Immunodeficiency in Bloom’s Syndrome
Bloom’s syndrome (BS) is an autosomal recessive disease, caused by mutations in the BLM gene. This gene codes for BLM protein, which is a helicase involved in DNA repair. DNA repair is especially important for the development and maturation of the T and B cells. Since BLM is involved in DNA repair, we aimed to study if BLM deficiency affects T and B cell development and especially somatic hypermutation (SHM) and class switch recombination (CSR) processes. Clinical data of six BS patients was collected, and immunoglobulin serum levels were measured at different time points. In addition, we performed immune phenotyping of the B and T cells and analyzed the SHM and CSR in detail by analyzing IGHA and IGHG transcripts using next-generation sequencing. The serum immunoglobulin levels were relatively low, and patients had an increased number of infections. The absolute number of T, B, and NK cells were low but still in the normal range. Remarkably, all BS patients studied had a high percentage (20–80%) of CD4+ and CD8+ effector memory T cells. The process of SHM seems normal; however, the Ig subclass distribution was not normal, since the BS patients had more IGHG1 and IGHG3 transcripts. In conclusion, BS patients have low number of lymphocytes, but the immunodeficiency seems relatively mild since they have no severe or opportunistic infections. Most changes in the B cell development were seen in the CSR process; however, further studies are necessary to elucidate the exact role of BLM in CSR.
KeywordsImmunodeficiency bloom’s syndrome lymphocyte DNA repair somatic hypermutations class switch recombination
Bloom’s syndrome (BS) is an autosomal recessive disease, caused by mutations in the BLM gene located at 15q26. This gene codes for BLM protein, which is a DNA helicase involved in DNA replication and repair. BS is characterized by predisposition to malignancy, prenatal growth retardation, gastro-esophageal reflux, café-au-lait spots, characteristic butterfly-shaped erythema, and immunodeficiency [1, 2]. The immunodeficiency is characterized by low serum immunoglobulins and different infections. Otitis media is a very common infection among BS patients, especially in children. Also, up to 20% of the BS patients had pneumonia.  However, the pathophysiology behind the immunodeficiency in BS has not yet been elucidated.
It is known that DNA repair defects can result in immunodeficiency since DNA repair is essential for the development of antigen receptors expressed on B and T lymphocytes. These antigen receptors are formed by recombination of the variable (V), diversity (D), and joining (J) genes on the antigen receptor loci. During this V(D)J recombination process, DNA double-strand breaks (DSBs) are introduced near the V, D, and J genes; the DNA ends are processed and eventually ligated by the non-homologous end-joining (NHEJ) DNA repair pathway . When T and B cells express a functional antigen receptor, also called a T cell receptor (TR) and B cell receptor (BCR), they can migrate to the periphery where they can encounter an antigen . After antigen encounter, B cells can further divaricate their BCR by introducing somatic hypermutations (SHM) or by class switch recombination (CSR). SHM increase the affinity of the BCR for their antigen, and CSR changes the effector function of the secreted BCR, also called immunoglobulin or antibody. Both SHM and CSR rely on DNA repair. SHM is initiated by AID, which deaminates cytosine (C) into uracils (U), creating a mismatch with the guanine (G) on the complementary strand [5, 6]. These U:G mismatches can be resolved using three different pathways: base excision repair (BER), mismatch repair (MMR), or replication. During BER, the U is removed and replaced by a random nucleotide by an error-prone polymerase, resulting in transition and transversion mutations at G/C bases [7, 8]. Mutations at A/T bases can occur via the MMR pathway when multiple bases surrounding the U:G mismatch are removed and filled by the error-prone polymerase eta, which introduces errors at A/T pairs specifically at WA/TW motifs [9, 10, 11]. Finally, if the U:G mismatch is not repaired before the DNA is replicated, the U will be recognized as a thymine (T), resulting in a C to T transition mutation. During CSR, AID introduces U:G mismatches in the switch regions upstream of the constant genes in the IGH locus . Subsequently, proteins of the BER pathway introduce DNA DSBs, which can be repaired by NHEJ or by alternative end-joining (A-EJ) [13, 14, 15, 16, 17].
