Genotyping of congenital hyperinsulinemia of infancy in Iranian population; The first larg report from Iran

Introduction: Congenital hyperinsulinemia of infancy (CHI) is one of the causes of hypoglycemia during infancy. While transient hypoglycemia could be considered as a common metabolic abnormality among neonates; however, only 1% of these infants will develop repetitive or persistent episodes of hypoglycemia (1). During CHI, increased level of insulin within infant’s blood decreases free glucose and result in neurologic dysfunction. Repeated episodes of hypoglycemia can also result in neurologic damage including mental retardation and developmental delay (2). According to the heterogeneous nature of CHI, not all the infants will have the same clinical presentation and histological findings. Mutation in some specific genes which regulate insulin secretion are considered to be responsible for development of CHI. ABCC8 and KCNJ11 are the two most common genes involved in CHI(3). These genes encode subunits of ATP sensitive K+ (ATP) channel in beta cells. The impaired activity of these channels is seen with ABCC8 and KCNJ11 mutations (2). Both of these genes are located on 11p15.1 and separated by few kilo bases. Different types of mutations including missense, nonsense, frameshift and insertions or deletions inactivate these genes. Determination of such mutations in candidate genes are useful in various ways including choosing the best treatment approach (4). Diazoxide is a K+ (ATP) channel agonist and the first line of therapy in most of the CHI patients. Those patients who are not responsive to diazoxide may use octreotide as an alternative treatment. Surgical therapy will be reserved for those CHI patients who are not responsive to medical therapies. Recessive mutations in ABCC8 and KCNJ11 genes is related to be unresponsive to some therapies including diazoxide while compound heterozygotes may have better response to treatment (4). In contrast to recessive mutations, dominant mutations will have better treatment response (4). Many case reports and case series are reporting new mutation in ABCC8 and KCNJ11 genes in different populations. Moreover, the prevalence of specific mutations in each population seems to be unique. While the genotyping of CHI has not been widely studied in Iranian population, in present report we discussed the mutations in ABCC8 and KCNJ11 genes as the most common genes involved in CHI. Materials & Methods: The present cross sectional study which has been approved by Mashhad University of Medical sciences ethic committee, includes pediatric patients who were referred to pediatric ward of Imam Reza Hospital, Mashhad, Iran from 2008 to 2017. All patients who were enrolled in present study were diagnosed as refractory Hyperinsulinemic Hypoglycemia and their parents had filled an informed consent from. Refractory Hyperinsulinemic Hypoglycemia was diagnosed according to low plasma glucose level (8mg/kg/min) and positive glycemic response (> 1.5mmol/L). In this study, infants with refractory Hyperinsulinemic Hypoglycemia did not respond to a dose of 41 mg/kg daily diasoxide and 91 mg/kg octreotide and 41 mg/kg dextrose.In children with resistant hyperinsulinemic hypoglycemia, treatment with Sirolimus (BIOCON, India) with a dose of 0.5mg of body surface area per day was considered. The dose of Sirolimus was gradually increased to reach serum level of 41 ng/ml within 5 to 6 week. Then, the infants were followed for 6 month. Patients underwent molecular diagnosis for refractory Hyperinsulinemic Hypoglycemia after genetic counselling. Sampling and DNA extraction In order to perform molecular tests, 3 ml of venous blood was taken from all subjects and their parents in EDTA tubes. Genomic DNA was extracted using salting out method. DNA samples’ quality and quantity were examined by Nano-Drop and gel-electrophoresis and stored in freezer at -70˚ C. PCR-sequencing Mutational analysis of coding and flanking intronic regions of the KCNJ11, HNF4A, HADH and ABCC8 geneswere performed by sanger sequencing following PCR fragment amplifications.Results: Twenty three pediatric patients enrolled in present study. Among these population, 5 infants did not have ABCC8, HNF4A or KCNJ11 gene mutation. Among other 18 infants, 14 were female and 4 were male. The mean age of genetic diagnosis was 18.6 days in those who had ABCC8 or KCNJ11 mutation. Most of the infants had their genetic tests at birth. The patient with the most delayed genetic diagnosis was the patient number 7 who was diagnosed 3.5 months after birth. Among those infants who did not have mutations in ABCC8 and KCNJ11 genes, the most delayed genetic testing was performed at age 3.5 years while other 4 infants were tested at birth. Most of the infants had mutation in ABCC8 gene (table 1). A missense and aberrant splicing mutation in ABCC8 gene (c.2041-21G> A) was seen in 3 infants. Other common mutations were frameshift mutation (c.3438dup) in ABCC8 gene and missense mutation (c.287-288 delins TG) in KCNJ11 gene. According to the American College of Medical Genetics (ACMG) guidelines, most of the genetic variants in our population were reported as a variant with uncertain significance.Conclusion & discussion: CHI is a rare disease with considerable mortality and morbidity if left untreated. The impact of genotyping of this heterogeneous disease is demonstrated and was the purpose of this study. According to this study results, more than half of this study’s population with clinical diagnosis of CHI have mutation in ABCC8 gene. Additionally, most of these infants had mutations with uncertain significance in ABCC8 or KCNJ11 genes.Recurrent or persistent hypoglycemia is the first clue for diagnosis of CHI. The presentation of CHI usually begins at neonatal period and some types of CHI including focal CHI can present later in life. Less than half of the children with CHI have the genetic form CHI (7). At least mutations in 9 genes considered to be related with CHI. ABCC8 and KCNJ11 genes carries most of the mutations which cause CHI. Mutations in both KCNJ11 and ABCC8 genes, which are located on 11p15.1, characterize by defective Kir6.2 and SUR1 subunits of ATP dependent K channels in B-cells (8). KATP Channel mutations results in various degrees of dysfunction ranging from partial to complete loss of channel function in B-cells (9). Recessive, dominant mutations in both of these genes can cause CHI. These inactivating mutation will unable B-cells to efflux K through these channels and inappropriate membrane depolarization will occur. The following unregulated entry of calcium ion alongside of inappropriate polarized membrane will end up in insulin release in to plasma(7).Many studies from around the word evaluated the heterogeneous genotype of CHI patients. It is noteworthy to mention that even in same country, a wide range of mutations in ABCC8 and KCNJ11 genes can be seen. As summarized in Table 1, only small number of mutations has been previously reported in the literature. Only few studies from our country, Iran, evaluated the genotyping of CHI patients. Recently a cross sectional study from Iran evaluated 6 patients with CHI. In contrast to their study, the only similar mutation among our patients is c.96C> G mutation in ABCC8 gene (6). They reported that one of their patients had this mutation in homozygous form while our patient had this mutation in compound heterozygous form. Another study from Iran had evaluated 23 patients with hyperinsulinemic hypoglycemia (5). They have demonstrated that after screening for ABCC8, HADH, HNF4A, KCNJ11, GLUD1 and GCK, only 4 patients did not show any mutation in these genes. Most of their patients had mutation in HADH gene and ABCC8 gene was the next gene with frequent mutations. The only similar mutation among this study and ours is the splicing mutation in ABCC8 gene (c.2041-21G> A) (5). They reported that their female heterozygous patient had died at age 3 months. In our population we have reported a compound heterozygous male with this mutation and 3 homozygous infants. The c.2041-21G> A mutation was the most common mutation in our population.

کلیدواژه ها:

Congenital hyperinsulinemia of infancy ، Insulin ، KCNJ11 ، ABCC8 ، Sequencing