التحليل الجزيئي للجين CYP21A2 في المرضى العراقيين المصابين بتضخم الغدة الكظرية الولادي == Molecular Analysis of CYP21A2 Gene in Iraqi Patients with Congenital Adrenal Hyperplasia
Author name:
رقية غياث ياسين العبيدي
Supervisor name:
بسام موسى صادق الموسوي
Abstract:
Background : Congenital adrenal hyperplasia (CAH) is a group of autosomal - recessive disorders that result from a genetic defect in the pathway of steroidogenesis. The most common type is 21 - hydroxylase enzyme deficiency (21 - OHD), which accounts for 90 - 95% of cases and is categorized into two clinical forms : severe classic early onset (salt wasting and simple virilizing) and milder non - classic late onset forms. The molecular analysis of mutations affecting CYP21A2 gene coding for 21 - OH enzyme was not tried earlier in Iraqi patients.Aim of Study : To analyze the commonly expected mutations affecting CYP21A2 gene causing 21 - hydroxylase and thus congenital adrenal hyperplasia in Iraqi children and to determine the frequency of each type and their clinical impact.Patients, Materials & Methods : Fifty - four children with a clinical diagnosis of CAH were recruited from the Pediatric Endocrine Consultation Clinic / Children Welfare Hospital - Baghdad and the Genetic Consultation Clinic - Teaching Laboratories / Medical City - Baghdad during the period between September 14th, 2014 and March 14th, 2015. The clinical diagnosis of 21 - hydroxylase deficiency was made when the patients presented with ambiguous genitalia in females with or without dehydration, or vomiting and dehydration with normal male phenotype early in life, or pseudo - precocious puberty later in life.Peripheral blood samples were collected from all patients. Two samples were taken from those with ambiguous genitalia; one for chromosomal analysis and sex determination and the other for DNA analysis. For the other cases, a single sample was taken for DNA analysis.The other tests, namely electrolytes and hormonal assessment were sent for to be performed by another laboratory (governmental or private).Two cases with ambiguous genitalia showing male karyotypes were excluded from this study as they do not fit in the criteria of 21OHD.Molecular analysis of CYP21A2 gene was performed by PCR amplification using biotinylated primers and hybridization of amplification products to a test strip containing allele - specific oligonucleotide probes immobilized as an array of parallel lines. The bound biotinylated sequences are detected using streptavidin - alkaline phosphatase and color substrates according to the manufacturers’ instructions.Results : CYP21A2 gene was analyzed by CAH StripAssay® for detection of 11 common point mutations, and 50% of deletions and conversions in all 52 cases. Mutations (point mutations and deletions/conversions) were detected in 36 (69.2%) cases while 16 (30.8%) cases had undetected mutations. The most frequent mutations were : large deletions / conversions in 11 (21.15%), Q318X in 7 (13.46%), I2 Splice in 6 (11.54%), I172N in 5 (9.6%), Del 8bp in 1 (1.9%), V281L in 2 (3.85%), P453S in 1 (1.9%), R483P in 1 (1.9%) and multiple mutations in 2 cases (3.85%). Among those with large deletions/conversions, deletion/conversion (P30L, I2Splice, Del8bp) found in 3 (5.8%) cases, Deletion/conversion (P30L, I2 Splice, Del8bp, I172N) in 2 (3.85%) cases, Deletion/conversion (Cluster E6, V281L, L307 frameshift, Q318X, R356W) in 4 (7.7%) cases, complete gene deletion in 2 (3.85%) cases.Four mutations (namely P30L, R356W, Cluster E6, L307 frameshift) were not detected in any of our cases.Most cases demonstrated good correlations between genotypes and phenotypes. In the salt wasting group, the correlation was found in all cases with homozygous genotype, while among the simple virilizing group, the concordance of genotype/phenotype was found in all cases of group B (5 cases with homozygous I172N) and the single case in group A (homozygous I2 Splice), while it was absent in group null (one case withhomozygous deletion/conversion) and in group C (one case with homozygous V281L).Conclusions : 1. The majority of 21 - hydroxylase deficiency cases were females presented early in life with ambiguous genitalia with/without salt losing crisis, while a smaller number of patients were males with normal genitalia and salt wasting with/without precocious puberty; the patients have a high rate of parental consanguinity.2. There is a diversity of mutations affecting CYP21A2 gene in Iraqi 21 - hydroxylase deficiency patients, with a good percentage of undetected mutations.3. The diagnosis of 21 - OHD in CAH patients will be made more accurate when based on results of molecular testing.4. The data of molecular studies are useful in genetic counseling, in prenatal diagnosis and offering accurate treatment plan during pregnancy and in detection of carriers in families having previously affected child, diminishing the complications for these families
التقييم الجزيئي لعوامل الخطورة الوراثية لامراض القلب والاوعية الدموية لعينة من المرضى العراقيين المصابين بنقص التروية القلبية الحاد == Molecular Assessment of Cardiovascular Genetic Risk Factors among a Sample of Iraqi Patients with Ischemic Heart Diseases
