Evolution of Graves’ Disease during Immune Reconstitution following Nonmyeloablative Haploidentical Peripheral Blood Stem Cell Transplantation in a Boy Carrying Germline SAMD9L and FLT3 Variants

Graves’ disease, characterized by hyperthyroidism resulting from loss of immune tolerance to thyroid autoantigens, may be attributable to both genetic and environmental factors. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a means to induce immunotolerance via an artificial immune environment. We present a male patient with severe aplastic anemia arising from a germline SAMD9L missense mutation who successfully underwent HSCT from his HLA-haploidentical SAMD9L non-mutated father together with nonmyeloablative conditioning and post-transplant cyclophosphamide at 8 years of age. He did not suffer graft-versus-host disease, but Graves’ disease evolved 10 months post-transplant when cyclosporine was discontinued for one month. Reconstitution of peripheral lymphocyte subsets was found to be transiently downregulated shortly after Graves’ disease onset but recovered upon antithyroid treatment. Our investigation revealed the presence of genetic factors associated with Graves’ disease, including HLA-B*46:01 and HLA-DRB1*09:01 haplotypes carried by the asymptomatic donor and germline FLT3 c.2500C>T mutation carried by both the patient and the donor. Given his current euthyroid state with normal hematopoiesis, the patient has returned to normal school life. This rare event of Graves’ disease in a young boy arising from special HSCT circumstances indicates that both the genetic background and the HSCT environment can prompt the evolution of Graves’ disease.


Introduction
Autoimmune Graves' disease, featuring a failure to tolerate thyroid-derived antigens, is characterized by hyperthyroidism and biochemically elevated thyroxine or suppressed thyrotropin together with the presence of thyroid-stimulating thyrotropin receptor antibodies [1,2]. Genetic predisposition and nongenetic factors have been linked to Graves' disease, resulting in overproduction of thyroid hormones by thyroid epithelial cells due to overactive thyrotropin receptor. Together with drastically amplified intrathyroidal cytokine production by infiltrating immune cells, thyrotropin receptor overactivity further activates and sustains inflammation to alter the behavior of thyroid epithelial cells [2][3][4]. Allogeneic hematopoietic stem cell transplantation (HSCT) procedures, including cytoreductive therapies to condition and modulate graft-host interactions, represent artificial platforms for immune reconstitution and tolerance induction. However, significantly, chronic graftversus-host disease (GVHD) may develop if immune tolerance induction is aberrant [5]. Several reports have described Graves' disease as having evolved following allogeneic HSCT for aplastic anemia, and they proposed associated factors for the rare coincidence including GVHD, use of rabbit antithymocyte globulins (rATG), and adoptive transfer of donor pathogenic lymphocytes [6][7][8][9][10][11]. Here, we report a case of coincident Graves' disease during post-transplant immune reconstitution in a boy displaying bone marrow failure and carrying germline SAMD9L and FLT3 variants. This patient had undergone father-to-son haploidentical peripheral blood stem cell (PBSC) transplantation with nonmyeloablative conditioning and high-dose post-transplant cyclophosphamide (PTCy) treatment.

Case Report
The patient, an eight-year-old boy and the sole child of nonconsanguineous parents with Chinese Han ethnicity, was diagnosed with severe aplastic anemia in October 2019. Treatments were initiated soon after diagnosis, including intravenous immunoglobulin (1 g per kg; 2 doses in October 2019), eltrombopag (up to 75 mg/day administered from October 2019 to April 2020), rATG (thymoglobulin 2.5 mg/kg/day for 5 days in October 2019), cyclosporine (administered by titrating the serum level between 200 and 300 ng/mL from October 2019), and intermittent granulocyte-colony-stimulating factors. The hematologic response was deemed inadequate, with a transfusion dependency requiring red blood cells to be provided monthly and platelets biweekly. Persistently low neutrophil counts (ranging between 0.35 and 0.75 × 10 9 /L) were also observed despite bone marrow cellularity increasing from <1% at diagnosis to 5-10% (July 2020). No physical anomalies or laboratory evidence of cytogenetic changes or Fanconi anemia were detected in July 2020 upon transfer to Koo Foundation Sun Yat-Sen Cancer Center, Taipei, although morphological myelodysplasia in the bone marrow was a concern (data not shown). Whole-exome sequencing revealed a germline SAMD9L c.3800G>T; p.(Cys1267Phe) mutation with a very low population allele frequency, which was validated by Sanger sequencing (Figure 1a-c). The patient's bone marrow failure syndrome was assumed to be linked to this mutation. Both parents displayed normal hematopoiesis despite the patient's mother also carrying the same SAMD9L mutation. Telomere length was also found to be short in the patient relative to an age-matched control ( Figure 1d).
