دراسة التغيرات الجزيئية وعدم الاستقرار الكروموسومي في امراضية اضطراب طيف التوحد في العراق

الباحث

زينب عبد الرضا العلي

المشرف

أ.د. عقيل عباس ياسين; أ.د. عرفات الدجيلي

الجامعة

جامعة الكوفة

الكلية / القسم

الطب / علم الأمراض

الشهادة

دكتوراه

السنة

2022

لغة الرسالة

انكليزي

الكلمات المفتاحية

Autism Spectrum Disorder;Oxytocin;Chromosomal Instability;Gene Polymorphism

اضطراب طيف التوحد اضطراب نمائي عصبي يتميز بضعف التفاعل الاجتماعي وتأخر اللغة والسلوك التكراري، ويبدأ عادة قبل سن الثالثة، ولا تزال آلية حدوثه غير مفهومة بشكل كامل رغم الأدلة القوية على دور الوراثة فيه. شملت هذه الدراسة (دراسة حالة - شاهد) 60 مريضاً بأعمار 3-16 سنة و60 طفلاً سليماً كمجموعة سيطرة، وصُنف المرضى وفق الدليل التشخيصي والإحصائي للاضطرابات العقلية (الإصدار الخامس) إلى حالات خفيفة (39) ومتوسطة (13) وشديدة (8). تمت دراسة الشذوذات الكروموسومية، ومستوى هرمون الأوكسيتوسين في مصل الدم، وتعدد الأشكال الجيني لجينات GABRG3 وRELN ومستقبل الأوكسيتوسين OXTR بتقنية PCR الخاصة بالأليل، والتعبير الجيني لجين FOXL1 وجين FOXP2 (عبر الجينات الهدف PHOX2B وNURR1) بتقنية PCR في الزمن الحقيقي. أظهرت النتائج أن نسبة الذكور إلى الإناث بين المرضى 5:1، ولم تظهر شذوذات كروموسومية إلا في حالة واحدة متوسطة الشدة هي متلازمة كلاينفلتر (47,XXY). كان مستوى الأوكسيتوسين في مصل المرضى (149.08 بيكوغرام/مل) أعلى معنوياً من مجموعة السيطرة (75.03 بيكوغرام/مل)، وأظهر تحليل منحنى ROC قيمة تشخيصية جيدة له (AUC=0.807) بحساسية 80% ونوعية 72.1% عند قيمة فاصلة 83.8 بيكوغرام/مل. وبينت دراسة التنميط الجيني وجود فروق معنوية في الأنماط الجينية CC وCT لمستقبل الأوكسيتوسين، والأنماط TT/TA والأليلات T/A لجين GABRG3 (rs208129)، والنمط TC لجين RELN (rs736707) بين المرضى ومجموعة السيطرة. كما ارتفع التعبير الجيني لجين FOXL1 لدى المرضى مقارنة بالأصحاء، وانخفض تعبير الجينين PHOX2B وNURR1 مما يشير إلى فرط تعبير جين FOXP2 الذي قد يكون سبباً في ضعف اللغة لدى المرضى. خلصت الدراسة إلى أن التغيرات الوراثية مهمة لفهم الأسس البيولوجية لسلوك التوحد، وأن قياس مستوى الأوكسيتوسين مع التغيرات الجينية لمستقبله يمكن أن يشكل اختباراً بسيطاً مزدوج المؤشر للتشخيص والمتابعة وتقييم شدة المرض، وأن المتغيرات الجينية لجينات GABRG3 وRELN ترتبط باضطراب طيف التوحد لدى المجتمع العراقي.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in social interactions, language delay and repetitive behavior of interest. ASD starts usually before the age of three years. Thus, it is unlikely that scientists and researchers may identify a single cause or reveal the precise nature for its etiology in near future due to the nature of this disorder and its pathogenesis is poorly understood so far. ASD particularly heritable, according to compelling data. However, the genetic pathways that underpin this heritability have yet to be identified. This was a case-control study that included 60 patients age betwen 3 - 16 years 6.66 ± 3. 051(mean ± SD )and the control group comprised 60 3and 16 years5.76 ± 2.38 (mean± SD). Participants were met the diagnostic criteria of autism according to the Diagnostic and statistical manual of mental disorders (5th ed.) and sub-grouped into mild (n=39), moderate (n=13) and severe autistic patients (n=8).The controls were normally developing, healthy children, unrelated to the autistic subjects. We studied firstly the chromosomal abnormalities in ASD ,secondly the oxytocin plasma level in autistic patient and control ,third SNPs polymorphism of GABAG3,RELN and OXT receptor gene by allele specific PCR , fourth the FOXL1 gene expression in ASD and control by real time PCR and lastly FOXP2 gene expression by detection of its parget gene (PHOXB2and NURR1gene)by real time PCR. The present study revealed that the male to female ratio is in the range of 5:1, the percent of male autistic patients and female autistic patients were 83.34% and 16.66%. The ages of the mild ASD patients were ranged between 3-16 years. Fourteen