Author name:
وسام جاسم محمد
Supervisor name:
بسام موسى صادق الموسوي
Abstract:
Ischemic heart disease (IHD) is a major cause of morbidity and mortality worldwide; its incidence is increasing in developing countries. It is estimated that 17.5 million individuals die from CVD each year, accounting for 31% of all deaths worldwide; more than 75% of these deaths occur in low to middle income population. Understanding the pathogenesis of IHD has been modified over the years and many new genetic risk factors have been recognized. Attention is now focused towards understanding the genetic basis of IHD. Enormous effort has been invested in understanding the genes and specific DNA sequence variations responsible for this heritability and genetic polymorphisms might be risk factors that predispose to IHD.Aim of Study : To analyze the genetic risk factors among Iraqi patients with acute IHD and to determine the frequency of each type of mutation / polymorphism.Patients, Materials & Methods : This is a cross sectional study that recruited 56 patients admitted to the Cardiac Care Unit (CCU) of Ibn Al - Nafees Teaching Hospital with a clinical diagnosis of acute ischemic heart disease (myocardial infarction and unstable angina) during a two - month period between December 8th, 2015 and February 8th, 2016. All cases <50 years with acute MI or angina were included, while those >50 years and those with documented hyperlipidemia were excluded.Demographic and clinical data of the enrolled patients were reported. Two - three ml of peripheral blood samples were aspirated from all recruited patients and collected in K2EDTA tube to be store at - 20oC for further DNA analysis. Molecular analysis to detect 12 commonIIImutations/ polymorphisms, namely : FV G1691A (Leiden), FV H1299R (R2), Prothrombin C20210A, Factor Xlll V34L, β - Fibrinogen - 455 G - A, PAI - 1 4G/5G, GPllla L33P (HPA - 1), MTHFR C677T, MTHFR A1298C, ACE l/D, Apo B R3500Q, and Apo E2/E3/E4 was performed by PCR amplification using biotinylated primers and hybridization of amplification products to a test strip containing allele - specific oligonucleotide probes immobilized as an array of parallel lines. The bound biotinylated sequences were detected using streptavidin - alkaline phosphatase and color substrates according to the manufacturers’ instructions.Results : The age range of patients was 18 - 50 years, with a mean ±SD of 40±7 years. The vast majority of enrolled cases were males (54/56 (96.4%). The traditional risk factors were frequently encountered in the current study (hypertension 21.4%, diabetes 26.8%, smoking 75%, family history of IHD 48.2%, and previous attack of ischemia 23.2%). Serum troponin was positive in 72.2% of cases. The study found that the genotype frequencies of 12 genetic mutations / polymorphisms were as follows : MTHFR A1298C and C677T were the highest reported mutations among the study group (62.5%) and (50%) respectively, followed by β - fibrinogen gene mutation detected in (46.5%), and PAI - 1 4G/5G polymorphism which was detected in (75%) of patients, while PAI - 1 4G/4G was detected in (16.1%) of patients. Homozygous ACE D/D polymorphism was present in 35.7% in our cases and heterozygous HPA - 1(a/b) polymorphisms was present in 28.6%. The E4 allele of Apo E gene was present in 7.1% of the studied cases.IV Heterozygous factor XIII (FXIII) V34L variant was detected in 21.4% patients, while homozygous state was detected in 3.6% patients, i.e. 25% of selected cases had Leu allele, Heterozygous FV R2 was detected in 12.5%, and factor V Leiden mutation was detected in 1.8%, while no abnormal homozygous alleles were detected. Prothrombin G20210A mutation were detected in 1(1.8%) patient. Neither heterozygous nor homozygous states for the mutant Apo B allele were detected in this study. The study showed no statistically significant difference between age group I (<40 years) and age group II (40 - 50 years), but the study showed higher frequency for some genes like PAI - 1(4G) and Apo E4 alleles in group I than group II (100% versus 85.3%) and (13.6% versus 2.9%) respectively, while HPA - 1 (a/b) polymorphism was higher frequent in group II than group I (35.3% versus18.2%).Subgroup analysis of the studied traditional risk factors (hypertension, diabetes mellitus, smoking, family history of ischemic heart disease) showed that β - Fibrinogen mutation had higher frequency in smoker patients than nonsmokers (50% versus 35.71%), the D allele of ACE gene was more frequent in hypertensive than in non - hypertensive patients (91.7% versus 79.5%), and higher frequency of HPA - 1b allele in diabetic than non - diabetic patients 46.7% versus 22%).Genetic risk score (0 - 16) was established according to the number of risky alleles detected in each case; the results showed that all patients had at least 2 genetic risk factors and none had more than 8; the study also showed that patients with 4 or more risky genes represented 82.14% of the studied patients, and that the risk of IHD increases in those who carry 4 or more genetic risk factors when associated with at least one traditional risk factor.
