Genetic Testing of Maturity-Onset Diabetes of the Young Current Status and Future Perspectives

Diabetes is a global epidemic problem growing exponentially in Asian countries posing a serious threat. Among diabetes, maturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders that occurs due to β cell dysfunction. Genetic defects in the pancreatic β-cells result in the decrease of insulin production required for glucose utilization thereby lead to early-onset diabetes (often <25 years). It is generally considered as non-insulin dependent form of diabetes and comprises of 1–5% of total diabetes. Till date, 14 genes have been identified and mutation in them may lead to MODY. Different genetic testing methodologies like linkage analysis, restriction fragment length polymorphism, and DNA sequencing are used for the accurate and correct investigation of gene mutations associated with MODY. The next-generation sequencing has emerged as one of the most promising and effective tools to identify novel mutated genes related to MODY. Diagnosis of MODY is mainly relying on the sequential screening of the three marker genes like hepatocyte nuclear factor 1 alpha (HNF1α), hepatocyte nuclear factor 4 alpha (HNF4α), and glucokinase (GCK). Interestingly, MODY patients can be managed by diet alone for many years and may also require minimal doses of sulfonylureas. The primary objective of this article is to provide a review on current status of MODY, its prevalence, genetic testing/diagnosis, possible treatment, and future perspective.

to D20S16. The monogenetic nature of MODY diabetes was confirmed after the identification of mutation in glucokinase (GCK) gene (32). The non-sense gene mutation (P.Q268X) in hepatocyte nuclear factor 4 alpha (HNF4α) gene was reported as the cause of MODY5 (11). At present, 14 MODY types specified by mutation in respective 14 genes with their etiologies are known.

PRevALeNCe OF MODY
Maturity-onset diabetes of the young is not the prevalent diabetic form in the world population and is usually misclassified with type 1 and type 2 diabetes (33,34). In UK, 80% of MODY patients are misdiagnosed as type 1 or type 2 diabetes (35). In developed countries, the reported frequency of MODY is 1-2% (36). Amed et al. (37) reported the minimum incidence rate of MODY in Canadian population that is 0.4 cases per 100,000 children and youth of <18 years age. However, it has been reported to be highly prevalent in certain communities including Pima Indians (38), Nauru population (39), and southern India (40). Mohan et al. (41) reported a high prevalence of MODY in Asian Indian patients. According, to Pihoker et al. (42), the minimum prevalence of MODY increases to 2.1/100,000 individuals of <20 years of age.
Till now, 14 subtypes of MODY with distinct genetic etiologies have been identified. The relative prevalence of MODY subtypes vary greatly with MODY2 and MODY3 being the most prevalent ( Table 2). The relative frequency of each MODY type varies due to different recruitment data and ethnic variability.

HNF4α-MODY (MODY1)
Hepatic nuclear factor 4 alpha (HNF4α) is an important gene located on chromosome 20 and plays a significant role in regulating the expression of liver and pancreatic β-cells (63,64). Mutation in exon 4 (Thr 130 → Ile) and exon 7 (Gly 268 → Thr) of HNF4α gene is responsible for causing MODY1 (8,9). HNF4α regulates the proteins essential for glucose transport metabolism as well as lipoprotein biosynthesis in liver cells (65)(66)(67). HNF4α mutation causes decline in insulin production except during neonatal period, where it results in hyperinsulinemic hypoglycemia thereby exhibit phenotype similar to MODY3 (8,9,68). MODY1 has the penetrance of less than 5% where most carriers exhibit diabetic signs before 25 years of age and about 15% patients show diazoxide-responsive hyperinsulinemic hypoglycemia history (69,70). HNF4α mutation also leads number of pancreatic disorders like decline in high-density lipoprotein (HDL), triglyceride levels and increment in low density lipoprotein (LDL), respectively.

GCK-MODY (MODY2)
Glucokinase enzyme also named as hexokinase D belongs to hexokinase gene family which acts as glucose sensor in pancreatic β-cells (71,72) thus, playing critical role in glucose homeostasis. This enzyme is constitutively expressed and catalyzes the initial rate limiting step in the glycolytic pathway by ATP dependent phosphorylation of glucose to glucose-6-phosphate (71) in presence of Mg ions. GCK gene mutations upregulating the insulin secretion and resetting the glucose threshold results in enhanced fasting glucose level (73). Ellard et al. (74) reported GCK mutations in 5-6% of women who were suffering from gestational diabetes. The frequent heterozygous inactivating GCK mutations are responsible for the second most common type of MODY termed as MODY2 (75). MODY2 is characterized by mild persistent fasting hyperglycemia and low glucose-stimulated insulin secretion (75

