Selective IgG2 deficiency due to a point mutation causing abnormal splicing of the C{gamma}2 gene

Yaofeng Zhao, Qiang Pan-Hammarström, Zhihui Zhao, Sicheng Wen and Lennart Hammarström

Division of Clinical Immunology, Department of Laboratory Medicine, F79, Karolinska University Hospital Huddinge, Karolinska Institute, SE-141 86, Stockholm, Sweden

Correspondence to: Y. Zhao; Email: Yaofeng.Zhao{at}labmed.ki.se


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The mechanism underlying selective IgG subclass deficiency is largely unknown in humans. We have previously reported the acquisition of a complete IgG2 deficiency in a leukemia patient after bone marrow transplantation. Southern blot analysis showed a deletion including the C{gamma}2 and C{gamma}4 genes on one chromosome in the donor, suggesting that the remaining C{gamma}2 gene allele was silent. In the patient and his two IgG2 deficient brothers, the silent C{gamma}2 gene showed both germ-line transcription and switch recombination and no structural defects were found in the intronic promoter or the switch region of the gene. However, an A->G transition in the fourth nucleotide in the 5' portion of intron 1 was identified. Transfection of artificial constructs into the human B cell lines demonstrated that this A->G transition inactivated the normal splice site, and instead, a cryptic splice site in the CH1 exon was used in RNA post-transcriptional processing, leading to a 16 bp deletion of the {gamma}2 CH1 exon. This aberrantly spliced RNA that is mostly derived from germ-line transcription in vivo was also detected in both homozygous and heterozygous individuals carrying this mutation. These findings suggest a novel genetic mechanism as the cause of IgG subclass deficiency in selected patients.

Keywords: class switch recombination, germ-line transcription, immunoglobulin


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The human immunoglobulin heavy chain constant region gene (IGHC) locus is located on chromosome 14 and organized as a gene cluster consisting of nine functional genes (µ–{delta}{gamma}3–{gamma}1–{psi}{varepsilon}{alpha}1–{psi}{gamma}{gamma}2–{gamma}4–{varepsilon}{alpha}2) (1). The four {gamma} genes, {gamma}1, {gamma}2, {gamma}3 and {gamma}4, encode IgG1, IgG2, IgG3 and IgG4, respectively. In normal individuals, ~66% of the total serum IgG is IgG1, followed by IgG2 (24%), IgG3 (7%) and IgG4 (3%) (2). While IgG1 and IgG3 appear early in ontogeny (3), IgG2 develops much later and adult levels of IgG2 are not reached until 5–10 years of age. Although selective Ig class or subclass deficiencies have been extensively documented, the underlying genetic basis is still largely unknown. In a few cases, the deficiency has been shown to be due to homozygous deletions of the corresponding C region genes (4,5). However, in most instances, the etiology remains unknown. In a recently reported case of IgG2 deficiency, the defect was shown to be caused by a homozygous one-base insertion in exon 4 of the C{gamma}2 gene, leading to a lack of expression of membrane bound IgG2 (6,7) and failure of expansion of IgG2 producing cells.

We have previously reported an acute lymphoblastic leukemia patient who developed IgG2 deficiency after receiving a bone marrow graft from his IgG2 deficient, HLA identical brother (8). Southern blot analysis of their immunoglobulin heavy chain constant region gene loci showed that the maternal haplotype lacked both the C{gamma}2 and C{gamma}4 genes (8), whereas the paternally derived 25 kb C{gamma}2 allele was silent due to an unknown defect. Here, we report a novel mechanism in the latter for the development of IgG subclass deficiency in human.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient
Patient OLS, who had been bone marrow transplanted (BMT) due to acute lymphoblastic leukemia, developed a profound IgG2 deficiency (<0.2 g/l) after BMT, while he had an IgG2 level in the lower normal range (1.2 g/l) before BMT (8). Our previous data showed that the patient's two brothers (one being the BMT donor) both had IgG2 levels below detection level (<0.2 g/l) and the patient's sister had a markedly reduced IgG2 level (0.4 g/l). The IgG2 level of the patient's father was normal (1.9 g/l), whereas the IgG2 level in his mother (1.0 g/l) was slightly below the normal range (8).

Total Ig measurements and IgG subclass analysis
Total serum levels of IgM, IgA, IgG1, IgG2, IgG3 and IgG4 of the patient and his relatives were measured again, 14 years after the bone marrow transplantation. The measurements were performed by nephelometry. Serological typing for the G2m(n+) and G2m(n–) allotypes was performed as described previously (9).

