First Report of Novel Heterozygous WRAP53 P.ala522glyfs*8 Mutation Associated Dyskeratosis Congenita.

Dyskeratosis congenita (DC) is a genetic condition caused by the alteration of the telomerase complex. It has variable phenotype ranging from idiopathic pulmonary fibrosis (IPF) to bone marrow (BM) failure, which in turn can evolve into myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML).1, 2 Among various mutations of the telomerase enzyme complex associated with DC, WD repeat-containing protein antisense to TP53 (WRAP53) is known to be homozygous, with autosomal recessive inheritance.3 In the present study, we report in a patient the first case of a novel heterozygous WRAP53 mutation, shared with the father, who developed features of DC, including MDS and later AML. We also describe tolerance to intensive chemotherapy, including allogeneic haematopoietic stem cell transplantation (HSCT) for MDS and AML. A 33-year-old male was referred to our clinic for evaluation of pancytopenia. The patient reported a few months of progressive fatigue but was otherwise asymptomatic. His history and physical examination was significant for early greying of hair at the age of 12 years and short stature (142 cm). The remainder of the examination, including a thorough skin and nail examination by dermatology, was unremarkable. Family history was significant for IPF in his father, an undefined bone cancer in his maternal grandfather and melanoma in his paternal grandfather. A BM biopsy was consistent with MDS, with a cellularity of 15–20% and 10–15% blasts. The karyotype was normal and next-generation sequencing (NGS) did not identify any clinically significant mutations. Chromosomal breakage analysis, to test for Fanconi anaemia, was negative. Telomere length and DC-associated gene sequencing was obtained, revealing telomere length below the first percentile and a novel, heterozygous WRAP53 mutation (c.1564dup; p.Ala522Glyfs*8). Family members were then tested with the same panel, revealing that his father harboured the same WRAP53 mutation as well as a telomerase reverse transcriptase (TERT) c.484G>A (p.Val162Met) mutation. His mother, sister and nephew did not harbour the WRAP53 mutation (Fig 1). Both the patient (Figure S1, Johns Hopkins Medical Laboratories) and the father (Figure S2, Repeat Diagnositcs) had documented abnormal telomere length pattern, determined using flow fluorescent in situ hybridisation (FISH). Diagnosis of myeloid neoplasm associated with telomere biology disorders was made. He was started on azacitidine and referred for a HSCT evaluation. Following two cycles of azacitidine, a BM biopsy revealed stable disease with 15% blasts. He then received a conditioning regimen consisting of alemtuzumab, fludarabine, cyclophosphamide, and total body irradiation (TBI; 200 cGy)4 in preparation for an 11/12 human leucocyte antigen (HLA)-matched unrelated donor HSCT with a mismatch at allele B. Graft-versus-host disease (GVHD) prophylaxis consisted of mycophenolate mofetil and cyclosporine. Pre-transplant pulmonary function testing revealed a forced expiratory volume in 1 s (FEV1) of 54%. BM biopsy at 30 days post-transplant demonstrated no evidence of disease with recovery of peripheral blood counts. The patient developed mild acute GVHD of the skin at day 104 post-transplant, which resolved with topical steroids. Infectious complications included recurrent episodes of Clostridioides difficile as well as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)/coronavirus disease 2019 (COVID-19) for which the manifestations included persistent fevers and mild dyspnoea without supplemental oxygen requirement. Circulating blasts were noticed on day 275 post-transplant and a subsequent BM biopsy revealed AML. Karyotype was normal and the NGS revealed an NRAS proto-oncogene, GTPase (NRAS) p.Q61H mutation. An echocardiogram demonstrated a left ventricular ejection fraction of 45–50%. Re-induction chemotherapy was started consisting of fludarabine, cytarabine and filgrastim (FLAG), which was well tolerated with expected haematological toxicities. Peripheral blood counts recovered in the fourth week of therapy and a repeat BM biopsy demonstrated 2% blasts by morphology but 8% focal clustering by cluster of differentiation 34 (CD34) staining. At 2 weeks later, he underwent a donor leucocyte infusion. The patient had a normal blood picture for 1 month at which point there was concern for relapse/refractory disease with appearance of peripheral blood blasts. Another BM biopsy confirmed the diagnosis, this time with a complex karyotype. The patient was then treated with decitabine and venetoclax as salvage therapy. Complete remission with incomplete platelet recovery (75 000) with minimal residual disease negativity by flow cytometry was achieved after the first cycle. The patient remains in remission with