Deconstructing common variable immunodeficiency by genetic analysis

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency. Patients have recurrent bacterial infections and an increased risk of developing autoimmune diseases, lung damage, and selected cancers. Since 2003, four genes have been shown to be mutated in CVID patients: ICOS, TNFRSF13B (encoding TACI), TNFRSF13C (encoding BAFF-R) and CD19. Heterozygous mutations in TNFRSF13B are also associated with CVID, whereas the other three genes are purely recessive. Recent genetic linkage studies have also identified possible loci for dominant CVID genes on chromosomes 4q, 5p and 16q. These findings markedly improved the genetic diagnosis of CVID and point towards new strategies for future genetic studies. In addition, some CVID genes might be relevant to more common diseases such as asthma and stroke.     

Immunoglobulin class-switch recombination deficiencies

Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.     

The genetics of the hereditary xeroderma pigmentosum syndrome

All living organisms are constantly exposed to endogenous or exogenous agents that can cause damage to the genomic DNA, leading to the loss of stable genetic information. Fortunately, all cells are equipped with numerous classes of DNA repair pathways which are able to correct many kinds of DNA damage such as bulky adducts, oxidative lesions, single- and double-strand breaks and mismah.The importance of these DNA repair processes is attested by the existence of several rare but dramatic hereditary diseases caused by defects in one of their repair pathways. These diseases are usually associated with early onset of malignancies confirming the direct relationship between unrepaired DNA lesions, mutations or chromosomal modifications and cancer incidence. Among these hereditary diseases the UV-hypersensitive ones have been particularly well studied and the xeroderma pigmentosum (XP) is probably the best known syndrome up to now in terms of genetics and biochemistry.     

DNA single-strand breaks and neurodegeneration

The association of human genetic disorders with defects in the DNA damage response is well established. Most of the major DNA repair pathways are represented by diseases in which that pathway is absent or impaired, including those responsible for repairing DNA double-strand breaks. Conspicuous by their absence, however, have been human disorders associated with defects in the repair or response to DNA single-strand breaks (SSBs). However, three papers have recently associated hereditary spinocerebellar ataxia with mutations in genes connected with SSBR. The emerging links between SSBR and neurodegeneration are discussed.     

Age-dependent usage of double-strand-break repair pathways

A DNA double-strand break (DSB) can be repaired by any of several alternative and competing mechanisms. The repaired sequences often differ from the original depending on which mechanism was used so that the cell's "choice" of repair mechanism can have profound genetic consequences. DSBs can accumulate with age , and human diseases that mimic some of the effects of aging, such as increased susceptibility to cancer, are associated with certain defects in DSB repair . The premeiotic germ cells of Drosophila provide a useful model for exploration of the connection between aging and DNA repair because these cells are subject to mortality and other age-related changes , and their DNA repair process is easily quantified. We used Rr3, a repair reporter system in Drosophila, to show that the relative usage of DSB repair mechanisms can change substantially as an organism ages. Homologous repair increased linearly in the male germline from 14% in young individuals to more than 60% in old ones, whereas two other pathways showed a corresponding decrease. Furthermore, the proportion of longer conversion tracts (>156 bp) also increased nearly 2-fold as the flies aged. These findings are relevant to the more general question of how DNA damage and repair are related to aging.     

Unique DNA Repair Gene Variations and Potential Associations with the Primary Antibody Deficiency Syndromes IgAD and CVID

Background

Despite considerable effort, the genetic factors responsible for >90% of the antibody deficiency syndromes IgAD and CVID remain elusive. To produce a functionally diverse antibody repertoire B lymphocytes undergo class switch recombination. This process is initiated by AID-catalyzed deamination of cytidine to uridine in switch region DNA. Subsequently, these residues are recognized by the uracil excision enzyme UNG2 or the mismatch repair proteins MutSα (MSH2/MSH6) and MutLα (PMS2/MLH1). Further processing by ubiquitous DNA repair factors is thought to introduce DNA breaks, ultimately leading to class switch recombination and expression of a different antibody isotype.

