Werner Syndrome (WS; OMIM entry no. 277700; https://omim.org/entry/277700) is a rare progeroid syndrome associated with a number of aging phenotypes. WS often occurs in consanguineous families and affects ~1 in 1 million individuals in the general population (1–3). Patients with WS often exhibit adult-onset progeria and have an increased risk of developing cancer (4). At birth, patients with WS do not present any clinical symptoms; the lack of pubertal growth spurts are generally the first symptom identified (5). Over time, the typical characteristics of WS become evident, including a ‘bird-like’ appearance, cataracts, canities, ulcerations around the ankles and certain patients develop hypogonadism with atrophic genitalia and infertility (6). The average life span of affected individuals is 54 years (5).

WS is caused by mutations in the Werner syndrome RecQ like helicase gene (WRN), which was first cloned in 1996 (7). WRN is located on chromosome 8p11-p12, spanning ~250 kb and consists of 35 exons, 34 of which are protein coding (7). WRN is generally considered to follow an autosomal-recessive pattern of inheritance (8). WRN, coding a 180 kDa multifunctional nuclear protein, belongs to the family of RecQ type helicases (9). Sequence analysis and subsequent biochemical analysis have revealed that human WRN possesses helicase and exonuclease functions (10). It serves a role in DNA replication, transcription, repair, recombination and heterochromatin maintenance (including telomere maintenance), indicating that one of the major causes of WS pathogenesis may be associated with genomic instability (11). In the absence of functioning WRN, cells accumulate potentially toxic DNA intermediates or critically short telomeres, which induce genetic instability, misexpression and mutagenesis (12–14). In addition, they may drive cell loss and produce tissue-specific defects (15). Compromised cell or tissue structure and function leads to two seemingly divergent outcomes: Senescence and neoplasms (16,17). To date, the majority of disease-inducing mutations in WRN are truncating mutations (5).

In addition to WRN, a small number of heterozygous mutations in the lamin A/C gene (LMNA) have been revealed to produce similar phenotypes to WS, which suggests that LMNA may be an underlying disease-inducing gene in WS (3,18,19). LMNA encodes nuclear intermediate filaments, lamin A and lamin C (1). A previous study demonstrated that WS patients with LMNA mutations exhibited a younger onset when compared to patients with classical WS (5). In addition, a number of studies have suggested that barrier to autointegration factor 1 (BANF1), zinc metallopeptidase STE24 (ZMPSTE24) and DNA polymerase Δ1 (POLD1) may be involved in progeroid syndrome (15,20,21).

The present study investigated the potential causative gene in a consanguineous family with WS from Northwest China. A novel homozygous splice-site mutation (c.IVS28+2T>C) was identified in intron 28 of WRN in the proband and co-segregated with the affected WS family members. To the best of our knowledge, this mutation has not been reported in previous studies, nor was it identified in our previous control cohorts or the single nucleotide polymorphism (dbSNP) database (https://www.ncbi.nlm.nih.gov/projects/SNP/) and the Exome Variant Server database (http://evs.gs.washington.edu/EVS/).

The present study investigated the case of a patient with WS (IV:2) born to consanguineous parents (family members III:2 and III:3) in a family comprised of four generations with 11 living members. The proband, with a ‘bird-like’ face, husky voice, canities and ankle ulcers, conforms to the typical phenotypes associated with WS. The present study investigated the potential causative genes among all family members. Sequence analysis of WRN, LMNA, BANF1, ZMPSTE24 and POLD1, identified a previously unreported homozygous splice-site mutation in intron 28 (c.IVS28+2T>C) of the WRN gene in the proband (IV:2) and co-segregated with the affected family members (Fig. 1C). No further relevant mutations were identified by direct sequencing of the genes for LMNA, BANF1, ZMPSTE24 and POLD1. In addition, the cDNA sequencing results from the proband identified deletion of WRN exon 28 (74 nucleotides; Fig. 1D). This newly discovered c.IVS28+2T>C mutation was not identified in the 200 control cohorts that our group studied previously (23). In addition, this mutation was not present in the dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/) or Exome Variant Server databases (http://evs.gs.washington.edu/EVS).

The WRN gene encodes a nuclear protein which belongs to the family of RecQ type helicases and comprises of five functional domains including an exonuclease region, a helicase region, a RecQ C-terminus consensus region, an RNase D consensus region and a nuclear localization signal region (NLS) (27). According to previous studies, homology-dependent recombination repair (HDR) may be used to repair DNA damage while suppressing gene loss or rearrangement. In addition, WRN appears to serve a role late in the HDR process when recombinant molecules are topologically disentangled for segregation to daughter cells (28,29), as well as in the maintenance of telomere length and the suppression of telomere sister-chromatid exchanges (30,31). WRN may also be involved in non-homologous DNA-end joining, base-excision repair, DNA-damage signaling and transcription (15). Zhang et al (11) revealed that the progressive heterochromatin disorganization observed in WRN-deficient mesenchymal stem cells underlies cellular aging (32). Thus, loss of WRN may disrupt genetic stability, lead to cell aging and death. Therefore, it may be pivotal in human aging and the development of WS.

The present study revealed that the novel mutation (c.IVS28+2T>C) causes a change in the splicing pattern, leading to a deletion of 74 nucleotides in the mRNA. It suggested that the substitution and subsequent skipping of exon 28 may produce a frameshift transcript, resulting in the absence of the NLS domain. The NLS domain is essential for WRN protein targeting to the nucleus via the nuclear pore complex (33). In addition, the majority of previously reported causative WRN mutations also give rise to a lack of NLS at the C-terminus of the protein (Fig. 2) (5,34).

During this research, the wife of the proband (IV:1) was pregnant. It was speculated that the infant (V:2) may be a carrier without any pathological phenotype. However, amniocentesis or shotgun sequencing of maternal plasma DNA is required to produce accurate and convincing results to verify this. As cancer predisposition is a key feature in WS, the proband may have a higher risk of cancer development and should therefore have regular health examinations.

In conclusion, the present study identified a novel homozygous splice-site mutation (c.IVS28+2T>C) in a four generation family with WS. The present identification of a novel mutation expands the spectrum of known WRN mutations (only 87 mutations were identified as of February 2015; http://www.hgmd.cf.ac.uk/ac/search.php) and it may contribute to novel approaches to genetic diagnosis and counseling of families with WS.