Missense mutations in the pancreatic beta-cell ATP-sensitive potassium channel Kir6.2: A case study of Pakistani patient of neonatal diabetes.
This case study describes clinical and molecular genetic data of a 45 days old male patient of neonatal diabetes mellitus. PCR amplification followed by DNA sequencing revealed two point mutations at positions 67A greater than G and 1009G greater than A in KCNJ11 gene encoding Kir6.2 protein, a component of the beta-cell ATP-sensitive potassium (KATP) channel which is a key component involved in insulin secretion.
KEY WORDS: Single Nucleotide Polymorphism; Nonsynonymous mutation; Genetic variation.
How to cite this article: Kiran S, Khan IA, Salman A, Azim MK, Fawwad A. Missense mutations in the pancreatic beta-cell ATP-sensitive potassium channel Kir6.2: A case study of Pakistani patient of neonatal diabetes. Pak J Med Sci 2012;28(1):213-216
Neonatal diabetes mellitus (NDM) is a rare condition with an estimated incidence of 1:400,000 and 1:500,000 live births in England and Germany respectively.1,2 It is defined as persistent hyperglycemia occurring within the first 6 months of life, lasting for more than two weeks.3 Patients diagnosed with diabetes in the first 6 months of life are more likely to have monogenic neonatal diabetes rather than type 1 diabetes which is of autoimmune origin.4
There are two main types of NDM, i.e. transient (TNDM) or permanent (PNDM).5 The TNDM is a clinically defined group affecting approximately 50% of children with NDM. It remits in infancy or early childhood. Relapse in childhood or adolescence occurs in up to 50% of cases.6
The PNDM is a group affecting 40-50% cases with no remission.7 Defining genetic etiology of this rare condition has not only given insights into clinical classification and disease mechanism, but has also influenced treatment.8 Mutations of the KCNJ11 gene have been identified as the most common genetic etiology in patients with PNDM.9 The KCNJ11 gene (present on chromosome 11) is comprised of 3409 base pairs and has a single exon flanked by two introns.10 There are 5 splice variants of this gene (ENSEMBL ID: ENSG00000187486).
Identification of genetic polymorphism in KCNJ11 gene is important in the diagnosis of NDM.11 Mutations in KCNJ11 and ABCC8 genes which encode the Kir6.2 and SUR1 subunits of the pancreatic ATP-sensitive potassium channel respectively have been implicated in the genesis of PNDM.9
Here, we report a case of 45 days old male child having neonatal diabetes mellitus. Genetic studies revealed two point mutations in KCNJ11 gene of the patient.
Forty five days old child presented with generalized seizures for four days with no history of fever, vomiting or diarrhea. He was the first child to his mother, born through normal vaginal delivery by traditional birth attendant without any complications in the Khyber Pakhtoonkhwa province of Pakistan. Birth weight was not recorded. The parents were having non-consanguineous marriage and there was no family history of diabetes. The new-born was well before he presented with fits to a general practitioner. There was no previous history of seizures.
At the time of presentation his weight was 3.8 kg. Fits were controlled by giving Inj Valium (0.3 mg/
Table-I: Names and sequences of the primers used for the PCR amplification of the KCNJ11 gene.
kg IV stat) and Inj Phenobarbitone 15 mg/kg IV stat. On investigations, his random blood sugar was found to be 530 mg/dl with no evidence of ketones on urine analysis. Blood sugar was brought down to 230 mg/dl by giving repeated boluses of regular insulin. Blood sample of the subject was collected for molecular genetic analysis to detect genetic variation. He was discharged home on one unit of regular insulin once a day. The patient then lost to follow up for a month, and had severe gastroenteritis which ultimately lead to his death as he remained untreated for circumstantial reasons.
To characterize the KCNJ11 gene polymorphism, the genomic DNA of the patient was purified from the whole blood followed by DNA amplification by PCR using primers mentioned in Table-I. The PCR was performed using 0.5 mM primers, 100 ng template DNA, 2.5 ul of high fidelity DNA polymerase mix (Fermentas Inc. Lithuania) and 0.2mM of dNTPs. The PCR conditions were as follows; initial denaturation at 94oC for 2 min, denaturation at 94oC for 45 sec, annealing at 51oC for 45 sec and extention at 68oC for 2 min (35 cycles). The primer pair KCN1F-KCN3R was used to amplify 1.8 kb region while other two primer pairs (Table-I and Fig.1) were used for internal amplification. The primer pairs KCN1F-KCN1R and KCN3F-KCN3R produced PCR products of ~500 bp while primer pair KCN1F-KCN3R produced an amplicon of 1.8 kb. The obtained amplicons were subjected to DNA sequencing using the same primers. Autamated DNA sequencing was done by Genetic Analysis System CEQ 8000 (Beckman Coulter Inc. USA).
