleotide 1-430, 326-879, 772-1260, 1182-1635 and 304-923, respectively. I t n r n ~ o ~ M oar~al~sis. t Cytosolicfractionsfrom the B-cell lines were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Proteinswere blotted to polyvinylidine difluoride (PVDF) membrane and detected with specific antibodies against p67-p11o.r and p47-pho.r, as previously described.’ lsolntion of RNA nrld r w t l w s i s of cDNA. Total RNA was isolated from B-cell lines with an acid guanidinium thiocyanate-phenolchloroform method asdescribedpreviously.” The first strandof cDNA was synthesized from20 ng total RNA using Moloney murine leukemia virus reverse transcriptase primed with 30 pmoles of random hexamer mixture. Subsequently, cDNA for p67-pho.~ was amplified by polymerase chain reaction (PCR)with the oligonucleotide primers shown in Table I. The PCR was performed for 30 cycles using the following conditions: denaturation at 94°C for 1 minute, annealing at 52°C for 1 minute. and extension at72°C for 30 seconds. PCR productsofp67-phoxcDNAwere run on l 9 lowmelting agarose gel, excised from the gel, andpurified using a glass powderbased procedure (Gene Clean 11 Kit: Bio 101, Inc, La Jolla, CA). Subsequently the PCR products of p67-phr~x cDNA were sequenced directly. Grnonlic DNA. GenomicDNAwasisolatedfromtheEBV Bcell lines. PCR was performed with the specific primers designed forexon8andadjacent intron sequence(5’-TCCGATCAGTCAGAAATGCGG-3’assenseprimer,andS’-GTGTCAGCCCCACCACAGTC-3’ as antisense primer). and for exon 9 and adjacent intron sequence (S’-TTGCAGTTAGATGTGGAGTT-3’ as sense primer, and S’-AACTCAACTGGTTCAAGGTAGTTG-3‘ antisense primer). The PCR products were purified with 2 9 low melting agarose gel and subcloned directly into a TA cloning vector (pCR 11: Invitrogen. San Diego, CA). The recombinant plasmids were prepared from transformant Esrhrrichia coli strains with an alkalineSDS method. Singlr-srrrmd con!fonrlcrrion polymorphirrn. About 100 ng each of PCR products in 12 pL of Tris-borate buffer (TBE) containing l 9 Ficoll 400. 0.259 BromophenolBlue.0.25%Xylene cyano1 were denatured at 80°C for 5 minutes, cooled on ice and run on a cooled 10% polyacrylamide gel at 4°C as described by Hongyo et al.”’ Subsequently,the gel wasstainedwiththesilver(2D-Silver Stain 11; Daiichi Pure Chemicals, Inc. Tokyo, Japan). DNA sequencing. Cyclesequencingreactionswereperformed using Taq Dye Deoxy Terminator Cycle Sequencing Kit (PerkinElmer,Chiba,Japan).Sequencingwasperformedusinganautomated sequencer (AB1 model 373A: Perkin-Elmer). A total of 1 p g of the cloned plasmid or 80 ng of the PCR products were used as template. RESULTS The patient was diagnosedwith CCD by the lack of superoxide dismutase sensitive cytochrome c reducing activity of his neutrophils on stimulation. In contrast to the cytosol of the EBV-transformed B-cell lines from normal volunteers, which supported the superoxide generating activity of neutrophil membrane in vitro, that from the patient did not support activity. Normal levels of the large and small subunits of cytochrome bSsRwere found in the patient’s neutrophils by Western blots. Cytosol of the patient’s neutrophils and EBV-transformed B-cell line contained a significant amount of p47-phox, but no detectable p67-phox protein (Fig I ) . Total RNA was isolated from thepatient’sEBV-transformed B-cell linesand used to obtainfirst-strand cDNA by reverse transcription, then amplified with a set of four overlapping sense and antisense primers designed to cover the entire coding sequenceof p67-phox (Table 1). As shown in Fig 2, PCR products using two sets of primers, 112 and 7/8, were detected, but not the product of primers 314 (lane F ) and 5/6 (laneG). The result suggests there wasa deletion in theregionbetweennucleotides 326and 1260. Subsequently, another set of primers (9/10) that span nucleotides 304 to 923were synthesized and the cDNA amplified to find the deletion in the p67-phox cDNA. The fragment obtained fromthe patient’s RNAwassingleand shorter than that from control RNAby about 200 bp (lanesI and J). By direct sequence of the fragment, the deleted part was determined l 2 PMN Fig 1. lmmunoblot analysis of transformed B-cell lines, cytosolic fraction, with antibodies against p67-phox and p47-phOX. Lane l,Xlinked CGD;lane 2, this case; PMN, normal neutrophil. From www.bloodjournal.org by guest on June 17, 2017. For personal use only. TWO EXONSKIPPING IN p67-ph0x-DEFlClENT CGD