RNA samples and reverse transcriptase-polymerase chain reaction
Total RNA was isolated from peripheral blood samples using a RNeasy® minikit (Qiagen, Valencia, CA), and subjected to cDNA synthesis with a First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Reverse-transcriptase PCR (RT–PCR) was conducted to detect the mature transcripts of the C{gamma}2 gene employing primers JHS (5'-CCTGGTCACCGTCTCCTCA-3') and HA1 (5'-ACTCGACACAACATTTGCG-3'). A nested PCR was also performed to detect germ-line transcripts using the primers IS-new (5'-GGCCGAGCTGTGATTTCCTA-3') and IgG2-Hingeas (5'-GTGGGCACTCGACACAACAT-3'), IS1 (5'-TCTCAGCCAGGACCAAGGAC-3') and HA1. The primers JHS, HA1 and IS1 were designed based on a previous publication (6), to which the PCR conditions are also referred. To confirm the transcription of mature {gamma}2 transcripts in the patient's family members, another nested PCR was performed using two pairs of primers JHS and IgG2-Hingeas, JHS1 (5'-GTCACCGTCTCCTCAGCCT-3') and HA1.

Amplification and sequencing of the intronic exon (I{gamma}2), switch region (S{gamma}2), constant region (C{gamma}2) and transmembrane region encoding exons (m{gamma}2) of the {gamma}2 gene
The I{gamma}2 region was amplified by a nested PCR using primers I{gamma}A (5'-GTCCATGGAGAGGCCCAGGTATTGAGAG-3'), I{gamma}B (5'-GTCCATGGACTCCCATCCCCACACA-3'), I{gamma}C (5'-ACGTCGACTTCCCTCCTCCCATCT-3') and S{gamma}2B (5'-ACGTCGACGCCCTTGTTCACCTTCAGTT-3') which were all designed based on published sequences (10). Hs{gamma}3A and Hs{gamma}3B were used to amplify the S{gamma}2 region (11). The entire C{gamma}2 was amplified using C{gamma}2-L (5'-GTCCATGGCCCTGCCTGGACCCTCGT-3') and C{gamma}2-P (5'-ACGTCGACAGACTCGGCCTGACCCAC-3').(12) The primers employed to amplify the m{gamma}2 region were IgG2-CH2Ls (5'-CATCTCTTCCTCAGCACCACCTG-3') and IgG2-TM2Las (5'-GGCCTGGGTGATGGCTGAGCAT-3') (6,12). All the resulting PCR fragments were gel purified, cloned into a pGEM-T vector and sequenced. To confirm the A->G mutation in intron 1, a short fragment encompassing the mutation site was amplified and directly sequenced using the primers IgG2-CH1s (5'-CCGTGCCCTCCAGCAACTTC-3') and IgG2-Hingeas.

Constructs and cell transfections
Genomic DNA samples from the patient and a healthy control were used to amplify the DNA fragment encompassing the mutation site using primers IgG2-CH1s-XhoI (5'-TTTTCTCGAGCCGTGCCCTCCAGCAACTTC-3') and IgG2-Hingeas-SalI (5'-TTTTGTCGACGTGGGCACTCGACACAACAT-3'). The resulting 525 bp PCR products were cloned into a pCI-Neo vector using the XhoI and SalI sites. The final construct containing the G mutation was termed pCIG2D, and the control construct containing the wild-type A nucleotide was termed pCIG2C. Ten micrograms of either plasmid was used to transfect two human B cell lines, BL2 and DG75. The protocols employed for cell culture and transfection have been described previously (13). The cells were collected 48 h after transfection and total RNA was extracted. About 5 µg of RNA was employed to synthesize first-strand cDNA using a First-Strand cDNA Synthesis Kit and a pCI-neo derived primer (SV40-polyA-as, 5'-ATTCTAGTTGTGGTTTGTCC-3'). The primers, IgG2-CH1s-XhoI and IgG2-Hingeas-SalI were employed to detect the processed mature RNA transcripts.

Detection of Sµ-S{gamma}2 switching fragments
Approximately 200 ng of genomic DNA from peripheral blood lymphocytes were subjected to a first round of PCR amplification using the primers Sµ1 and S{gamma} common (11,14). One microliter of the first PCR reaction was used as a template in the second round of amplification. The primers used were Sµ5 (11,14) and S{gamma}2as (5'-GTCTGCAGTGTGGCTGCTCTGCTCTGAT-3'). The second round of amplification was carried out using 35 cycles of 94°C, 50 s; 60°C, 50 s; 72°C, 1 min following the denaturing step at 94°C for 3 min.

