Prevalence of UL97 gene mutations in cytomegalovirus reactivation in the colon associated with or without ulcerative colitis

Background: Cytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC) and in immunocompromised patients, and may be associated with poor prognosis. Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV colitis remains to date unknown. The present study aimed to investigate the prevalence of CMV UL97 gene mutations in Japanese patients with colonic CMV infection associated with or without UC. Methods: Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-xed paran-embedded samples were used for nested polymerase chain reaction (PCR) amplication of the UL97 gene. Sanger DNA sequencing of the PCR products was performed. Results: UL97 mutations were frequently detected in codons T75A (95.5%), Q126L (86.4%), and D605E (86.4%), and less frequently (<10%) in L228P, D263G, A53S, R137C, A140V, G188S, A674T, and T675A. However, no known GCV resistance mutations were found. There was no signicant difference between the ratio of mutations in patients with and without UC. Conclusions: We did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. In contrast, T75A, Q126L, and D605E mutations may be used as genetic markers for CMV in East Asian countries.


Background
Human cytomegalovirus (CMV) infections cause signi cant morbidity and mortality in immunocompromised hosts, such as patients who have undergone solid organ or bone marrow transplantation, human immunode ciency virus (HIV)-infected patients, and children with congenital immunode ciencies [1,2]. In patients with are ups of refractory ulcerative colitis (UC) and sometimes in immunocompromised patients, CMV reactivation in the colon is common and may be associated with poor prognosis [3]. In immunocompetent individuals, CMV usually causes self-limiting mild hepatitis, mononucleosis, or subclinical infection [4]. Antiviral treatment with ganciclovir (GCV) has been highly recommended for CMV reactivation, along with anti-tumor necrosis factor (anti-TNF) monoclonal antibody therapy, without losing time, even though there is some debate recently in are ups of refractory UC [5][6][7][8][9].
GCV, a 2′-deoxyguanosine nucleoside analog, was used as the rst-line drug for the treatment of CMV disease and for prophylaxis in groups at high risk for CMV infection. However, prolonged therapy with GCV can lead to the development of GCV-resistant mutations [10,11]. GCV is selectively phosphorylated by a viral protein kinase homolog, a product of the UL97 gene [12,13]. Approximately 90% of GCV resistance results from mutations in that gene [13]. Well characterized GCV resistance mutations at UL97 codons 460, 520, and 590-607 impair the phosphorylation of GCV that is necessary for its antiviral activity, presumably by altering substrate recognition [14][15][16]. Despite their lack of association with GCV resistance, some additional mutation sites of UL97 have also been reported [17,18].
Previous studies have revealed the frequency of drug-resistant CMV in organ transplant patients, bone marrow transplant patients, and HIV-infected patients, and have discussed algorithms for antiviral therapy [11,19]. However, there are no data regarding the prevalence of UL97 gene mutations in colonic CMV infection associated with or without UC. One of the clinical questions is whether or not the uniform use of GCV is appropriate for initial antiviral therapy. The aim of this study was to investigate the prevalence of GCV resistance-conferring UL97 gene mutations in Japanese patients with colonic CMV infection associated with or without UC, especially in GCV-naive patients.

Study design
This was a single-center, retrospective study conducted in patients with colonic CMV infections with and without UC.
Patients and specimens Twenty-two GCV-naive patients who were diagnosed with colonic CMV infection in our hospital between 2012 and 2018 were enrolled in this study. Fifteen patients with CMV infection in the colon had UC, and seven patients had other diseases. Frozen biopsy samples or formalin-xed para n-embedded samples were used for nested polymerase chain reaction (PCR) ampli cation of the UL97 gene.
DNA puri cation from the samples Frozen biopsy samples were used for DNA extraction using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD). Formalin-xed para n-embedded samples were puri ed genomic DNA using the QIAamp DNA FFPE Tissue Kit (Qiagen). The extractions were carried out according to the manufacturer's instructions.

