RESEARCH ARTICLEOFFICIAL JOURNAL

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Primary Immunodeficiency Caused by an ExonizedRetroposed Gene Copy Inserted in the CYBB Gene

Martin de Boer,1 Karin van Leeuwen,1 Judy Geissler,1,2 Corry M. Weemaes,3 Timo K. van den Berg,1 Taco W. Kuijpers,1,2

Adilia Warris,3 and Dirk Roos1∗

1Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam 1066 CX, The Netherlands;2Emma Childrens Hospital, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; 3Department of Pediatrics and theNijmegen Institute for Infection, Immunity and Inflammation, Radboud University Medical Centre, Nijmegen, The Netherlands

Communicated by David N. CooperReceived 1 August 2013; accepted revised manuscript 10 January 2014.Published online 29 January 2014 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22519

ABSTRACT: Retrotransposon-mediated insertion of a longinterspersed nuclear element (LINE)-1 or an Alu elementinto a human gene is a well-known pathogenic mechanism.We report a novel LINE-1-mediated insertion of a tran-script from the TMF1 gene on chromosome 3 into theCYBB gene on the X-chromosome. In a Dutch male pa-tient with chronic granulomatous disease, a 5.8-kb, incom-plete and partly exonized TMF1 transcript was identifiedin intron 1 of CYBB, in opposite orientation to the hostgene. The sequence of the insertion showed the hallmarksof a retrotransposition event, with an antisense poly(A)tail, target site duplication, and a consensus LINE-1 en-donuclease cleavage site. This insertion induced aberrantCYBB mRNA splicing, with inclusion of an extra 117-bpexon between exons 1 and 2 of CYBB. This extra exoncontained a premature stop codon. The retrotranspositiontook place in an early stage of fetal development in themother of the patient, because she showed a somatic mo-saicism for the mutation that was not present in the DNAof her parents. However, the mutated allele was not ex-pressed in the patient’s mother because the insertion wasfound only in the methylated fraction of her DNA.Hum Mutat 35:486–496, 2014. C© 2014 Wiley Periodicals, Inc.

KEY WORDS: gene copy retroposition; chronic granuloma-tous disease; X-chromosome inactivation; CYBB; TMF1

IntroductionTransposable DNA elements are discrete DNA sequences that can

be transported to or duplicated at other regions in the genome. Thedriving force of this process in the human genome is the activity oflong interspersed nuclear elements (LINEs), which act by reverse-transcribing RNA and inserting the cDNA into the genome at a newlocation. There are about 100 LINE-1 copies (six hot, L1-Ta family)that are functional in retrotransposition, via target-primed reversetranscription (TPRT). TPRT occurs in 1:20 to 1:200 births [Ewing

Additional Supporting Information may be found in the online version of this article.

Contract grant sponsors: CGD Research Trust, London, UK; EURO- CGD grant from

the E-RARE program of the European Union.∗Correspondence to: Dirk Roos, Sanquin Research, Plesmanlaan 125, Amsterdam

1066 CX, The Netherlands. E-mail: d.roos@sanquin.nl

and Kazazian, 2010]. Each complete LINE-1 element measures 6 kbin length and contains two open reading frames: ORF1 encodesan RNA-binding protein and ORF2 a protein with endonucleaseand reverse-transcriptase activities. This process of retrotransposi-tion takes place with LINE-1 mRNA in a cis preference [Esnaultet al., 2000; Wei et al., 2001], but can also occur with RNA fromother sources, such as Alu repeats [Mitchell et al., 1991; Makałowskiet al., 1994; Boeke, 1997; Dewannieux et al., 2003], SVA (short in-terspersed nuclear elements [SINE], Variable Number of TandemRepeats [VNTRs], and Alu) elements [Shen et al., 1994; Kajikawaand Okada, 2002; Ostertag et al., 2003; Vorechovsky, 2010], or U6small nuclear RNAs [Buzdin et al., 2002]. For this reason, LINE ele-ments are called autonomous and the others, which need the help ofLINEs, nonautonomous. Together, these mobile elements accountfor �30% of our genome.

Retrotransposition starts with cleavage of the consensus retro-transposition entry site TTAAAA sequence between the A and Tin the minus strand by the endonuclease activity of the LINE-1ORF2 protein, followed by hybridization of the poly(A) tail to theTTTT sequence on the minus strand. Elongation by the reverse-transcriptase activity of the ORF2 protein then generates a cDNAcopy of the mRNA to be inserted. A second cut is made by the ORF2endonuclease in the complementary strand 7–20 bases downstreamof the first cut. Due to this unequal cleavage, a duplication on bothsides of the insertion is formed.

LINE-1-mediated retrotransposition can lead to insertion of ge-netic material within existing genes, both of LINE-1 itself [Kazazianet al., 1988; Hancks and Kazazian, 2012] as well as of nonau-tonomous elements [Wallace et al., 1991; Deiniger and Batzer, 1999;Ostertag et al., 2003; Chen et al., 2005] and as such cause human ge-netic diseases. Such LINE-1-driven insertions can disrupt the codingsequence of genes, lead to aberrant mRNA splicing or cause changesin genetic information by unequal homologous recombination be-tween retroposed elements [Deiniger and Batzer, 1999; Chen et al.,2006].

In principle, cellular mRNA can also be retrotransposed, thusgiving rise to pseudogenes characterized by a lack of introns.Indeed, approximately 8,000 processed pseudogenes have beenidentified in the human genome [Zhang et al., 2003]. However,this process is thought to occur only rarely, because retrotrans-posed gene transcript insertion has not been reported as a re-cent source of human pathology. We now report about a Dutchpatient with chronic granulomatous disease (CGD) caused by aLINE-1-driven 5.8-kb insertion of a partly processed, incompletemRNA from the TMF1 gene (MIM #601126) on chromosome 3into the CYBB gene (MIM #300481) on the X-chromosome. This

C© 2014 WILEY PERIODICALS, INC.

insertion created a new exon containing a stop codon in CYBBmRNA.