The patient's treatment course was complicated by stomatitis (alleviated by valacyclovir treatment, without recurrence after drug withdrawal on day +68), an episode of uncharacterized low-grade febrile upper airway illness with self-limited azotemia (creatine levels increased to 1.52 mg/dL on day +55, but returned to a baseline level of 0.48 mg/dL 3 days later), and right-side epididymo-orchitis 6 months post-transplant that was resolved via a short course of orally administered cephalosporin. No other significant infections, GVHD, sinusoidal obstruction syndrome, respiratory distress, or other organ dysfunctions were encountered.
The patient's treatment course was complicated by stomatitis (alleviated by valacyclovir treatment, without recurrence after drug withdrawal on day +68), an episode of uncharacterized low-grade febrile upper airway illness with self-limited azotemia (creatine levels increased to 1.52 mg/dL on day +55, but returned to a baseline level of 0.48 mg/dL 3 days later), and right-side epididymo-orchitis 6 months post-transplant that was resolved via a short course of orally administered cephalosporin. No other significant infections, GVHD, sinusoidal obstruction syndrome, respiratory distress, or other organ dysfunctions were encountered.
However, Graves' disease arose dramatically 10 months post-transplant, corresponding to 1 month after tapering and discontinuation of cyclosporine treatment ( Figure 2). Graves' disease was diagnosed when the boy presented with restlessness, rapid speech, poor sleep, orbital swelling, and persistent tachycardia (resting pulse of up to 130 beats/min compared to a baseline of 90-100). An investigation of thyroid function revealed markedly elevated free thyroxin (fT4; >7.77 ng/dL, normal range 0.8-2.3) and total triiodothyronine (T3; 488.90 ng/dL, normal range 90-240), but undetectable thyroid-stimulating hormone (TSH; <0.005 µIU/L, normal range 0.7-6.4). Ultrasonography of the thyroid showed heterogeneous parenchymal echotexture with hyperemia throughout the gland (Figure 3), compatible with Graves' disease. The patient also displayed markedly elevated levels of antibodies to thyroglobulin (479.0 IU/mL, normal < 115), thyroid peroxidase (293.3 IU/mL, normal < 5.61), and thyrotropin receptors (64.0%, normal 0.0-14.0%), all compatible with the diagnosis of Graves' disease. The patient's post-transplant course of immune reconstitution was also affected due to downregulation of lymphocyte subsets soon after Graves' disease diagnosis, which was more prominent for non-B cell lineages ( Figure 2). Immunoglobulin levels remained within normal ranges throughout the post-transplant period (data not shown).  (Figure 3), compatible with Graves' disease. The patient also displayed markedly elevated levels of antibodies to thyroglobulin (479.0 IU/mL, normal < 115), thyroid peroxidase (293.3 IU/mL, normal < 5.61), and thyrotropin receptors (64.0%, normal 0.0-14.0%), all compatible with the diagnosis of Graves' disease. The patient's post-transplant course of immune reconstitution was also affected due to downregulation of lymphocyte subsets soon after Graves' disease diagnosis, which was more prominent for non-B cell lineages ( Figure 2). Immunoglobulin levels remained within normal ranges throughout the posttransplant period (data not shown).  In investigating the patient's genetic predisposition to Graves' disease, we identified a FLT3 c.2500C>T; p.(Arg834Ter) mutation in both the patient and donor (Figure 1a-c). This mutation occurs at a rare population frequency (Figure 1b) and is predicted to elicit premature termination in tyrosine domain 2, indicating that it may result in a truncated protein.
