CD8+ T‐cell senescence and skewed lymphocyte subsets in young Dyskeratosis Congenita patients with PARN and DKC1 mutations

Abstract Background Dyskeratosis congenita (DC) is a syndrome resulting from defective telomere maintenance. Immunodeficiency associated with DC can cause significant morbidity and lead to premature mortality, but the immunological characteristics and molecular hallmark of DC patients, especially young patients, have not been described in detail. Methods We summarize the clinical data of two juvenile patients with DC. Gene mutations were identified by whole‐exome and direct sequencing. Swiss‐PdbViewer was used to predict the pathogenicity of identified mutations. The relative telomere length was determined by QPCR, and a comprehensive analysis of lymphocyte subsets and CD57 expression was performed by flow cytometry. Results Both patients showed typical features of DC without severe infection. In addition, patient 1 (P1) was diagnosed with Hoyeraal‐Hreidarsson syndrome due to cerebellar hypoplasia. Gene sequencing showed P1 had a compound heterozygous mutation (c.204G > T and c.178‐245del) in PARN and P2 had a novel hemizygous mutation in DKC1 (c.1051A > G). Lymphocyte subset analysis showed B and NK cytopenia, an inverted CD4:CD8 ratio, and decreased naïve CD4 and CD8 cells. A significant increase in CD21low B cells and skewed numbers of helper T cells (Th), regulatory T cells (Treg), follicular regulatory T cells (Tfr), and follicular helper T cells (Tfh) were also detected. Short telomere lengths, increased CD57 expression, and an expansion of CD8 effector memory T cells re‐expressing CD45RA (TEMRA) were also found in both patients. Conclusion Unique immunologic abnormalities, CD8 T‐cell senescence, and shortened telomere together as a hallmark occur in young DC patients before progression to severe disease.


| BACKG ROU N D
Dyskeratosis congenita (DC), a telomere disorder, is characterized by the classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia. 1 It was first described by Zinsser in 1906.

Subsequent detailed confirmation by Engman in 1926 2 and Cole
in 1930 3 led to its designation as Zinsser-Engman-Cole syndrome.
Patients with DC with extremely short telomeres (<1st percentile according to age) are at an increased risk of bone marrow failure (pancytopenia), pulmonary fibrosis, malignancy, and other medical problems. Hoyeraal-Hreidarsson syndrome (HHS) is a clinically severe variant of DC that typically presents early in childhood with cerebellar hypoplasia, 4,5 immunodeficiency, progressive bone marrow failure, and intrauterine growth retardation (IUGR). 6 In recent years, pathogenic germline mutations associated with DC and related disorders have been identified in at least 14 different telomere biology genes. [7][8][9][10] Mutations in DKC1 occur with a relatively high frequency in patients with the classical DC phenotype. Pathogenic variants of PARN are also known to cause telomere shortening and result in DC. Autosomal recessive inheritance of PARN variants was reported in 2015 and is extremely rare, with less than ten cases reported. Autosomal dominant inheritance of pathogenic variants of PARN can present as pulmonary fibrosis and/or bone marrow failure. Thirty percent of all DC patients continue to be genetically uncharacterized. 11 Immunodeficiency is a major clinical feature of DC that can lead to significant morbidity and premature mortality. Lymphocytes are highly proliferative and are therefore particularly vulnerable to a decrease in telomerase activity. B and NK cells have been reported to be decreased in DC patients. 12,13 However, no detailed immunophenotyping has been performed in patients with DC.

| Human subjects
Both patients were under treatment at the Children's Hospital of Chongqing Medical University. Clinical data were collected during patient visits. In addition, healthy controls were recruited. All research studies were approved by the Medical Ethics Committee of the Children's Hospital of Chongqing Medical University.

| Telomere length assessment and mutation analysis
Genomic DNA was isolated from whole blood using the QIAamp DNA mini kit (Qiagen Inc) according to the manufacturer's instructions. Total RNA was isolated from peripheral blood mononuclear cells (PBMCs) using the AxyPrep blood total RNA miniprep kit (Axygen Biosciences), and cDNA was synthesized using the EvoScript Universal cDNA Master (Roche). The relative telomere length (RTL) was measured from DNA from PBMCs using quantitative multiplex real-time polymerase chain reaction (QPCR), as previously described. 14 In-house reference values were obtained from PBMCs of age-matched healthy donors. The RTL was calculated as the median from at least three independent runs. Whole-exome sequencing (WES) and direct sequencing were used to detect mutant sites, and the resulting protein structures were analyzed with Swiss-Model and Swiss-PdbViewer 15,16 based on the crystal structure of the protein database molecule 2a1s for PARN and 3uai for DKC1. Phylogenetic conversation was assessed using Bioedit.

| Analysis of CD57 expression
The expression of CD57 in T cells was also analyzed by flow cytometry. 22 Isolated PBMCs were incubated with anti-human CD4 (PE-Cy7), anti-human CD8 (BV421), and anti-human CD57 (FITC) for 30 minutes on ice. After washing, cells were detected on a FACSCanto II flow cytometer.

| Statistical analysis
The data were shown as mean ± standard deviation (SD), and Student's t test was used to determine the statistical significance of telomere length. Three independent experiments were performed.