CYBB encodes a glycoprotein with a molecular mass of 91 kD thathas a central function in the NADPH oxidase enzyme in phagocyticleukocytes. This protein is known as gp91phox or NOX2 and con-tains an NADPH-binding site and an FAD molecule in its cytosolicpart and two heme groups in its transmembrane region. It formsa dimer with p22phox, another transmembrane protein in phago-cytes; this dimer formation is essential for stable expression of bothproteins. NADPH oxidase generates superoxide after activation ofphagocytes, for example, by pathogen uptake. This activation in-duces translocation of three cytosolic NADPH oxidase components(p40phox, p47phox, and p67phox) to the gp91phox/p22phox dimer in themembrane that surrounds the cellular compartment with the in-gested pathogen (the phagosome). As a result, the NADPH-bindingsite in gp91phox becomes accessible for NADPH, and electrons fromNADPH are transported within gp91phox from the cytosolic side tofree oxygen on the phagosomal side. Superoxide is then releasedwithin the phagosome, a process that is essential for killing themicroorganisms [Roos et al., 2007].

Defective NADPH-oxidase structure or activation leads to animmunodeficiency disorder termed CGD, and the most frequentcauses of this disease are hemizygous disabling mutations in CYBB,leading to X-linked CGD (MIM #306400) [Roos et al., 2007; Vanden Berg JM et al., 2009; Roos et al., 2010]. Insertions in CYBB areknown but form a minority of all X-CGD mutations [Roos et al.,2010]. Among them, two cases with LINE-1 insertions leading toaberrant CYBB mRNA splicing have been detected [Meischl et al.,2000; Brouha et al., 2002]. The mutation we now found is LINE-1 driven, as evidenced by an antisense poly(A) tail, a target-siteduplication and a consensus LINE-1 endonuclease cleavage site, butdoes not involve retrotransposition of LINE-1 itself or of an Alu,SVA, or U6 element. Instead, an incomplete, partly exonized mRNAcopy of the TMF1 gene from chromosome 3 was inserted in intron1 of CYBB, leading to aberrant splicing and premature terminationof protein synthesis. The TMF1 retrocopy was found only in theCGD patient and his mother, but not in his maternal grandparents.This proves that the process was a recent event and shows thatretroposition of cellular RNA is still shaping the human genome.

Methods

Ethics

The study was approved by the Sanquin Blood Supply Institute,Amsterdam, The Netherlands. Informed consent was obtained fromthe patient, his sister, parents, and grandparents.

Neutrophils

Neutrophils were purified from heparinized blood as describedpreviously [Roos and De Boer, 1986].

Nitroblue Tetrazolium Slide Test

Nitroblue tetrazolium (NBT) slide test was performed as previ-ously described [Meerhof and Roos, 1986].

Amplex Red Assay

Purified neutrophils were incubated at a concentration of106 cells/ml in Hepes buffer containing 5.5 mM glucose 1.0 mMCaCl2, 0.6 mM MgCl2 and 0.5% (w/v) human albumin. The neu-trophils were stimulated with phorbol myristate acetate (PMA)(100 ng/ml), and hydrogen peroxide release was measured withthe Amplex Red assay (Molecular Probes, Thermo Scientific, SunnyVale, CA) on a Tecan plate reader (Mannedorf, Switzerland) [Zhouet al., 1997].

7D5 FACS Staining

Neutrophils were incubated with the Moab 7D5. This 7D5 Moabis directed against gp91-phox in the membranes of phagocytes.FITC-labeled goat-anti-mouse-Ig (Invitrogen, Thermo Scientific)was used as secondary antibody, as described before [Weening et al.,2000].

Western Blot

108 Neutrophils were pretreated with 10 mM diisopropyl fluo-rophosphate for 10 min. The cells were then centrifuged for 20 secat full speed in an Eppendorf centrifuge (13,000g), and resuspendedin lysis buffer (PBS containing 1x complete EDTA-free Protease In-hibitor Cocktail [Roche Applied Science, Penzberg, Upper Bavaria,Germany]) and SDS sample buffer 1:1, and incubated for 30 min at95°C. Samples were run on a 12.5% bis–Tris gel and subsequentlyblotted on a nitrocellulose membrane. The blot was incubated withMoabs 48 and 449 for staining of the gp91phox and p22phox pro-teins, respectively [Verhoeven et al., 1989], or with MoAb clone10 anti-p47phox from Santa Cruz Biotechnology (Dallas, TX; cat.no. SC-17845) and pAb rabbit-anti-human p67phox from UpstateBiotechnology (Lake Placid, NY; cat. no. 07-002). Visualizationwith secondary antibodies was performed on the Odyssey (LI-COR,Lincoln, NE).

Preparation of RNA and DNA

Total RNA of the patient and a healthy control was purified frommononuclear leukocytes by the QIAamp RNA Blood Mini Kit (Qi-agen, Sciences, Frederick, MD). Genomic DNA was isolated fromcirculating blood leukocytes with the Puregene kit (Qiagen) accord-ing to the manufacturer’s instructions.

Southern Blotting

Southern blotting, with 8 μg of genomic DNA, was performed aspreviously described [Bolscher et al., 1991], with a full-length CYBBcDNA probe PSVCGD obtained from Dr. Mary Dinauer [Royer-Pokora et al., 1986].

