Case report: A novel mutation in RTEL1 gene in dyskeratosis congenita

Dyskeratosis congenita (DKC), also known as Zinsser–Cole–Engman syndrome, is a telomeropathy typically presenting as a triad of leukoplakia, nail dystrophy, and reticular hyperpigmentation. Reported genetic mutations linked to DKC include DKC1, TINF2, TERC, TERT, C16orf57, NOLA2, NOLA3, WRAP53/TCAB1, and RTEL1. Homozygous, compound heterozygous, and heterozygous mutations in RTEL1 (RTEL1, regulator of telomere elongation helicase 1) gene on chromosome 20q13 are known to cause autosomal dominant as well as recessive DKC. Pathogenic variants of RTEL1 gene in DKC patients include c.2288G>T (p. Gly763Val), c.3791G>A (p. Arg1264His), and RTEL p. Arg981Trp. We report a novel homozygous variant of RTEL1, transcript ID: ENST00000360203.11, exon 24, c.2060C>T (p.Ala687Val), in a patient of DKC presenting with leukoplakia, dystrophic nails, reticulate pigmentation, and positive family history of a similar phenotype. The novel variant, reported as a variant of uncertain significance, may therefore be considered diagnostic for DKC in a Pakistani population.


Introduction
DKC is an inherited bone marrow failure syndrome due to telomere biology disorder and presents with heterogenous clinical manifestations (1). Spectra of clinical features vary from the classical triad of reticulated pigmentation of skin, nail dystrophy, and oral leukoplakia to life-threatening complications including bone marrow failure, solid tumor malignancies, pulmonary and liver fibrosis, and immunodeficiencies (2,3).
The diagnosis of DKC in a proband requires a combination of classical clinical features and molecular criteria; this includes either demonstration of shortened telomere length established via automated multicolor flow or identification of one of the genes known to cause DKC depending on the pattern of inheritance. Genes responsible for the disorder and reported in the literature include DKC1, TINF2, TERC, TERT, C16orf57, NOLA2, NOLA3, WRAP53/TCAB1, and RTEL1 (4). These mutations are encountered in about 80% of patients meeting the diagnostic criteria of DKC. RTEL1 (regulator of telomere elongation helicase 1) gene prevents the loss of telomere during cell division, playing a pivotal role in telomeric DNA repair and replication. Mutations in RTEL1 gene result in loss of genomic stability, shortened telomere lengths, and resultantly DKC phenotype. Molecular testing in most centers comprises targeted gene sequencing through either a customized mutigene panel or a comprehensive genomic testing. Severe variants of DKC have been associated with homozygous mutation in RTEL1 gene and very short telomeres. Similarly heterozygous mutations are commonly encountered in cases of familial pulmonary fibrosis (5). While identification of one of the variants of uncertain significance does not confirm the diagnosis, it does not exclude the disease per se. We, hereby, report a case of a young male patient with characteristic phenotypic abnormalities associated with DKC, a significant family history of similar phenotypic abnormalities, and a homozygous mutation in RTEL1, reported as a variant of uncertain significance (VUS).
2 Case report

Patient information and clinical findings
Our patient was seen initially by a general physician (GP), in 2015 at the age of 14 years, complaining of failure to gain weight. There was no history of fever, cough, chronic diarrhea, loss of appetite, weight loss, skin rash, or joint pains. On examination, the patient's weight was below the third centile for his age. Complete blood counts showed mild thrombocytopenia in 2015 (WBC 3.9 × 10 9 /l, absolute neutrophil count 1.95 × 10 9 /l, hemoglobin 13.1 g/dl with MCV 95 Fl, and platelet count of 85 × 10 9 /l). At that time, GP evaluated the patient on lines of metabolic and endocrine dysfunction. All investigations conducted including liver function tests, renal function tests, serology for hepatitis B and C, thyroid profile, and insulin-like growth factor 1 were within normal limits. Based on history of poor weight gain, celiac screen was advised and his anti-tissue transglutaminase IgA levels were 14.8 (U/ml) (negative <12, borderline (6)(7)(8)(9), and positive >18). The patient was started on a gluten-free diet. His duodenal mucosal biopsy revealed total villous atrophy suggestive of celiac disease (Marsh stage 3b). (microscopic exam: villous to crypt ratio <1:1, increased intraepithelial lymphocytes, and a subepithelial tissue infiltrate composed of lymphocytes and plasma cells). The patient remained on a gluten-free diet till 2018, but thrombocytopenia persisted and no increment in weight was seen. He continued to visit local physicians and was advised to start hematinic and multivitamins. Due to persistent thrombocytopenia, the patient was referred to our center for further evaluation and management.

