The International Registry of Werner syndrome (www.wernersyndrome.org) has been providing molecular diagnosis of the Werner syndrome (WS) for the past decade. The present communication summarizes, from among 99 WS subjects, the spectrum of 50 distinct mutations discovered by our group and by others since the WRN gene (also called RECQL2 or REQ3) was first cloned in 1996; 25 of these have not previously been published. All WRN mutations reported thus far have resulted in the elimination of the nuclear localization signal at the C-terminus of the protein, precluding functional interactions in the nucleus; thus, all could be classified as null mutations. We now report two new mutations in the N-terminus that result in instability of the WRN protein. Clinical data confirm that the most penetrant phenotype is bilateral ocular cataracts. Other cardinal signs were seen in more than 95% of the cases. The median age of death, previously reported to be in the range of 46-48 years, is 54 years. Lymphoblastoid cell lines (LCLs) have been cryopreserved from the majority of our index cases, including material from nuclear pedigrees. These, as well as inducible and complemented hTERT (catalytic subunit of human telomerase) immortalized skin fibroblast cell lines are available to qualified investigators.

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Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

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Skin fibroblast-like (FL) cells from patients with Werner’s syndrome (adult progeria) regularly demonstrate frequent pseudodiploidy involving variable structural rearrangements that are clonal: variegated translocation mosaicism (VTM). Ninety-two percent of 1,538 metaphases from 29 independent strains derived from five patients with Werner’s syndrome demonstrated this cytogenetic abnormality. In contrast, only eight (8.4%) of 95 non-Werner’s syndrome FL cell cultures demonstrated VTM: seven with low-grade VTM (approximately 5% of 300 metaphases), and one with VTM affecting 90–100% of metaphases. Unlike the cytogenetic abnormalities observed in the terminal stages of normal FL cell cultures, VTM occurs throughout the entire lifespan of Werner’s syndrome cultures. Ten of the identifiable break points in 1,005 banded metaphases accounted for 27% of all definable rearrangements. Baseline sister chromatid exchanges were not increased. Co-cultivation of Werner’s syndrome and normal strains did not induce VTM in the normal strain. The relationship between VTM and the reduced growth potential of Werner’s syndrome FL cells is not yet understood, nor is the relationship between these in vitro abnormalities and the presumptive single gene defect that causes the progeroid clinical manifestations of Werner’s syndrome.

Fanconi anemia (FA) is a genetically and phenotypically heterogenous autosomal recessive disease associated with chromosomal instability and hypersensitivity to DNA crosslinkers. Prognosis is poor due to progressive bone marrow failure and increased risk of neoplasia, but revertant mosaicism may improve survival. Mechanisms of reversion include back mutation, intragenic crossover, gene conversion and compensating deletions/insertions. We describe the types of reversions found in five mosaic FA patients who are compound heterozygotes for single base mutations in FANCA or FANCC. Intragenic crossover could be shown as the mechanism of self-correction in the FANCC patient. Restoration to wildtype via back mutation or gene conversion of either the paternal or maternal allele was observed in the FANCA patients. The sequence environments of these mutations/reversions were indicative of high mutability, and selective advantage of bone marrow precursor cells carrying a completely restored FANCA allele might explain the surprisingly uniform pattern of these reversions. We also describe a first example of in vitro phenotypic reversion via the emergence of a compensating missense mutation 15 amino acids downstream of the constitutional mutation, which explains the reversion to MMC resistance of the respective lymphoblastoid cell line. With one exception, our mosaic patients showed improvement of their hematological status during a three- to six-year observation period, indicating a proliferative advantage of the reverted cell lineages. In patients with Fanconi anemia, genetic instability due to defective caretaker genes sharply increases the risk of neoplasia, but at the same time increases the chance for revertant mosaicism leading to improved bone marrow function.

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.

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