Single patient in GCK-MODY family successfully re-diagnosed into GCK-PNDM through targeted next-generation sequencing technology
Single patient in GCK-MODY family successfully re-diagnosed into GCK-PNDM through targeted next-generation sequencing technology
Karolina Antosik 0 1 2 3 4 5
Piotr Gnys 0 1 2 3 4 5
Elisa De Franco 0 1 2 3 4 5
Maciej Borowiec 0 1 2 3 4 5
Malgorzata Mysliwiec 0 1 2 3 4 5
Sian Ellard 0 1 2 3 4 5
Wojciech Mlynarski 0 1 2 3 4 5
0 Department of Clinical Genetics, Medical University of Lodz , Lodz , Poland
1 Managed by Massimo Porta
2 To the Editor
3 Institute for Biomedical and Clinical Science, University of Exeter Medical School , Exeter , UK
4 Department of Paediatrics , Diabetology and Endocrinology , Medical University of Gdansk , Gdan ́sk , Poland
5 Department of Paediatrics , Oncology, Haematology and Diabetology , Medical University of Lodz , 36/50 Sporna Str., 91-738 Lodz , Poland
In light of recent findings concerning the next-generation sequencing (NGS) of our previously reported patient, we feel it necessary to briefly review our research and provide updated results. In March 2011, we described the case of a female infant diagnosed with a heterozygous p.Gly223Ser mutation in the glucokinase gene (GCK) [1]. Heterozygous mutations of the GCK gene usually cause a mild clinical phenotype characterized by moderately elevated fasting hyperglycemia with slightly elevated levels of glycated hemoglobin, although the clinical course of diabetes may be highly variable. In contrast, homozygous or compound heterozygous mutations in this gene result in early onset of diabetes in the initial days of life, as well as pronounced hyperglycemia, ketoacidosis, and a severe clinical condition [2].
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Although ten family members of the patient are also
heterozygous carriers of the p.Gly223Ser mutation, she was
the only one who did not present the heterozygous
GCKMODY phenotype. In fact, she exhibited severe
hyperglycemia (765 mg/dl), dehydration, glucosuria, and
ketoacidosis (pH 7.09, BE 14 mM) on the day after her
birth. For the first 72 h after diagnosis, she was treated with
0.1–0.3 units/kg/h intravenous insulin for persistent
hyperglycemia: the mean value from that period equaled
340 mg/dl.
During this time, her DNA was directly sequenced using
Sanger’s method to identify homozygous or compound
heterozygous mutations in the GCK gene using an ABI
3130 genetic analyser and DNA Sequencing Analysis
Software (Applied Biosystems, Foster City, CA, USA).
Sequencer software v4.1.4 (GeneCodes, Ann Arbor, MI,
USA) was used for the comparative analysis of evaluated
sequences. In addition, the use of Sanger’s sequencing and
multiplex ligation-dependent probe amplification technique
(MLPA) did not detect mutations or deletions in other
known genes associated with monogenic diabetes. Only the
heterozygous GCK p.Gly223Ser mutation was detected.
To further investigate the cause of such diversions from
the expected phenotype, the DNA was reanalysed by
nextgeneration sequencing in the reference laboratory in
Exeter, UK. A targeted next-generation sequencing assay
performed using an Illumina HiSeq 2000 sequencer
(Illumina, San Diego, CA, USA) [3] indicated the presence of a
second missense mutation in the GCK gene, c.1236A[G,
which resulted in the amino acid substitution of glutamic
acid to lysine at position 256 (p.Glu256Lys). This mutation
was not visible in the results of the previous Sanger‘s
sequencing analysis and is now known to represent part of
a compound heterozygous genotype resulting in PNDM
(Permanent Neonatal Diabetes Mellitus). Reanalysis of the
Fig. 1 Genetic evaluation of the patient with GCK-PNDM originally
previously reported [1]. a Family pedigree of the PNDM patient with
compound heterozygous mutations in the GCK gene (No. 5109),
N wild type, M mutation; b chromatograms obtained by direct
sequencing according to Sanger, presenting a previously reported
p.Gly223Ser mutation (top chromatogram) and a newly identified
p.E256K de novo mutation (bottom chromatogram)
patient’s sample by Sanger’s sequencing revealed a rare
single nucleotide polymorphism located in the DNA
sequence covered by one primer used for PCR
(rs573845006 reported in dbSNP build 142), which was in
cis with the p.Glu256Lys mutation. This SNP resulted in
allele dropout during PCR and previous misdiagnosis.
Finally, a repeated Sanger’s sequencing of the GCK gene
with redesigned primers also confirmed the p.Glu256Lys
mutation (Fig. 1).
The p.Glu256Lys mutation was not inherited from the
patient’s mother who is a carrier for the p.Glu223Ser
mutation. We were not able to obtain biological material
from the father of the patient. Since he did not report any
symptoms of glucose metabolism disorders at the age of
38 years (fasting glucose 76 mg/dl; HbA1c 5.1 %; OGGT
normal) and no family history of diabetes, and the
p.Glu256Lys substitution is known in literature as causal
for GCK-MODY phenotype, one may speculate that this
mutation occurred as de novo in our PNDM patient. This is
unusual finding leading to GCK-PNDM.
This case reinforces the need to re (...truncated)