PCR

The 13 exons of the CYBB gene with their adjacent intronic se-quences (exon 1 + promoter) were amplified with the appropriateprimer combinations as described before [Weening et al., 2000]. Inshort, genomic DNA (50–500 ng) was amplified by means of theRapid Cycler (Idaho Technology, Salt Lake City, UT) with 50 cyclesof 95°C for 5 sec, 60°C for 30 sec, and 72°C for 15 sec and slope S9.The reaction volume of 15 μl contained 2 U of Taq DNA polymerase

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(Promega, Fitchburg, WI) and 2 U of TaqStart antibody (ClontechLaboratories, Mountain View, CA).

A mutation-specific quantitative PCR was designed with a for-ward primer in the TMF insert within the first intron of CYBB anda reverse primer 3′ of this insert in the first intron of CYBB. Theresults were corrected for the differences in DNA input, either bymeasuring the DNA concentration or by a reference-quantitativePCR performed around exon 8 of CYBB with the same DNA sam-ples. For primer sequences, see Supp. Table S1. PCR amplificationwas performed on a LightCycler instrument (Roche, Almere, TheNetherlands), and analyzed with software version 3.5. The reactionwas performed with Lightcycler Faststart DNA Master PLUS SYBRGreen I (Roche Diagnostics, Indianapolis, IN). The annealing tem-perature used for the target PCR was 65°C. The reaction mixtureconsisted of 4 μl of DNA, 1 μl of forward primer (10 μM), 1 μl ofreverse primer (10 μM), and 4 μl of SYBR Green I mix, in a totalvolume of 20 μl. A standard curve was created by 10-fold dilutionsof the DNA of the patient.

For amplification, the following LightCycler protocol was used.The chemical cleft of the Taq polymerase was removed by prein-cubation for 10 min at 95°C. The template was amplified for 45cycles, 5 sec at 95°C, 30 sec at 65°C or 60°C, and 15 sec at the endof each cycle at 72°C, when fluorescence was measured. At the endof 45 cycles, a melting curve was generated to determine the uniquefeatures of the DNA amplified. Subsequently, the PCR product waspurified and sequenced by Bigdye Terminator Sequencing, and thesequence was verified with BLAST to determine its specificity.

RT-PCR

Total RNA was reverse transcribed with the SuperscriptTM IIIFirst strand Synthesis System for RT-PCR (Invitrogen), with oligo-dT. The CYBB cDNA was amplified by PCR in three overlappingfragments as described before [Dinauer et al., 1989]. A quantita-tive PCR was performed with the same cDNA samples, with beta-glucuronidase as a reference.

Long-Range PCR

Long-range PCR products, spanning intron 1 of CYBB, of thepatient and a healthy control, were obtained by the Expand LongTemplate PCR System (Roche Applied Science) according to themanufacturer’s recommendations. The PCR conditions were 94°Cfor 2 min, 10 cycles of 94°C for 10 sec, 52°C for 30 sec, 68°C for12 min, followed by 20 cycles of 94°C for 15 sec, 52°C for 30 sec,68°C for 12 min, with an elongation of each cycle with 20 sec at68°C, followed by a final elongation at 68°C for 7 min. For PCRprimer sequences, see Supp. Table S1.

Sequencing

The full sequence of the insertion was obtained by DNA sequenc-ing of a long amplicon around the insertion in intron 1 of theCYBB gene, with primer walking. The PCR products were purifiedwith the GFX PCR DNA Gel Band Purification kit (GE Healthcare,Bio-Sciences, Uppsala, Sweden). The purified templates were cycle-sequenced by means of the Big Dye Terminator Cycle SequencingReady Reaction kit v1.1 (Applied Biosystems, Thermo Scientific)and run on an ABI 3130 XL Automated DNA Sequencer (AppliedBiosystems). For primer sequences, see Supp. Table S1. Sequenceanalysis was performed by means of the Sequence Analysis, Se-

quence Navigator, and Auto Assembler software (all from AppliedBiosystems). Primer sequences used for sequencing are listed inSupp. Table S1.

HUMARA Assay

The HUMARA assay was performed as described previously[Wolach et al., 2005].

Separation of Methylated and Nonmethylated DNA

Genomic DNA from neutrophils or mononuclear leukocytes wassheared into fragments of 0.4–2.0 kb in a nebulization device (Beck-man Coulter, Fullerham, CA) for 1 min with 210 kPa of N2 pressure.The EpimarkTM Methylated DNA Enrichment kit (New EnglandBiolabs, Ipswich, MA) with MBD2-Fc bound to Protein A mag-netic beads was used to separate methylated DNA from nonmethy-lated DNA. The unbound fraction contained the nonmethylatedDNA, and the methylated DNA was eluted from the magnetic beadsby incubation at 65°C in water, after several washings with washbuffer. The purity of both fractions was checked with methylated-and nonmethylated-specific quantitative PCR; the fractions con-tained more than 98% methylated and nonmethylated DNA, re-spectively. The DNA concentration was measured with the Quant-iT

TMPicoGreen R© dsDNA Assay Kit (Life Technologies, Thermo

Scientific), on a Tecan plate reader. The samples were diluted to aDNA concentration of 25 ng/μl. Both methylated and nonmethy-lated DNA fractions were then subjected to the mutation-specificquantitative PCR.

