Hyperglycemia and Hypoglycemia via the GCK Gene
- Summary and Pricing
- Clinical Features and Genetics
- Bi-Directional Sanger Sequencing
- Deletion/Duplication Testing Via Array Comparative Genomic Hybridization
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The great majority of tests are completed within 18 days.
The GCK mutation detection rate is widely variable depending on the age and clinical selection criteria of the target population (Osbak et al. Hum Mutat 30(11):1512-1526, 2009; Pihoker et al. J Clin Endocrinol Metab 98(10):405540-62, 2013). In a study of 82 children with incidental hyperglycaemia, 43% were found to carry GCK mutations (Feigerlová et al. Eur J Pediatr 165(7):446-452, 2006). The GCK mutation detection rate in a large cohort of patients with PNDM is unavailable because mutations have been only reported in limited individual cases. Defects of GCK represent a rare cause of PNDM. In a cohort of 298 diazoxide-nonresponsive CHI patients studied at the Hyperinsulinism Center in The Children’s Hospital of Philadelphia (CHOP), approximately 2% (7 of 298) were identified to have GCK mutations via DNA sequencing (Snider et al. J Clin Endocrinol Metab. 98(2):E355-63, 2013). In another cohort of 300 CHI patients studied in the United Kingdom, no GCK mutations were found in 105 diazoxide-nonresponsive or 183 diazoxide-responsive patients (Kapoor et al. Eur J Endocrinol 168(4):557-564, 2013). In a study of CHI patients enrolled from three European referral centers, the overall prevalence of GCK-related non-syndromic CHI was approximately 1%. In the medically responsive group, the prevalence of GCK-caused CHI was approximately 7% (Christesen et al. Eur J Endocrinol 159(1):27-34, 2008).
Deletion/Duplication Testing via aCGH
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The great majority of tests are completed within 28 days.
Defects of glucokinase (hexokinase 4) can cause either hyperglycemia or hypoglycemia depending on underlying mutations of its encoding gene GCK (Osbak et al. Hum Mutat 30(11):1512-1526, 2009). Inactivating (loss-of-function) GCK mutations lead to two types of hyperglycemia: maturity-onset diabetes of the young (MODY) and permanent neonatal diabetes mellitus (PNDM). On the other hand, activating (gain-of-function) GCK mutations have been associated with hyperinsulinemic hypoglycemia, also known as congenital hyperinsulinism (CHI).
MODY is the most common type of monogenic diabetes, accounting for up to 5% of young adults diagnosed with diabetes (Owen. Clin Med 13(3):278-281, 2013; McDonald et al. Ann Clin Biochem 50(Pt 5):403-415, 2013). MODY typically presents in lean young adults before 25 years with an autosomal dominant family history. MODY patients have continuous production of endogenous insulin, absence of beta-cell autoimmunity and absence of signs of insulin resistance. As this non-insulin dependent type of diabetes is frequently misdiagnosed as Type 1 or Type 2 diabetes, a timely and accurate molecular diagnosis of MODY is essential to treatment decisions, prognosis, family screening and obstetric management of gestational diabetes (Pihoker et al. J Clin Endocrinol Metab 98(10):405540-62, 2013; Ellard et al. Diabetologia 51(4):546-553, 2008). Mutations in the GCK, HNF1A and HNF4A genes account for up to 80% of all MODY cases. GCK-MODY (MODY, type II or MODY2) represents a milder form of disease with a mild, asymptomatic and non-progressive fasting hyperglycaemia from birth without the requirement of pharmacological treatment. GCK-MODY is the most common form of MODY in children, accounting for about 30% of total MODY cases in the UK (Owen, 2013).
Neonatal diabetes mellitus, another type of monogenic diabetes, is defined as insulin-requiring hyperglycemia within the first 6 months (or even beyond 6 months) of life and is often associated with intrauterine growth retardation (Njølstad et al. N Engl J Med 344(21):1588-1592, 2001; Njølstad et al. Diabetes 52(11):2854-2860, 2003; Massa et al. Hum Mutat 25(1):22-27, 2005; Colombo et al. J Clin Invest. 118(6):2148-2156, 2008). About half of affected neonates have transient diabetes while the rest have permanent neonatal diabetes mellitus (PNDM; OMIM# 606176). Some severe PNDM patients may have neurological complications including developmental delay, epilepsy and neonatal diabetes (also termed DEND syndrome) (Molven et al. Expert Rev Mol Diagn 11(3):313-320, 2011). GCK-PNDM is caused by complete deficiency of glucokinase and thus represents the severe end of GCK- associated hyperglycemia.
