Ligase-4 Deficiency Causes Distinctive Immune Abnormalities in Asymptomatic Individuals

Abstract

Purpose: DNA Ligase 4 (LIG4) is a key factor in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway needed for V(D)J recombination and the generation of the T cell receptor and immunoglobulin molecules. Defects in LIG4 result in a variable syndrome of growth retardation, pancytopenia, combined immunodeficiency, cellular radiosensitivity, and developmental delay.

Methods: We diagnosed a patient with LIG4 syndrome by radiosensitivity testing on peripheral blood cells, and established that two of her four healthy siblings carried the same compound heterozygous LIG4 mutations. An extensive analysis of the immune phenotype, cellular radiosensitivity, telomere length, and T and B cell antigen receptor repertoire was performed in all siblings.

Results: In the three genotypically affected individuals, variable severities of radiosensitivity, alterations of T and B cell counts with an increased percentage of memory cells, and hypogammaglobulinemia, were noticed. Analysis of T and B cell antigen receptor repertoires demonstrated increased usage of alternative microhomology-mediated end-joining (MHMEJ) repair, leading to diminished N nucleotide addition and shorter CDR3 length. However, overall repertoire diversity was preserved.

Conclusions: We demonstrate that LIG4 syndrome presents with high clinical variability even within the same family, and that distinctive immunologic abnormalities may be observed also in yet asymptomatic individuals.

Keywords: DNA repair; Ligase 4 deficiency; immune repertoire; microhomology-mediated end-joining; telomere length.

Fig. 1. Two compound heterozygous LIG4 mutations are found in the index patient and two of her siblings

Diagram of the pedigree and annotation of LIG4 genotypes. The mutation c.1345A>C (K449Q) was found on one allele in the father, and c.2440C>T (R814X) in the mother. Both mutations were inherited by siblings P1, P2, and P3. III.3 is heterozygous for the c.2440C>T mutation. The symbol “+” denotes the wild type allele.

Fig. 2. Increased sensitivity to ionizing radiation (IR) and short telomere length in genotypically affected siblings

Fibroblasts of P2 were irradiated with 5Gy and γH2AX foci were counted in at least 50 cells using a Fluoview FV 1000 confocal microscope (Olympus) with an UplanApo 60x/1.2na water lens (Olympus) at given time points after IR. Numbers of γH2AX foci/cell are compared to the established LIG4-deficient cell line 411BR [11] and a healthy control (a). PBMCs from patients P1-P3 and two controls C1-C2 (healthy siblings III.2 and III.3) were irradiated with 10Gy, and γH2AX was assessed by flow-cytometry. The γH2AX fold induction was calculated by dividing the mean fluorescence intensities (MFI) of unirradiated cells by the MFI of irradiated cells of each time point (* p≤0.05) (b). Telomere length in lymphocytes (c) and granulocytes (d) is expressed relative to healthy age-matched controls. The colored lines represent percentile thresholds. In two controls, granulocyte event numbers were too low to measure the telomere length with confidence.

Fig. 3. Evaluation of TRB and IGH repertoire diversity shows modest clonal expansions

The Shannon Entropy index (H) was used to measure repertoire diversity in unique TRB (a) and IGH (b) sequences in each patient and controls. Groups of 3 controls and 3 patients (P1-P3) are shown, black bars indicate mean values, colored bars the SD. The top 100 most abundant unique TRB (c) and IGH (d) clones were considered, and their frequency was reported as referred to the cumulative number of sequences in the whole data set. Horizontal bars indicate mean value (***, p≤0.001). The cumulative frequencies of total TRB (e) and IGH (f) sequences are plotted against the cumulative frequencies of unique sequences in controls (C1-C3) and patients (P1-P3).

Fig. 4. The TRB repertoire of LIG4-deficient patients P1-P2 shows minimal skewing

The global usage of unique TRB sequences is demonstrated for 3 healthy controls and P1-P3 on a heat map showing TCRBV genes joint with TCRBJ genes. The frequency of gene usage is color-coded, using yellow and red to indicate rare and most common pairing of V-J genes, respectively. Blank boxes indicate absence of that specific rearrangement in the total population of sequences obtained.

Fig. 5. Shorter length of the CDR3 regions in LIG4-deficient individuals

The relative frequency with which CDR3 sequences of indicated length (expressed as number of nucleotides, nt) occur is expressed as percentage of total (a) and unique (b) TRB CDR3 sequences, and total (c) and unique (d) IGH CDR3 sequences for P1-P2, as compared to mean control values. Error bars on control columns indicate SD.

Fig. 6. Sequences obtained from LIG4-deficient individuals show significantly reduced N nucleotide addition between joined V(D)J gene elements

The percentage of unique sequences with indicated numbers of N nucleotides added are shown for TRB (a, b) and IGH (c, d). N1 nucleotides are added between V and D or V and J gene elements, and N2 nucleotides between D and J elements. No N nucleotide addition is indicated as 0, each 3 additional N nucleotides added are shown in one bar up to more than 16 (16+) N nucleotides; error bars indicate SD (* p≤0.05; ** p≤0.01).

Fig. 7. MHMEJ of V(D)J elements is more frequently used in LIG4-deficient patients than in controls

MHMEJ was found in joins of V-D and D-J elements of TRB sequences (a). The diagram shows the frequency of specific joins mediated by MH, expressed as percentage of all unique sequences. Corresponding sequences found in the joint region are shown below. Some joins of TRBV5.1, TRBV5.4, TRBV5.6 with TRBD1 had 1nt deletion. In contrast, MHMEJ was found only in three joins of the IGH locus, and almost exclusively in P2 (b). Some sequences had a 1nt insertion (indicated in blue) in the corresponding J segment.