DNA typing of the G2m(n+) and G2m(n–) allotypes
To confirm the results of the serological typing of the IgG2 allotypes, a genomic fragment encompassing the {gamma}2 CH2 exon and part of CH3 exon was amplified using the primers IgG2-CH2Ls and IgG2-CH3as2 (5'-CTGGTTCTTGGTCATCTCCTCCTCC-3') and directly sequenced. To examine the expression of different alleles at the RNA level, 1 µl of blood-derived cDNA was subjected to a first round of amplification using the primers IgG2-RLPCRs (5'-CAGCAACTTCGGCACCCAGAC-3') and IgG2-CH3as1 (5'-AGGAAGAAGGAGCCGTCGGAGT-3'). A second round of PCR was subsequently performed using the nested primers C{gamma}2-upper (5'-GCGCAAATGTTGTGTCGAGTG-3') and IgG2-CH3as2. The resulting 427 bp PCR fragment was gel-purified and directly sequenced using both the C{gamma}2-upper and IgG2-CH3as2 primers.

Screening of the A->G mutation in different populations using site-specific PCR amplification
A site-specific PCR was developed to determine whether the A to G transition is present in different ethnic populations such as Swedes (102 samples), Chinese (92 samples) and Gambians (96 samples). The primers used for screening were IgG2-cG (5'-GTGGACAAGACAGTTGGTcG-3') and IgG2-Hingeas (5'-GTGGGCACTCGACACAACAT-3'), where the IgG2-Hingeas exactly matches the target sequence, and the last second nucleotide of IgG2-cG was modified to a C instead of the original G. The modification of the former primer ensures that the mutated G allele can be specifically amplified under PCR conditions of 94°C, 3 min, then 30 cycles of 94°C, 30 s; 60°C, 30 s; 72°C 30 s.

Computational analysis
BLAST searches were conducted using the National Center for Biotechnology Information BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). Sequence editing, alignments and comparisons were performed using the DNASTAR package (DNAstar, Madison, WI).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Quantification of serum immunoglobulin levels and serological typing of IgG2 allotypes
Immunoglobulin levels of the patient and his family members are presented in Table 1. Consistent with our previous report (8), only trace amounts of IgG2 could be detected in the patient and his two brothers. The IgG2 levels in the mother and sister were below the normal range, whereas the patient's father had a normal concentration of serum IgG2. The serologically detected IgG2 allotype in the patient before bone marrow transplantation (BMT) and his father was G2m(n+), whereas G2m(n–) was observed in the mother and sister. No IgG2 allotype was discernible in the two IgG2 deficient brothers and in the patient after BMT.


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Table 1. Serum immunoglobulin levels (g/l)

 
RT–PCR detection of germ-line and mature C{gamma}2 transcripts
A nested PCR was employed to detect germ-line transcripts and showed that the I{gamma}2 exons in the patients and his two brothers could be transcribed and processed (Fig. 1A, lanes 4, 5 and 6, respectively). A human JH derived primer, JHS, and a C{gamma}2 hinge region specific primer, HA1, were used to detect mature transcripts of IgG2 (Fig. 1B, lanes 4, 5 and 6, respectively). No mature IgG2 heavy chain transcripts (or only trace amounts) could be detected in the patient and his two IgG2 deficient brothers (Fig. 1B). To confirm this finding, another nested PCR was conducted, showing the same result (Fig. 1C). It also showed that the weak bands in the patient's two brothers in the first PCR (Fig. 1B, lanes 5 and 6) were due to non-specific amplification.



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Fig. 1. RT–PCR detection of germline and mature {gamma}2 transcripts. (A) Amplification of germline transcripts. (B) Amplification of mature {gamma}2 transcripts. In both (A) and (B), 1, father; 2, mother; 3, sister; 4, patient; 5, first brother (donor); 6, second brother. (C) A nested PCR amplification of mature {gamma}2 transcripts. 1, father; 2, mother; 3, sister; 4, patient; 5, first brother (donor); 6, second brother; 7, an unrelated IgG2 deficient patient; 8, healthy control.

