In a Welsh woman and a French woman with Seckel syndrome-10 (SCKL10; 617253), Payne et al. (2014) identified compound heterozygosity for frameshift mutations in the NSMCE2 gene: the first was a 1-bp deletion (c.345delT; chr8.126,194,424delT, GRCh37) between the N-terminal helix bundle and the SP-RING domain, resulting in a premature termination codon (Ser116LeufsTer18); the second was a 4-bp insertion (c.700_701insAGGG; 617246.0002), causing a premature termination codon that removes the 14 C-terminal amino acids of the protein (Ala234GlufsTer4) and disrupts the C-terminal alpha helix. The parents in both families were heterozygous for the mutations; haplotype analysis confirmed that both patients inherited the same rare 2 haplotypes within a region greater than 510 kb, suggesting shared common ancestral haplotypes. Neither of the mutations or haplotypes were found in a sample of 54 controls from Great Britain, in 4,190 internal control exomes and genomes, or in the 1000 Genomes Project database (July 10, 2012); however, the 1-bp deletion was found in heterozygous state in 2 of 6,250 individuals in the NHLBI Exome Sequencing Project database (April 2013; allele frequency, 0.00016). Analysis of cDNA from patient dermal fibroblasts showed no evidence of the Ser116LeufsTer18 species, suggesting that its mRNA is unstable; however, immunoblotting of A549 cells in which the 1-bp deletion was transiently expressed showed low levels of the truncated protein. In contrast, the product of the 4-bp insertion was detected at levels close to those of wildtype, but its levels decayed more quickly in cyclohexamide-treated HeLa cells, consistent with reduced protein stability. Auto-SUMO-ligase activity of Ser116LeufsTer18 was virtually undetectable in A549 cells, whereas the Ala234GlufsTer4 mutant exhibited a level of auto-SUMOylation similar to that of wildtype. Analysis of the cellular phenotype of patient dermal fibroblasts showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (see 604610) after hydroxyurea-induced replication fork stalling, compared to wildtype cells; these nuclear abnormalities were restored by expression of wildtype NSMCE2. In addition, the dwarf phenotype of morphant zebrafish with reduced Nsmce2 mRNA could be attenuated by coinjection of human wildtype NSMCE2, but coinjection of either mRNA variant identified in these patients failed to rescue the morphant phenotype.
