A unique homozygous WRAP53 Arg298Trp mutation underlies dyskeratosis congenita in a Chinese Han family

Dyskeratosis congenita (DC) is an inherited telomeropathy characterized by ectodermal dysplasia, bone marrow failure (BMF), cancer predisposition and other somatic abnormalities. The typical triad of nail dysplasia, skin pigmentation and oral leukoplakia has clinical diagnostic value; DC is also associated with the risk of developing trilineage BMF, pulmonary fibrosis, liver fibrosis and cirrhosis, malignancies, developmental delays, and stenosis of the esophagus, urethra, or lacrimal ducts [1‐4]. However, the clinical symptoms of DC are highly heterogeneous, making it difficult to diagnose DC with confidence based on clinical features alone.

A significant advance in the understanding of the pathogenic mechanism of DC came from the discovery that dyskerin, a protein encoded by DKC1, which was the first gene identified in X-linked DC patients [5], was associated with telomerase and that dyskerin mutations caused a reduction in telomerase RNA component (TERC) and telomere length [6]. After these findings, studies over the next 10 years showed that DC was fundamentally a telomere disease in which patients showed premature loss of telomere repeats, and the loss of functional telomere structure leading to early cell death/senescence and a wide variety of clinical features [6‐8]. To date, there are eleven reported DC-associated genes (DKC1, TERT, TERC, TINF2, WRAP53, NOP10, NHP2, CTC1, RTEL1, PARN and TPP1) [5, 9‐16] that are responsible for the function or maintenance of telomeres [17, 18]. The inheritance of DC is genetically heterogeneous, including autosomal dominant inheritance (TERT, TERC, TINF2, TPP1 or RTEL1), autosomal recessive inheritance (TERT, WRAP53, NOP10, NHP2, CTC1, PARN or RTEL1), and X-linked inheritance (DKC1) [19, 20].

In 2011, compound heterozygous WD repeat containing antisense to TP53 (WRAP53) mutations were identified in 2 unrelated DC patients with autosomal recessive inheritance [21]; that study was the only report thus far of WRAP53 mutations in DC. The two unrelated probands presented the classical triad of skin pigmentation, oral leukoplakia, and nail dysplasia, and the peripheral lymphocytes of the probands had shortening telomere lengths. Parents and siblings of the probands harbored heterozygous WRAP53 mutations and had normal telomere lengths, showing that it was an autosomal recessive inheritance [21]. WRAP53 is associated with telomerase and is required for its delivery to the telomeres during the S phase [22]. WRAP53 is associated with TERC, TERT, dyskerin, and small Cajal body RNAs [22] and mediates the localization of H/ACA class RNAs to Cajal bodies through the recognition of a CAB box [22, 23]. Knockdown of WRAP53 causes the loss of TERC and dyskerin from the Cajal bodies, mislocalization of TERC in the nucleolus, and progressive telomere shortening [21, 22].

In this work, we identified a unique homozygous DC-associated WARP53 Arg298Trp mutation with an autosomal recessive inheritance mode in a DC patient who was an offspring of a consanguineous marriage.

In this study, four individuals from a 2 generation family were enrolled for investigation of an underlying DC-related gene mutation. The recruited individuals consisted of the proband (IV-1, 30 years), his asymptomatic sister (IV-2, 34 years), his asymptomatic consanguineous father (III-1, 58 years) and mother (III-2, 55 years). Pedigree analysis showed an autosomal recessive inheritance mode (Fig. 1a).

The clinical presentation of DC is highly variable, and little is known about the physiopathological mechanisms underlying that variability. The clinical diagnosis of DC may be challenging due to different ages of onset, varied clinical features and genetic heterogeneity. In this study, we identified a DC patient with a unique homozygous WRAP53 mutation with consanguineous parents. The onset age of the patient with the homozygous WRAP53 mutation was 27 years (which was relatively late), and he presented classic symptoms, BMF and liver cirrhosis.