of them were the children of consanguineous marriage (35.89%) and six of them had a positive family history accounting to (15.38%)(95%CI: 0.4180-2.2381;OR: 0.967; P=0.938; fit=2(5.12). No chromosomal abnormalities were detected among them. On the other hand, 13 patients who were classified as moderate in autistic severity ,four out of the 13 were the product of consanguineous marriages (30.76 %) with only one patients had a positive family history accounting to ( 07.69%)( 95%CI:0.2114-2.7876; OR: 0.767; P= 0.687; fit = 3(23.07) . One ASD patients showed obvious chromosomal aberration accounting to 1.66% from the total ASD and 7.69% from the only 13 moderate cases. Eight patients who were classified as a sever ASD subjects, 4 of them were the children of consanguineous marriages accounting to (50%). ( 95%CI; 0.3924-7.6035; OR: 1.7273; P = 0.469; fit = 3(37.5). No chromosomal abnormalities were found. The chromosomal study revealed that only one moderate case showed chromosomal abnormalities which was 47,XXy Klinefelters syndrome with normal parent chromosomal analysis. the ELISA measurements of serum OXT showed the serum OXT levels in the patients with ASD mean± SD (149.08±8.24 pg/ml) were significantly higher (p=0.003) in comparison to the healthy control group mean± SD (75.03±6.38 pg/ml). In mild cases the oxytocin level mean± SD165.02±11.13, the moderate cases the oxytocin level mean± SD 152.78±13.04 and for sever cases the oxytocin level mean± SD 129.08±9.38. Remarkably, significant difference in the oxytocin levels estimated in the serum of the aforementioned categories of ASD group was evidenced. p-value 0.032. The ROC analysis of OXT serum data presented as AUC, best cut off values, specificity, and sensitivity. OXT exhibited AUC values higher than 0.8 in the overall ASD patient population (AUC=0.807), and similar values in mild (AUC =0.793) moderate (AUC=0.889), and severe autistic patients (AUC=0.795). The best cutoff in pediatric serum samples of 83.8 pg/mL OXT offers a diagnostic accuracy of ASD at sensitivity and specificity levels of 80% and 72.1% respectively. Within the stratified population of ASD, the cutoff was highest in mild patients (87.2 pg/mL) and lowest in severe cases (71.4 pg/mL). The highest test accuracy occurred in patient with moderate ASD according to sensitivity and specificity indicators. The oxytocin level in cooperated with age of the patient showed there no correlation found between oxytocin level and age of patient. Genotype study inferred that there was no significant difference could be evidenced neither in the genotypes nor in the alleles among the two stratified ASD patients with the mild and severe but significant for moderate one correlated with plasma oxytocin level. There was a significant difference in CC and CT genotype between patients and control and none significant difference for TT genotype and C and T allele between patients and control. OR for CT was 2.315. So CT genotype more with patient also TT genotype, While CC genotype more in control. The distribution of the genotype CC and CT was significantly higher among the male autistic patients than that of the female autistic patients. Conversely there was no significant difference could be evidenced neither in the genotypes nor in the alleles distribution with severity. Result of studied the SNP of GABRG3 gene rs208129 revealed that there was significant difference in the genotypes TT/TA but not for the genotypes AA .Additionally, highly significant difference was remarked in the alleles T/A among the autistic patients and control, the OR for TT genotype was 4.33 so it more expressed in autistic patient than control groups .while TA genotype was for control groups .Also the T allele more distributed in autistic