Congenital hyperinsulinism (CHI) is a clinically and genetically heterogeneous condition characterized by hypoglycemia (Glaser et al. GeneReviews, 2003; Arnoux et al. Early Hum Dev 86(5):287-294, 2010). The age of disease onset ranges from the neonatal period with severe forms to infancy or childhood with milder forms. Severe patients typically have extremely low serum glucose while milder cases present with variable hypoglycemia. Affected newborns also develop nonspecific symptoms including seizures, apnea, hypotonia, and poor feeding. Severity of disease manifestations can vary within the same family.
The GCK gene has 10 coding exons that encode glucokinase (hexokinase 4), which phosphorylates glucose to produce glucose-6-phosphate in glucose metabolism pathways (Osbak et al., 2009). Playing a crucial role in the regulation of insulin secretion, glucokinase has been termed the glucose sensor in pancreatic beta-cells. GCK defects throughout the whole gene include missense, nonsense, regulatory, splicing site mutations, small deletion/insertions while intragenic exon-level deletions/duplications are rare (Human Gene Mutation Database).
MODY is inherited in an autosomal dominant manner. So far, there are about 12 different genes associated with MODY-like phenotypes (Molven et al., 2011). Alongside HNF1A, HNF4A and HNF1B, GCK is among the most frequently involved genes in MODY (Owen, 2013). Approximately 85% of documented pathogenic GCK mutations are associated with MODY (Human Gene Mutation Database). Among these MODY-associated GCK mutations, about 60% are missense changes. Partial or whole gene deletions have been found to be rare in GCK-MODY (Ellard et al. Diabetologia 50(11):2313-2317, 2007; Garin et al. Clin Endocrinol 68(6):873-878, 2008). Phenotypes are notably similar for all MODY-associated GCK mutations (Ellard et al., 2008).
PNDM is commonly caused by mutations in the genes ABCC8 and KCNJ11, which encode the subunits of the beta-cell ATP-sensitive potassium (KATP) channel (Molven et al., 2011). Another relatively frequent cause of PNDM are mutations in the insulin gene (INS). Defects of GCK represent a rare cause of PNDM. GCK-PNDM is inherited recessively and only limited cases have been reported. PNDM-associated GCK defects include missense, nonsense and frameshift mutations, either homozygous or compound heterozygous which result in complete deficiency of glucokinase activity (Osbak et al., 2009).
CHI is genetically caused by defects in genes involved in regulation of insulin secretion from pancreatic beta-cells (Kapoor et al. Eur J Endocrinol 168(4):557-564, 2013; Snider et al. J Clin Endocrinol Metab 98(2):E355-363, 2013). GCK-related congenital hyperinsulinism (OMIM# 602485) is inherited in an autosomal dominant manner. Dominant activating GCK mutations are the third most common genetic cause of CHI (Snider et al., 2013; Glaser et al. N Engl J Med 338(4):226-230, 1998). GCK mutations have been found in both diazoxide-responsive (Christesen et al. Eur J Endocrinol 159(1):27-34, 2008) and diazoxide-unresponsive CHI patients (Snider et al., 2013). De novo GCK mutations in CHI are very common. All germline GCK mutations found in CHI patients are missense substitutions with a clustering at a confined region termed the allosteric activator site, which is remote to the substrate-binding site. Notably, a postzygotic mosaic GCK mutation (c.1361_1363dupCGG/p.Ala454dup) has been found in a patient’s pancreatic, but not peripheral blood, DNA (Snider et al., 2013). Eight other genes have also been associated with CHI including two KATP genes ABCC8 and KCNJ11, GLUD1, HADH, SLC16A1, HNF4A, HNF1A and UCP2 (Glaser et al., 2003; Kapoor et al.; Snider et al., 2013).
Testing is accomplished by amplifying each of the 10 coding exons and ~20 bp of adjacent noncoding sequence, then determining the nucleotide sequence using standard dideoxy Sanger sequencing methods and a capillary electrophoresis instrument. It also includes targeted testing of a promoter regulatory mutation c.-557G>C (Gasperíková et al. Diabetes 58(8):1929-1935, 2009). We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.
Indications for Test
Candidates for this test include:
1) Patients with mild fasting hypoglycemia and pregnant women with gestational diabetes (Ellard et al., 2008).
2) Infants with neonatal diabetes, especially if some first degree relatives have a mild form of diabetes or glucose intolerance (Molven et al., 2011; Njølstad et al., 2001).
3) Patients with CHI, especially when ABCC8 and KCNJ11 are normal (Snider et al., 2013; Christesen et al., 2008).