Methodology/Principal Findings

Defects in AID and UNG2 have been shown to result in the primary immunodeficiency hyper-IgM syndrome, leading us to hypothesize that additional, potentially more subtle, DNA repair gene variations may underlie the clinically related antibody deficiencies syndromes IgAD and CVID. In a survey of twenty-seven candidate DNA metabolism genes, markers in MSH2, RAD50, and RAD52 were associated with IgAD/CVID, prompting further investigation into these pathways. Resequencing identified four rare, non-synonymous alleles associated with IgAD/CVID, two in MLH1, one in RAD50, and one in NBS1. One IgAD patient carried heterozygous non-synonymous mutations in MLH1, MSH2, and NBS1. Functional studies revealed that one of the identified mutations, a premature RAD50 stop codon (Q372X), confers increased sensitivity to ionizing radiation.

Conclusions

Our results are consistent with a class switch recombination model in which AID-catalyzed uridines are processed by multiple DNA repair pathways. Genetic defects in these DNA repair pathways may contribute to IgAD and CVID.     

Human MSH6 deficiency is associated with impaired antibody maturation

Ig class-switch recombination (Ig-CSR) deficiencies are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect, defective Ig-CSR may also be associated with impaired somatic hypermutation (SHM) of the Ig V regions. Although the mechanisms underlying Ig-CSR and SHM in humans have been revealed (at least in part) by studying natural mutants, the role of mismatch repair in this process has not been fully elucidated. We studied in vivo and in vitro Ab maturation in eight MSH6-deficient patients. The skewed SHM pattern strongly suggests that MSH6 is involved in the human SHM process. Ig-CSR was found to be partially defective in vivo and markedly impaired in vitro. The resolution of γH2AX foci following irradiation of MSH6-deficient B cell lines was also found to be impaired. These data suggest that in human CSR, MSH6 is involved in both the induction and repair of DNA double-strand breaks in switch regions.     

Common variable immunodeficiency: the immune system in chaos

Common variable immunodeficiency (CVID) is a heterogeneous disorder that is associated with low serum-immunoglobulin concentrations, defective specific-antibody production and an increased susceptibility to bacterial infections of the respiratory and gastrointestinal tracts. In spite of the identification of genes that are associated with several known primary immunodeficiencies, the basic immunologic and molecular defects of the majority of patients with CVID have remained obscure. Most of the studies aimed at understanding the immunopathogenesis of CVID suggest that this condition is primarily a T-cell disorder, although renewed attention on the genetic linkage and haplotype analysis in families of patients with CVID and on the role of dendritic cells and B cells has revealed several interesting features. This new information should assist in understanding the pathogenesis of CVID and improving the therapeutic strategies.     

Assessing somatic hypermutation in Ramos B cells after overexpression or knockdown of specific genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination. Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase η). However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes. Ramos - a Burkitt lymphoma cell line that constitutively undergoes SHM - has been a popular cell-line model to study SHM. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).     

Decreased DNA repair in familial Alzheimer's disease

Alterations in the capacity of a cell to repair DNA lesions play an important role in a number of human diseases. We and others have demonstrated defective DNA repair of alkylation damage in cells from patients with Alzheimer's disease. It has been hypothesized that this defect is related to the cause of Alzheimer's disease and results in the accumulation of lesions in the central nervous system neurons. One prediction of this hypothesis is that in dominantly inherited Alzheimer's disease, the repair defect will be present in half of the offspring of affected patients long before they develop symptoms of the disease. In order to test the hypothesis that decreased DNA repair is responsible for familial Alzheimer's disease and their at-risk offspring we have studied DNA repair in these individuals after exposure of lymphoblasts to alkylating agents. Our results indicate that cell lines from affected patients repair significantly less damage in 3 h than cell lines from healthy controls. A small number of at-risk individuals were also studied and some of these had lower levels of repair, although more cell lines from individuals in this group must be studied. These findings provide further support for defective DNA repair playing a role in the pathogenesis of Alzheimer's disease.     