 
Amplification and sequencing of I{gamma}2 region
The I{gamma}2 regions from all family members were amplified and sequenced (Fig. 2). No structural defects were found. The I{gamma}2 regions in the mother, sister and two brothers all shared the same sequence as the I{gamma}2 region from the patient (GenBank accession number: AY372691). Two I{gamma}2 alleles were identified in the father, where one allele displayed a single nucleotide difference at position 435 (G to A transition, GenBank accession number: AY372691), the other was identical to the allele shared by the other family members.



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Fig. 2. The genomic structure of the human C{gamma}2 gene. The bold black line indicates the cloned genomic fragments. I{gamma}2: I region; S{gamma}2: {gamma}2 switch region; CH1, CH2 and CH3: CH1, CH2 and CH3 encoding exons; H: hinge region encoding exon; M1, M2: transmembrane region encoding exons. Arrows indicate locations of the primers used in this study.

 
Amplification and sequencing of the S{gamma}2 region and detection of Sµ-S{gamma}2 switch fragments
We subsequently amplified the S{gamma}2 region from the patient (11). The resulting 1.2 kb, S{gamma}2 containing fragment, was cloned and sequenced (GenBank accession number: AY372692) (Fig. 2). A comparison with a previously published S{gamma}2 sequence (10) showed that they are slightly different, displaying 15 single nucleotide, one dinucleotide and one trinucleotide insertions or deletions, and three single nucleotide polymorphisms. We further employed a nested PCR approach (11,14) to analyze whether switching to {gamma}2 occurred in the patient and his two brothers. Rearranged Sµ-S{gamma}2 fragments could be amplified from both the patient (Fig. 3C) and his two brothers (data not shown), suggesting that the S{gamma}2 regions are functional and that the {gamma}2 gene could still be utilized, indicating that switching to the {gamma}2 gene in the patient and his two brothers was essentially normal.



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Fig. 3. Detection of Sµ-S{gamma}2 switch fragments by a nested PCR. (A) Patient's father; (B) patient's mother, (C) patient; (D) healthy control. The PCR fragments from 10 PCR reactions (lanes 1–10) differ in their sizes and represent random amplification of in vivo switched clones.

 
Amplification and sequencing of the transmembrane region encoding exons
To specifically amplify the transmembrane region of the C{gamma}2 gene, IgG2-CH2Ls and IgG2-TM2Las were used to obtain a PCR fragment spanning the CH2, CH3 and transmembrane exons (Fig. 2). The transmembrane exons were deduced by sequencing the cloned PCR product. Only a single nucleotide change was found in the M1 exon (GenBank accession number, M1 exon: AY372693; M2 exon: AY372694) as compared to the previously published sequence (6). It does however not cause an amino acid alteration. No structural defect in splice sites was found in either of the M1 and M2 exons. Since the previously published M1 and M2 exon sequences did not include the branchpoint sites, we sequenced the upstream region of these exons in the patient, demonstrating that the branchpoint sites for both the M1 and M2 exons were normal (data not shown) and conform to conserved sequences in mammals (1517).

Amplification and sequencing of the C{gamma}2 gene in the patient
An ~1.8 kb DNA fragment encompassing the whole C{gamma}2 constant region was amplified from the patient (Fig. 2) and sequenced (GenBank accession number: AY372690). As compared to the previously published C{gamma}2 sequence (12), three nucleotide replacements, one in the 5' flanking region of the first C{gamma}2 exon (the CH1 encoding exon), one in the CH1 exon and one in intron 1 (the intron between CH1 and the hinge encoding exons) were observed (Fig. 4). In addition, a single-nucleotide insertion in the 5' flanking region, upstream of CH1, was also identified (Fig. 4). The first two nucleotide replacements, and the single-nucleotide insertion in the 5' flanking region found in the patient were also observed in healthy controls, suggesting that they represent normal sequence polymorphisms in the human population. Furthermore, the C->G mutation in exon 1 does not alter the encoded amino acid and is, most likely, a silent mutation. The A to G mutation in intron 1 was potentially significant, as it is located in the 5' splice site of intron 1, although it does not change the conserved GT nucleotides required for RNA splicing. To confirm the presence of this mutation in the patient and other family members, a short fragment encompassing the mutation site was amplified using primers IgG2-CH1s and IgG2-Hingeas, and directly sequenced. The results show that in the patient and his two brothers, the remaining single copy of the C{gamma}2 gene carries a 5' splice site composed of GTGGG, in contrast to GTGAG in healthy controls. Both the patient's father and sister appear to be heterozygous for this site. The single C{gamma}2 copy in the mother showed a normal GTGAG sequence.