Additionally, testing is indicated for family members of patients who have known GCK mutations.
|Official Gene Symbol||OMIM ID|
|Hyperinsulinemic Hypoglycemia, Familial 3||602485|
|Maturity-Onset Diabetes Of The Young, Type 2||125851|
|Permanent Neonatal Diabetes Mellitus||606176|
|Congenital Hyperinsulinism Sequencing Panel|
|Maturity Onset Diabetes of the Young (MODY) Sequencing Panel|
- Genetic Counselor Team - email@example.com
- Wuyan Chen, PhD - firstname.lastname@example.org
- Arnoux J.B. et al. 2010. Early Human Development. 86: 287-94. PubMed ID: 20550977
- Christesen, H. et al. (2008). “Activating glucokinase (GCK) mutations as a cause of medically responsive congenital hyperinsulinism: prevalence in children and characterisation of a novel GCK mutation.” Eur J Endocrinol 159(1):27-34. PubMed ID: 18450771
- Colombo, C. et al. (2008). “Seven mutations in the human insulin gene linked to permanent neonatal/infancy-onset diabetes mellitus.” J Clin Invest 118(6):2148-2156. PubMed ID: 18451997
- Ellard, S. et al. (2007). "Partial and whole gene deletion mutations of the GCK and HNF1A genes in maturity-onset diabetes of the young." Diabetologia 50(11):2313-2317. PubMed ID: 17828387
- Ellard, S. et al. (2008). "Best practice guidelines for the molecular genetic diagnosis of maturity-onset diabetes of the young." Diabetologia 51(4):546-553. PubMed ID: 18297260
- Feigerlová, E. et al. (2006). “Aetiological heterogeneity of asymptomatic hyperglycaemia in children and adolescents.” Eur J Pediatr 165(7):446-452. PubMed ID: 16602010
- Garin, I. et al. (2008). "Haploinsufficiency at GCK gene is not a frequent event in MODY2 patients." Clin Endocrinol 68(6):873-878. PubMed ID: 18248649
- Gasperíková, D. et al. (2009). "Identification of a novel beta-cell glucokinase (GCK) promoter mutation (-71G>C) that modulates GCK gene expression through loss of allele-specific Sp1 binding causing mild fasting hyperglycemia in humans." Diabetes 58(8):1929-1935. PubMed ID: 19411616
- Glaser B. 2003. Familial Hyperinsulinism. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301549
- Glaser, B. et al. (1998). "Familial hyperinsulinism caused by an activating glucokinase mutation." N Engl J Med 338(4):226-230. PubMed ID: 9435328
- Human Gene Mutation Database (Bio-base).
- Kapoor R.R. et al. 2013. European Journal of Endocrinology / European Federation of Endocrine Societies. 168: 557-64. PubMed ID: 23345197
- Massa, O. et al. (2005). “KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes.” Hum Mutat 25(1):22-27. PubMed ID: 15580558
- McDonald, T. et al. (2013). “Maturity onset diabetes of the young: identification and diagnosis.” Ann Clin Biochem 50(Pt 5):403-415. PubMed ID: 23878349
- Molven, A. et al. (2011). "Role of molecular genetics in transforming diagnosis of diabetes mellitus." Expert Rev Mol Diagn 11(3):313-320. PubMed ID: 21463240
- Njølstad, P. et al. (2001). "Neonatal diabetes mellitus due to complete glucokinase deficiency." N Engl J Med 344(21):1588-1592. PubMed ID: 11372010
- Njølstad, P. et al. (2003). “Permanent neonatal diabetes caused by glucokinase deficiency: inborn error of the glucose-insulin signaling pathway.” Diabetes 52(11):2854-2860. PubMed ID: 14578306
- Osbak, K. et al. (2009). "Update on mutations in glucokinase (GCK), which cause maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemic hypoglycemia." Hum Mutat 30(11):1512-1526. PubMed ID: 19790256
- Owen, K. (2013). "Monogenic diabetes: old and new approaches to diagnosis." Clin Med 13(3):278-281. PubMed ID: 23760703
- Pihoker C, Gilliam LK, Ellard S, Dabelea D, Davis C, Dolan LM, Greenbaum CJ, Imperatore G, Lawrence JM, Marcovina SM, Mayer-Davis E, Rodriguez BL, et al. 2013. Prevalence, characteristics and clinical diagnosis of maturity onset diabetes of the young due to mutations in HNF1A, HNF4A, and glucokinase: results from the SEARCH for Diabetes in Youth. J. Clin. Endocrinol. Metab. 98: 4055–4062. PubMed ID: 23771925
- Snider K.E. et al. 2013. The Journal of Clinical Endocrinology and Metabolism. 98: E355-63. PubMed ID: 23275527
- Bi-Directional Sanger Sequencing
- Deletion/Duplication Testing Via Array Comparative Genomic Hybridization
Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.
As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
Deletion/Duplication Testing Via Array Comparative Genomic Hybridization
Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.
PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.
Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.
This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.
aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.
Breakpoints, if occurring outside the targeted gene, may be hard to define.
The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.
myPrevent - Online Ordering
- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.