Compound heterozygous WRAP53 mutations were identified in 2 unrelated patients with DC [21] and, until now, that was the only report about WRAP53 mutations in DC patients. In this study, we revealed a homozygous WRAP53 mutation in a DC patient with consanguineous parents. WRAP53 is a scaffold protein that acts as a platform for the assembly of large protein and RNA complexes to facilitate their interactions. WRAP53 mediates the localization of the specific H/ACA class RNAs to Cajal bodies through the recognition of a CAB box [22, 23]. WRAP53 joins in the activation of telomerase and is associated with the delivery of telomerase to the telomeres during the S phase [22]. WRAP53 is involved in DNA repair, the maintenance of Cajal bodies, and telomere elongation [22, 25]. Knockdown of WRAP53 causes mislocalization of TERC to the nucleolus, the loss of TERC and dyskerin from Cajal bodies, and progressive telomere shortening over time [21, 22]. Mutations in WRAP53 cause the misdirection of telomerase RNA to the nucleoli, inhibit the localization of the telomeres to Cajal bodies, and prevent telomerase from elongating the telomeres, which results in the shortening of the telomeres. WRAP53 contains five WD40 repeats that appear to be critical to its function. WD40 repeats are a protein structure, and they mediate a variety of cellular processes by serving as a scaffold for multiple interactions between molecules via flexible loops. In our case, the mutated region of WRAP53 was in the second WD40 domain. Disease-associated mutations in WRAP53 are all located in that highly conserved residue and are predicted to change the function of the protein [26]. Arg298 is in the very highly conserved residue of the WRAP53 protein. We used three bioinformatics tools to analyze the impact of the Arg298Trp mutation on the structure and function of WRAP53.The mutation was predicted to be damaging, with a score of 1.000, protein destabilizing and disease causing. A previous study showed that DC-associated WRAP53 mutations contribute to a marked reduction in endogenous WRAP53 levels and prevent it from localizing to Cajal bodies [21]. The proband’s parents and sister had a heterozygous WRAP53 mutation and had no clinical symptoms, indicating that WRAP53 mutation has an autosomal recessive inheritance mode, which agrees with the previous report [21].

DC patients have shorter telomeres than healthy individuals, but there is substantial variation in different patients [27]. Flow-FISH has been widely used to detect telomere length in the leukocytes of DC patients, and telomere lengths in lymphocytes less than the first percentile has been shown to be a sensitive and specific test to differentiate DC patients from healthy individuals [28]. In this study, we showed that the telomere length in peripheral blood lymphocytes from the proband was significantly shorter, corresponding to less than the 1st percentile of age-matched controls, which implies that a telomere length less than the 1st percentile could be used as a significant marker for DC in the clinical setting. The proband’s asymptomatic sister, father, and mother had heterozygous WRAP53 Arg298Trp mutations; the telomere lengths in the peripheral blood lymphocytes from the sibling and the mother were less than the 10th percentile of age-matched controls, and the father had a normal telomere length. In a previous report, parents with heterozygous WRAP53 mutations also had normal telomere lengths [21], suggesting that heterozygous WRAP53 mutation may not be a critical factor affecting telomere length. In fact, telomere inheritance, chronic inflammatory diseases, confounding environmental factors and lifestyle can all affect the mean PBMC telomere length of an individual.

DC is a multiorgan disorder, and BMF and liver cirrhosis are the severe complications. The DC patient with the homozygous WRAP53 mutation had marked hypocellularity of the BM, liver cirrhosis, portal hypertension, marked splenomegaly, and had no response to danazol, glucocorticoid and/or CsA. BMF in DC patients does not respond to immunosuppressive therapy, including CsA and antithymocyte globulin [29]. In total, 50%–70% of DC patients respond to androgens, achieving red blood cell and/or platelet transfusion independence [30‐32]. The DC patient with the homozygous WRAP53 mutation did not respond to danazol, and the liver cirrhosis, portal hypertension and marked splenomegaly may be some of the factors influencing that lack of response. In addition, the patient had esophageal varices and seriously decreased platelet levels (often could not receive platelet transfusion in a timely manner because of his poor economic situation), and he often suffered from gastrointestinal hemorrhage, which may have further aggravated the symptoms and affected the absorption of the drugs.

In conclusion, our study is the first to report a unique homozygous WRAP53 mutation site in a DC patient. The DC patient harboring the homozygous WRAP53 Arg298Trp mutation had shortened telomeres, classic clinical symptoms, BMF, liver cirrhosis and poor response to therapy.