Dissertation committee:
Xριστίνα Κανακά -Gantenbein, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Κυριακή-Αθηνά Καραβανάκη, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Εμμανουήλ Καναβάκης, Ομότιμος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Αλεξάνδρα Σολδάτου, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γεώργιος Βάρτζελης, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Λυδία Κόσσυβα, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γεώργιος Μαστοράκος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Diabetic Neuropathy (DN), although one of the most common complications of Type 1 (T1DM) and Type 2 (T2DM) diabetes mellitus in adult life, is the least recognizable complication in clinical practice. While neuronal lesions begin early in childhood, diabetic neuropathy is rarely became a clinical problem, although clinical DN is about 3-5% of the pediatric population. However, autonomic diabetic neuropathy (ADN) and peripheral neuropathy (PDN) significantly reduce the quality of life and increase the risk of cardiovascular death. Disease duration and glycemic control are the most recognized risk factors for the development of microangiopathic complications in patients with diabetes mellitus (Corrêa-Giannella ML et al., 2008). However, risk factors appear to be under the control of genetic predisposition (Doria et al., 2010). Thus it is observed that in some patients with low glycemic control there are many years left without effect, while others develop complications soon despite maintaining normoglycaemia (Veves A και συν., 2007, Diabetes Control And Complications Trial Research Group, 1997, Alcolado J και συν., 1998, Harjutsalo V και συν., 2004, Monti MC και συν., 2007). Therefore, it was considered important to look for genetic factors, which may be involved in the pathogenesis of diabetic complications. The Z-2 polymorphism of the AKR1B1 gene, which is the first enzyme station in the polyol pathway, has been found to be associated with autonomic diabetic neuropathy.
The purpose of this study was to evaluate the incidence of pathological markers of autonomic and peripheral neuropathy in children and adolescents with T1DM and consequently their impact on this population. In addition to determine the frequency of Z-2 / Z-2 homozygous polymorphism of the AKR1B1 gene and to correlate with markers of autonomic and peripheral neuropathy.
This study population consisted of 106 T1DM patients over 8 years of age with a disease duration over 2 years and 102 matched controls of the same age and sex. A complete physical and neurological examination was conducted in every subject. Metabolic control was assessed with mean HbA1c value (average of the last year’s ΗbA1c recordings).
Then, to evaluate the autonomic nervous system, the pupil dilation in darkness was measured using a portable polaroid camera (Pupillometer). The study of the autonomic nervous system was then completed with the postural blood pressure test (PBPT). Postural blood pressure test (PBPT) was performed in patients and controls. Orthostatic hypotension was defined as a systolic BP decrease of at least 20 mmHg or a diastolic BP decrease of at least 10 mmHg after three minutes of standing from supine to upright position. Orthostatic systolic pressure difference was defined as supine systolic pressure minus upright systolic pressure. Orthostatic diastolic pressure and pulse differences were defined accordingly.
To assess the peripheral nervous system, the vibration sensitivity threshold (VST) was measured with a thumb biomarker, index finger, forefinger and outboard hammer. Then, patients were submitted to electromyography (EMG), to determine the speed of stimulation treatment and the potential produced in the medial, peroneal, and gastrocnemius nerve. Finally, to determine the polymorphism of the AKR1B1 gene, we first isolated the DNA of patients from peripheral blood lymphocytes according to QIAGEN's QIAmp DNA Mini Blood kit protocol. PCR was followed, using specific primers for the AKR1B1 gene (ARpr1, ARpr2). Sequencing was then performed, as following Sanger sequencing, using ABI ANALYZER 3130 (Applied Biosystems), and finally the structure of the microsatellite sequence alleles was determined and therefore homozygous and homozygous polymorphisms were identified among the patients and controls. Analysis of the study data showed that 34.34% of the patients had abnormal pupil dilation in the dark (PD) as an indicator of autonomic neuropathy compared with 3.3% of controls. In patients, PD was associated with age (r = 0.16, p = 0.038), HbA1c: (r = 0.23, p = 0.048) and disease duration (r = 0.20, p = 0.022). In particular, there has been a progressive and significant increase in the proportion of pathologic PD associated with worsening diabetic regulation. There was also a significant increase in the proportion of abnormal PD as the duration of the disease increased. A statistically higher proportion of patients, compared to controls, had abnormal VST at each test site. Adolescence was associated with a higher percentage of abnormal VST values in the lower limbs (adolescent vs children, left 18.64% vs 2.8%, p = 0.001, right: 20.33% vs 0.0%, p <0.001). Consequently, lower extremity VST was abnormal mainly in adolescents and less frequently in children with T1DM.
T1DM patient homozygotes of AKR1B1 polymorphism Z-2 had a higher rate of pathological PD (24.32% vs 9.23%, p = 0.047). Pathologic value on electromyography (EMG) was present in 39.6% of patients with T1DM. There was a significant correlation between PD and EMG in patients with T1DM (r = 0.34, p = 0.008). Patients with T1DM with omogygous polymorphism Z-2/Z-2 of the aldose reductase gene (AKR1B1) had a higher incidence of abnormal EMG (21.7% vs 2.8%, p = 0.032) and abnormal PD (62.5% vs 37.5%, p = 0.023) in relation with healthy controls.
T1DM1 homozygous patients of AKR1B1 polymorphism Z-2 had abnormal VST in 12.98% vs 20% of patients without abnormal VST. So, no statistically significant difference was found in the homozygous patients and VST as an indicator of peripheral neuropathy (12.98% vs 20%, p = 0.047). However, VST is a screening method, with no significant reliability, whereas EMG is the most reliable (gold standard) method for detecting subclinical peripheral neuropathy.
The results of our study show that in children and adolescents with diabetic neuropathy is primarily subclinical, regardless of the quality of metabolic control and the duration of the disease. However, the disease begins in childhood and there is a need for vigilance by clinicians for early diagnosis of this complication, as it is the least recognizable complication in clinical practice in both pediatric patients with T1DM, as well as in adults patients with DM. Polymorphism of the homozygous AKR1B1 gene is a reliable indicator of the future appearance of autonomic and peripheral diabetic neuropathy (DN). Therefore, children with T1DM and homozygous Z-2/Z-2 polymorphism of gene AKR1B1make up a high-risk group for the development of DN and should have excellent diabetic regulation and regular monitoring for early detection and prevention of DN.