Nested PCR ampli cation
All PCR primers were purchased from Merck KGaA (Darmstadt, Germany). Nested PCR was performed with KOD FX neo (TOYOBO CO., LTD, Osaka, Japan). The primer sets used in the nested PCR for this study are shown in Fig. 1  Kit (Qiagen) was used to purify the PCR products. Reaction products were stored at − 20 °C until they were analyzed by 2% agarose gel electrophoresis or puri ed for DNA sequencing.

Patient characteristics
The demographic information for 22 GCV-naive patients enrolled in this study is shown in Table 2. The mean age was 59.5 years. Fifteen patients had UC and seven patients had other diseases. In non-UC patients, the background diseases were malignant melanoma (one patient), myelodysplastic syndrome (one patient), sarcoidosis (one patient), ischemic colitis (one patient), autoimmune hepatitis (one patient), and hemodialysis (one patient). A comparison of the patient characteristics between the UC and non-UC groups are shown in Table 3. In the comparison of the two groups, serum albumin was signi cantly lower in the non-UC group. Among the treatments, there was a signi cant difference only in the use of 5aminosalicylate (5-ASA) ( Table 3). Among the UC patients, steroid therapy was used for ve patients (33.3%), immunosuppressive therapy was used for seven patients (46.7%), anti-TNF therapy was used for ve patients (33.3%), and 5-ASA was used for 10 patients (66.7%). The clinical UC activity of the enrolled patients assessed by the Rachmilewitz index was 8.75 ± 5.39 (Supplemental Table 1).  To identify the mutation of the UL97 gene in GCV-naive patients, Sanger DNA sequencing of the PCR products was performed. Compared with the wild-type GCV-sensitive Towne strain as a reference, we were able to detect several mutations of the UL97 gene. Typical results of identi ed UL97 genetic mutations are shown in Fig. 2. Overall, in 22 patients with CMV reactivation in the colon, UL97 mutations were frequently detected in codons T75A (95.5%), Q126L (86.4%), and D605E (86.4%), and less frequently in L228P (9.0%), D263G (9.0%), A53S (4.5%), R137C (4.5%), A140V (4.5%), G188S (4.5%), A674T (4.5%), and T675A (4.5%). T75A and Q126L have not been reported previously (Table 4). However, no known GCV resistance mutations were found in our series. We attempted to examine whether the frequency and type of UL97 mutations in GCV-naive patients were different between UC and non-UC patients. As shown in Table 4, there was no statistically signi cant difference between the ratio of the mutations between the UC group and non-UC group.