Since the FLT3 stop mutation rs76428106-C, which also results in a truncated protein, has been linked to autoimmune thyroid disease [12], in April 2022, we conducted relevant tests for the donor, which revealed normal telomere length (Figure 1d), fT4 of 1.68 ng/dL, T3 of 121.20 ng/dL, TSH of 1.740 μIU/L, nondetectable antinuclear antibody, normal levels of antibodies to thyroglobulin (19.7 IU/mL), and thyrotropin receptors (6.0%), but borderline elevated antithyroid peroxidase antibodies (7.8 IU/mL). Susceptibility to Graves' disease among different ethnic groups has been associated with increased frequencies of specific HLA haplotypes [13][14][15][16][17][18][19][20][21], and we identified the donor as carrying HLA-B*46:01 and HLA-  In investigating the patient's genetic predisposition to Graves' disease, we identified a FLT3 c.2500C>T; p.(Arg834Ter) mutation in both the patient and donor (Figure 1a-c). This mutation occurs at a rare population frequency (Figure 1b) and is predicted to elicit premature termination in tyrosine domain 2, indicating that it may result in a truncated protein. Since the FLT3 stop mutation rs76428106-C, which also results in a truncated protein, has been linked to autoimmune thyroid disease [12], in April 2022, we conducted relevant tests for the donor, which revealed normal telomere length (Figure 1d), fT4 of 1.68 ng/dL, T3 of 121.20 ng/dL, TSH of 1.740 µIU/L, nondetectable antinuclear antibody, normal levels of antibodies to thyroglobulin (19.7 IU/mL), and thyrotropin receptors (6.0%), but borderline elevated antithyroid peroxidase antibodies (7.8 IU/mL). Susceptibility to Graves' disease among different ethnic groups has been associated with increased frequencies of specific HLA haplotypes [13][14][15][16][17][18][19][20][21], and we identified the donor as carrying HLA-B*46:01 and HLA-DRB1*09:01 and the patient as carrying HLA-C*07 (Table 1). Neither the patient nor the donor hosted HLA haplotypes reported to be protective against Graves' disease, such as HLA-DRB1*07:01 and HLA-DQA1*02:01 (DR7) (data not shown) [18,19]. Initiating and titrating carbimazole (antithyroid) therapy was begun immediately upon detecting hyperthyroidism, but the trialed rapid tapering failed, as shown in Figure 2, and the boy now requires an oral dosage of 2.5 mg carbimazole twice daily to maintain his euthyroid status. Moreover, follow-up thyroid ultrasonography at 5 months ( Figure 3) and 1 year (data not shown) of carbimazole treatment revealed characteristics similar to those at the time of Graves' disease diagnosis. Follow-up at 6 and 12 months of carbimazole treatment, respectively, indicated that levels of antithyroglobulin antibodies (1062.0 and >4000 IU/mL), thyroid peroxidase (214.6 and 632.1 IU/mL), and thyrotropin receptors (54.0% and 24.0%) remained high compared to those measured when Graves' disease was diagnosed. No proptosis, periorbital edema, scleral injection, or lid retraction was observed, evidencing inactive or mild orbitopathy. Furthermore, there were no indications of dermatopathy or acropathy. Absolute numbers of lymphocyte subsets recovered during carbimazole treatment (Figure 2), indicating a revival of immune reconstitution. The patient's telomere lengths improved 1.5 years post-transplant compared to those of a pre-transplant sample (Figure 1d). The boy has returned to school and has remained transfusion-independent since August 2020, with hematological assessment in 2022 revealing a neutrophil count of 2.07-2.95 × 10 9 /L, hemoglobin of 13.0-13.8 mg/dL, and a platelet count of 204-209 × 10 9 /L.

