Identication and Clinical Characteristics Analysis of HNF-1A Mutations p.I27L, p.S487N and p.G574S in Chinese Patients with MODY3

Background: Due to the rarity, the type 3 of maturity-onset diabetes of the young (MODY3) has not been explored comprehensively. The study aimed to describe and analyze the molecular and clinical characteristics of MODY3, which could help physicians understand the subtype of diabetes and increase the diagnosis rates in the future. Methods: Ten unrelated patients with suspected maturity-onset diabetes of the young (MODY) were included in the study based on information on family history and onset age. Sanger sequencing was used to identify cases with mutations in the hepatic nuclear factor 1A gene (HNF-1A). Results: Five patients were identied with MODY3, three cases (60%) with p.I27L mutation, one case (20%) with p.S487N mutation, and one case (20%) with p.G574S mutation. The average onset age was (23.00±3.00) years and the average age of diagnosis was (28.67±9.29) years. Most patients had typical clinical symptoms (polydipsia, polyuria, polyphagia, and weight loss). The main complications included diabetic ketoacidosis (DKA, 3/5,60%), diabetic macroangiopathy (2/5,40%), diabetic peripheral neuropathy (DPN,3/5,60%), diabetic nephropathy (DN,1/5,20%) and diabetic retinopathy (DR,1/5,20%). Four patients (80%) had fatty liver. The average body mass index (BMI) (26.39±4.67) kg/ (cid:0) , triglyceride (TG 2.95±1.43 mmol/L) and low-density lipoprotein (LDL-C 3.37±0.65 mmol/L) were beyond normal value. The glycosylated hemoglobin A1c (HbA1c 12.45±4.60 %), fasting plasma glucose (FPG 10.10±3.57 mmol/L) and postprandial plasma glucose (PPG 21.88±2.53 mmol/L)


Background
Maturity-onset diabetes of the young (MODY) is a monogenic dominant type of diabetes with the characters of early onset and impaired insulin secretion [1]. It is estimated to account for 1-2% of all patients diagnosed with diabetes, however, due to the similarity of clinical features, it is challenging to distinguish MODY with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) [2]. Patients with MODY still have some functional beta cells after three to ve years of diagnosis while the insulin is at a complete loss in T1DM [2]. It is still controversial whether patients with MODY have positive pancreatic antibodies [3,4]. Contrary to patients with T2DM, patients with MODY lack insulin resistance symptoms such as hypertension [5]. The contrast is more distinct in adolescents [2]. MODY is divided into more than 10 subtypes based on different mutation sites, of which the type 3 of maturity-onset diabetes of the young (MODY3) is the most common subtype [6].
MODY3 results from mutations in the hepatic nuclear factor 1 A gene (HNF-1A) located on chromosome 12q24 [7]. HNF-1A encodes for a transcription factor expressed in liver and pancreas cells in the regulation of glucose transport and metabolism [6,8]. The onset age and severity of the disease vary within patients with MODY3 [9]. Several modifying genes have been identi ed, which might explain the heterogeneity of MODY3 [10]. Environmental factors, such as exposure to diabetes in utero also in uence the age at onset of MODY3 [1]. It is unclear whether the type of the HNF-1A mutation contributes to the phenotype variability of the disease [9,11]. At present, sulfonylureas are the rst-line therapies owning to the high sensitivity of MODY3 to sulfonylurea medication [2]. Microvascular and macrovascular complications are observed with the same risk in patients with MODY3 as in T1DM and T2DM [12,13].
The mutation sites and clinical features of MODY3 in China have not been investigated comprehensively due to the rarity of the disease. Besides, due to the lack of awareness of this subtype of diabetes, MODY3 is often misdiagnosed in the real clinical practice. In this study, we identi ed patients with MODY3 to analyze the molecular and clinical characteristics, which could help doctors to make a correct diagnosis of MODY3 in the future.

Subjects
Genetic testing was performed on ten unrelated patients with suspected MODY3 in the endocrinology department of the A liated Hospital of Yunnan University/the Second People's Hospital of Yunnan Province from September 2018 to December 2019. The suspected MODY3 were identi ed based on two criteria as previously reported [14]. (a) autosomal dominant inheritance pattern through at least two generations; (b) diagnosis of diabetes under the age of 25 years in at least one family member. A detailed medical history was quizzed and the peripheral venous blood was collected after obtaining written informed consent from the patients or their guardians. The blood volume was 5ml, it was anticoagulated by EDTA. The institutional review boards of the Second People's Hospital of Yunnan Province.

DNA Extraction
EDTA anticoagulated venous blood samples were collected from all study subjects and the genomic deoxyribonucleic acid (DNA) was isolated from whole blood by proteinase K digestion followed by phenol-chloroform extraction. Subsequently genomic DNA was precipitated in ethanol. Those passed the test were placed at 4℃ or -20℃ for a long-term storage, but those failed passed the extraction would be re-extract. The extracted DNA was measured with an ultraviolet spectrophotometer, and the concentration was ≥ 50ng/ul and 260/280 = 1.7-2.0 as the DNA ampli cation template.