HNF1α-MODY (MODY3)
The hepatocyte nuclear factor 1 alpha (HNF1α) gene plays an important role in regulating expression of insulin gene in the mature β-cell and glucose transporter GLUT2 gene (82,83). Fajans (12) revealed the association of HNF1α gene mutation with MODY 3 diabetes by conducting linkage analysis of R-W pedigree. Hyperglycemia due to HNF1α mutations can be controlled with sulfonylureas for a number of years (84). Anuradha et al. (85) studied the gene polymorphism of HNF1α and showed the high prevalence of amino acid polymorphism at codon (Ala 98 → Val) which is associated with MODY3 diabetes. Earlier, it was demonstrated that patients with HNF1α mutations develop diabetes after the first decade, preceded by abnormal glucoseinduced insulin secretion (86). MODY3 is characterized by a progressive reduction in insulin secretion. It has been shown that carriers of HNF1α mutation show increased β-cell apoptosis (87).

NeUROD1-MODY (MODY6)
Neurogenic differentiation-1 gene (NEUROD1) transcription factor is significant role in pancreatic and neuronal development and is functionally expressed in both pancreatic and neurons cells. However, its impairment results in autosomal dominant inheritance mutations similar to type 2 (16,96,97). Modified activity of NEUROD1 gene in carriers of Arg 111 → Leu and insertion of cytosine residue in a polyC tract in codon 206 (206 + C) of exon2 were largely responsible for development of diabetes (16). Mutation in basic-loop-helix transcription factor NEUROD1 is responsible for MODY6 (75).

KLF11-MODY (MODY7)
Krueppel-like factor 11 (KLF11) gene is located on chromosome 2 and plays important role in pancreatic β-cell functioning (98,99). KLF11 mutation affects the pancreatic β-cell function by modulating the expression of certain free radical scavengers such as catalase and superoxide dismutase (SOD). The expression level of these antioxidant enzymes is reported to be low in pancreatic islets; however, the over expression of these enzymes protects β-cells against glucolipotoxicity (100). The occurrence of several uncommon variants Ala 347 → ser, Thr 220 → Met, and Gln 62 → Arg of KLF11 gene associated with familial diabetes which severely disrupts transcriptional machinery was analyzed by genetic analysis and mutation in KLF11 results in MODY termed MODY7 (17).

CeL-MODY (MODY8)
The main ingredient of pancreatic juice is carboxyl-ester lipase (CEL) encoded by carboxyl-ester lipase gene (9q) and plays an important role in newborns by digesting milk and hydrolyzing dietary esters in duodenum (101,102). A single base deletion resulted in the altered C-terminal sequencing, thus causing the monogenetic disease, i.e., MODY8. On the other hand, a single base insertion (1686delT; C563fsx673) causes polygenic diabetes resulted by truncation of the CEL (103), thus suggesting a frame shift deletion mutation in variable number tandom repeats (VNTR) in the 11th exon of CEL gene. In addition, common as well as rare CEL mutations also affect exocrine and endocrine functioning of pancreas leading to MODY diabetic syndrome (MODY8).

PAX4-MODY (MODY9)
Paired box gene 4 (PAX4) is a transcription factor which is a member of PAX family located on chromosome 7 and regulates fetal development, cancer growth, commitment of progenitor cells to various islet cells and also represses the promoter activity of insulin and glucagon (104,105). PAX4 is required for the regeneration of β-cells in adults and its mutation blocks or inhibits β-cell proliferation (106). Two PAX4 gene mutations viz Arg 164 → trp and IVS7-IG>A were reported as the cause of monogenetic form of diabetes in Thai population termed MODY 9 (19).

iNS-MODY (MODY10)
The insulin gene (INS) which is located on chromosome 11p15.5 encodes the proinsulin precursor of insulin, its mutation cause defect in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and thus leads to MODY10 (107,108

bLK-MODY (MODY11)
Lymphocyte kinase tyrosine kinase (BLK-B) encoding the receptor protein tyrosine kinase, plays a significant role in thymopoiesis in immature T cells (109). Drebin et al. (110) cloned human homolog of murine BLK and concluded that like murine BLK gene human BLK is expressed only in β-cells. The obese feature of diabetes was reported to be linked with chromosome 8p23 gene locus (111). BLK expression is altered in response to this Ala 71 → Thr substitution (22). The BLK gene mutation essentially affects MIN6 B-cells (a highly differentiated B-cell line) and is responsible for MODY11 (22).

