Craniofrontonasal syndrome (CFNS) is an X-linked developmental disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Females have frontonasal dysplasia and coronal craniosynostosis (fusion of the coronal sutures); in males, hypertelorism is the only typical manifestation. Here, we show that the classical female CFNS phenotype is caused by heterozygous loss-of-function mutations in EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases. In mice, the orthologous Efnb1 gene is expressed in the frontonasal neural crest and demarcates the position of the future coronal suture. Although EFNB1 is X-inactivated, we did not observe markedly skewed X-inactivation in either blood or cranial periosteum from females with CFNS, indicating that lack of ephrin-B1 does not compromise cell viability in these tissues. We propose that in heterozygous females, patchwork loss of ephrin-B1 disturbs tissue boundary formation at the developing coronal suture, whereas in males deficient in ephrin-B1, an alternative mechanism maintains the normal boundary. This is the only known mutation in the ephrin͞Eph receptor signaling system in humans and provides clues to the biogenesis of craniosynostosis.

Somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes are dependent upon activation-induced cytidine deaminase (AID)-induced mutations. The scaffolding properties of proliferating cell nuclear antigen (PCNA) and ubiquitylation of its residue K164 have been suggested to play an important role organizing the error-prone repair events that contribute to the AID-induced diversification of the Ig locus. We generated knockout mice for PCNA (Pcna ؊/؊ ), which were embryonic lethal. Expression of PCNA with the K164R mutation rescued the lethal phenotype, but the mice (Pcna ؊/؊ tg K164R ) displayed a meiotic defect in early pachynema and were sterile. B cells proliferated normally in Pcna ؊/؊ tg K164R mice, but a PCNA-K164R mutation resulted in impaired ex vivo CSR to IgG1 and IgG3, which was associated with reduced mutation frequency at the switch regions and a bias toward blunt junctions. Analysis of the heavy chain V186.2 region after NP-immunization showed in Pcna ؊/؊ tg K164R mice a significant reduction in the mutation frequency of A:T residues in WA motifs preferred by polymerase-(Pol ), and a strand-biased increase in the mutation frequency of G residues, preferentially in the context of AID-targeted GYW motifs. The phenotype of Pcna ؊/؊ tg K164R mice supports the idea that ubiquitylation of PCNA participates directly in the meiotic process and the diversification of the Ig locus through class-switch recombination (CSR) and somatic hypermutation (SHM).T o mount an effective antibody response, mice and humans create a highly diverse repertoire of antigen binding sites through the rearrangement of the germ line variable (V), diversity (D), and joining (J) Ig locus. Following interaction with antigen, B cells in the germinal centers (GCs) of secondary lymphoid organs express activation-induced cytidine deaminase (AID). AID, together with other enzymes, causes a very high rate (10 Ϫ5 -10 Ϫ3 /base pair/generation) of point mutations in Ig V regions resulting in the affinity maturation and the changes in fine specificity required to produce protective antibodies (1, 2). AID also initiates class-switch recombination (CSR) by mutating the switch regions (SRs) that are located just 5Ј of the constant region genes (3, 4). CSR allows antibodies to be distributed throughout the body and to carry out a wide variety of effector functions. AID deaminates deoxycytidines (dC) in single-stranded DNA in the V and SRs to generate deoxyuridine (dU) (1, 2). However, more than half of the mutations in the V and SRs of mice and humans are in A:T bases and are not the result of the direct biochemical action of AID. Rather, these mutations arise during a second phase of SHM and result from the error-prone base excision repair (BER) and mismatch repair (MMR), both of which are recruited to the dU:dG mismatch generated by AID (1, 2, 4).When critical MMR genes are deleted from mice, most of the mutations in A:T in the V region no longer occur, suggesting that MMR is responsible for the majority of the mutations that ar...