Sequencing and Sequence Analysis
All exons and anking intron regions of the HNF-1A gene were ampli ed from the genomic DNA samples by the polymerase chain reaction (PCR) according to published methods, and primers for amplifying the speci c fragments of the promoter region were synthesized based on the human mtDNA cambridge sequence (provided by Kunming Shuo Qing Biotechnology Co. LTD Table 1). To ensure the quality of the DNA sequences, PCR products were sequenced at least twice in both directions. Sanger sequencing technology was used to select the PCR ampli ed fragment. DNA sequences were edited using DNASTAR's SeqMan software (DNASTAR Inc., Madison, WI, USA). According to the cDNA sequence of the gene provided by NCBI and the amino acid sequence of the encoded protein, the mutation types of HNF-1A gene and its impact on the encoded amino acid were clari ed. Table 1 Primers for speci c fragments of HNF-1A gene promoter region.

Statistical analysis
Statistical analysis was performed using SPSS software (version 23). Data were expressed as mean ± standard deviation (SD), and as percentages (%).

Results
The mutations of HNF-1A

Islet function
The islet function was shown by the insulin and C-peptide release levels, from best to worst: C-III:7, A-III:4, B-III:9, E-III:8, D-III:8. The basal secretion of insulin and C-peptide was low. The level of insulin and C-peptide reached the peak at 120 minutes. The multiply growth was not enough, and there was no obviously decrease at 180 minutes (Fig. 4).

Discussion
Our study, a population-based investigation, estimates the prevalence of MODY3 and describes clinical features of the disease. Previous studies mainly discussed MODY3 in the European population [15]. Few studies have focused on the incidence and characters of this subtype of diabetes in China [16]. Meanwhile, MODY3 is often misdiagnosed asT1DM orT2DM owning to the similar symptoms [2]. Our study is of great signi cance for doctors to reduce the frequency of wrong diagnosis and provide appropriate treatment for patients with MODY3 in the future.
Fifty percent clinically suspected MODY cases had missense mutations in HNF-1A, p.I27L, p.S487N and p.G574S. The result showed that patients with mutations located in the transactivation domain (B-III:9 and C-III:7) were diagnosed later than those carrying mutations in the dimerization/DNA-binding domains (A-III:4, D-III:8 and E-III:8), which is consistent with previous studies [9]. Our study showed no negative correlation between the duration of MODY3 and the islet function. Patient B-III:9 had MODY3 for 15 years, the longest period among ve patients, however, the islet function of that patient was better than patient D-III:8 and patient E-III:8 who only suffered from MODY3 for 4 years and 1 year separately. The duration of the disease was the same in patients A-III:4, C-III:7 and E-III:8, but the islet function of patient C-III:7 was obviously the best. The difference might be caused by different mutation sites. Mutations in the transactivation domain may have less effect on the islet function, which need to be veri ed by future research. Another impact of mutation site is the risk of angiopathy in MODY3. Both patients with mutations in the transactivation domain had angiopathy secondary to MODY3, opposite to patients with mutations in the dimerization/DNA-binding domains. The study indicated patients with mutations in the transactivation domain could have later onset age and better islet function, but could also have higher susceptibility of complications such as angiopathy.
The onset age in the study is similar to the prior literature [5]. Previous research showed that patients with MODY3 lacked clinical features of insulin resistance, such as obesity and dyslipidaemia, which was different from patients with T2DM [2,5]. However, insulin resistance existed in the ve MODY3 patients in the current study. The average BMI, TG and LDL-C was over normal value. All patients had dyslipidemia. Four patients (80%) had fatty liver. As all patients in our study were from Yunnan province, they might carry some similar modifying genes which could be different to genes carried by patients from other districts in China and other parts in the world. The difference of clinical features in the present study might be caused by these speci c modifying genes. The overlap of clinical symptoms between MODY3 and T2DM brought the di culty in making a correct diagnosis. In our study, the average age of diagnosis was 4.2 years later than the average onset age. Before patients being diagnosed correctly, insulin was used in all ve patients, of which three patients also used antidiabetic drugs which not including sulfonylureas. The control of diabetes was poor as the average HbA1c, FPG and PPG were still beyond the normal range. Therefore, early diagnosis and appropriate treatment of MODY3 are vital for effective control of blood glucose and decreasing complications related to diabetes.
Genetic testing is speci c for MODY3, but due to the high expense, it is not feasible as a routine examination for all diabetic patients [17]. Previous research recommended genetic testing only for young diabetic patients with strong family histories of diabetes, which was also the criteria in our study to select suspected MODY3 patients [18,19]. However, it was estimated that more than half of MODY cases were missed in the real practice as some patients could not meet these criteria [20]. Therefore, sensitive and speci c biomarkers are needed to select proper patients for diagnostic genetic testing. N-glycan pro le and high-sensitivity C-reactive protein (hs-CRP) have been reported to have a role in identifying diabetic patients with high risk of carrying HNF-1A mutations, which could improve diagnosis rates of MODY3 [21]. At present, low dose sulfonylureas are recommended as rst-line therapy for patients with MODY3 [2,22,23]. Some recent studies reported that nateglinide alone or DPP-4 inhibitor linagliptin as add-on therapy to glimepiride could better control glycemic uctuations without increasing risk of hypoglycemia in MODY3 patients, which need to be veri ed by randomized multicenter trials [24,25].

Limitations
There are some limitations in our study. First, modifying genes inherited independently of MODY were not tested. Thus, the impact of modifying genes on the age at diagnosis could not be analyzed in the study. Second, we did not measure the level of hs-CRP in MODY3 patients and in those without MODY3 mutations. Therefore, we could not evaluate the role of hs-CRP to screen MODY3 patients from diabetic cases. Third, only ve patients with MODY3 were identi ed. Small samples might cause incomplete analysis of the in uence of mutation sites on the features of MODY3. The mutation sites and clinical features of MODY3 in Chinese population need more research from other districts in the future.