BLM is described to have a role in at least two DNA repair pathways involved in lymphocyte development: alternative end-joining and BER [18, 19, 20]. In addition, BLM can also stimulate DNA synthesis by pol eta . So far, there is no active role discovered for BLM during V(D)J recombination . In mice, there is a reduced CSR capacity and a shift to microhomology-mediated switch junction formation .
The aim of this study is to give insight of the immunodeficiency in BS and to discover the role of BLM in CSR and SHM in humans.
Cell Samples and Flow Cytometric Immunophenotyping
Peripheral blood samples and clinical data were collected from six patients with BS with informed consent and according to the guidelines of the Medical Ethics Committee of the Radboud University Nijmegen medical Center and Erasmus MC Rotterdam. Flow cytometric analysis of peripheral blood for the healthy controls and patient 5 was performed using 6-color labeling as previously described . For patient 1–4 and patient 6, an 8-color protocol was used with the following antibodies: CD24-PB (SN3) (Exbio, Vestec, Czech Republic), CD45-PO (HI30) (Invitrogen), IgD-FITC and IgM-PE (Southern Biotechnologies, Birmingham, AL), IgA FITC (IS1 1-8E10) (Mitenyl Biotech, Bergisch Gladbach, Germany), IgD-biotine (IA6-2) (Biolegend, San Diego, CA), CD45RO-FITC (UCHL1) (DAKO, Agilent Technologies, Glostrup, Denmark), CCR7-PE (Miltenyi Biotech), CD28-PE-Cy7 (CD28.2; e-Bioscience), CD19-PerCP-Cy5 (SJ25C1), CD27-ApC (LL128), CD38-APC-H7 (HB7), IgG-PE (G18-145), CD4-PB (RPA-T4), CD3-PerCP (SK7), and CD8-APC-H7 (SK1) (all from BD Biosciences, CA, USA). The absolute numbers were calculated BD Trucount™ tubes (BD Bioscience). The following CD19+ B cell subsets were defined: transitional B cells as CD24highCD38highCD27−IgM+IgD+, naïve mature B cells as CD27− CD24dimCD38dimIgM+IgD+, natural effector B cells as CD27+IgD+CD24dimCD38dim, and memory B cells as CD27+ IgD−CD24dimCD38dim. The following CD3+ T cell subsets were defined: CD8+ naïve T cells as CD8+CD45RO−CCR7+CD27+CD28+, CD8+ central memory T cells as CD8+CD45RO+CCR7+CD27+CD28+, CD8+ effector memory T cells as CD8+CCR7−. CD4+ naïve T cells as CD8−CD45RO−CCR7+CD27+CD28+, CD4+ central memory T cells as CD8−CD45RO+CCR7+CD27+CD28+, and CD4+ effector memory T cells as CD8−CCR7−.