Discussion
Here, we report the case of a young boy diagnosed with aplastic anemia who developed Graves' disease after rATG-containing immunosuppressive treatments and haploiden-tical HSCT from his father. Co-occurrence of aplastic anemia and Graves' disease, both rare disorders that display similar autoimmune pathogeneses, has been reported previously on several occasions, albeit under different circumstances. For instance, aplastic anemia can arise after Graves' disease and with or without administration of antithyroid medication [22,23]. Alternatively, Graves' disease may develop after aplastic anemia, with different mechanisms having been proposed [24][25][26][27]. For example, rATG, which has proven effective in treating steroid-resistant Graves' orbitopathy [28], has been implicated in promoting the development of Graves' disease following treatment for aplastic anemia with and without HSCT [24,26]. Adoptive transfer of a pathogenic clone from a donor suffering Graves' disease [6][7][8], as well as the process associated with GVHD [9], have been postulated as plausible mechanisms by which Graves' disease evolves upon allogeneic HSCT for aplastic anemia. Nevertheless, neither of those proposed mechanisms seem applicable in the case reported herein; although the donor did display a low titer of antithyroid peroxidase, he exhibited a euthyroid state and an absence of other tested autoantibodies. Indeed, Graves' disease may evolve 10 months to 8 years following HSCT through variable conditioning regimens, stem cell sources, treatment types, and with or without GVHD ( Table 2). We believe that HSCT platforms must lower the threshold level of stimulating anti-thyroid autoimmunity, meaning that, in association with other predisposing factors, Graves' disease evolves more frequently during immune reconstitution. Interestingly, in our patient's case, reconstitution of lymphocyte subsets was transiently downregulated as Graves' disease evolved, as shown in Figure 2.  , not available; PBSC, peripheral blood stem cells; rATG, rabbit anti-thymocyte globulin; SAA, severe aplastic anemia; TBI, total body irradiation; TLI, total lymphoid irradiation. * HSCT regimen includes conditioning-GVHD prophylaxis. Numbers following Cy, Flu, or TBI/TLI denote total mg per kg body weight given, total mg per m 2 body surface area given, and TBI/TLI, total irradiation given, respectively.
The pathogenesis of post-HSCT autoimmunity is believed to be multifactorial, involving genetic, infectious, hormonal, and other environmental factors [29]. Many variants of genes involved in immune responses (e.g., HLA, PTPN22, CTLA4, and IL2RA), thyroid function (e.g., TSHR, FOXE1), and other processes (e.g., LPP, TRIB2) have been linked to autoimmune thyroid dysfunctions [3]. A large single-center study focused on post-HSCT thyroid autoimmunity, including Graves' disease and Hashimoto thyroiditis, reported a 2.9% 5-year actuarial rate for autoimmune thyroid dysfunction post-allogeneic HSCT [20]. Thus, investigation of the genetic factors potentially contributing to the development of Graves' disease in our patient was warranted. We identified germline variants of FLT3 (carried by both the patient and his HSCT donor) and SAMD9L (carried solely by the patient), as well as HLA-B*46:01 and HLA-DRB1*09:01 (carried by the donor) and HLA-C*07 (carried by the patient) haplotypes, together potentially representing genetic predispositions for the evolution of Graves' disease post-HSCT treatment for aplastic anemia.
FLT3 encodes fms-related tyrosine kinase 3, a receptor that regulates differentiation and proliferation of hematopoietic progenitor and dendritic cells [30,31]. Notably, mutation at FLT3 residue D835 has been linked to an increased risk of developing acute myeloid leukemia [32,33]. Both our patient and his father carried a germline FLT3 p.R834* variant, which introduces a premature stop mutation at tyrosine kinase domain 2 and thus represents a loss-of-function germline mutation. Interestingly, the germline intronic rs76428106-C variant of FLT3, also representing a loss-of-function mutation, engenders the greatest risk of autoimmune thyroid disease [12]. It has been proposed that this latter intronic variant introduces a novel splice site to curtail FLT3 protein length, resulting in a compensatory increase in levels of its ligand so that it also behaves like a gain-of-function mutation [12]. It would be illuminating to establish if the germline FLT3 p.R834* variant identified in our patient induces similar effects.