Diagnostic assessment at AFBMTC/NIBMT
The patient presented to our institute in March 2021. A detailed family history revealed that he was born to a consanguineous marriage. He was the youngest of eight siblings with a history of early neonatal death of two siblings and death of one sibling at age of 4.5 years (cause unknown in all). He had an elder sister of 25 years with a history of chronic gastrointestinal (GI) complaints, rash, oral lesions, and nail abnormalities (Figures 1, 2) However, the blood counts of the sister were in normal limits and there was no history of bleeding or infections. All other family members were phenotypically normal. The patient had no significant surgical history. Psychosocial history was unremarkable. On examination, he was active, alert, thin built, and had a weight of 38.8 kg and height 157 cm, both of which were below the third centile for his age. Moreover, he had reticulated hyperpigmentation over the neck and upper chest, dystrophic nails, and leukoplakia ( Figure 3). His bone marrow examination showed a markedly hypocellular marrow for age with depressed trilineage hematopoiesis, absence of reticulin fibrosis, and infiltration. Cytogenetics revealed a 46 XY male karyotype. Based on family history, classical clinical features, and hypocellular marrow, a provisional diagnosis of inherited marrow failure syndrome most likely dyskeratosis congenita (DKC) was FIGURE 1 Summary of Clinical events before diagnosis. Keeping in view the positive family history and characteristic triad of leukoplakia, nail dystrophy, and skin pigmentation in the elder sister, a family screening through molecular testing was planned. Samples of the elder brother, sister, and mother were sent to Invitae for genetic testing. Interestingly, the sister was also found to be homozygous for a similar mutation, c.2060C>T in RTEL1 gene. Moreover, the elder brother and mother's samples also revealed a heterozygous variant of uncertain significance, c.2060C>T (p. Ala687Val) in RTEL1 gene. To obtain further supporting information regarding pathogenicity of the variant, relative telomere lengths of the proband, his sister, brother, and mother were carried out through qPCR using Richard M. Cawthon 2002 protocol (7). The proband (homozygous for the mutation) and his mother (heterozygous) were found to have a shortened telomere to single-copy gene ratio, whereas the brother and sister had normal  ratios. The variant c.2060C>T; A687V was predicted to be conserved missense mutation by Mutation Taster, PhyloP (3.8), and PhastCons (1). This variant could not change the polarity of amino acids as both alanine and valine are neutral and aliphatic amino acids. Functional conservation was also predicted from the physiochemical properties of the variant c.2060C>T, predicted by the ProtParam tool, as shown in the table (Table 1; Supplementary  File). Hence, the variant c.2060C>T could locally affect the protein structure whereas protein features would be conserved. However, RNA sequencing of the variant with functional assay of the protein could not be done because of financial/resource constraints.

Family tree 2.3.1 Therapeutic intervention and followup
Please refer to Figure 4 for details of family tree. The patient was started on oxymetholone (100 mg once a day) along with a tablet of folic acid (5 mg once a day). The patient took the treatment for 6 months (January 2022 to June 2022). His platelet counts remained stable (60,000-70,000/µl), and he has remained transfusion independent. On the last assessment in September 2022, his counts were WBC 3.5 × 10 9 /l, absolute neutrophil count 1.8 × 10 9 / l, hemoglobin 13.4 g/dl with MCV 95 fl, and platelet count of 70 × 10 9 /l. The patient is followed up every 4 weekly currently. At present, the plan is to continue oxymetholone and pursue with a nontransplant strategy as the patient has stable blood counts.
RTEL1 has a vital role in genomic stability through regulation of homologous recombination (HR). The gene is critical for mitotic and meiotic double-strand DNA repair and mutations resulting in enhanced susceptibility to chromosomal breakage (15-17). RTEL1 deficiency, despite being reported as a major genetic contributor, has not been extensively studied, and the specific clinical outcome with correlation to molecular landscape of the mutation is still under research.
Here, we report a case of DKC presenting with a classical DKC triad and a homozygous variant of RTEL1 c.2060C>T (p. Ala687Val). This sequence change replaces alanine, which is neutral and nonpolar, with valine, which is neutral and non-polar, at codon 687 of the RTEL1 protein. The variant was reported to be of uncertain clinical significance according to the American College of Medical Genetics and Genomics (ACMG) 2015. At the time this patient was tested, Invitae was using AlamutSplice for these splice predictors. Alamut uses three algorithms for these predictions, MaxEntScan (MES), SpliceSiteFinder-like (SSF-L), and NNSPLICE. At least two of the three scores must be significant for the mutant vs. the wild type to predict a gain of a new acceptor/donor splice site. A score is deemed significant if MES >0, SSF >70, and NNSPLICE >0.4. However, Splice AI did not predict the mutation to affect RNA splicing. Currently, it fulfills the moderate evidence of pathogenicity, PM2, supporting evidence of pathogenicity, PP1, and PP4 and hence does not meet criteria for pathogenic or likely pathogenic (18). Based on expanding ACMG variant classification guidelines into a general framework 2022  (22). Previously, pulmonary fibrosis without hematologic or immunological abnormalities was seen in cases of isolated heterozygous RTEL1 mutations (23-25).
Despite having a markedly hypocellular marrow, the patient has maintained a fairly stable ANC (lowest 1.32 × 10 9 /l) and Hb (12.3 g/ dl) with moderate thrombocytopenia (average platelet count6 0,000 to 70,000/µl, lowest platelet counts 43,000/µl) and certainly is not a candidate for HSCT at the moment. We plan to keep him on vigilant monitoring through follow-up by periodic complete blood counts and assessment of clinical examination for development of any complications. We would also need to find a potential donor for the patient in case a need for transplant arises. His elder brother, 36 years of age, despite being completely HLA matched with him, is heterozygous for RTEL1 mutation. HSCT from a clinically silent heterozygous family member is not recommended in DKC (26,27).
Our case reports that homozygous RTEL1 gene variant c.2060C>T (p.Ala687Val), can present with the classical DKC triad.
However, the c.2060C>T variant has NOT been reported in either the gnomAD or ClinVar database to date despite having phenotypic abnormalities in the Pakistani population as reported here. A general representation of South Asians toward the reporting of variant interpretation in genetic diseases is relatively poor compared with other populations with more extensive studies including those with European and American ancestries. Discrepancies in screening efficiency and lack of inclusion of South Asian variants in testing panels are an extension of the issue being expressed here and need to be addressed by increasing the pool of South Asian individuals.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Ethics statement
The studies involving human participants were reviewed and approved by Institutional Review Board Committee Armed Forces Bone Marrow Transplant Center. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Author contributions
HN and MK conceived and designed the study and drafted the manuscript. RI critically revised the manuscript. QC also contributed to the revision of the manuscript. All authors contributed to the article and approved the submitted version.