Repertoire Analysis of IGH Transcripts Using Next-Generation Sequencing
PBMC’s were isolated from peripheral blood or cord blood samples using Ficoll. mRNA was isolated using the Gen-Elute Mammalian total RNA miniprep kit from Sigma-Aldrich (St. Louis, MO). cDNA was created from 2 μg RNA using the Superscript II reverse transcriptase kit from Invitrogen (Paisley, UK). IGH rearrangements were amplified in a multiplex PCR using the forward VH1-6 FR1 (BIOMED-2) primers  and either the CgCH1  or the IGHA  reverse primer. The PCR products were purified and sequenced using Roche 454 sequencing as previously described . In short, PCR products were purified by gel extraction (Qiagen, Valencia, CA) and Agencourt AMPure XP beads (Beckman Coulter, Fullerton, CA). Subsequently, the PCR concentration was measured using the Quant-it Picogreen dsDNA assay (Invitrogen, Carlsbad, CA). The purified PCR products were sequenced on the 454 GS junior instrument according the manufacturer’s recommendations. Sequences were demultiplexed based on their multiplex identifier sequence and 40 nucleotides trimmed from both sides to remove the primer sequence using ARGalaxy  (https://bioinf-galaxian.erasmusmc.nl/argalaxy). Fasta files were uploaded in IMGT/High-V-Quest , and subsequently the IMGT output files were analyzed in ARGalaxy . Only productive sequences that were complete, without ambiguous bases, present twice or more, in which a C subclass could be defined, were included once in the analysis. All information on the FR1 region was excluded from the analysis since the forward primers used to amplify the transcripts were located in FR1. The age and number of sequences used for the analysis are listed in Supplemental Table 1. The percentage SHM was calculated per sequence by dividing the number of mutations in the CDR1-FR3 region by the number of nucleotides in the CDR1-FR3 region. The CDR3 region was excluded from the SHM analysis since it is not possible to distinguish true somatic mutations from N-nucleotides. In addition, the Immunoglobulin analysis tool (IgAT)  was used to determine the percentage of antigen-selected sequences. The data from P1 to P3 were compared to data obtained from six healthy children (7–15 years) and data from P4 to P6 were compared to ten healthy adults (31–55 years).
Characterization of Switch Recombination Junctions
The Sμ-Sα recombination fragments were PCR amplified from DNA derived from peripheral blood cells and sequenced as previously described [16, 32]. The pattern of CSR junctions was analyzed according to guidelines .
Principal Component Analysis
Z-scores were calculated by the sample score minus the mean of the controls divided by the standard deviation of the controls. Thereby, the mean and standard deviation of either the controls between 7 and 15 years of age (n = 6) or the adult controls (n = 10) were used. Since we had missing data for either IGHG or IGHA in three controls, we did not plot these controls in the graph. This resulted in 32 parameters for 13 controls and 6 BS patients. Subsequently, components and contributions were calculated and plotted using prcomp in R .
Age at analysis
Recurrent ENT infections
Growth hormone replacement therapy
Squamous cell carcinoma and colon carcinoma
Adenocarcinoma of the bowel
Monoclonal gammopathy of unknown significance (MGUS)
Polyp during colonoscopy
BS Patients Have Subnormal Numbers of Lymphocytes
Decreased Memory B Cells and Subnormal Levels of Immunoglobulins
Frequency and Repair of SHM Is Normal in the BS Patients
Class Switching to more Downstream Constant Regions Is Reduced in BS
Characterization of Sμ-Sα junctions
Total no. of junctions
P1 (9 years)
P2 (12 years)
P4 (35 years)
P5 (37 years)
P6 (46 years)
All BS patients
Selection of the BCR Repertoire Is Subnormal in BS Patients
BS Patients Cluster Differently from Healthy Controls
Immunodeficiency is one of the characteristics of BS; however, the immunodeficiency is not well described and the underlying mechanism is not clear. A long-term follow-up of two BS patients showed repeated prolonged middle-ear infections and upper respiratory tract infections several times per year from 2 till 14 years of age; after which, the frequency of infections decreased . In our cohort, two patients suffered from pneumonia, which is a common infection among BS patients . However, most other infections were minor and did react well on antibiotics, and no uncommon infections were noticed. So, although the infections are not severe, infections are more common in BS patients compared to healthy controls . Therefore, the question remains what causes this increased susceptibility for infections in these patients.
The longitudinal data of our cohort showed subnormal levels of serum immunoglobulin levels, especially for IgG and IgM. This decrease in serum immunoglobulin levels can either be explained by an intrinsic B cell defect or by a defect in stimulation of B cells by CD4+ T cells. In this cohort, the naïve B cell subsets were in the normal range, but the absolute numbers of natural effector B cells and memory B cells were significantly lower than the age-matched controls, which might suggest a maturation defect. Additionally, the CD4+ T cells were decreased. Combined, these two factors might contribute to increased numbers of infections observed in the BS patients.