decitabine and venetoclax (8 months currently). Dyskeratosis congenita is a rare hereditary disorder involving various mutations of the telomerase enzyme complex, which commonly results in BM failure and malignancies in addition to other multiorgan manifestations.2 Mutations leading to DC arise in the telomerase RNA component (TERC), TERT, dyskerin (a TERC-binding protein), TERF1-interacting nuclear factor 2 (TINF2; a component of the shelterin telomere protein protection complex) and telomerase Cajal body protein 1 (TCAB1), the WRAP53 gene product.5, 6 TCAB1 functions to localise the telomerase ribonucleoprotein complex to Cajal bodies that play an integral role in spliceosomal small nuclear ribonucleoprotein particle maturation and regeneration.7, 8 Mislocalisation of the telomerase complex to Cajal bodies secondary to various in vitro WRAP53 missense mutations across multiple gene domains have demonstrated shortened telomeres predictive of a DC phenotype.9 However, most documented mutations in TCAB1 that result in DC are contained in the same scaffolding domain, required for localisation of the enzyme complex to Cajal bodies.5 This case suggests that other mutations of the TCAB1 protein may cause the DC phenotype, including p.Ala522Glyfs*8, which introduces a frameshift mutation near the end of the protein. Furthermore, reports of WRAP53 mutations in DC have all been homozygous or compound heterozygous. In our present patient’s case, WRAP53 p.Ala522Glyfs*8 was heterozygous in nature, inherited from his father. To date, heterozygous WRAP53 mutations have not been considered sufficient to cause a DC-associated phenotype. To our knowledge, this is the first reported case of heterozygous WRAP53 associated with DC (abnormal telomere length by flow FISH), highlighting that absence of WRAP53 homozygosity should not rule out DC, if the phenotype is characteristic. In addition, only his father also had TERT p.Val162Met mutation. TERT Val162 is highly conserved, but in the case of our present patient’s father, it was replaced with a similarly sized and also hydrophobic methionine. Predictive algorithms of function alteration are either unavailable or do not agree on the potential impact of this mutation [SIFT:Deleterious; PolyPhen-2: Probably Damaging; Align-GVGD: Class C0 (VCV000971916·2 - ClinVar - NCBI nih.gov)]. In summary, currently we do not have evidence to assume TERT Val162Met is pathogenic in general, and it is certainly not in this family where our patient did have a clinical phenotype of DC and documented short telomere despite not having inherited this mutation. Lastly, the present case highlights tolerability of non-myeloablative pre-transplant conditioning as well as intensive anti-leukaemia chemotherapy agents in patients with short telomere syndromes. Reported complications of transplant, in recipients with DC, include increased risk of death due to myeloablative conditioning, risk of GVHD, life-threatening infectious complications, pulmonary fibrosis, hepatic and dermatological complications and risk of secondary malignancies. Whereas our patient did experience infectious complications, they were not life threatening. The GVHD observed was mild and most likely related to the addition of alemtuzumab to the preparative regimen, which has previously been shown to reduce the risk of GVHD. Furthermore, the non-myeloablative regimen used in this patient, which has been well tolerated as previously reported,4 incorporates dose reduction of agents (cyclophosphamide and TBI) to minimise organ-specific toxicity and excludes conventional agents associated with severe organ toxicity such as busulfan and melphalan. Partial pulmonary shielding during low-dose TBI may also explain the limited toxicity seen in this case. Lastly, early referrals to dermatology, hepatology, and otolaryngology for multidisciplinary care, including screening for secondary malignancies, were facilitated in this case and the patient continues to follow-up with them. Whereas the pre-transplant conditioning regimen has been well tolerated as previously reported,4 data on intensive chemotherapy, such as high-dose cytarabine as well as novel agents such as venetoclax to treat AML in patients with DC, are limited. These agents are associated with myelosuppression and their toxicity in patients with DC may have been thought to be more pronounced than that seen in routine practice. We demonstrate feasibility of administrating such agents in a patient with DC without excessive toxicities. Tylan Magnusson, Richard C. Godby, Kimo Bachiashvili and Omer Jamy contributed towards data collection, drafting, revising, and approving the manuscript. No funding sources to disclose. All authors have no relevant conflict of interest to disclose. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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