AbCC8-MODY (MODY12)
Before 40 years of age, the ABCC8 mutations were reported in the cohort of adult type 2 diabetic patients (112). The insulin secretion which regulates blood sugar levels is mediated by the ATP-binding cassette transporter subfamily C member 8 (ABCC8) encoding sulfonyl-urea receptor 1 ATP-binding cassette transporter subfamily C member 8 (ABCC8), and encodes sulfonyl-urea receptor 1 (SUR1) which is the subunit of K-ATP channel (ATP-sensitive potassium channel) found across β-cell membrane.

KCNJ11-MODY (MODY13)
The potassium channel, rectifying subfamily J, having member 11 (KCNJ11) which encodes human BIR (beta cell inward rectifier or Kir6.20) that is intron less coding gene was determined (114). Earlier, it was predicted that KCNJ11 gene mutations might result in neonatal diabetes. The sequence analysis revealed the occurrence of six new heterozygous missense mutations in 10 out of 29 patients and among these four patients exhibit Arg 201 → His mutation concluding that neonatal diabetes is caused by heterozygous mutations of the KCNJ11 gene (115). The onset and severity of diabetes occurring due to the missense KCNJ11 gene mutation is variable and spans up to three generations (116). Massa et al. (117) revealed the occurrence of five different heterozygous mutations including two novel mutations by screening the KCNJ11 gene in eight Italian patients and concluded that KCNJ11 gene mutations are the common cause of PNDM. The KCNJ11 gene mutation due to the disrupted subunit interaction results in severe conditions like channel inactivation, which was found to be associated with Arg 301 mutation that determinately cause hyperinsulinism (118).

APPL1-MODY (MODY14)
The knockdown mutation of APPL1 gene results in apoptosis and the overexpression resulted in dysmorphic phenotypes and delay in development, as was revealed from the previous reports of APPL1 gene expression in Zebra fish (119). In adapter protein, phosphotyrosine interaction domain and leucine zipper containing protein-1 (APPL1), both missense and non-sense heterozygous loss of function results in asp-to-asn (Asp 94 → Asn) substitution, while non-sense mutation results in Leu 552 → X (Leu 552 → X) substitution (7). The mutation in APPL1 gene was reported by Prundate et al. (7) as a cause of MODY14.

DiAGNOSiS/CLiNiCAL FeATUReS AND MANAGeMeNT
Some of the major diagnostic characteristics of MODY given by Vaxillaire and Froguel (62) are as follows: • Elevated glucose level (hyperglycemia) is diagnosed in early age (before 25 years) in one or two suspects of the diabetic family. • Autosomal dominant inheritance is showing vertical transmission through at least three generations. Similar phenotype shared by diabetic family members. • Insulin is not required by patients up to 5 years after initial diagnosis. As far as C-peptide level is concerned it tends to remain low even in patients on insulin therapy. • Functional impairment in the pancreatic β cells.
• Does not show the normal diabetic features like obesity, etc.
Besides the clinical features, probability of MODY occurrence can be calculated by using standardized MODY probability calculator (120) (Figure 2), and the clinical features may vary with the MODY type in consideration. However, clinical diagnostic characteristics of the most common MODY types are as follows.