Discussion
In the present study, we investigated the prevalence of CMV UL97 gene mutations in Japanese patients with CMV reactivation in the colon for the rst time. We revealed that several UL97 mutations were frequently detected, such as in codons Q126L (100%), T75A (93.3%), and D605E (80%). Q126L and T75A have not been reported previously. However, no known GCV resistance mutations were found in our series of GCV-naive patients, and, furthermore, there was no signi cant difference between the ratio of mutations in the UC and the non-UC patients. Our results suggest that it is not necessary to consider CMV drug resistance in the initial treatment of CMV colitis. The Q126L, T75A, and D605E mutations could be used as genetic markers for CMV in East Asian countries because UL97 mutations were suggested to have regional differences.
Human CMV remains the most common infection in solid organ recipients, HCT recipients, HIV-infected patients, and children with congenital immunode ciencies. It remains an important pathogen despite advances in the prophylaxis and acute treatment of CMV. The emergence of CMV resistance in a patient reduces the clinical e cacy of antiviral therapy, complicates therapeutic and clinical management decisions, and, in some cases, results in death of the patient. According to recent reports, the incidence of GCV resistance is 5-12% among solid organ recipients [20][21][22] and 31% in intestinal and multivisceral organ transplant recipients [23]. GCV resistance is 7.9% in HCT recipients from matched related or unrelated donors [21,24] and 14.5% in high-risk patients [25]. GCV resistance in HIV-infected patients is reported to be 19.5% [26].
Common mechanisms of CMV resistance to ganciclovir have been described chie y with UL97 mutations. GCV is an acyclic nucleoside analog of 2of oguetions. GC. In a multistep process dependent on both viral and cellular enzymes, GCV is converted to ganciclovir triphosphate, the chemical form that is active against CMV. The initial phosphorylation is catalyzed by an unusual protein kinase homolog encoded by the CMV UL97 open reading frame. Resistance to GCV arises from mutations in the UL97 gene. In several reports, numerous GCV-related mutations have been described. Most UL97 mutations conferring GCV resistance are strongly clustered at codons 460, 520, or 590 to 607 [16,27,28]. In daily medical care, timely results of resistance testing would be useful for making clinical decisions. If no drug resistance is identi ed, clinical management may focus on improving host defenses rather than switching antivirals. If there is con rmed genotypic evidence of resistance, the speci c mutation, host immune status, and disease severity should all factor into these decisions, to continue or intensify current treatment, to switch to a non-cross-resistant drug, to use drug combinations, or to try experimental drugs. Management algorithms have been proposed by several groups [29].
In this study, we did not detect the mutations of the UL97 gene associated with GCV resistance. Our results indicated that the uniform use of GCV is appropriate for initial antiviral therapy in CMV colitis associated with or without UC in GCV-naive patients, when antiviral therapy is needed, as there are no data regarding the prevalence of UL97 gene mutations in CMV colitis associated with or without UC. On the other hand, several mutations of the UL97 gene not associated with GCV resistance were detected, such as Q126L (100%), T75A (93.3%), and D605E (80%). The D605E variant of UL97 was rst described in 1 of 8 CMV isolates from an immunocompromised host in France [30], but it has not been commonly observed in the human CMV strains circulating in western countries. Some reports have shown that the D605E mutation has frequently been detected in only Asian countries, and is estimated at 91.8% in Japanese infants and children and 78% in Chinese transplant recipients [10,11]. Thus, they suggested that this mutation could be an important molecular marker of CMV evolution in East Asian countries. Q126L, T75A, and D605E mutations, which were detected in this study, could potentially be used as genetic markers for CMV in East Asian countries.
There are several limitations in this study. First, the present study included a small number of patients, all of whom were GCV-naive patients. Therefore, the results should be interpreted only in GCV-naive patients. It is speculated that a high rate of UL97 mutations may be identi ed in patients who have been treated with GCV for a longer period. Second, we used the Sanger sequence to detect the mutations in this study.
This cannot detect mutants that are present in < 10-20% of the viral population. Recently, several studies have been reported which detected the mutation of the CMV gene using a next-generation sequence (NGS) [31,32]. NGS methods have an improved ability to detect mixed populations and have been used to assess low-abundance variants. Third, we did not assess the mutation of DNA polymerase UL54 gene in this study, which is related to GCV resistance and cross-resistance to other antiviral drugs [33]. Therefore, further studies using NGS targeting both UL97 and UL54 in patients who have been treated with GCV are expected to provide more reliable evidence than this study.
Recognizing these limitations, the implications of our report are that no drug-resistant CMV strains were detected in the mucosa of the colon in patients with gastrointestinal CMV reactivation without a history of GCV administration, suggesting that it would not be necessary to consider CMV drug resistance at treatment initiation. In addition, these results were similarly observed when associated with UC and non-UC disease. Furthermore, the Q126L, T75A, and D605E mutations could potentially be used as genetic markers for CMV in East Asian countries. We look forward to further research in this area.

Conclusions
In the present study, we investigated the prevalence of CMV UL97 gene mutations in Japanese patients with CMV reactivation in the colon associated with UC and other diseases. Drug-resistant CMV strains were not detected in the colon of ganciclovir-naive patients, suggesting that it would not be necessary to consider CMV drug resistance at treatment initiation of CMV reactivation in the colon. Furthermore, Q126L, T75A, and D605E mutations could potentially be used as genetic markers for CMV in East Asian countries.

Declarations
Ethics approval and consent to participate: This study was conducted in accordance with the Declaration of Helsinki, and the study protocol was reviewed and approved by the Ethics Committee of Hamamatsu University School of Medicine, Japan (EG16-257). Informed consent was omitted, and information of this study was disclosed in the form of an opt-out on our hospital website. Information regarding the conduct of the research, including the objectives, was disclosed, and the research subjects were provided an opportunity to refuse inclusion in the research.

Consent for publication:
Not applicable.