Results

Short Clinical Report of the Patient

The patient is a young man, born in 1991. CGD was suspectedwhen the patient was 2 years old and suffered from an intra-abdominal abscess due to Staphylococcus aureus, requiring surgicaltherapy in addition to prolonged antimicrobial treatment. The NBTslide test confirmed the diagnosis of CGD. Subsequently, he sufferedfrom a life-threatening invasive pulmonary aspergillosis at the ageof 5 years, a milder episode of invasive pulmonary aspergillosis atthe age of 8 years, and again an invasive pulmonary aspergillosis atthe age of 11 years. All three episodes required prolonged antifun-gal therapy [Van‘t Hek et al., 1998; Verwey et al., 2003]. A cervicallymphadenitis was treated with combined antibiotic and surgicaltherapy at the age of 16 years. At the age of 20, he suffered from aperianal abscess treated with surgical drainage and prolonged an-tibiotic therapy. Preventive measurements since the diagnosis wasmade consisted of antibacterial and antifungal prophylaxis com-bined with subcutaneous interferon-gamma therapy. At present,the patient has reached the age of 22 years and is in good clinicalcondition.

Neutrophil Function Tests

The NBT test for superoxide production by individual neutrophilsand the Amplex Red test for hydrogen peroxide generation by a sus-pension of neutrophils showed that the patient had a total deficiencyof NADPH oxidase activity in his neutrophils, whereas his mother,sister, and maternal grandparents were normal in this respect(Table 1). A mosaic of oxidase-positive and oxidase-negative cells

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Table 1. NADPH Oxidase Characteristics of the Patient and His Family

NBT test (%)a Amplex Redb 7D5 binding (%)c HUMARA peaksd HUMARA ratio ± HpaIIe Mutation-specific PCR (%)f

Patient 0 0.01 4 187 n.a. 100Sister 98 n.t. n.t. 167,190 2.0→1.19 0Mother 100 2.05 100 187,190 1.57→2.2 15g

Father n.t. n.t. n.t. 167 n.a. n.t.Maternal

Grandmothern.t. 1.86 100 n.t. n.t. 0

Maternal grandfather n.t. 2.01 100 n.t. n.a. 0Normal controls

(n = 20)95–100 0.80–1.72 98–100 n.t. n.t. 0

aNBT slide test with PMA-activated neutrophils (100 cells counted), in % of cells with formazan deposits.bAmplex Red test with PMA-activated neutrophils (30 min, maximum slope), in nmoles H2O2/min 106 cells.cBinding of Moab 7D5 to an extracellular epitope of gp91phox on neutrophils, in % positive cells (Fig. 4).dPosition of peaks obtained in HUMARA assay, in units.eRatio of peak heights in HUMARA assay before and after treatment of DNA with HpaII restriction enzyme.fQuantitative, mutation-specific PCR results with DNA from mononuclear leukocytes, in % of CYBB exon 8 results.gMononuclear leukocytes 15%, neutrophils 14%, monocytes 13%, B lymphocytes 13%, T lymphocytes 22%, memory T lymphocytes 14%, saliva 8%–10%, buccal swap 10%–12%.n.t., not tested; n.a., not applicable

in the NBT test of the patient’s sister or mother was not detected,rendering it unlikely that they were carriers of an NADPH oxidase-inactivating mutation in CYBB. We consolidated this diagnosis witha Western blot, which showed no or very low amount of p22phox

and gp91phox in the patient’s phagocytes (Fig. 1A). His mother andsister had normal expression of both proteins. As these two subunitsare mutually required for stable expression, our observation impliesthat the patient carries a disabling mutation in either CYBA or CYBB[Roos et al., 2007; Van den Berg JM et al., 2009; Roos et al., 2010].

Genetic Testing

We sequenced the relevant regions of the patient’s CYBA geneand found no mutations. Also, in his CYBB exons and surroundingintron borders, we did not detect a mutation. We then performeda Southern blot analysis with blood DNA from the patient and hismother. Upon analysis of NsiI restriction fragments encompassingthe CYBB locus, we obtained a band pattern for the patient thatlacked a 4.4-kD fragment and displayed a novel 3-kD band (Fig. 1B).In contrast, his mother had restriction fragments identical to thehealthy controls and concordant with the location of NsiI restrictionsites in the locus. These data demonstrate that the patient carries aprivate deletion or insertion (see below) within a segment starting�1.3 kb upstream of the transcription start site and ending in thesecond intron of CYBB (fragment a in Fig. 1B).

The positive PCR results of exon 1 and exon 2 excluded thepossibility of a deletion of both exons. We developed a multiplexligation-dependent probe amplification assay, with probes coveringall exons and the promoter region to search for duplications. In thisassay, all probes gave a normal pattern in the patient (one copy of alltargets) and in the mother of the patient (two copies of all targets)compared with a male reference sample (not shown).

We then performed RT-PCR on the mRNA of the patient inthree overlapping fragments of the coding region, and found thatfragments 2 and 3 had a normal size. However, the first fragment,containing exon 1 to exon 7, showed three abnormal bands and noband of normal size (Figs. 2C and 3A). Sequencing showed that thelarger band contained an insertion of 117 bp between exon 1 andexon 2. The NCBI nucleotide database revealed that this insertionbelongs to part of exon 2, in reverse orientation, of the TMF1 genefrom chromosome 3 (3p21-p12) [Garcia et al., 1992]. One of thetwo minor bands in Figure 3A, lane 3, was identified as a fragmentfrom the AKAP8L transcript, but the other, weaker one, could notbe identified. These two PCR products are probably derived from

nonspecific amplification of cDNA caused by low expression of themutant CYBB mRNA. The amount of CYBB mRNA was quantifiedby lightcycler experiments in mononuclear leukocytes from the pa-tient, his mother, and a healthy control individual. In the patient, wefound 17% of fragment 1 and 29% of fragment 2 as compared withthe control (100%). In the mother, we found a normal amount ofboth fragments (Supp. Fig. S1). Fragment 3 was not tested. The lowexpression of the mutant CYBB mRNA may be due to nonsense-mediated mRNA decay.