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Fig. 4. Comparison of the C{gamma}2 gene cloned from the patient with a previously published C{gamma}2 gene sequence. JCg2: previously published C{gamma}2 sequence (accession number: J00230); PCg2: the C{gamma}2 sequence obtained from the patient. Identical nucleotides are indicated as stars, and non-identical nucleotides are underlined and in bold. The 16 bp sequence in the CH1 exon that is deleted in the mature {gamma}2 transcript due to the G mutation is underlined and in bold. Exon sequences are shown in uppercase letters, while intron sequences are shown in lowercase letters. Solid arrows indicate borders of exons. An empty arrow indicates the position of the A to G transition that causes abnormal RNA splicing. Note: only part of the C{gamma}2 sequence is presented in this figure.

 
The A->G point mutation causes an alternatively spliced {gamma}2 CH1 exon in vitro
To test whether the A->G mutation in intron 1 influences the splicing pattern of the IgG2 heavy chain transcripts, a DNA fragment spanning part of the CH1 exon, the entire intron 1 and the hinge exon was amplified from both the patient and a healthy control, and cloned into a mammalian expression vector, pCI-neo. The resulting recombinant plasmids were termed pCIG2D (containing the mutated G nucleotide) and pCIG2C (containing the wild-type A nucleotide) (Fig. 5A). Both the pCIG2D and pCIG2C plasmids were introduced into the human B cell lines DG75 and BL2 in order to analyze, using RT–PCR, how the C{gamma}2 derived fragments were processed after being transcribed. As shown in Fig. 5(B), the pCIG2D derived transcripts (lanes 1 and 3) are shorter than those derived from pCIG2C (lanes 2 and 4) in both of the human B cell lines used, suggesting an altered splicing pattern caused by the A->G mutation. Sequence analysis of these PCR products revealed that the pCIG2C derived transcripts were normally spliced, and that the CH1 exon was precisely fused to the hinge exon. However, the pCIG2D derived transcripts were 16 bp shorter, due to utilization of a cryptic splice site sequence in the CH1 exon. The 16 bp deletion in CH1 exon of the IgG2 heavy chain transcripts changes the open reading frame of the downstream sequence, introducing a premature translation termination codon (PTC) in the CH2 exon, resulting in a nonfunctional protein.



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Fig. 5. Splicing of artificial C{gamma}2 sequences in two human B cell lines. (A) Physical structure of pCIG2D and pCIG2C. (B) RT–PCR detection of the C{gamma}2 transcripts from the two artificial constructs in two human B cell lines: 1, pCIG2D in the DG75 human B cell line; 2, pCIG2C in the DG75 human B cell line; 3, pCIG2D in the BL2 human B cell line; 4, pCIG2C in the BL2 human B cell line.

 
RT–PCR detection of the abnormal transcript in vivo
To determine whether the abnormal transcript could be detected in vivo, total RNA samples, isolated from PBL from the patient and his family members, were subjected to RT–PCR following the procedure used in the above in vitro experiments. As shown in Fig. 6(A) (lanes 4–6), only the abnormal transcripts could be amplified from the patient and his two brothers, whereas only normal transcripts could be amplified from his parents and sister (Fig. 6A, lanes 1–3). The latter results are unexpected, as the father and sister both carry one mutated and one normal C{gamma}2 gene. To increase the sensitivity of detection, a second nested PCR was performed, using the primers IgG2-CH1s and IgG2-Hingeas. Still, however, no short transcripts could be amplified in samples from the patient's father and sister (Fig. 6B).



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Fig. 6. Detection of the splicing pattern of the C{gamma}2 gene in the patient's family. (A) The first round RT–PCR amplification using primers IgG2-CH1s-XhoI, IgG2-Hingeas-SalI. (B) The second round of RT–PCR amplification using primers IgG2-CH1s and IgG2-Hingeas, and the products of the first PCR reactions as templates. In both (A) and (B), 1, father; 2; mother; 3, sister; 4, patient; 5, first brother (donor); 6, second brother.

 
Sequence analysis, together with our previous data (8), showed that both the father and sister are heterozygous for the mutation. As the expressed IgG2 in the father appeared to be G2m(n+) and the mutated {gamma}2 allele encodes G2m(n–) (9), the father possesses both a normal G2m(n+) and a mutated G2m(n–) allele in his genome. This conclusion was also based on direct sequencing of the amplified genomic fragments from the father and sister. Consistent with serological typing of the IgG2 allotypes in the father, direct sequencing of the expressed {gamma}2 cDNA fragments from the father showed only the G2m(n+) encoding sequence. It again suggested that there is no mature mRNA transcript derived from the mutated C{gamma}2 copy in the father and sister, although germ-line transcription of the mutated C{gamma}2 gene in the father and sister could be still detected by nested PCR (data not shown).