M A B C 1843 D E F G H \I I .I Fig 2. Reverse transcriptase-PCR for p67-phox cDNA. PCR products amplified with the five sets of primers (1/2, 3/4, 5/6, 7/8, and 9/10] shown in Table 1. Lanes A and E,PCR product obtained with primer 1/2; lanes B and F,PCR product obtained with primer 3/4; lanes C and G, PCR product obtained with primer 5/6; lanes D and H,PCR product obtained with primer 7/8; lanes I and J, PCR product obtained with primer 9/10, Lanes A,B,C,D, and I, PCR products fromRNA prepared from controlEBV B cells; lanes E, F, G, H, and J, PCR products from RNA prepared from patient's EBV B cells. Lane M, DNA size marker &X174/Hae 111 digest. PCR products were run on 1.5% gel (the left panel) and 3% gel (the rightpanel). to be nucleotides 694 to 879, which correspond to the entire sequence of exons 8 and 9 (Fig 3). To investigate the cause of the deletion, genomic DNA fragments of exons 8 and 9 and adjacent introns were amplified from the patient's cell line and compared with those of a normal control. The PCR products fromthepatientand control were found to be the same length. However, the PCR product from the patient's specimen that includes the splice junctions of exon 9 exhibited different mobility when singlestrand conformational polymorphism analysis was performed (data not shown). The results suggested the mutation was located in the splice junction of exon 9. The fragment including exon 8 or 9 was subcloned into TA cloning vector. Patient a Each six clones were sequenced in full-length. As shown in Fig 4, there is a G to A replacement at position + 1 of intron 9, the first base of the splice consensus sequence in the splice doner site (5' splice site), which was found to be the sole mutation in the fragment. Matching of splice donor and acceptor sequences to the consensus can be quantified using a scoring system described by Shapiro and Senapathy.'" According to the formula they proposed, we calculated the scores for the splice donor and acceptor sites of the p67phox gene to see if the point mutation found in the splice donor site of intron 9 affected the matching score. As shown in Table 2, the score of the donor site (5' splice site) of intron 9 in normal p67-plrm is 82.7, whereas the G to A mutationat + l in the CGD gene was64.6. The acceptor a Control cwnY I mmnY A C G G G C A G G T A T G C A G b b Control 1 exon 7 exon 8 exon C) I cxnn In ..CTCTGCAACCACAG,GCAGCTGAGCCTC.....TGlTCAACGGGCAG,AAGGGCCTTGlTC.. Patient v eson 7 Patient A C G G exnny G C A I lnmnY G[A]T A T G C A G e3' I exon In ...GCCCCTCTGCAACCACAGAAGGGGClTGlTCCCT... Fig 3. Sequence analysis of the p67-phoxcDNA. (a) This patient; (b) alignment of the sequence of cDNA from control (upper) and this case (lower). Fig 4. Sequence analysis of genomic DNA. (a) Control sequence around theexon 9, intron 9 boundary; (b) sequence around theexon 9, intron 9 boundary from patient. From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 1844 AOSHIMA ET AL Table 2. Scores for Splice Site Sequence for the p67-phox Gene Donor Site Intron 1 Intron 2 Intron 3 Intron 4 Intron 5 Intron 6 Intron 7 Intron 8 Intron 9 Intron 10 Intron 11 Intron 12 Intron 13 Intron 14 Intron 15 81.4 96.7 87.6 95.4 86.3 86.3 95.4 100.0 82.7 (64.6) 81 .o 96.7 80.1 90.9 88.9 92.2 Acceptor Site 88.1 95.0 78.2 85.9 87.4 81.4 71.2 96.9 81.6 93.0 98.6 91.6 87.7 97.0 84.4 The scores for splice acceptor site were calculated from nucleotide sequences at the position from -14 to +l, and donor sites were from the sequence at the position from -2 to +6. The formula was proposed by Shapiro and Senapathy.”The score for aG to A mutation at +l in intron 9 is given in parentheses. site (3’ splice site)of intron 7 has a score of 71.2, the lowest score among all donor and acceptor sites in p67-phox. DISCUSSION Sequenceanalysis of cDNA from a CGD patient with p67-phox deficiency has shown that the patient’s mRNA for this protein lacks a region between nucleotides 694 and 879. The deleted nucleotides correspond to the entire sequence of exons 8 and 9 in the gene and to aminoacid residues 231 to 293. Nevertheless, even though thedeletion was in-frame, no protein was found in thepatient’s neutrophils or in EBVtransformed B-cell lines from the patient. The residues includeone of twoSrchomologysequence 3 consensus regions (SH3; residues 245 to 295) and a part of the prolinerich sequence(residues227to 234)presentin wild-type p67-phox. It is knownthat the SH3domain binds to the proline-rich sequence and the mutant protein may have lost its stability due to a loss of these sequences. The sequences, therefore, contributetoprotein stabilitythrough intra-or intermolecular association of p67-phox with itself or with other proteins such as p47-phox or p40-phox that contain similar sequences.”