CME INDIA Case Presentation by Dr. R. Anil Kumar, MD FRCP (Edinburgh) FICP FRSSDI WHO fellowship in Diabetology, Associate professor and HOD Diabetes and endocrinology, Karnataka institute of endocrinology and research, Bangalore.



CME INDIA Case Study

How Presented?

• DK, Male
• Age-15 years
• Weight-44 kg
• BMI-15.22
• WC-72 cm
• BP-100/70 MM
• Family history – father diabetic.
• New onset diabetes

19/5/22

• FPG-339 mg/dl
• PPPG-557 mg/dl
• HBA1c-13.8%
• C-peptide-1.81 nano gram/ml (A normal result is between 0.5 to 2.0 nanograms per milliliter)
• (Note: C-peptide is released in the circulation along with insulin, it has widely been used as a measure of insulin secretion to assess the pancreatic beta-cell function. The plasma concentration of C-peptide in a fasted state is 0.9 to 1.8 ng/ml)
• Fasting insulin 11.63mU/ml

• GAD Antibody-2.32 IU/ml (Negative = 0-4.9U/ml,Low Positive = 5-30U/ml )
• He was put on Basal Bolus (Inj Aspart – 4-4-0 U with Inj Degludec 0-0-12U)

Further evaluation on 4/6/22

• FPG-92 mg/dl
• PPPG-119 mg/dl
• C-peptide-4.75 nano gram/ml
• Stimulated C-Peptide-5.08 nano gram/ml
• Insulin was slowly tapered and stopped. He was put on glimepiride 0.5 mg od.

Scenario worth pondering on 6/8/22

• FPG-101 mg/dl
• PPPG-85 mg/dl
• HBA1c-6.1%
• Weight – 50 kg
• BMI -17
• C-peptide-5.67 nano gram/ml
• Stimulated C-Peptide-17.76 nano gram/ml

This all happened and Genetic Panel test was available

Coverage of MODY and Neonatal diabetes panel genes

Variant description

• A heterozygous missense variation in exon 4 of the GLIS3 gene (chr9:g.4118536T>C; Depth: 163x) that results in the amino acid substitution of Methionine for Isoleucine at codon 314 (p.Ile314Met; ENST00000381971.8) was detected
• OMIM phenotype: Neonatal diabetes mellitus and congenital hypothyroidism (OMIM#610199) is caused by homozygous or compound heterozygous mutations in the GLIS3 gene (OMIM*610192).
• This disorder is characterized by intrauterine growth retardation and onset of nonimmune diabetes mellitus within the first few weeks of life.
• Other features include renal parenchymal disease, primarily renal cystic dysplasia, and hepatic disease, with hepatitis in some patients and hepatic fibrosis, cirrhosis, exocrine pancreatic dysfunction, low-set ears, epicanthal folds, flat nasal bridge, long philtrum, and thin upper lip. Most patients exhibit developmental delay

Interpretation in this UNIQUE case

• Mutations in GLIS3 gene has previously been reported to be associated with development of maturity onset diabetes of the young. However, a second significant heterozygous variation in the GLIS3 gene was not detected. The sensitivity of NGS based assays to detect large heterozygous deletions/duplications is low and an alternate method is recommended. Based on the above evidence, this GLIS3 variation is classified as a variant of uncertain significance and has to be carefully correlated with the clinical symptoms.
• The significance/classification of the variant may change based on the genetic testing in parents and other family members.

Recommendations

• Sequencing the variant(s) in the parents and the other affected and unaffected members of the family is recommended to confirm the significance.
• Genetic counselling is advised for interpretation on the consequences of the variant(s).
• If results obtained do not match the clinical findings, additional testing should be considered as per referring clinician’s recommendations.

What is targeted gene sequencing?

• Selective capture and sequencing of the protein coding regions of the genome/genes is performed. Mutations identified in the exonic regions are generally actionable compared to variations that occur in noncoding regions.
• Targeted sequencing represents a cost-effective approach to detect variants present in multiple/large genes in an individual. DNA extracted from blood was used to perform targeted gene capture using a custom capture kit.

Emerging roles of GLIS3 in neonatal diabetes, type 1 and type 2 diabetes

How association between GLIS3 and diabetes corelated

Neonatal Diabetes

• These association studies between GLIS3 variants and diabetes in people are intriguing. However, the underlying mechanisms were largely unknown until Glis3 global and β cell-specific knockout mice were generated.
• Studies in three groups independently by generated Glis3-deficient mice with different strategies showed that GLIS3 is required not only for fetal islet differentiation but also for the maintenance of adult β cell function.
• There is reported GLIS3 and neonatal diabetes syndrome
• It was year 2003 when Taha and coworkers first described a rare clinical syndrome in a consanguineous Saudi Arabian family which was characterized by permanent neonatal diabetes, congenital hypothyroidism, polycystic kidneys, intrauterine growth retardation, facial anomalies, congenital glaucoma and hepatic fibrosis. Three affected children died at 10 days, 6 and 16 months of life, respectively (Taha et al. 2003, Senee et al. 2006).

