Discussion

MODY9 is a rare type of MODY caused by a PAX4 gene mutation. In vertebrates, the PAX family consists of nine members, which can be divided into four subgroups according to the specific composition of the structural domain. Among them, the subfamily IV (PAX4 and PAX6) is composed of paired and homologous domains [12]. PAX4 gene is located on 7q32.1 and contains 10 exons and 9 introns with a molecular weight of 7,851bp. Its encoding product is the paired box protein PAX-4. PAX-4 protein contains a paired domain (PD) and a homeobox domain (HD), both of which are potential DNA-binding domains (DBDS) [13]. PAX4 protein is widely distributed, mainly expressed in islet pancreatic cells, and plays an important regulatory role in the production of islet progenitor cells and the differentiation of isletβ -cells and islet δ -cells [11,14]. It can inhibit the expression of α -cell genes and promote the connection of endocrine cells with β- cells and δ -cells. As a key regulator of mammalian pancreatic development, PAX4 protein first appeared in endocrine progenitor cells at 9.5 days of embryos and then is selectively expressed in β -cells [15]. In β -cells, PAX4 protein can maintain the expression of pancreatic and duodenal homeobox 1, Pdx1 and NK6 homeobox 1, Nkx 6.1, both of which are important regulators of pancreatic β -cell development [16].
In animal experiments, heterozygous PAX4 knockout mice had almost no mature β -cells and δ -cells, and a large number of abnormalα -cells were found. All of which could lead to an increase in blood glucose levels, suggesting that PAX4 is a key regulator in the differentiation process of precursor cells into different islet cell [17]. Other research data showed that PAX4 homogenous variation can cause the destruction of PAX4 protein’s targeting ability, resulting in an reduced inhibitory function and an increase in α -cells, which promotes glucagon production and leads to increased blood sugar [18,19].
In 2007, Nattachet Plengvidhya et al. studied the pathogenic genes of 46 MODY probands (all Thai origin) applying DNA direct sequencing techniques [10]. They reported a missense mutation of the PAX4 gene with a variation of R164W and a shear mutation of PAX4 gene responsible for a IVS7-1variation in two diabetic families, respectively, being the first two reports on PAX4 mutation in relation to MODY and was defined as MODY9 [10]. Nattachet Plengvidhya et al. found that 4 members of the diabetic family with the R164W variation carried the same mutation. Three of which suffered from diabetes. The onset age of the proband was 20, while in the other two cases it was 29 and 50. No ketosis, nor ketoacidosis were detected at the onset of the disease. Another family member developed impaired glucose tolerance at age 14, which did not progress to diabetes. Patients in this group were treated by diet control or oral hypoglycemic drugs, and their blood glucose level were well controlled. 6 members of the diabetic family with IVS7-1 variation presented with diabetes, of which only the proband underwent gene sequencing analysis and was found to carry the mutation. Of the 6 members with diabetes, 1 developed retinopathy and kidney disease 10 years after diagnosis of diabetes, and 3 died of end-stage renal failure at the age of 52-53 years [10].
MODY9 is rarely seen in clinical practice, and only a few families have been reported so far. In 2011, Wakako Jo et al reported a Japanese family with MODY9 [20]. There were 2 patients with DM in this family, all of whom carried a deletion mutation in the third exon region of PAX4 C374-412del39. The proband’s disease onset was at the age of 15 with ketosis and had been treated with insulin since diagnosis. Another patient in the family, the father of the proband, was diagnosed at the age of 30 with mild diabetes, which was well controlled through diet management only. In 2018, Serena Pezzilli et al. reported a missense mutation of PAX4 c.593C>T (A198V) in a MODY9 Italian family with 4 diabetic patients. Among them, the proband and the sister carried Ala198Val missense mutation, while the proband’s mother and aunt did not undergo gene sequencing. The 4 members were diagnosed as early as 20 years old and as late as 60 years old. Three of them were treated with oral medication and their blood glucose was well controlled, while the other one received insulin treatment [21].
Three more missense mutations of the PAX gene were found in subsequent studies. The missense mutation of c.377A>G (p. D126G) and c.55C>T (p.R19W) was reported by Natalia Zubkova et al. [22]. C.92G>T (p.r.31l) missense mutation was reported by Aaron Chapla et al. in India [23]. So far, the inheritance pattern of MODY9 mutation has been mainly autosomal dominant.
In this study, we report a disease that started in infancy and was characterized by an autosomal dominant mutation, which was consistent with the characteristics of MODY. C.487C>T missense mutation was found in the 7th exon region of the PAX4 gene in children, leading to a variation of p.R163W in the amino acid sequence. The R163W variation is located in the homologous domain of the PAX4 gene, which is responsible for the binding of PAX4 protein to the target DNA sequence and is also associated with conserved amino acid residues between species. R163W variation causes the replacement of polar amino acids by non-polar amino acids, which is suspected to impair the inhibitory effect of PAX4 protein on insulin and glucagon promoters [24], leading to hyperglycaemia and diabetes. This mutation has not been reported in previously published papers. Genetic analysis of the family members showed that the father of the patient carried the same heterozygous mutation, while the mother presented with a normal genotype, suggesting that the mutation was autosomal dominant, and the inheritance pattern was consistent with previous literature reports.
The estimated frequency of C.487C>T missense mutation in the PAX4 gene in the normal population database is 0.00010, which is a low-frequency mutation. According to clinical manifestations and sequencing results, the patient was diagnosed with MODY9 monogenic diabetes. The father of the proband had no diabetes at that time, which may be related to epigenetic factors or incomplete genetic penetrance.
Previous literature showed that the onset age of MODY9 patients ranged from 14 to 50 years. The clinical manifestations of patients with the same mutation were also significantly different. Mild cases may only present with elevated fasting glucose levels or impaired glucose tolerance, while some cases manifest with typical symptoms of diabetes such as polydipsia, polyuria and emaciation, only few cases occur with ketoacidosis. In this study, the onset of illness occurred during early childhood, characterized by typical diabetic symptoms accompanied by ketoacidosis. In which case was a MODY9 with early onset and severe clinical symptoms. Islet autoantibody was negative, and fasting insulin and C-peptide levels were significantly reduced, which was consistent with the clinical characteristics of MODY9 reported in previous literature.
MODY9 is generally treated the same way as other types of MODY, mainly with diabetic diet, exercise and insulin therapy. Some patients are responsive to oral hypoglycemic agents. In some patients, treatment has to adjusted according to the progression of the disease. On the latest follow-up, the patient had been diagnosed with diabetes for 17 months. He was treated with basic and mealtime insulin injections and his blood sugar levels were in normal range ever since.
The clinical outcome of MODY9 patients varies greatly. In mild cases, the only symptom may be impaired glucose tolerance, without progression to diabetes, and in severe cases, renal or retinal complications may occur, or even death from end-stage renal failure.
In summary, in Chinese children, the heterozygous mutation of c.487C>T in the PAX4 gene can lead to the occurrence of MODY9. MODY9 patients differ in onset ages and clinical symptoms, making it difficult to distinguish them from other MODY subtypes. Here, we conclude that gene sequencing analysis is of great significance in the diagnosis and classification of MODY, and an accurate genetic diagnosis will enable patients to receive more specialized treatment.