Mitochondrial tRNALeu(UUR) gene mutation and maternally inherited diabetes mellitus in Pakistani population

2.1. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis 

The 422-bp Fragment of mitochondrial DNA amplified by PCR was digested by the restriction enzyme Apa1 (Fermentas Life Sciences) to identify any A-to-G mutation at np 3243 and electrophoresed on 4% agarose gel stained with Ethidium Bromide. DNA purification was conducted through standard kit method followed by DNA sequencing PCR. Finally, the PCR products were directly sequenced (Beckman Coulter, USA).

Plasma glucose was estimated by the enzymatic colorimetric method using glucose oxidase enzyme to oxidize glucose [11]. Glycosylated hemoglobin (HbA1c) was determined by micro column method (ion exchange chromatography) [12].

Obesity was defined as a body mass index (BMI) of 30kg/m2 or greater, body weight, excess was defined as BMI of 25kg/m2 or greater and low body weight was defined as a BMI less than 18.5kg/m2 [13].

Audiography was performed to detect the type and extent of hearing loss [14].

2.2. Statistical analysis 

All statistical calculations were conducted through computer software programme “Statistical Package for Social Sciences (SPSS)” for windows, version 15. Data was subsequently examined by Independent Sample T-test. The percentage was calculated based on the number of subjects showing positive results, over the total no of subjects analyzed in each categorized group.

3.1. Detection of A3243G mutation 

The amplified PCR product was 422 bp in length. After digestion with restriction endonuclease, ApaI, it was to be separated into 210 bp or 212 bp if it had a substitution of A to G at np 3243 to constitute the recognition site for ApaI. The results obtained from the less sensitive method, i.e., ApaI digestion technique (Fig. 1.1), were confirmed by direct sequencing (Fig. 1.2, Fig. 1.3, Fig. 1.4).

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Fig. 1.1. Electropherogram of Ethidium bromide stained 4% agarose gel for amplified 422 bp segment of mitochondrial DNA in control group. Lanes 1&2: patients with T2DM and deafness; lanes 3&4: patients with T2DM and no complaint of deafness; lanes 5&6: first-degree relatives of patients with T2DM and deafness group; lanes 7&8: non-diabetic controls.

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Fig. 1.2. Sequence alignment showing no A to G substitution at 3243 position in mtDNA of patient with T2DM & deafness.

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Fig. 1.3. Sequence alignment showing no A to G substitution at 3243 position in mtDNA of the first-degree relative of patient with T2DM & deafness.

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Fig. 1.4. Sequence alignment showing no A to G substitution at 3243 position in control group.

Mean plasma glucose and glycosylated hemoglobin levels in patients of type 2 diabetes mellitus were significantly higher (p<0.001) as compared with the control group. However no significant differences in plasma glucose and glycosylated hemoglobin were observed between the first-degree relatives of diabetic patients with control group (Table 1).

Table 1. Age of onset of T2DM and deafness, plasma glucose and Glycosylated hemoglobin levels of patients with type 2 diabetes mellitus and first-degree relatives of diabetics with control group.

	Group	Age of onset of T2DM (years) Mean±SD	Age of onset of deafness (years) Mean±SD	Plasma glucose (mmol/L) Mean±S.E.	Glycosylated hemoglobin %
	Diabetics (50)	35.86±5.23	47.56±6.4	13.36±0.94a	9.39±0.43a
	First-degree relatives of diabetics (50)	–	–	5.01±0.09	5.34±0.09
	Non-diabetic controls (50)	–	–	5.13±0.08	5.55±0.10

a p<0.001 as compared with normal control subjects (highly significant).

In patients with T2DM, BMI was found to be within the normal range. However, statistically, BMI was significantly less (p<0.05) in group 1 as compared with control subjects. However, no significant difference was observed between the first-degree relatives of diabetic patients with their control group.

In patients with T2DM, deafness was observed in 27 patients (54%). While 50% of the patients were dependent on insulin therapy for the control of the plasma glucose level, the remaining 50% patients were able to control blood glucose level with the help of oral hypoglycemic drugs (Table 2).

Table 2. Body Mass Index (BMI), deafness, Glycosuria and dependence on insulin therapy of patients with type 2 diabetes mellitus and first-degree relatives of diabetics with control group.

	Group	BMI Mean±S.E.	Deafness %	Glycosuria %	Insulin therapy %
	Diabetics (50)	22.94±0.39a	54	76	50
	First-degree relatives of diabetics (50)	24.20±0.52	NIL	NIL	NIL
	Non-diabetic controls (50)	24.52±0.53	NIL	NIL	NIL

a p<0.05 as compared with normal control subjects (significant).