GCK-MODY2
1. The fasting elevated glucose level observed is ≥5.5 mmol/l (98%) which is stable (from months up to years) and persistent (for at least three independent occurrences). 2. It has been observed that the Hb1Ac is typically just above the normal level and in some cases rarely exceeds 7.5% (3). HNF1α-MODY3 1. Lower insulinogenic index and lower early insulin response is observed in HNF1α mutants compared with the familial non-mutants even when blood glucose level is normal (86). 2. Patients are typically lean and without characteristic insulin resistance (121). 3. Positive β-auto-antibodies (IA-2) were reported in 25% (7/28) of HNF1α patients, and these patients develop diabetes much later than those with negative autoantibody. It was  further reported that in such patient's glycemia was difficult to control (122). 4. C-reactive protein level tends to be lower in MODY3 patients (123). 5. Glycosuria often observed in patients with low renal threshold due to diminished expression of high affinity low-capacity cotransporter 2 (SGLT2) which decreases the glucose reabsorption from kidney renal tubules (124).
For the genetic diagnosis of MODY, only those subjects are selected which fit into the standard clinical criteria of disease (125). For testing the monogenetic diabetes, DNA is isolated from the patient's blood sample which is then analyzed for mutation(s). Next-generation sequencing (NGS) strategies according to Ellard et al., (126), Johanson et al. (127) can be employed to identify MODY gene mutation. By direct sequencing, MODY can be diagnosed approximately with 100% sensitivity (128). According to Nyunt et al. (129) suspected cases when not diagnosed by routine screening technique can be detected by multiple ligase dependent probe amplification (MLPA) technique. There are some biomarkers that effectively differentiate MODY from other forms of diabetes and are thus effectively used for accurate testing. MicroRNAs possibly can be used as biomarkers of HNF1α-MODY, as it has been observed that the inducible expression of Pro 291 → fsinsC-HNF1A in INS-1 cells resulted in a significant upregulation of three miRNAs (miR-292-3p, miR-103, and miR-224) (130). Furthermore, the elevated expression levels of miR-103 and miR-224 could be detected in the serum of HNF1α-MODY carriers compared with MODY-negative family controls. The high HDL cholesterol level in MODY3 patients acts as the biomarker for differentiating MODY from type 2 diabetes (131). Sanger sequencing is the most reliable method for MODY diagnosis (70). A correct molecular diagnosis helps in the optimal treatment of disease. As β-cell function deteriorates over time, pharmacologic therapy becomes necessary to prevent diabetes-related complications. Once the gene mutation is accurately detected, only then the first degree relatives with MODY symptoms can be accurately diagnosed with the disease (132). Maturity-onset diabetes of the young patients can be managed by diet alone for many years and sulfonylureas is also recommended to be very effective for managing glucose level for more than 30 years (12). Patients diagnosed with GCK mutations usually need no treatment, but require medication in case of pregnancy and gestational diabetes mellitus (GDM). HNF1α and HNF4α MODY patients are particularly sensitive to sulfonylureas, but in the later stages should be supplemented with the addition of insulin (133). In HNF1α MODY patients (134) reported higher mean A1C levels in those taking insulin (7.5%) vs. oral agents (6.7%). Patients with HNF1α and HNF4α mutations are typically sensitive to sulfonylureas. Low-dose sulfonylureas (20-40 mg gliclazide regularly) can aid in sustaining the MODY patients for decades (135). Drugs like nateglinide and liraglutide lower the postprandial glucose levels in HNF1α MODY subjects (136,137). The pathophysiology associated with MODY gene mutations and various possible treatments for different MODY types are shown in (Table 3).