Retroposition

We performed long-range PCR on genomic DNA of the patient,with a forward primer on exon 1 and a reverse primer at the start ofintron 2. This resulted in a PCR product of the expected 2-kb size inthe control sample, but no product was obtained with the patient’sDNA. We then used a forward and reverse primer on the novel exonsequence with several forward and reverse primers on intron 1 ofthe CYBB gene. One combination of primers gave a PCR productin the patient’s sample; the control sample gave no product, as onewould expect. Sequencing with walking primers from both sides ledto the finding of the breakpoint. Due to the presence of a poly(A) tailof about 100 bp, primer walking could only be continued from oneside into the inserted sequence. This resulted in the finding of an in-sertion in intron 1 of CYBB of about 5,800 bp of a retrogene derivedfrom a truncated TMF1 transcript (TMF1r), in reverse orientation,within a consensus LINE-1 endonuclease cleavage site TTAAAA. Thecomplete insertion sequence is shown in Supp. Figure S2. A target-site duplication (GAGTCTTTGAGTTTT) is present on both sidesof this insertion, which is characteristic for retrogene insertions. Theofficial notation of this mutation is as follows: insertion of T(100)+ Chr3:69170850-69167134 + 69171534-69171392 + 69174753-69174648 + 69175717-69175591 + 69176436-69176333 + 69180403-69179199 + 69184125-69183786 + GAGTCTTTGAGTTTT be-tween ChrX:37640282 and ChrX:37640283. The sequence of thisinsertion has been submitted to the GenBank human muta-tion database at http://www.ncbi.nlm.nih.gov/WebSub (accessionnumber KJ027511), and to the dbVar database at http://www.ncbi.nlm.nih.gov/dbvar/studies (accession number nstd89).

TMFr is derived from a multi-exon gene (TMF1, on chromo-some 3p12-p21; Garcia et al., 1992), and originated via RNA-basedretroposition [Weiner et al., 1986]. The parental template transcript(TMF1p) that gave rise to TMF1r, is a semiprocessed splice variantdisplaying several features that probably favored its retroposition

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Figure 1. Cellular and genetic features of the patient and his family. A: Western blot of the neutrophil lysates of two healthy CYBB+/+ controls(lanes 1 and 3), the patient (lane 2), and another X-linked CGD patient with a c.1555G>T (p.Glu519X) mutation in CYBB (CYBB─ control; lane 4). Theblot was incubated (left side) with Moabs 48 and 449, which detect the gp91phox and p22phox proteins, respectively, or (right side) with antibodiesspecific for p47phox or p67phox. On the extreme left and right, the size markers. B: (top). CYBB gene structure. The exons are indicated as blackvertical blocks, the 3′UTR as a blue block. The restriction fragments induced by NsiI are indicated in the middle and termed a–g. B: (lower part).Southern blot of NsiI-digested genomic DNA from leukocytes of the patient’s mother, the patient, and four normal individuals (CYBB+/+ controls).The fragments are indicated a–g, as in the upper part of this figure. The blot shows in the patient’s DNA an abnormal band at about 3 kD and thelack of a band at about 4.4 kD, as well as a normal restriction pattern in the other samples. C: Pedigree of the patient’s family. The patient’s motheris indicated as CYBB+/− healthy female with methylated CYBB− X-chromosome in some of her cells (− indicates carrying the CYBB mutation). Thepatient’s father is deceased.

(Fig. 2A). First, TMF1p did not utilize the consensus (AATAAA)polyadenylation [poly(A)] site present at the 3′ end of TMF1 butone of two upstream, nonconventional poly(A) sites (AGTAAA andAATATA) located in intron 8 of TMF1 (Supp. Fig. S2). The poly(A)site in intron 8 must compete with the upstream 5′ donor splice sitein intron 8, for pairing with the 3′ acceptor splice site of intron 7.The poly(A) site in this case won this competition, and exon 8 wasdefined as the terminal exon [Martinson 2011]. The use of this al-ternative poly(A) site implies that during processing, TMF1p lackedthe 3′ part of the transcript (exon 9–17). Together with the notionthat weak polyadenylation sites are associated with inefficient splic-ing of terminal introns in eukaryotes [Rigo and Martinson, 2008],this may explain why TMF1p retained intron 7 as well (Fig. 2A).

TMF1p was reverse transcribed and integrated on the oppositestrand of the first intron of CYBB (Fig. 2B). TMF1r carries a novelNsiI restriction site, which explains the restriction profile we ob-tained with the patient’s DNA (Fig. 1B) (this restriction site pro-duces a novel visible 3-kb band together with a novel 7.2-kb bandthat is masked by a preexisting band at 7.2 kb on our blot).

During splicing of the mutated transcript, cryptic acceptor(CAG), donor (GTA), and branchpoint (TATTAAC, 24 bp upstreamof the acceptor site) sites are activated, resulting in the insertion of

part (117 bp) of exon 2 of the TMF1 retrogene in reverse orientationbetween exon 1 and 2 of the CYBB transcript (Supp. Fig. S3). Thisexonization produced a novel fusion transcript carrying a prema-ture stop codon TAG at the second codon in the novel exon, whichdramatically truncates the encoded protein (the novel peptide—ifexpressed—would be 16 amino acids long, whereas the wild-typegp91phox protein contains 570 amino acids). Taken together, ourdata indicate that TMF1r insertion leads to a defective CYBB allele,which is in turn responsible for the immunodeficiency we observed.However, we note that this defective allele may still produce somefunctional protein by relying on conventional CYBB splice sites,which would be consistent with the residual 4% of gp91phox ex-pression we observed in the patient’s neutrophils by FACS analysis(Fig. 4, not detectable on the Western blot shown in Fig. 1A). In con-trast, his mother and maternal grandmother had 100% neutrophilswith normal gp91phox expression.

