Immunodeficiency in Bloom’s Syndrome

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 [24]. 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 CD24^highCD38^highCD27⁻IgM⁺IgD⁺, naïve mature B cells as CD27⁻ CD24^dimCD38^dimIgM⁺IgD⁺, natural effector B cells as CD27⁺IgD⁺CD24^dimCD38^dim, and memory B cells as CD27⁺ IgD⁻CD24^dimCD38^dim. 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⁻.

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 [25] and either the CgCH1 [26] or the IGHA [27] reverse primer. The PCR products were purified and sequenced using Roche 454 sequencing as previously described [28]. 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 [29] (https://​bioinf-galaxian.​erasmusmc.​nl/​argalaxy). Fasta files were uploaded in IMGT/High-V-Quest [30], and subsequently the IMGT output files were analyzed in ARGalaxy [29]. 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) [31] 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).

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 [33].

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 [34].

Patient characteristics are shown in Table 1. We describe three children (9–12 years) and three adult BS patients (36–49 years). The adult BS patient P4 (SuS or SuSc), P5 (MP or MaPa), and P6 (GC) have been described previously when they were in their childhood [35, 36]. All patients have a low birth weight, and birth length indicating a growth deficiency. The characteristic butterfly-shaped erythema was present in all patients. Recurrent ear infections were present, which is a well-known phenomenon in BS [1]. Two adult patients had a double solid malignancy, both were treated with success. Patient 5 was diagnosed with a monoclonal gammopathy of unknown significance (MGUS), which did not deteriorate in a multiple myeloma so far. In addition, patients suffered from different infections like bronchitis, uveitis, and two out of five patients had pneumonia. However, the patients in this cohort did not have opportunistic infections, sepsis or other life-threatening manifestations, or a serious failure of the immune system.

To elucidate the effect of BLM deficiency on the lymphocyte development, we analyzed the lymphocyte subsets using flow cytometry. We compared the absolute numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells with previously published data from 71 healthy children (5–16 years of age) and 22 healthy adults [37]. The absolute numbers of B- and NK cells in the BS patients were in the range of the healthy controls (HC) (Fig. 1a). The absolute number of T cells was significantly lower in the BS children compared to HC, and low in the adult BS patients (Fig. 1b). The CD4+ T cells were significantly reduced in all BS patients, while the CD8+ T cells were low but in the normal range. The absolute number of CD4+ and CD8+ naïve, effector memory (Tem), and central memory (Tcm) populations were reduced compared to the HC (Fig. 1c). However, the distribution of the naïve, Tem, and Tcm populations was relatively normal in most of the BS patients, except for P4 who had a strong decrease in CD4+ and CD8+ naïve T cells (Fig. 1c). Together, these data showed that BS patients had subnormal levels of T cells.

While the total number of B cells were in the normal range, the absolute number of transitional and naïve mature B cells was normal or slightly increased, and natural effector B cells and memory B cells decreased compared to HC (Fig. 2a). Since this suggests that B cell maturation might be impaired in the final stages of differentiation, we analyzed the levels of serum immunoglobulins over the past decades (0–40 years of age). The IgM, IgG, and IgA levels were all low but mostly at the border of the normal range during the years (Fig. 2b). Only P5 has a relative peak in both IgG, IgM, and IgA at 31 years of age, which is unexplained so far (Fig. 2b) The low concentration of total IgG was not caused by a decrease in one of the IgG subclasses, since all the serum IgG subclasses were at the lower border of the normal range (Supplemental Fig. 1).

Since BLM is involved in BER and has been described to stimulate DNA syntheses by pol eta, we analyzed the frequency and repair patterns of SHM in switched B cells. We used next-generation sequencing to sequence IGHG and IGHA transcripts from the six BS patients and compared the data to age-matched controls (see Supplemental Table 1 for overview for the number of sequences used for the analysis). The median frequency of SHM in both IGHG and IGHA transcripts was in the normal range of the age-matched healthy controls (Fig. 3a). Subsequently, we studied the location and repair patterns of the SHM, which provide information about the repair pathway that is used to introduce the mutation during SHM. Similar to the healthy controls, around 35% of the SHM were located in RGWY/WRCY (AID) and around 20% in WA/TW (pol eta) motif, indicating that the targeting of SHM is not aberrant in the BS patients. Then we investigated the repair patterns of the SHM in more detail. U:G mismatched resolved by the MMR pathways lead to mutations at A/T location, and BER is the dominant pathway for transversion mutations at G/C locations. In the BS patients, 39–47% of the mutations were located in A/T locations, which is comparable to the HC. Moreover, the percentages of transitions and transversions at G/C location in the BS patients were also within the normal range. This indicated that the targeting and repair mechanisms in SHM are not affected by the BLM deficiency.

Besides V(D)J recombination and SHM, DNA repair is also crucially important for CSR. We determined the percentages of rearrangements having a certain Cg or Ca constant gene. For both children and adult BS patients, the frequency of IGHG transcripts with Cg1 and Cg3 constant genes were increased compared to age-matched controls. These data suggests that switching to the downstream Cg2 and Cg4 constant genes is reduced in the BS patients. The frequency of Ca1 and Ca2 constant genes was normal in the BS children, but the adults have a slightly higher frequency of Ca2 (Fig. 4).

Since these data indicated that CSR might be hampered in BS patients, we analyzed the repair of the switch junction to determine if BLM has a role in CSR. During CSR, AID deaminates multiple cytosines in the switch regions upstream of the constant genes. Since the switch regions contain many repeats, the DSBs are often repaired using small homologous region (microhomology). The BS patients had a significant increase of 7–9 microhomology in the switch junction; however, the average microhomology use was not different in the BS patients we analyzed (Table 2).

Previous studies have shown that the antigen-experienced B cells have shorter CDR3 length and express less often a rearrangement with the IGHV4-34 gene compared to naïve B cells [38, 39]. Similar to HC, the CDR3 length was shorter and the IGHV4-34 usage was reduced in the IGHG and IGHA transcripts derived from the BS patients compared to IGH rearrangements derived from naïve B cells from HC (Fig. 5a and b). In addition, to these changes, BCR rearrangements from antigen-experienced B cells are selected for replacement mutations in the CDR3 region, since they can increase the affinity for their antigen, and against replacement mutations in the FR regions since they can negatively influence the stability of the antibody protein. Therefore, we compared the replacement/silent (R/S) mutation ratio in the IGHG and IGHA transcripts of the BS patients with healthy controls. The R/S ratio seemed lower in the IGHG transcripts but in the normal range in the IGHA transcripts (Fig. 5c). Additionally, we also used the Immunoglobulin Analysis tool (IgAT) which calculates for each rearrangement whether it has undergone antigen selection based on the number of replacement mutations in relation to the total number of mutations. The BS patients had a lower frequency of antigen-selected sequences compared to the age-matched controls, which was significant in the adult BS patients (Fig. 5d). These data suggest that antigenic selection of the B cells is affected by the BLM deficiency.

The data obtained from the IGHG and IGHA transcripts showed that BS patients have small changes in the subclass distribution (Fig. 4) and antigen selection (Fig. 5). To see if the BS patients deviate from the HC, we performed a principal component analysis (PCA) on all the data obtained from the IGHG and IGHG transcripts as displayed in Figs. 3, 4, and 5. This analysis showed that the HC cluster together and that the BS patients indeed separate from the HC (Fig. 6a). The contribution of each parameter of principal component 1 (PCA1) and PCA2 are shown in Fig. 6b. So although we did not find major differences in the B cell repertoire of the BS patients, their repertoire characteristics are clearly deviate from HC.