GeNeTiC TeSTiNG MeTHODOLOGieS
Different genetic testing methodologies like linkage analysis and direct sequencing were used by different workers for investigating gene mutations associated with MODY diabetes, which are summarized below ( Table 4). Fajans (12) by linkage analysis investigated 360 members of the diabetic family (R-W Pedigree) by taking into account MODY patients. Linkage analysis confirmed that the HNF1α gene which is located on chromosome 12q24.2 and is responsible for causing MODY3. Association of the GCK gene mutations with the early-onset diabetes has been reported with the help of linkage analysis (32). Froguel et al. (32) reported hyperglycemia in 125 out of 304 examined subjects, with 33% exhibiting clinical characteristics of maturity early-onset diabetes termed MODY2. In addition to this, they also analyzed that there was statistically significant evidence of genetic heterogeneity with an estimated 45-95% of the 16 families showing linkage to GCK. Yamagata et al. (8,9) isolated and partially sequenced the human HNF4α gene in R-W pedigree; following genetic screening identified thr130ile mutation in exon 4 and Gly 268 → Thr mutation in exon 7 of the HNF1α gene. Finally, confirmed the association of early-onset diabetes with HNF4α gene mutation.
Stoffers et al. (13) on the basis of linkage analysis, mode of transmission and phenotypic variations suggest the occurrence of fourth type of early-onset diabetes termed MODY 4 caused due to mutation of IPF1 gene locus. Stoffers et al. (13) reported that pancreatic agenesis is caused by homozygous mutation in IPF, which results in exocrine pancreatic insufficiency and permanent neonatal diabetes (PND). Malecki et al. (16) by direct sequencing of DNA samples from 94 subjects with type 2 diabetes, verified that the disrupted activity of NEUROD1 gene in carriers of Arg 111 → Leu (arginine to leucine substitution at codon 111) and insertion of cytosine residue in a polyC tract in codon 206 (206 + C) in exon 2 of NEUROD1 were largely responsible for development of type 2 diabetes. The NEUROD1 results in autosomal dominant inheritances similar to type 2 diabetes (16). Neve et al. (17)  WES approach In the diabetic families a missense asp94-to-asn (D94N) and non-sense leu552-to-X (L552X) APPL1 gene mutations were frequently observed (7) 8  46 Thia type 2 diabetic patients of <35 years of age by PCR-single-stranded conformation polymorphism (PCR-SSCP), this was succeeded by direct screening of PAX4 mutations; final observation of variants was performed by restriction fragment length polymorphism. Two unusual mutations Arg 164 → Trp and IVS7-IG > A of PAX4 gene were identified. R164W mutation was shown to be associated with diabetes, by affecting the function of PAX4 gene (suppressing the insulin and glucagon promoter action). Edghill et al. (21) in European population observed INS mutation in 33 of 141 probands (diagnosed at < 6 months age), 2 of 86 (diagnosed between 6 and 12 months age) and none of 58 (diagnosed between 12 and 24 years of age) by PCR-single strand sequencing. Edghill et al. (21) observed that 46% of the complications were due to the three already described mutations viz Arg 24 → Asp, Phe 48 → Cys, and Arg 89 → Cys and also identified six unusual mutations: His 29 → Asp, Leu 35 → Pro, Gly 84 → Arg, Cys 96 → Ser, Ser 101 → Cys, and Tyr 103 → Cys. Borowiec et al. (22) by using the techniques of immune florescence staining, western blotting, sequencing, RT-PCR, examined 6 MODY families and identified 410 sequence variations in 732 kb of genomic sequence at 8p23. Based on the results observed-five mutations with the frequency of <1% comprises four non-coding mutations and Ala 71 → Thr substitution, they summarized that BLK expression is altered in response to this Ala 71 → Thr substitution.
Bowman et al. (23) sequenced ABCC8 gene in 85 patients (BMI < 30 kg/m 2 ) with no familial diabetic history and observed ABBC8 mutations in 7 (8%) probands. In addition, to four unique mutations (Glu 100 → Lys, G 2ly14 → Arg, Gln 985 → Arg, and Asn 125 → Asp), some previously reported heterozygous mutations were also reported in four patients. Final results showed that only four probands fulfill the clinical criteria of early-onset diabetes termed MODY 12 which was having clinical pathologies identical to that of HNF1α MODY and can be diagnosed before 25 years of age. Bonnefond et al. (24) performed WES (Agilent-sureselect capture/Illumina Golden Gate assay) and linkage analysis in three affected and one unaffected subjects in MODY-X family, and observed 69 mutations with only one mutation (p.Glu 227 → Lys) in KCNJ11 gene associated with diabetes (LOD-score of 3.68). Thus, confirming KCNJ11 gene mutation as the cause of earlyonset diabetes termed MODY13. Prundate et al. (7) studied loss-of-function mutations in APPL1 gene in 60 families (52 from US and 8 from Italy) by following WES approach. However, in Italian families, normal subjects of >55 age were also included in the study. Both missense and non-sense heterozygous lossof-function mutations have been reported to occur in APPL1 gene, and missense mutation results in asp-to-asn (Asp 94 → Asn) substitution while non-sense mutation can result in Leu 552 -to-X (Leu 552 → X) substitution.

CONCLUSiON AND FUTURe PeRSPeCTive
Researchers have been effectively working to investigate the genetic determinants and pathophysiology of MODY by means of various powerful approaches (based on genome and cell biology). These studies have been very promising and insightful which has greatly extended our understanding of both normal and pathologic β-cell biology as well as regulation of insulin secretion in humans. At clinical level, MODY requires correct and accurate methods of diagnosis as well as to distinguish it from diabetes type 1 or type 2 to avoid the unnecessary insulin or sulfonylureas treatment, which may severely affect the patient's health.
In future, translational biology and integrative genomic research and studies on both monogenic and polygenic forms of diabetes are required, which will not only provide novel insights but, also broaden our understanding in terms of pathophysiology, treatment and cure of diabetes. Currently, application of NGS has emerged a key factor to decipher the large amount of known genetic defects underlying pancreatic islet β-cell dysfunction in diabetic patients. As there is increased rate of MODY across the globe, NGS can serve as one of the potential and rapid molecular based diagnostic tool as well as to identify novel genetic etiologies of familial or atypical early-onset diabetes. Transcriptomic and secretomic analysis related to MODY may provide a significant molecular insight into the disease and its procurement. Besides genomic approaches, stem cell technology can also be another novel and effective disease model to study and therapy for the diabetes. Use of pluripotent stem cells (PSCs) can help us to better understand the molecular mechanisms underlying different forms of diabetes (MODY). In addition, metabolomic profiling can also be one of the important approaches to study and differentiate MODY with other diabetes.

AUTHOR CONTRibUTiONS
PF and BG conceived and designed this review. PF, KN, US, and TH collected the literature for this review. PF, KN, and SA wrote the manuscript draft. BG, SA, and SM edited this manuscript. All the authors gave final shape to this manuscript.