The obtained sequence data was analyzed using Lasergene software package (DNA Star Inc., USA), Staden R12 and Clustal W.13
Fig.1 shows amplified band of KCNJ11 gene (1.8 kilo bases). DNA sequencing was performed by using automated Genetic Analysis system (Beckman Coulter Inc. USA). Analysis of sequencing results revealed single nucleotide polymorphism at two positions i.e. 67A greater than G and 1009G greater than A.
The case we are reporting is not a very commonly encountered clinical condition. These patients usually present with hyperglycemia, failure to thrive or Diabetic Ketoacidosis (DKA) due to inadequate insulin production.14 As defined in NDM, marked hyperglycemia in the first month of life, no ketosis despite marked hyperglycemia made this provisional diagnosis possible in our patient. Because the anthropometry was not recorded at birth, we cannot comment whether the baby was of normal birth weight or Small for Gestational Age as usually reported in such cases.
Studies have shown that approximately one-third to one-half of all cases of PNDM are due to mutations in KCNJ11.15 Along with mutations in KCNJ11, the genes GCK and GLUT2 are also related to NDM. The GCK is a glycolytic enzyme that acts as a glucose sensor in pancreatic b-cells and plays important role in the regulation of insulin secretion while GLUT2 encodes glucose-transporter protein.11
The KCNJ11 gene encodes for Kir6.2 protein, a component of the beta-cell ATP-sensitive potassium (KATP) channel which is a key component involved in insulin secretion.11
The non-synonymous SNPs at two positions i.e. 67A greater than G and 1009G greater than A detected in this case would result in amino acid substitutions Glu23Lys and Ile337Val (Fig. 2 and 3). Activating mutations in KCNJ11 cause permanent neonatal diabetes due to overactive Potassium ATP channels, resulting in reduced insulin secretion.9
Identification of a KCNJ11 mutation has implications on clinical management. Many patients can be successfully treated with oral sulphonylureas rather than insulin.16 Sulphonylureas have affinity to SUR1 and stimulate insulin secretion by an ATP-independent mechanism and can successfully replace insulin therapy to achieve a better metabolic control.9 The effectiveness of oral sulfonylurea in improving glyceamic control of diabetic patients due to KCNJ11 mutations after transfer from insulin therapy has been confirmed by many reports16 but a molecular diagnosis is required before the use of sulfonylurea therapy in neonatal diabetes is considered.17
We are thankful to Dr. Pir Alam, Associate Dia-betologist from Mardan, Pakistan for referring the above mentioned case.
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Shazia Kiran, Ishtiaq A. Khan, Ahmed Salman, M. Kamran Azim and Asher Fawwad
1. Shazia Kiran, MBBS, DDM, MRCGP (Int) Research Officer, Research Department, 2. Ishtiaq A. Khan, PhD, Assistant Professor, 3. Ahmed Salman, MRCP (UK), Assistant Professor Medicine, Department of Medicine, 4. M. Kamran Azim, PhD, Assistant Professor, 5. Asher Fawwad, M Phil, Assistant Professor, Senior Research Scientists, Research Department, 2,4: Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences. University of Karachi, Karachi, Pakistan. 1,3,5: Baqai Institute of Diabetology and Endocrinology Baqai Medical University, Karachi, Pakistan., Correspondence: Asher Fawwad, M Phil, Assistant Professor (BMU), Senior Research Scientists, Research Department, Baqai Institute of Diabetology and Endocrinology, Baqai Medical University. Plot No. 1-2, II-B, Block 2, Nazimabad, Karachi-74600, Pakistan. E-mail: email@example.com, Received for Publication:May 4, 2011, 1st Revision Received: October 26, 2011,
2nd Revision Received: November 1, 2011, Final Revision Accepted: November 20, 2011
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|Publication:||Pakistan Journal of Medical Sciences|
|Article Type:||Clinical report|
|Date:||Mar 31, 2012|
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