Unusual Germline DSP2 Gene Accounts for All Apparent V-D-D-J Rearrangements in Newborn, But Not Adult, MRL Mice

Abstract

Anti-dsDNA autoantibodies in MRL mice contain a higher than average frequency of atypical complementarity-determining regions 3, including those made with D-D rearrangements. It has been reported that MRL mice have an intrinsically high frequency of creating VDDJ rearrangements; however, we show in this study that the majority of these apparent D-D rearrangements in B cell progenitors can be accounted for by a very novel germline DH gene in mice of the Ighj haplotype. This gene has the appearance of a D to D rearrangement due to the duplication of 9 bp common to most DSP2 genes. Germline DSP2 genes from Ighj mice were amplified, cloned, and sequenced, showing the presence of this novel gene as well as a new allele of a conventional DSP2 gene. Sequencing of D-J rearrangements revealed that Ighj mice also have a different allele of DFL16.1 and apparently lack DFL16.2. Despite the existence of this new DSP gene, analysis of VDJ rearrangements from adult bone marrow pre-B cells of MRL/lpr mice still revealed the presence of complementarity-determining region 3 containing apparent D-D joinings in 4.6% of the sequences. C3H pre-B cells had 4.2% of sequences with apparent VDDJ rearrangements, and BALB/c pre-B cells had ∼2%. DDJ intermediates were also observed, but at a lower frequency. However, strikingly, no VDDJ rearrangements were observed in newborn sequences, suggesting the process of assembly of VDJ rearrangements is fundamentally different in newborn mice vs adult mice.

Paucity of V-D-D-J Rearrangements and VH Replacement Events in Lupus Prone and Nonautoimmune TdT−/− and TdT+/+ Mice

“…We previously reported a novel V H 7183 gene in MRL/lpr and C3H mice, both of which are Igh j haplotype (25). In this study, we have found this V H gene in another lupus-prone strain, NZB, which is also Igh j for most of the V H locus (56).…”

“…Furthermore, there have been reports of unusually high frequency of D-D rearrangements in two different strains of lupus-prone mice (5,6). We previously reported that we could only identify ϳ2-4% of rearranged sequences as having potential D-D rearrangements in CDR3 from adult bone marrow pre-B cells in both lupus-prone and nonautoimmune strains (25). We further showed that newborn sequences from these same strains displayed no D-D rearrangements (25).…”

“…We previously reported that we could only identify ϳ2-4% of rearranged sequences as having potential D-D rearrangements in CDR3 from adult bone marrow pre-B cells in both lupus-prone and nonautoimmune strains (25). We further showed that newborn sequences from these same strains displayed no D-D rearrangements (25). Hence, the probability that the potential second D segment in the CDR3 sequences was fortuitous identity of N region addition with a D gene seemed a reasonable concern, which we address in this current study.…”

“…We previously showed that 2-4% of CDR3 sequences from adult pre-B cells contained sequences in which a second potential D segment could be observed (25). We arbitrarily defined a second D segment as 5 consecutive bp that were not accounted for by V or J genes or P nucleotides and that completely matched a germline D gene.…”

“…cDNA was made with SuperScript reverse transcriptase (Invitrogen Life Technologies) using oligo(dT) primers. PCR conditions and primers for amplification of V H 7183, V H J558, and V H S107 rearrangements were performed as described previously (25). PCR products were gel purified, and the eluted DNA was ligated into pBluescript or Topo (Invitrogen Life Technologies) vectors.…”