We used the Website http://astlab.tau.ac.il/index.php to investi-gate whether an explanation could be found for the novel exon tobe inserted into the CYBB mRNA. The acceptor splice site used gavethe second best score (79.32), 6 bp downstream of a potential ac-ceptor splice site with a slightly higher score (79.63) (the 3′ splicesite TTTTTTTTTTTCAG/G [/ indicates the intron/exon junction]

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Figure 2. Genetic events causing the patient CYBB gene defect. A: (top) gene structure of TMF1 on chromosome 3; (middle) parental transcriptof TMF1 (TMF1p), a semiprocessed splice variant of TMF1 with a poly(A) sequence attached to part of intron 8; (bottom) semiprocessed TMF1pwith unprocessed introns 7 and 8. B: (top) Gene retrocopy of TMF1p (TMF1r) reverse transcribed and in reverse order integrated into intron 1 ofCYBB on the X-chromosome of the patient. In red, novel splicing of the patient’s CYBB induced by the retrocopy; (bottom) CYBB–TMF1r fusionmRNA product containing a novel 117-bp exon (with a TAG stopcodon at position 2) between the original exon 1 and exon 2 of CYBB. Translationof this fusion mRNA might lead to a short gp91phox peptide, and hence to CGD. C: Details of the CYBB–TMF1r genomic insertion site. From 5′ to 3′:exon 1 of CYBB including 5′UTR, part of intron 1 of CYBB, target-site duplication GAGTCTTTGAGTTTT, poly(A) tail attached to intron 8 sequenceof TMF1p, part of intron 8 of TMF1, exon 8 of TMF1, intron 7 of TMF1, exons 7–3 of TMF1, exon 2 of TMF1 containing a novel splice acceptor CAGsequence followed by TTT (Phe) and TAG (stop), another 111 bp and a novel splice donor GTA sequence, exon 1 of TMF1 including 5′UTR, target-siteduplication CAGTCTTTGAGTTTT, retrotransposition entry site TTTTAA, remainder of intron 1 of CYBB, exon 2 of CYBB, intron 2 of CYBB. The totalsize of the TMF1r insertion is about 5.8 kb. The original NsiI fragment of 4.4 kb is changed by the insertion into a 10.2-kb fragment that is cut byNsiI into two fragments with sizes of about 3 and 7.2 kb due to an extra NsiI restriction site in the insertion (indicated at the bottom). In red, novelsplicing of the patient’s CYBB induced by the retrocopy.

will give a score of 100). If the acceptor splice site with the high-est score were used, this would lead to expression of an in-frameprotein without a stopcodon and with 37 extra amino acids ingp91phox, which would probably be without catalytic function butmight show immune reactivity with anti-gp91phox and thus con-tribute to the low expression of this protein in the patient (Fig. 4).The donor splice site gave the highest score (81.63) (5′ splice-siteCAG/GTAAGT will give a score of 100). The branch-point sequenceis also indicated in Supp. Figure S4. Splicing enhancer binding sites

[Fairbrother et al., 2002] are twice as abundantly present in the novelexon as binding sites for silencer-splicing regulators: score RESCUE-ESE for the novel exon is 20/1.17 = 17.1%, score FASS ESS, hex 2 set,for the novel exon is 10/1.17 = 8.55%; PESE score is 10/1.17 = 8.55%,PESS score is 4/1.17 = 3.42% [Wang et al., 2004] (Supp. Fig. S4). Bothbinding sites are also twice as abundantly present in the novel exonas in an exon with an average composition [Kralovicova and Vore-chovsky, 2007]. Of the splicing enhancers SRSF1, SRSF2, SRSF5,and SRSF6, only SRSF1 is expressed in neutrophils, whereas SRSF1

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Figure 3. PCR results. A: RT-PCR results from a healthy control andthe patient. Lane 1 is from a reaction with water instead of cDNA. Lanes2 and 3 show amplicons from the first fragment comprising exon 1–7.Lane 4 is again from a reaction with water instead of cDNA. Lanes 5and 6 show amplicons from the second fragment comprising exon 6–10. In the last lane, the 1-kb Generuler from Fermentas was loaded. Thepatient sample gave three bands in the first fragment (lane 3); no band ofnormal size was found in the patient. The upper band proved to containthe TMF1p insertion; the lower two bands proved to be nonspecific (noCYBB sequence). The second fragment from the patient’s cDNA (lane 5)gave the same product as that obtained with the healthy control sample(lane 6). B: Mutation-specific PCR on separated fractions of methylatedand nonmethylated DNA from the mother’s neutrophils and PBMCs.Primers were chosen on the novel exon (forward) and downstream ofthe inserted retrogene on intron 1 (reverse) of the CYBB gene. Onlysamples containing the insertion gave a PCR product of about 340 bp.The results show the mutation to be present only in the fractions withmethylated DNA (Lightcycler results and analysis of the PCR productson gel are shown in Supp. Fig. S5).

and SRSF2 are both expressed in monocytes (GeneCards R© databaseand our own observations). Two SRSF1 and three SRSF2 splicingenhancer binding sites are present in the novel exon; one and two,respectively, are present in exon 2 of CYBB. For comparison, thesplicing regulator elements in exon 2 of CYBB are also depicted inSupp. Figure S4.

Inheritance

We found the TMF1 insertion in leukocyte as well as in salivaDNA from the patient. We then developed an insertion-specificquantitative PCR and found that the patient’s leukocyte DNA was100% positive for this insertion in CYBB intron 1 (with CYBB exon8 = 100%). To our surprise, we also found a weak positive reactionin the DNA from his mother’s leukocytes. We then quantified thisin her leukocyte subsets and found these to be 13%–22% positivein this test (Table 1), and also DNA from her saliva and from abuccal swap was found 10%–12% positive. DNA from mononu-clear leukocytes of the patient’s sister, maternal grandmother, andmaternal grandfather was completely negative in this test.