4.1. A3243G mutation near the region of tRNALeu(UUR) gene in Pakistani population 

The Pakistani population provides a valuable resource for mapping mtDNA mutations, as this population is still not explored with respect to any mitochondria associated disease as compared to other populations in advanced countries. All 50 patients described herein have at least three to four dominant clinical features characteristic of MIDD. The results of the search for A3243G mutation in mitochondrial tRNALeu(UUR) gene in subjects with type 2 diabetes mellitus in Pakistani population identified none of the individual as a carrier of this mutation indicating that the variation within mtDNA 3035–3456 is not the major cause of type 2 diabetes mellitus in the selected Pakistani population in this study. We were unable to identify A3243G mutation among those diabetics who reported having a maternal history of diabetes, deafness and insulin dependence.

It should not be excluded that other gene variation may also play a potential role in the etiology of this disease. Pathogenic mutations can occur at almost any site throughout the mitochondrial genome; hence comprehensive screening requires an analysis of the entire mtDNA molecule [15]. Moreover, the level of mutated mtDNA in relation to the wild-type mtDNA (% Heteroplasmy) varies between tissues, being high in post-mitotic tissues such as endocrine tissues like pancreas, brain and skeletal muscles, and low in rapidly dividing tissues, such as blood leukocytes [16]. The reliable detection of mitochondrial DNA mutations in T2DM requires thousands of well-characterized samples. Large population based samples are needed to provide an epidemiological context and to characterize susceptible genes. Moreover, direct sequencing of isolated mitochondrial proteins may provide evidence as to whether alterations in the primary structure are, indeed, involved in the pathogenesis of MIDD. We studied 50 patients and there was clear maternal transmission of diabetes in each pedigree. Our comprehensive search for mtDNA defects, therefore, allows us to conclude that mechanisms other than inherited defects of mtDNA must be contributing to the maternal transmission of diabetes in these families [17].

Hyperglycemia is a specific sign of diabetes mellitus and HbA1c concentration is a strong tool to diagnose the disease [18], [19], [20].

4.2. Insulin dependence and BMI 

Mostly in T2DM, obesity is one of the causes of insulin resistance [21]. However, the patients in this study had low BMI as compared with controls, so there must be some other cause of insulin dependence like pancreatic β cells defect that limits their ability to secrete or produce insulin [22]. There is evidence that uncontrolled hyperglycemia in MIDD is due to the impaired ability of β cells to secrete insulin and respond to the elevated plasma glucose level, not peripheral insulin resistance as in case of maturity onset type 2 diabetes mellitus of the youth (MODY). This impairment may reflect an underlying genetic variation in the genes that control β cell glucose sensing. There is also evidence that patients with T2DM may have fewer β cells than non-diabetics. This decrease in β cell mass could result from an initial failure to produce an adequate number of β cells during the development or increase in β cell impairment. This decline in insulin production capacity leads to an eventual failure to respond to oral hypoglycemic therapy and requires insulin therapy [23], as in case of present study.

4.3. Deafness 

An auditory defect is a progressive bilateral sensorineural hearing loss that first affects high frequency sound waves. The most characteristic audiographic feature is that of sloping, with flat profiles in advanced cases [24]. Mitochondrial pathology plays an important role in both inherited and acquired hearing loss. Inherited mitochondrial mutations have been implicated in syndromic and non-syndromic hearing loss [25].

To our knowledge, no other mechanism has been described that leads to maternal transmission of both diabetes and deafness. However, sensorineural deafness, like diabetes, is a common condition and can arise from a large number of autosomal – inherited gene defects. It is quite possible, therefore, that these 27 patients with MIDD phenotype were from pedigrees that carried separate defects, one causing maternally inherited diabetes and the other leading to deafness [26].

One of the limitations of this study is that the percentage of mtDNA with mutation varies from tissue to tissue. Unfortunately, the heteroplasmy of the mutation is considered to be much lower in peripheral leukocytes, and highest in the affected tissues. Moreover, heteroplasmy levels in leukocytes also decline upon aging by ∼0.7% per year [27]. In the present study, leukocytes were isolated for DNA extraction. It has probably hampered the detection of this mutation. In this regard, the pancreas would be the best source of tissue for the examination of the A3243G mutation in patients with diabetes. However, pancreatic biopsy is virtually impossible as a part of routine screening for logistic reasons. Moreover, due to the limited funds, sequencing of entire mtDNA was not possible, so we could not look for other possible mutations.