| Clinical phenotype and history of DC patients
The clinical characteristics of the two patients evaluated in this study are summarized in Table 1. P1 was born at term to healthy non-consanguineous parents from China and was delivered by Cesarean section at full term, weighing 2.5 kg and was vaccinated at birth with BCG without adverse effects. He initially developed oral lesions at the age of 2 years. Nail dystrophy and skin pigmentation were first documented at age 3 and 7, respectively ( Figure 1A). P2 was born to non-consanguineous parents with an uneventful family history and had a healthy older sister. Post-natal development was normal and was vaccinated with no adverse effects. The patient was referred to us for pancytopenia with petechia at the age of 7 years. At the same time, the patient was found to have the classical triad of DC symptoms ( Figure 1C), but the onset of the symptoms is unknown. Further investigation of bone marrow cytology indicated aplastic anemia. As in P1, microcephaly and growth retardation were also found. Moreover, the patient was diagnosed with a fungal infection of the skin. After treatment with erythrocyte and platelet infusion, the patient's erythrocyte and platelet count increased, but did not return to normal levels.

| Severe telomere shortening and computational analysis of mutations
Both patients and the parents of P1 exhibited short telomeres

| Analysis of lymphocyte subsets
Both patients showed an increased frequency of T cells but a de-

| Analysis of B-cell subsets
We used the methods in the literature to further distinguish the subsets of B cells. 18,19 Naïve B cell numbers were decreased in both patients ( Figure 5A,B). Moreover, more detailed clustering revealed that the proportions of switch memory (sm) B cells and transit B cells were increased in P1 ( Figure 5C,D), and the proportion of CD21 low B cells was significantly increased in both patients ( Figure 5E). Both CD21 -CD27 -B cells and CD21 -CD38 -B cells were increased in P1, while only CD21 -CD27 -B cells were significantly increased in P2 (Figure 5F,g).

| Analysis of CD4 T-cell subpopulations
The proportion of Treg was increased in both patients ( Figure 6A), mainly due to an increase in resting and non-sup Treg, when Treg was subdivided into CD45RA − Foxp3 + activated Treg (aTreg), CD4 + CD45RA + Foxp3 + resting Treg (rTreg), and CD45RA−Foxp3− non-suppressive Treg ( Figure 6B,C). In addition, the expression of CD25, FoxP3, and cytotoxic T lymphocyte-associated-4 (CTLA-4) was increased in active Treg cells from P1, but in P2, only the expression of CD25 was slightly increased ( Figure 6D). In addition, we further analyzed the percentage of Tfh, Tfr, and their subgroups by using the gating method as shown in the Figure 6E and found in P1, but not P2, the percentage of Tfh was increased, and the percentage of Tfr was decreased ( Figure 6E,F). The expression of PD-1 on Tfh was increased in P1 only ( Figure 6E,F)

| Expression of CD57 in CD4 and CD8 T cells
The expression of CD57 was increased both in CD4 and CD8 T cells in P1, but in P2, it was only increased in CD8 T cells ( Figure 7A-C). Analysis of the percentage of CD57 + cells as well as the mean fluorescence index (MFI) for CD57 expression showed a similar trend ( Figure 7D).  However, P1 showed a persistent increase in IgE and a decrease in IgM, while P2 showed an increase in IgA (Table 1) It is known that with every cell division, 50-200 bases of DNA are lost due to incomplete terminal replication of the daughter strand. As cell division continues, the telomeres shorten, and F I G U R E 5 Characterization of B-cell subsets. A, Peripheral blood CD19 + CD27 -B cells were gated into CD38 low (naïve, N), CD38 int (prenaïve, Pre-N), and CD38 hi (transitional, Tr) subsets. B, Both P1 and P2 showed a decreased naïve cell population, and transitional B cells were increased in P1 compared with age-matched controls. C, B cells were assessed for the distribution of IgM hi , marginal zone-like (Mz-like), and switch memory (sm) B cells after staining for the expression of CD19, CD27, and IgM. D, P1 showed an increase in the proportion of sm B cells and a slight decrease in IgM hi cells, while P2 showed normal frequencies of these subsets. E-F, Both P1 and P2 showed significantly increased percentages of CD21 -B cells. Increased proportions of CD21 -CD27 -B cells and CD21 -CD38 -B cells were found in P1, while CD21 -CD27 -B cells were increased in P2 once a critical length is reached, cell senescence/apoptosis will be triggered. 33 DC is caused by germline mutations in genes associated with regulation of telomere length, resulting in very short telomeres. Both patients in our study had short telomeres, consistent with previous reports. 11,24,34 Because the telomere length is highly variable according to race, age, and disease state, the cell surface marker CD57 is routinely used to identify terminally differentiated, senescent cells with reduced proliferative capacity and altered functional properties. 22 The expression of CD57 was dramatically elevated in CD4 and CD8 T cells in both patients in our study. Given the reduced telomere lengths, the increased expression of CD57 on CD8 T cells, and the increased frequency of CD8 TEMRA, our findings suggest that CD8 T-cell senescence may be another hallmark of DC.

TA B L E 2 Immunological phenotype
In summary, we provide a thorough analysis of the clinical symptoms, genetic mutations, and immunophenotype in two cases of typical DC. Our study provides additional information on immune dysfunction and CD8 T senescence may be hallmark of DC in young patients without severe immunodeficiency or definite molecular diagnosis, especially 30% of DC patients reported were without of known pathogenic genes. Given the extremely low disease incidence of DC, more case studies are needed to draw more solid conclusions.
F I G U R E 6 Characterization of CD4 T-cell subsets. A, The frequencies of total Treg in P1 and P2 were increased compared with HC. B, Detailed phenotyping of Treg subsets by flow cytometry. C, The increase in total Treg was mainly due to an increase in the proportion of resting and non-sup Treg. D, The expression of CD25, FoxP3, and CTLA-4 was increased in activated Treg cells in P1, but only slightly elevated CD25 expression was found in P2. E, Detailed method for identifying Tfh, Tfr, Th1/2/17, and Th1/2/17-like cells by flow cytometry. F, P1 showed an increased percentage of Tfh and increased Tfh expression of PD-