تقييم الطفرات الوراثية ل BRAF, IDH1 وفقدان 1p/19q في الاورام الدبقية للمرضى القراقيين : دراسة نسيجية مناعية كيميائية مع التهجين الموضعي المتفلور == Assessment of BRAF, IDH1 mutations and 1p/19q loss in Gliomas in Iraqi patients. Immunohistochemical and Fluorescence in Situ Hybridization study
الكشف الجزيئي عند مرضى HLA - B27 والمصلي لـ التهاب الفقار المقسط مع بعض الارتباطات السريريه == Molecular and Serologic Detection of HLA - B27 Among Ankylosing Spondylitis Patients With Some Clinical Correlations
Author name:
ميادة محمد علي عبد الهادي الضايف
Supervisor name:
بسام موسى صادق الموسوي
Abstract:
مرض التهاب الفقار المقسط (AS)هو مرض التهابي مزمن يصيب المفاصل العجزية الحرقفية، العمود الفقري وليس من النادر المفاصل المحيطيه، كما يؤثر على 0.1 - 1% من عامة الناس. السبب الدقيق لـ AS غير معروف لكن تم تحديد استعداد وراثي قوي لـ AS مع ارتباط قوي بـ HLA - B27 الايجابيه، HLA - B27 يوجد في > 90% من المرضى AS البيض و50 - 80% من المرضى AS غير البيض. هناك عدة طرق للكشف عن HLA - B27 كالطرق المصلية (طريقة مقايسة الممتز المناعي المرتبط بالانزيم(ELISA) وطريقة اختبار السمية للخلايا اللمفاوية الصغيرة(MLCT) والتقنيات الجزيئية (القائمة على الحمض النووي مثل PCR).كانت الطرق المصلية في البداية هي الاختبارات القياسية للكشف عن المتسضدات HLA - B27 ولكن استبدلت بالطرق الجزيئيه على اساس الحمض النووي لان الطرق المصلية لها عيوب عديدة كان تكون حساسة للتنظيم السفلي اوالتغييرات التوافقية للمستضدات، لا يمكنها اكتشاف فروق بنية البروتين الناتجة عن تعدد اشكال النوكليوتيدات الاحادي او المحدود ، لها نتائج سلبيه كاذبة او ايجابية كاذبة لعدة اسباب مثل التفاعل المتصالب للاجسام المضادة مع مستضدات فئة HLA class I المختلفة بسبب التشابه الواسع النطاق داخل الطبقه، وتغيير الحواتم المستضدي، تلوث الصفائح الدموية او كريات الدم الحمراء. تحدث هذه التغييرات في بنية البروتين (المستضد) في كثير من الاحيان استجابة للعدوى المتزامنة، والعلاج بالعقاقير والحالة الصحية للمريض.لاتتاثر التقنيات القائمة على تفاعل البلمرة المتسلسل (PCR) بهذه التغييرات في بنية البروتين، وبالتالي فهي اكثر تمثيلا لحالة منتج الجين (الحاضر/ الغائب)؛ بالاضافة الى انها قادرة على اكتشاف اختلاف اساسي واحد في تسلسل DNA بين الاليلين، ويمكن تطبيقه على اي مصدر من الحمض النووي الجيد بغض النظر عن قابلية الخليه. الهدف من الد راسة : 1. الكشف الجزيئي ل HLA - B27 عند المرضى المصابين بالتهاب الفقار المقسط بواسطة طريقة تفاعل البلمرة المتسلسل بالوقت الحقيقي.2. المقارنة بين التقنيات المصلية والجزيئية في التحقق من حالة HLA - B27.3. تقييم علاقة HLA - B27 مع بعض المتغيرات السريريه.