Consistent with reported pediatric myelodysplastic and bone marrow failure syndromes [34,35], our patient also carried a germline SAMD9L p.C1267F mutation, which is ultra-rare in terms of population allele frequency (Figure 1b). Gain-of-function SAMD9L mutations have been shown to impair multiple pathways and result in enhanced proliferative inhibition and repression of protein translation elongation in primary hematopoietic cells [35,36]. HSCT treatment, either from unrelated cord blood or matched sibling/unrelated bone marrow, has been reported as successful for four of six cases of pediatric myelodysplastic syndromes with germline SAMD9L mutations, including one with reduced intensity conditioning [37]. In our patient, bone marrow failure was successfully corrected by nonmyeloablative haploidentical PBSC transplantation from his father, who does not have the SAMD9L p.C1267F variant. It remains to be determined how interactions between the host cells carrying SAMD9L p.C1267F/FLT3 p.R834* mutations and the donor cells carrying the FLT3 p.R834* mutation resulted in Graves's disease evolving post-HSCT. The donor, who carries both FLT3 p.R834* mutation and HLA-B*46:01/HLA-DRB1*09:01 haplotypes, has not shown any manifestation of Graves's disease to date.
In conclusion, the interplay between genetic (the FLT3 variant carried by the patient and the FLT3/HLA-B*46:01/HLA-DRB1*09:01 variants carried by his PBSC donor) and environmental factors arising from the process of immune reconstitution post-haploidentical PBSC transplantation may prompt loss of immune tolerance to thyroid autoantigens, which may have resulted in the development of Graves' disease in our patient.

Identification of Variants by Whole-Exome Sequencing (WES)
Informed consent was explained and obtained from both the patient and his parents prior to this study. Peripheral blood from the patient and his parents was collected before PBSC transplantation of the patient. One additional peripheral blood sample was obtained from the patient 1.5 years after PBSC transplantation. Genomic DNA from whole blood was isolated using a Wizard genomic DNA purification kit (Promega, Madison, WI, USA) for WES. A DNA library was prepared using a Roche KAPA HyperExome kit (Roche, Basel, Switzerland), and sequencing was performed using a NovaSeq 6000 system (Illumina, San Diego, CA, USA). The sequencing data were aligned to the GRCh37 (hg19) reference genome to identify genetic variants. Mutations were verified by Sanger sequencing. A fragment of the SAMD9L gene containing the SAMD9L c.3800G>T mutation was amplified by PCR using 5 -TCTCCTAGAAGCTGCGGAAA-3 and 5 -TGCTGCAGTAGGAAGGCATA-3 primers, with the former also used for Sanger sequencing. A fragment of the FLT3 gene containing the FLT3 c.2500C>T mutation was amplified by PCR using 5 -CACAAAGAACTGCAGCCACC-3 and 5 -GCCCAAGGACAGAT GTGATG-3 primers. The DNA fragment of the correct size (559 bp) was isolated from the agarose gel for Sanger sequencing using the 5 -CACAAAGAACTGCAGCCACC-3 primer.

Determination of HLA Haplotypes
Clinical HLA types including low-resolution 2-digit (the donor) and high-resolution 4digit (the patient) were obtained by Micro SSP™ HLA DNA Typing Trays from LinkSeq TM HLA-ABCDRDQB1 kit with Applied Biosystems TM Standard Block (One Lambda, Inc. West Hills, CA, USA). Next-generation sequencing-based HLA genotypes were extracted from donor and patient WES data using the HLAscan application [38]. In brief, WES sequence reads were aligned with human genome reference hg19 (UCSC browser). Each read in aligned results was classified into a different HLA gene classes via HLAscan application using exon sequence from ImMunoGeneTics project (IMGT)/HLA database (https://www.ebi.ac.uk/ipd/imgt/hla/ (accessed on 4 December 2019)). Reads were aligned with exons 2, 3, 4, and 5 of HLA class I genes, and exons 2, 3, and 4 of HLA class II genes. Allele types were then determined based on the numbers and distribution patterns of the reads for each reference target.

Terminal Restriction Fragment Assay
Leukocyte telomere length was analyzed using terminal restriction fragment assay, as described previously [39]. In brief, leukocyte genomic DNA was digested by Rsa I and Hinf I restriction enzymes and then resolved by pulsed-field gel electrophoresis. Telomeric DNA was detected by in-gel hybridization using a [ 32 P]-labeled telomeric probe.