Prevalence of Latent Autoimmune Diabetes of Adult (LADA) among Type 2 Diabetes Mellitus (D.M.2) in Karbala

Objective: the aim of the study is to determine the prevalence of LADA among group of clinically diagnosed type 2 diabetes mellitus. Methodology: sample size equal to 280 patients with type 2 D.M. were subjected in this study, participating patients were consecutively recruited from Diabetes outpatient clinic in AL-Hussein Teaching Hospital in Karbala from June, 2013 through January, 2014. A clinical questioner containing personal data, family history, type of diabetes, type of treatment, FBS, HA1C, BMI, diabetes duration& complications of diabetes. For analysis of data Prevalence of LADA among D.M.2 cases was calculated as the proportion of LADA cases in the D.M.2 cases. The Chi square test (X) was used to assess the significance (P-value) of differences in frequencies of categorical variables. Results: prevalence of LADA among D.M.2 patients was 12.1%. A significant difference in family history, BMI, FBS, HA1C and duration of D.M was found between the studied groups. Conclusion: The prevalence of LADA is clinically underestimated among D.M2 patients and age of onset of diabetes should no longer be considered as a valid way to differentiate diabetes. Recommendation: the study recommends screening for islet cell autoantibodies as GAD65 for all patients with diabetes at onset or beginning of disease. Key word: GAD65, LADA, diabetes mellitus.


INTRODUCTION
Diabetes mellitus is a group of metabolic diseases in which a patient has increase blood sugar, because the pancreas does not secret enough insulin, or unresponsiveness of cells to the insulin that is produced (1) .Diabetes mellitus is categorized into general categories: type1, type2, gestational diabetes and other specific types.The specific types are group of a few dozen individual causes (1) .Type 2 diabetes is characterized by impaired β-cell function and may be accompanied with changes of the immune system (2) .Latent autoimmune diabetes in adults (LADA) or Type 1.5 diabetes has some clinical features of type 2 diabetes & shows immunological abnormalities similar to those in type 1 diabetes, such as glutamic acid decarboxylase antibody (GADA) (3) .So far it is not understood why disease progression in LADA is slower than in type 1 diabetes despite the immunological similarities.Insulin secretion was reported to be intermediate in LADA compared with type 1 and type 2 diabetes, whereas metabolic syndrome was similar in type 1 diabetes and LADA (4) .Type 1.5 diabetes is usually diagnosed after the age of 35 years and there is no immediate requirment for insulin therapy (5) .Approximately 10% to 30% of adults with type 2 diabetes test positive for autoantibodies, depending on the age and ethnicity of the study group (6) .The immune-mediated destruction of beta-cells in type 1.5 diabetics leads to insulin dependency more rapidly than in type 2 diabetes, but the more attenuated genetic and immune factors associated with type 1.5 diabetes as compared with type 1 diabetes lead to an older age at onset and a slower progression to insulin dependency (7) .The Clinical characteristics predictive of type 1.5 diabetes include: Age < 50 years, acute symptoms of hyperglycemia (polydypsia, polyuria, or unintentional weight loss), body mass index < 25 kg per m², family history of autoimmune disease (thyroid disorders, celiac disease, type 1 diabetes, rheumatoid arthritis or any other form autoimmune disorder), personal history of autoimmune disease (thyroid disorders, celiac disease, type 1 diabetes, rheumatoid arthritis or any other form of autoimmune disorder).The presence of at least two of these clinical features (LADA risk score ≥ 2) was found to have 90 % sensitivity and 71 % specificity for identifying diabetic patients affected by type 1.5 diabetes.Patients with one or no feature were unlikely to have LADA (8) .
Type 1.5 diabetes and type 2 diabetes populations can be distinguished from each other based on clinical features, but a large degree of overlap exists between the two types of diabetes.Hence, the use of immunogenetic markers, in particular the measurement of autoantibodies, remains the gold standard for identifying type 1.5 diabetic patients.Identification of these patients is clinically relevant to their management as the early use of insulin resulted in β-cell preservation in several pilot studies (9) .Type 1.5 diabetes is diagnosed by the presence of pancreatic autoantibodies, such as glutamic acid decarboxylase (GAD) antibodies in an adult initially presenting with non-insulin dependent diabetes (10) .