GLIS3 and type 1 diabetes

• Common T1D is an autoimmune disorder that arises from the action of a combination of genetic and environmental.
• Barrett and coworkers first identified the association between a GLIS3 variant rs7020673 and common T1D in European populations. They found that the region of strong linkage disequilibrium at chromosome 9p24.2 that harbors only a single gene, GLIS3.
• Awata and coworkers reported that low frequency GLIS3 A908V variant was associated with the resistance to T1D in Japanese population. The underlying mechanism remains elusive. Given that GLIS3 mRNA is also moderately expressed in human thymus, they proposed that the protective variant GLIS3 908V might more efficiently induce central or peripheral immune tolerance than wild-type GLIS3 908A (Awata et al. 2013).

GLIS3 and type 2 diabetes

• Dupuis and coworkers first identified the GLIS3 variant rs7034200 as one of nine newly identified loci associated with fasting glucose in T2D trait, but not associated with body mass index (BMI), blood pressure or lipid profile in participants of European descent (Dupuis et al. 2010).
• This finding has been replicated in Danes (Boesgaard et al. 2010), Chinese (Hu et al. 2010, Liu et al. 2011, Dou et al. 2016) and South Asians (Rees et al. 2011), with a borderline association with T2D in a Japanese population (Fujita et al. 2012).
• The glucose-raising allele GLIS3 rs7034200 A was also strongly associated with impaired β-cell function both in non-diabetic adults (Dupuis et al. 2010, Hong et al. 2014) as well as in healthy children and adolescents (Barker et al. 2011), which suggests an age-independent effect of this locus on inter-individual differences in glucose levels from childhood onward.
• Two other GLIS3 variants, rs7041847 and rs10814916, has been  reported to confer the susceptibility to T2D in East Asians (Cho et al. 2012, Sakai et al. 2013) and in Chinese (Li et al. 2013), respectively.
• In contrast to GLIS3 rs7034200, a risk SNP for T2D in multi-ethnic populations, GLIS3 rs7041847, appears to confer T2D susceptibility only in East Asians
• Notably, GLIS3 rs2380949 was found to be associated with insulin clearance in Hispanic Americans (Goodarzi et al. 2013), which suggests that GLIS3 may play a dual role in regulating both insulin production and clearance.

What is GLIS3?

• GLIS3 is a member of the Kruppel-like zinc finger family of transcription factors, presenting high expression in beta-cells.
• GLIS3 plays a key role in the differentiation of pancreatic cells during embryonic development, especially in the development of mature islets.
• Some rare GLIS3 mutations lead to the neonatal diabetes and hypothyroidism syndrome.
• Glis3 activates the insulin gene transcription directly and indirectly by interacting with beta-cell specific transcription factors (MafA, NeuroD1, and Pdx1) in rat insulinoma cells).
• Glis3 expression may also be necessary for beta-cell survival since Glis3 knockdown in rat insulinoma cells increased proinflammatory cytokines- and palmitate-induced beta-cell apoptosis (Nogueira et al., 2013). Dysfunction of these pathways may be implicated in the development of both T2DM and T1DM (Wen and Yang, 2017).
• Glis3 is possibly involved in the compensatory IR-induced proliferation and expansion of beta-cells in mice, which when disrupted may lead to T2DM (Wen and Yang, 2017; Yang et al., 2013).

Quick Take-Aways

• Many times, in clinical practice, we get cases, which look like Type 1 DM, LADA or T2DM.
• Apart from clinical profile and family history, in appropriate cases C-peptide, Serum Insulin and GAD65 give enough input to think differently to apply precision medicine in Diabetes
• Genetic Panel tests are rarely advised due to poor awareness about its importance, high cost and limited availability.
• This case is an eye opener, genetic panel test provided a new understanding of a rare association.
• Precision technology is the need of hour to manage patients appropriately.

References:

1. Richards S. et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, Genet Med., 17(5):405-24, 2015. 2.
2. Kalia S.S. et al., Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics. Genet Med., 19(2):249-255, 2017.
3. Shakhtshneider EV et.al., Polymorphism of the GLIS3 gene in a Caucasian population and among individuals with carbohydrate metabolism disorders in Russia. BMC Res Notes. 2018 Apr 2;11(1):211.

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