Screening for the A->G mutation in different ethnic populations
To determine whether the A to G mutation can be found in the general population, a site-specific PCR approach was developed. Using the primers IgG2-cG and IgG2-Hingeas, only individuals with the G mutation would yield PCR products. No mutation was found in 102 Swedish, 92 Chinese and 96 Gambian DNA samples, nor in four additional samples from patients with selective IgG2 deficiency (data not shown), suggesting that the A to G transition is a rare mutation in the human population.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously reported a patient who developed a complete IgG2 deficiency after bone marrow transplantation using his IgG2 deficient brother as a donor (8). The patient's two IgG2 deficient brothers both possess a 50–70 kb deletion in their IGHC loci that was inherited from their mother, encompassing the genes encoding IgG2 and IgG4 (del G2-G4) (8), indicating that the remaining copy of the C{gamma}2 gene in their IGHC loci, inherited from their father, was silent. However, the underlying cause of the latter was previously unknown. In this study, we found that a point mutation in the intron between the CH1 and hinge exons caused the IgG2 deficiency in this family.

The point mutation identified in the single C{gamma}2 gene copy in lymphocytes from the patient was an A to G transition in the 5' splice site of the intron between the CH1 and hinge exons, changing the wild-type site TGgtgaga to TGgtggga, thus abrogating appropriate splicing. Instead, a potential splice site, AGgtggac, 16 bp upstream in the CH1 exon, was used to generate a shorter IgG2 heavy chain transcript. As shown in Fig. 4, the 16 bp deletion (GTGGACAAGACAGTTG) in the CH1 exon altered the reading frame of the sequences downstream, introducing a PTC in the CH2 exon, resulting in a nonfunctional protein. The shorter transcript could also be detected in vivo in the patient and his two brothers (Fig. 6). Obviously, the abnormal transcripts detected by RT–PCR in the patient and his two brothers should mainly be derived from germ-line {gamma}2 transcripts, as no, or only trace amounts of the mature VDJC containing transcripts could be detected (Fig. 1B and C). These data thus explain the IgG2 deficiency in the patient and his two brothers, as they only carry the mutant C{gamma}2 gene, implying that the IgG2 deficiency in the studied family was due to a structural defect in the C{gamma}2 gene itself.

The finding that {gamma}2 switch recombination occurred at the DNA level in the patient and his two brothers, consistent with the detected, albeit lower, germ-line transcription which is essential for class switch recombination, indicates that the mutant C{gamma}2 gene could potentially be transcribed. However, in the three IgG2 deficient individuals in the studied family, no, or very low, levels of such transcripts could be detected even by two rounds of RT–PCR amplification. mRNA harboring a PTC is usually subjected to a rapid degradation in eukarotyic cells due to RNA surveillance or nonsense-mediated decay (1820). This also applies to mammalian lymphocytes (2125) and nonsense-mediated RNA decay is involved in many human diseases (26,27). It is thus likely that the abnormal IgG2 transcripts were rapidly eliminated in these IgG2 deficient individuals and probably also in the father and sister.

A comparison of sequence of the C{gamma}2 gene with other three human IgG subclass genes shows that the potential splice site AGgtggac is present in the CH1 encoding exons of all four C{gamma} genes (28). It is thus likely that any mutation that abolishes or influences the 5' splice site of intron 1 may result in abnormally spliced mature RNA transcripts of the affected IgG subclass gene. This mechanism may therefore be involved in additional patients with selective IgG subclass deficiencies.


    Acknowledgements
 
The authors want to thank Professor Per Ljungman, Department of Hematology, Karolinska University Hospital Huddinge, Sweden, and the patient's family for the blood samples. We are also indebted to Dr Gerda G. de Lange, Red Cross Blood Transfusion Service, Amsterdam, The Netherlands, for allotyping of serum samples.


    Abbreviations
 
BMT   bone marrow transplantation
IGHC   immunoglobulin heavy chain constant region
PTC   premature translation termination codon

    Notes
 
Transmitting editor: A. Radbruch

Received 10 August 2004, accepted 29 October 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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