PATIENTS AND METHOD
A 280 individual were enrolled in this study at the period from June, 2013 through January, 2014.All patients were selected randomly from Diabetes outpatient clinic in AL-Hussein Teaching Hospital in Karbala; these patients were clinically diagnosed as type 2 D.M, 135 males &145 females with age range from 30-73 years, and duration of disease between 1 month -25 years.Descriptive variables of patients included: name, age, gender, type of diabetes, type of treatment (insulin, OHD, diet or mixed), FBS, HA1C, BMI, complications they were suffered due to D.M. and duration of disease.Serum samples were taken from all patients and subjected to ELISA analysis by GAD65 ELISA kits (CUSABIO BIOTECH CO., LTD.USA) which is a solid phase enzyme immunoassay based on the sandwich technique, in which two monoclonal antibodies are directed against separated antigenic determinants on the GAD65 molecule.

Figure1. Prevalence of LADA among 280 D.M.2 cases
Figure 1 shows that out of the 280 patients with type II D.M., 34 had LADA, GAD65 positive this giving a prevalence of LADA among D.M.2 patients equal to (12.1%).

DM2 LADA
The comparison of baseline characteristics of studied groups as it shown in table 1 revealed: A significant differences in family history (P=0.001);Positive family history was more frequent in D.M.2 than LADA cases; 211 (85.5%) vs. 21 (61.8%),respectively.
A highly significant difference in BMI; D.M.2 cases were more likely to be overweight and obese than LADA cases, (P<0.001).
No significant differences had been found in age, gender, type of treatment or complications in between both groups, in all comparison , P>0.05.

Figure2. Comparison of mean BMI values of studied group
The mean BMI of D.M.2 group was 27.8± 3.6 kg/m 2 , of LADA group was 25.7±3.5, it had been significantly found that patients in D.M.2 group had the higher BMI value than LADA.The comparison of baseline characteristics of studied groups as it shown in table 1 revealed: A significant differences in family history (P=0.001);Positive family history was more frequent in D.M.2 than LADA cases; 211 (85.5%) vs. 21 (61.8%),respectively.
A highly significant difference in BMI; D.M.2 cases were more likely to be overweight and obese than LADA cases, (P<0.001).
No significant differences had been found in age, gender, type of treatment or complications in between both groups, in all comparison , P>0.05.

Figure2. Comparison of mean BMI values of studied group
The mean BMI of D.M.2 group was 27.8± 3.6 kg/m 2 , of LADA group was 25.7±3.5, it had been significantly found that patients in D.M.2 group had the higher BMI value than LADA.

Mean BMI
The comparison of baseline characteristics of studied groups as it shown in table 1 revealed: A significant differences in family history (P=0.001);Positive family history was more frequent in D.M.2 than LADA cases; 211 (85.5%) vs. 21 (61.8%),respectively.
A highly significant difference in BMI; D.M.2 cases were more likely to be overweight and obese than LADA cases, (P<0.001).
No significant differences had been found in age, gender, type of treatment or complications in between both groups, in all comparison , P>0.05.

Figure2. Comparison of mean BMI values of studied group
The mean BMI of D.M.2 group was 27.8± 3.6 kg/m 2 , of LADA group was 25.7±3.5, it had been significantly found that patients in D.M.2 group had the higher BMI value than LADA.

Figure3. Proportional distribution of complications in DM2 and LADA groups
LADA cases have 17.7% retinopathy and 2.9% diabetic foot.