patient. There was no significant difference in the genotypes TT/TA/AA for SNP rs208129 in GABRG3 gene in relation to the gender .Only autistic patients with the moderate symptoms showed the genotype AA. However, the other two genotypes TA and TT did not exhibit any significant difference. Both allele A and T exhibit a significant difference with the severity of disease ,p-value 0.007. The study RELN gene SNP rs736707 conferred that TC was significant difference and OR was2.626 so TC genotype more in patient. there was no significant difference in the genotypes TT/CC nor for alleles T/C among the autistic patients in relation to control group, there was no significant difference in the genotypes TT/TC/CC for SNP rs736707 in RELN gene in relation to the gender nor for allelic distribution nor for severity. The expression of FOXL1 among study participants were quantitated on the transcriptional level through qRT-real time PCR. The expression of FOXL1 was significantly higher among autistic patients than that of control participants .The expression fold in autistic patient was 0.02-100.52, mean± SD was 45.25 ± 27.02 and median 43.9.And for control the expression fold 0-43.64, mean± SD was9.52±11.82 and median 6.78. Up-regulation of FOXL1in autistic patients than control one. Estimation of GIT symptoms with severity of disease showed significant difference with severity . Down regulation of PHOX2B and NURR expression among autistic patients and control participants Conversely, there was no significant difference in the expression of PHOX2B and NURR1 among autistic patients and control participants. So the FOXP2 overexpressed as it target genes(PHOX2B ,NURR1) down-regulated. The study concluded the following: ASDs are heterogeneous in terms of behavior. The genetic alterations seem to be important in understanding biological links to behavior in autism. It has been shown through the present study that ASD patients had higher risk in male more than female, suggesting role of sex chromosomes in autistic disorders, chromosomal rearrangement is crucial in the genesis of autism, chromosomal abnormalities have supplied a lot of information on the candidate gene and a potential autism-related molecular pathway.Language impairments represent a frequent feature of the autism phenotype. FOXP2 overexpression may be the cause of Language impairments.The measurement of peripheral OXT levels showed high in autistic patient than control. The measurement of peripheral OXT levels and OXTR genetic alterations provide a simple dual-biomarker test to gauge social functioning in the ASD patient for diagnostic, monitoring and support purposes.The results obtained from ROC curve demonstrated that these parameters can be used in future as good markers for diagnosis and prognosis and to assess the severity of autism.The stratification of patients with ASD into severity categories shows significant differences both in OXT levels and OXTR (rs2268491, C/T) genotype and allele distributions, that can be sex dependent. OXT based therapies will require personalized medicine tactics to correctly identify patients with ASD who require neuropeptide boosting in social settings.Variants of GABRG3 gene SNP rs208129 are associated with ASD in Iraq population, which is likely role of SNPs in etiology ASDs. In the Iraqi population, variants of RELN gene SNP rs736707 the are linked to ASD, suggesting that SNPs play a role in the pathology of ASDs. Up-regulation of FOXL1in autistic patients subjected to this study that in turn may involve in the pathogenesis of autism.

طلب نسخة من الملف

الملفات غير منشورة على الموقع. اترك بياناتك وسيتواصل معك موظفو قسم الشؤون الثقافية والفنون لإرسال النسخة مجاناً.

الاسم

رقم الهاتف

البريد الإلكتروني

الغرض من الطلب (اختياري)

إرسال الطلب