BLM is involved in DNA repair and may have a role in the DNA repair-dependent processes during lymphoid development. Previous studies have shown that BLM does not have a role in V(D)J recombination [23, 41, 42], and we also did not observe differences in V, D, and J gene usage, and the number of deletions, palindromic (P)-nucleotides, and non-templated (N)-nucleotides (data not shown). The role of BLM in SHM has not been studied extensively. A study in a small number of rearrangements (12 IGHV sequences obtained from two BS patients) showed normal frequency of SHM and distribution of transition and transversion mutations in the BS patients . Based on these results, Sack et al. conclude that de immunoglobulin hypermutation is normal in BS and that BLM plays no significant role in the process of SHM. In this study, we showed in a large number of rearrangements obtained from six BS patients low but normal frequency of SHM and normal SHM patterns. Together, these data suggest that BLM is not essential for SHM.
In mice models, there is no crucial role for BLM in the mechanism of CSR identified . In this study, we showed that the memory B cells were low in three of the six BS patients, and the serum immunoglobulins levels were subnormal. The IGHG transcripts in humans showed particularly Cg3 and Cg1 gene usage with an increased use of short microhomology in the switch regions. This suggests that B cells in BS patients switch less to the more downstream Cg2 and Cg4 constant genes. Switching to these more downstream constant genes happens during the course of an immune response and requires CD4+ T cell stimulation [44, 45]. This indicates that the process of class switch recombination is disturbed in BS patients, which can either be caused by the low CD4+ T cells or an intrinsic B cell defect. Previous studies have shown that lymphocytes of Blm-deficient mice and BS patients (including P4, P5, and P6) have reduced proliferation capacity, which likely also contribute to reduced switching to the more downstream constant genes since these processes are dependent on B cell proliferation [23, 35, 36, 46, 47].
However, the immunodeficiency cannot be fully explained by an impaired CSR, because IgM is also produced at subnormal level, which is independent of CSR.
T cells in the BS patients were low in absolute numbers and percentages, for both CD4+ and CD8+. It is most likely that the process is already disturbed in the thymus since all T cell subsets are reduced compared to the HC. This suggests a possible role of BLM in the development of T cells. This was also earlier demonstrated in mice models, where mice with a conditional knockout of Blm in the T cell lineage have severely reduced thymocyte numbers . The reaction of human T cells on phytohemagglutinin lymphocyte stimulation is disturbed and also stimulation by pokeweed mitogen is mostly decreased, which is an indication of less growth of B and T cells [35, 36]. Our results showed a relative increase of effector memory T cells compared to naïve and central memory T cells. So far, we can conclude that there is a disturbance in the development of T cells, which results in lower T cell levels, as is shown in our patient series. This reduced number of T cells could explain the low number of immunoglobulins and memory B cells, as CD4+ T cells stimulate SHM, CSR, and B cells to produce immunoglobulins.
We showed BS patients suffer from relatively mild infections, which might be explained by the subnormal level of T, B, and NK cells and immunoglobulins. The B cell repertoire data on SHM, subclass distribution, and antigen selection of the B cells showed that the BS patients did not have great differences, but they clearly deviated from HC. Most importantly, despite the multiple disruptions, the immunodeficiency has a relatively mild character and functions in the laboratory and clinically at an acceptable level.
We would like to thank Dr. Mirjam van der Burg for critically reading the manuscript, Andrea Björkman and Likun Du for analyzing the switch regions, and Anne Bras for help with the principal component analysis.
MS, CW, and HIJ wrote designed research and wrote the paper. JZ, SP, IP, PH, and HIJ performed experiments and analyzed data. SH, CW, MvD provided patient material, collected clinical data, and critically read the manuscript.
Compliance with Ethical Standards
Conflict of Interest
The authors declare no conflict of interest.
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