-” When sequence in the vicinity of exons 8 and 9 in p67phox genomic DNA was analyzed, a homozygous substitution of G to A at position + l of intron 9 was found as the sole mutation. The results suggest that the mutated splice donor site of intron 9 influenced site recognition and caused skipping of the upstream exon(s). Point mutations affecting splice donor sites often cause the loss of an entire upstream exon.34 In the case of CGD, mutations located in the genes for gp91-phox, p22-phox. and p67-phox that include nucleotide changes at positions + 1 or + 2 of the splice donor sites of introns have been reported to cause the skipping of the immediate upstream Decreased interaction of a ribonucleoproteinparticle involvedinsplicingwiththe splice donor site of an intron may influence its binding to the splice acceptor site of the preceding intron, resulting in a skip of the intervening exon.”’ In the present case, both exons 8 and 9 were skipped due to a point mutation at the splice donor siteof intron 9. Such a mutation has been reported in only a limited number of cases. A point mutation caused by ultraviolet irradiation at theacceptorsite ofintron 1 in the hypoxanthine-guanine phophoribosyltransferase gene resulted in a loss of exons 2 and 3, or exon 2 alone.34 In maple syrup urine disease, the E1P subunit of the branched-chain a-keto acid dehydrogenase (BCKDH) wasreported to have a point mutation at the donor site of intron 5 that is responsible for the loss of exons 5 and 6.” Most of the mRNA for BCKDH in this disease lacked exon 5, but some normallyspliced and two exonskipped mRNA were also found. Althoughthe mechanism of twoexonskipping isnot known, the low matching scores of the splicing sequences to the consensus atthe acceptorsite of intron7 and the mutated splice donorsite of intron 9in thepresent case seem to affect site recognition. Shapiro and Senapathy” proposed that such matching scores area useful wayto predictrecognition of the splice site. In the BCKDH case, matching scores to the consensus at the mutated donor and the acceptor sites of intron 5 were 70.4 and 78.1, respectively. In p67-phox deficiency, the low scores of the acceptor site of intron 7 (71.2) and the mutated donor site of intron 9 (64.6) are as low as that of themutatedsitein the BCKDH case. The splicing machinery may not be able to recognize sequences with these low scores quite as well, resulting in the deletion of one or more exons. If actualsplice sites have alow score,othersequence signals have been proposed that direct splicing in addition to the splice junction sequence signal^.^' These regulatory elements, termedsplicing enhancers, arelocated downstream and stimulate splicing efficiency for binding of the splicing machinery. Since theacceptor site ofintron7has a very lowscore in thepresent case, it is possiblethatsuch an enhancer element is present downstream, but is affected by the mutation at the splice donor site of intron 9 or by exon 9 skipping. In addition to the splice site sequences andpossible enhancer sequence, the size of an exon seems to influence exon skipping.” Exon 8 of the p67-phox gene has only 44 bases, andthis small size may also contribute to the skipping of this exon. Further studiesmay help clarify the unusual two exon skipping mechanism that led to CGD in this patient. REFERENCES 1. Smith M, Cumutte T: Molecular basis of chronic granulomatous disease. Blood 77:673, 1991 2. Kobayashi S, Imajoh-Ohmi S, Kuribayashi F, Nunoi H, Nakamura M, Kanegasaki S: Characterization of the superoxide-generating system in human peripheral lymphocytes and lymphoid lines. J Biochem 117:758, 1995 3. 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For personal use only. 1996 88: 1841-1845 Two-exon skipping due to a point mutation in p67-phox--deficient chronic granulomatous disease M Aoshima, H Nunoi, M Shimazu, S Shimizu, O Tatsuzawa, RT Kenney and S Kanegasaki Updated information and services can be found at: http://www.bloodjournal.org/content/88/5/1841.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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