A role for Mlh3 in somatic hypermutation

Somatic hypermutation (SHM) and class switch recombination (CSR) allow B cells to make high affinity antibodies of various isotypes. Both processes are initiated by activation-induced cytidine deaminase (AID) to generate dG:dU mismatches in the immunoglobulin genes that are resolved differently in SHM and CSR to introduce point mutations and recombination, respectively. The MutL homolog MLH3 has been implicated in meiosis and DNA mismatch repair (MMR). Since it interacts with MLH1, which plays a role in SHM and CSR, we examined these processes in Mlh3-deficient mice. Although deficiencies in other MMR proteins result in defects in SHM, Mlh3(-/-) mice exhibited an increased frequency of mutations in their immunoglobulin variable regions, compared to wild type littermates. Alterations of mutation spectra were observed in the Jh4 flanking region in Mlh3(-/-) mice. Nevertheless, Mlh3(-/-) mice were able to switch to IgG3 or IgG1 with similar frequencies to control mice. This is the first instance where a loss of a DNA repair protein has a positive impact on the rate of SHM, suggesting that Mlh3 normally inhibits the accumulation of mutations in SHM.     

The "Dutch DNA Repair Group", in retrospect

The "Dutch DNA Repair Group" was established about 35 years ago. In this brief historical review some of the crucial decisions are described that have contributed to the relative success of the research of this group. The emphasis of the work of this group has been for many years on the genetic analysis of nucleotide excision repair (NER) and genetic diseases based on defects in this repair process: xeroderma pigmentosum (XP), Cockayne syndrome and trichothiodystrophy.     

A new pathway that regulates 53BP1 stability implicates cathepsin L and vitamin D in DNA repair

Genomic instability due to telomere dysfunction and defective repair of DNA double-strand breaks (DSBs) is an underlying cause of ageing-related diseases. 53BP1 is a key factor in DNA DSBs repair and its deficiency is associated with genomic instability and cancer progression. Here, we uncover a novel pathway regulating the stability of 53BP1. We demonstrate an unprecedented role for the cysteine protease Cathepsin L (CTSL) in the degradation of 53BP1. Overexpression of CTSL in wild-type fibroblasts leads to decreased 53BP1 protein levels and changes in its cellular distribution, resulting in defective repair of DNA DSBs. Importantly, we show that the defects in DNA repair associated with 53BP1 deficiency upon loss of A-type lamins are due to upregulation of CTSL. Furthermore, we demonstrate that treatment with vitamin D stabilizes 53BP1 and promotes DNA DSBs repair via inhibition of CTSL, providing an as yet unsuspected link between vitamin D action and DNA repair. Given that CTSL upregulation is a hallmark of cancer and progeria, regulation of this pathway could be of great therapeutic significance for these diseases.     

Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication.     

Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitro DNA repair assay and an Escherichia coli readout system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.     

Chromosomal radiosensitivity in common variable immune deficiency

From more than 500 tumours reported in human primary immune deficiencies a majority has been observed in two disorders: ataxia telangiectasia (A-T) and common variable immune deficiency (CVID). Since both diseases have an increased risk of lymphomas/leukaemias and gastrointestinal tumours, suggesting a common risk factor, and the cells derived from A-T patients exhibit an increased chromosomal radiosensitivity we analysed chromosome damage in the G₂ lymphocytes of 24 CVID pateints and 21 controls after X-irradiation in vitro.

There was a significant difference in mean aberration yields between patients and controls. Three CVID patients had yields higher than the mean + 3SD of the controls. Six patients but only one control had yields higher than the mean + 2SD of controls. The patient with the highest chromosomal radiosensitivity subsequently developed a lymphoma. Repeat assays on the same blood sample, with a 24-h delay in setting up the second culture, showed as much variability for control donors as the variation between control donors although for CVID patients inter-individual variation was greater than the difference between results of repeat samples. There was a weak positive correlation between radiosensitivity and age of donor. Chromosomal radiosensitivity of five patients with X-linked hypogammaglobulinaemia was not different from healthy donors.

The mean mitotic index (MI) for unirradiated samples from CVID patients was significantly lower than for controls and there was an inverse relationship between MI and aberration yields in the patients, but not in controls. We suggest that the defect in CVID patients that reduces response to mitogenic stimuli may have mechanism(s) in common with those involved in cellular repair processes.