Thus, we found a mutation in the patient’s CYBB gene on theX-chromosome, but only 10%–20% presence of this mutated al-lele in his mother’s DNA. Moreover, we found no indication forexpression of the mutation in the mother’s neutrophils. AlthoughCYBBwt/mut females do not usually suffer from CGD, their phagocytepopulations often display heterogeneous patterns of gp91phox ex-pression and superoxide production due to random X-chromosomeinactivation [Roos et al., 2007; Van den Berg JM et al., 2009; Rooset al., 2010]. However, the sister, the mother, and the maternal grand-mother had phagocyte profiles characteristic of CYBBwt/wt individ-uals (see above), and these homogenous profiles were not caused byskewed X-chromosome inactivation, as assessed by an HUMARAassay. This test was performed on blood DNA from the patient, hisparents, and his sister. The patient and his father showed one peak,diminished after HpaII treatment of the DNA. His mother and sis-ter showed two peaks, with peak height ratios that did not changedramatically by HpaII treatment (Table 1). This indicates that themother and the sister have two X-chromosomes, one of which isinactivated (methylated) in part of their leukocytes. There is no ex-treme skewing of methylation of one of both X-chromosomes inthese women. Moreover, the patient’s sister has not inherited theX-chromosome that in her brother carries the mutation in CYBB.

We considered the possibility that the mother of the patient car-ried the mutation only in a fraction of her cells, on an inactivatedX-chromosome. Indeed, when we repeated the mutation-specificquantitative PCR on separated fractions of methylated and non-methylated DNA from her neutrophils and mononuclear leuko-cytes, we found the mutation for 98%–99% in methylated DNA(Fig. 3B, Supp. Fig. S5).

DiscussionThe LINE-mediated retrotransposition relies on two key en-

zymes, an endonuclease and a reverse transcriptase, encoded byLINEs. Although this enzymatic machinery has a cis preferencefor LINE transcripts, it may also capture mRNA and consistentlyproduced thousands of gene retrocopies during mammalian evolu-tion [Vinckenbosch et al., 2006; Baertsch et al., 2008; Cordaux andBatzer, 2009; Gogvadze and Buzdin, 2009; Kaessmann et al., 2009].Gene retroposition has been recognized as an important sourceof genetic innovation during mammalian evolution [Brosius 1991;Brosius and Tiedge 1995; Kaessmann et al., 2009]. Indeed, several

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Figure 4. Binding of mAb 7D5 to gp91phox on neutrophils. A: Dotplots of neutrophils incubated with mouse IgG1 or with mAb 7D5 against gp91phox,followed by FITC-labeled goat-anti-mouse-Ig. X-axis, scatter of cells; Y-axis, FITC signal. Upper panels, cells incubated with mouse IgG1; lowerpanels, cells incubated with mAb 7D5. Left panels, control neutrophils; right panels, patient neutrophils. B: Histograms of neutrophils incubatedwith mouse IgG1 or with mAb 7D5, followed by FITC-labeled goat-anti-mouse-Ig. X-axis, FITC signal; Y-axis, number of cells. Upper panel left,control neutrophils; upper panel right, neutrophils from the patient’s mother; lower panel left, neutrophils from the patient’s maternal grandmother;lower panel right, neutrophils from the patient. White area, histogram obtained with mouse IgG1; dark area, histogram obtained with 7D5.

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mammalian retrocopies have been shaped into functional retro-genes by positive Darwinian selection and now fulfill novel—orenhanced—functions in various biological processes, includingspermatogenesis and neurotransmitter metabolism [Burki andKaessmann, 2004; Marques et al., 2005]. Also, several mammalianretrogenes were previously shown to have gained expression via ex-onization in host gene/retrocopy fusion transcripts [Bradley et al.,2004; Vinckenbosch et al., 2006]. Often, these mRNAs comprise 5′

untranslated exons from the host gene and only translate the ret-rogene open reading frame [Vinckenbosch et al., 2006], but otherchimeric transcripts (such as the TRIM5/CypA fusions in Old Worldor New World monkeys) encode fusion proteins with fascinating bi-ological properties [Sayah et al., 2004; Wilson et al., 2008].

Retrocopies of mRNA studied so far are relatively old and carriedby all individuals in a given species. However, a recent study pro-vided a remarkable example of a polymorphic retrogene in dogs,which was artificially selected during dog domestication because itcauses anatomical features that were attractive to breeders (namely,a short-, curved-leg phenotype trait termed chondroplasia) [Parkeret al., 2009]. This canine retrogene, which originated from a growthfactor gene FGF4, is a paradigmatic example illustrating how a singlegene retroposition can potentially lead to an immediate impact onthe phenotype of its carrier, in this case via gene dosage alterationduring skeletal development. Also, in humans and other mammals,retrocopy insertion polymorphisms have recently been identified[Ewing et al., 2013]. Our study provides yet another mechanism fora (human) retrocopy with immediate phenotypical consequences,namely, the disruption of a gene with a major role in the immuneresponse, via exonization into a defective fusion transcript.