DISCUSSION
LADA is not a rare disease, and many subjects are still under diagnosed.Without awareness, a correct diagnosis of LADA is not easy.Adults with LADA may initially be diagnosed as having type 2 diabetes based on their age, particularly if they have risk factors for type 2 diabetes such as a strong family history or are obese.80% of persons initially diagnosed with type 2 but test positive for GAD progress to insulin dependency within 6 years (some sources say between 3-12 years after diagnosis) (11) .
Our study shows that from the 280 diabetic patients, there were (34) GADA positive, the prevalence of LADA was 12.1%.This result is in line with what was obtained by Olufunmilayo study (12) who report prevalence rate equal to 14%, also reports from Ghana where documented prevalence rates for LADA amongst people being managed for type 2 DM was 13.5% (13) and the study of Lutale et al., showed a level of islet cell positivity 7.3% (14) .However other studies done in Korea found lower prevalence rate of LADA 4.3% (15) & 4.7% (16) .Seissler and Scherbaum demonstrated the relative high frequency of this form of diabetes (approximately 20%) among type 2 diabetic patients in the age range 25-44 years.This difference could be attributed to the difference in population ethnicity and age of onset of the disease (17) .
Regarding demographic characteristics of the studied groups, although no significant difference in age between LADA & D.M.2 Majority of the subjects with LADA in this study were in 41-50, 51-60 & ˃60 age categories (30/34 cases) and less than 11.7% were in other age categories.This observation suggests that LADA increases with increasing age decade, confirming result observed by Olufunmilayo study (12) & Carlson et al. study (18) that older age was an important risk factor for LADA as for Type 2 DM and this may suggest a potential role for insulin resistance in the pathogenesis of LADA.Similarly Chinese study found that the prevalence of LADA slowly increased with age up to 60 years and was high in individuals aged 50-59 years (19) .
Our study shows that the presence of LADA is non sex specific although the higher percentage (58.8%) of patients positive for antiGAD autoantibodies in this study were females, this may be due to the fact that autoimmune diseases are more common in females than males and the logical cause for this difference would be the sex hormones, females might respond more to conventional antigens due to sex hormones.This goes with other studies as Olufunmilayo study who found (64%) of subjects positive for antiGAD autoantibodies were females (12) & same result was clarified by Qi et al. (19) .
A highly significant difference in BMI between LADA & D.M.2 cases; D.M.2 cases were more likely to be overweight and obese than LADA cases this is due to insulin resistant in those patients.This finding is similar to what was obtained by Genovese et al. as they concluded that, LADA patients are non obese in contrast to those who are actually type 2 DM as they are mostly obese with BMI >30 (20) .Although 52.9% of LADA patients are overweight in our study, this may be due to the fact that they have low levels of insulin improper treatment with oral hypoglycemic drugs.The proportion of overweight among subjects with LADA in this study was higher than those in normal weight category and this suggests insulin resistance as possible contributory factor in the pathogenesis of LADA amongst our patients.The mean BMI of our study LADA patients was 25.7 kg/m², which is lower than that of Western studies (27.5 to 32 kg/m²), (21) .This result is in agreement with report done by Yul Hwangbo in Korea that found mean BMI was 25.3 kg/m² (15) and this may be explained due to different features related to different ethnic groups.
Significant positive family history for D.M.2(85.8%),although high percent of LADA (61.8%) have positive family history for type 2 D.M., Olufunmilayo study reveal that 39% of LADA patients have positive family history for type 2 D.M. (12) .
Duration of diabetes was highly significant longer in GAD negative than LADA group; this goes with studies done in Korea & Nigeria where longer duration of disease reported in D.M.2 (15; 12) .
Regarding insulin treatment 23.5% of LADA patients were already on insulin at the time of the study for glycaemic control, although statistically result is not significant.Other studies found that 15%, 37% of LADA cases were on insulin treatment at study time (15; 12) respectively, and this may be due to autoimmune destruction of the β-cells which has been reported to be present at diagnosis of diabetes in LADA patients (6) .
A higher proportion of subjects with LADA had evidence of microvascular complications of diabetes namely retinopathy while D.M.2 cases had more macrovascular complications.The foregoing would suggest that macrovascular complications were more frequent in D.M.2 patients, while LADA subjects manifest predominantly microvascular complications especially retinopathy.The poor indices of long term glycemic control in LADA may account for the observed higher percentage of LADA subjects with evidence of microvascular DM complications.This is in agreement with finding of study from Turkey (22) , Reports from Western Finland (19) & study of Olufunmilayo (12) .

Figure 1
Figure1.Prevalence of LADA among 280 D.M.2 cases Figure 1 shows that out of the 280 patients with type II D.M., 34 had LADA, GAD65 positive this giving a prevalence of LADA among D.M.2 patients equal to (12.1%).