Only one example is known of a contemporary 330-bp insertionof a transcript (without protein-coding ability) from a gene-poorregion on chromosome 11 into an exon of the dystrophin gene onthe X-chromosome, inducing exon skipping, creating a prematurestop codon, and leading to Duchenne muscular dystrophy [Awanoet al., 2010]. However, this insertion was later shown to be derivedfrom a sequence downstream of a LINE-1 element situated at chro-mosome 11q22.3 [Solyom et al., 2012]. Thus, the likely explanationfor this mutagenic insertion is a LINE-1-mediated 3′ transductionwith severe 5′ truncation, and thus another example of LINE-1-mediated autotransposition. We have also searched for a LINE-1sequence in or around the TMF1 gene, but found none. Therefore,the mutation we describe is most probably caused by a real LINE-1-mediated translocation of mRNA. More distantly in the past, a2,667-bp sequence from a gene on chromosome 6 (ORF 68) has beenretroposed into the SLC25A13 gene, resulting in citrin deficiency inJapanese, Chinese, and Korean patients [Tabata et al., 2008]. Likethe retrotransposed insertion we found, this 2,667-bp sequence wasinserted in an intron and created a novel exon with a stop codon.

The LINE-1-driven insertion of incomplete, partly exonizedTMF1 cDNA proves that insertion of retroposed copies and theirexonization can occur together. In fact, the exonization of the TMF1mRNA was not complete when the reverse transcription and theinsertion already took place, probably due to the use of an alter-native poly(A) site leading to inefficient exonization. Furthermore,eukaryotic cells readily export intron-free mRNA into their cyto-plasm but intron-containing RNAs tend to remain in the nucleus[Reed, 2003]. We thus propose that TMF1p colocalization with theLINE machinery in the nucleus further facilitated its retroposition.

The event found by us also gives insight into the splicing process,because the splicing of the TMF1 transcript was “caught in the act”of splicing when it was reverse transcribed by the LINE machinery.Intron 7 and part of intron 8 were still present when the TMF1transcript integrated into the CYBB gene. This means that the order

of introns that are spliced out follows the same direction as, andprobably during, the transcription process, from 5′ to 3′.

Four examples of retained introns in a retrogene have been foundbefore in mice and rats. Mice and rats have two, equally expressed,nonallelic genes encoding preproinsulin (genes I and II). Gene I wasgenerated by an RNA-mediated duplication-transposition event in-volving a transcript of gene II. Gene I has a single 119-bp intron,whereas gene II contains a second 499-bp intron [Soares et al., 1985].Mice also have a partially processed methylene-tetrahydrofolate re-ductase pseudogene (Mthfr-ps) that is missing intron 1 but haspartial retention of intron 2 [Leclerc et al., 2003]. This pseudo-gene arose by retrotransposition of a misspliced Mthfr transcript.Mice also have two other semiprocessed pseudogenes, ψRev-erbβ

and ψLHR1, derived from semiprocessed RNA transcripts with re-tained but modified intron sequences [Zhang et al., 2008]. Perhapsweak poly(A) signals may be present also in the other retrogenesmentioned above in which the last intron has been retained.

TMF1 encodes TATA element modulatory factor 1 (TMF1or TMF), also called androgen receptor coactivator of 160 kDa(ARA160). This promoter protein can enhance DNA transcriptioneither in a direct way, by binding to TATA elements in the promoterregion of genes and in this way promoting RNA polymerase II ac-tivity [Garcia et al., 1992], or in an indirect way by binding to theandrogen receptor, thus enhancing androgen-dependent transac-tivation [Hsiao and Chang, 1999]. Moreover, TMF1 also interactswith the small GTPase Rab6 and in this way regulates retrogrademembrane traffic from endosomes to the Golgi apparatus and fromthere to the endoplasmic reticulum [Yamane et al., 2007; Milleret al., 2013]. TMF1 RNA and protein have been found in many hu-man tissues, among them blastocyst cell lines and embryonic stemcells [Su et al., 2004]. However, it is not known whether TMF1 isexpressed in early embryogenesis.

The origin of the mutation we found must be sought in the earlyembryonic development of the patient’s mother. It is known thatLINE-1-mediated retrotransposition can indeed occur very earlyin human embryonic development [Van den Hurk et al., 2007].We found that 12%–15% of the CYBB from the patient’s mothercontained the insertion, a low amount that escaped detection onthe Southern blot (Fig. 1B). In the DNA of her parents, the mu-tation was absent. However, in the patient’s mother, this insertionwas found exclusively in the fraction of methylated DNA (Fig. 3B).Probably, the retroposition took place in one cell at the 8-cell stageof her embryonic development, and subsequently in this cell theX-chromosome with this insertion was inactivated by methylation.This explains the presence of the mutation in only 12%–15% of themother’s DNA and why she had a normal phenotype. This expla-nation assumes that the X-chromosome inactivation process takesplace already at the 8-cell stage, or that in a later stage the same (mu-tated) X-chromosome was inactivated in all daughter cells from theoriginal cell in which the retroposition took place. There is discus-sion about the embryonic stage in which human X-chromosomeinactivation takes place, but recent evidence points to a start of thisprocess already at the 8-cell stage [Van den Berg IM et al., 2009].In contrast, there are no indications for imprinted inactivation ofX-chromosomes with a genetic insertion. Thus, we favor the firstexplanation. During formation of the mother’s germline cells, hermethylated X-chromosome was reactivated during meiosis; the mu-tation was included in some of her oocytes and subsequently passedon to her son but not to her daughter. Whether the mutation affectsthe fertility of the patient is not known yet. In principle, the mu-tation can be passed on to later generations, as happens with otherX-CGD patients. Prenatal diagnosis of offspring is available, so newpatients with this mutation can be avoided.

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Acknowledgments

The authors thank Dr. Nicolas Vinckenbosch, Dr. Henrik Kaessmann,and Dr. Manyuan Long for suggestions and comments concerning thismanuscript, and Anton Tool and Michel van Houdt for technical support.MdB designed and carried out experiments; KvL and JG carried out exper-iments; CMW and AW took care of the patient and contacted his family;TKvdB and TWK discussed the results; and DR discussed the results andwrote the manuscript.

Disclosure statement: The authors declare no conflict of interest.

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