المرضى والمواد وطرق العمل : اجريت دراسة مستعرضة على 83 مريضا عراقيا بالغا شخصوا في عيادة استشارات الروماتيزم والتاهيل في مدينة الطب - بغداد / العراق. واجريت تقارير بيانات المرضى والاختبارات اللاحقة بعد موافقة المريض. سجلت البيانات الديموغرافية والسريرية لجميع المرضى. حددت frequency of HLA - B27 لجميع المرضى المسجلين بطريقة تفاعل البلمرة المتسلسل بالوقت الحقيقي باستخدام HLA - B27 RealFast™ kit وكذلك بطريقة مقايسة الممتز المناعي المرتبط بالانزيم (ELISA) باستخدام (human leukocyte antigen) ELISA kit.سحبت 3 - 5 مل عينات دم من المرضى الداخلين بالدراسة وقسمت الى عينتين : 1.5 - 2 مل جمعت في الانبوب العادي plane tube وتركت لتتجلط لاختبار (ELISA) والذي اجري في المختبرات التعليمية في مدينة الطب، وجمعت عينة دم اخرى بحجم 2.5 - 3 مل في انبوبK2 EDTA tube وحفظ مجمدا لتحليل الحمض النووي الذي اجري في المختبر المركزي للصحة العامه.النتائج : كان متوسط عمر المريض اعمار المرضى (±الانحراف المعياري) هو 38.4±9.8 سنة (المدى 16 - 62 سنه)، ونسبة الذكور الى الاناث هي 10.86/1، المرضى الذكور كان عددهم 76 ومرضى الاناث 7. متوسط عمر المرض من المرضى هو 9.08±32.13 سنة وكان متوسط عمر المرض للمرض هو 6.5±5.8 سنه. لوحظ التهاب الارتكاز في 44 (53%)، كان التهاب المفاصل الاطراف حاضرا في 14 (16.9%)، وكان التهاب العنبة موجودا في 12 (14.5%)، وامراض القلب والاوعية الدموية والصدفية وجدت في مريض واحد (1.2%) لكل منهما. كشف وجود HLA - B27 في 55 )66.3%) من المرضى من خلال real time PCR و28 (33.7%) خالة المتبقية كانوا حالات سلبية لـHLA - B27. كان ظهور المرض مبكرا وكانت مدة المرض اطول في المرضى الايجابيين لـHLA - B27 (قيمة p <0.05 لكل منهما). كانت هناك علاقة ذات دلالة احصائية بين HLA - B27 الايجابية مع الجنس والتهاب العنبة (p= 0.002, 0.032، على التوالي)، ولم تكن هناك ارتباطات ذات دلالة احصائية لـ HLA - B27 الايجابية مع العمر للمريض عند التشخيص ، وتاريخ العائلة، والتهاب الارتكاز ، والتهاب مفاصل الاطراف ، وESR وCRP والنتائج الاشعاعية وانواع العلاج. اظهرت حالة sHLA - B27 بواسطة ELISA لـ 83 حالة 32 حالة ايجابية (38.6%). عند مقارنة نتائج كلا الاختباريين باستخدام نتائج PCR كسيطرة، كانت 11 حالة نتائج ايجابية كاذبه و34 حالة كانت نتائج سلبية كاذبه، حساسية ELISA هي 38.18% والخصوصية 60.71%، وهذه الطريقة تكشف عن HLA - B27 القابل للذوبان، لذلك يمكن ان تتاثر بعدة عوامل بما في ذلك العلاج المستمر للمريض وصحة المريض. من بين 83 حالة، هناك 28 حالة (33.7%) لديها اختبار المصلي الاولي للكشف عن HLA - B27 باختبار microlymphocytotoxicity (MLCT)؛ اوضحت المقارنة بين نتائج ELISA وMLCT مع real time PCR (المستخدم كسيطرة) ان طريقة ELISA اظهرت 13 (46.4%) حالة هي نتائج سلبية كاذبة و3 (10.7%) حالات هي نتائج ايجابية كاذبة (حساسية = 60.6% ، خصوصية = 72.72% ، القيمة التنبؤية الايجابية (PPV)= 86.95%) في حين MLCT اظهرت 9 (32.1%) حالة كانت نتائج سلبية كاذبة و2 (7.1%) حالة هي نتائج ايجابية كاذبة (حساسية = 68.96% وخصوصية = 80% ، PPV = 90.9%). الاستنتاجات : • يتفوق كشف real time PCR لحالة HLA - B27 على التقنيات المصليةMLCT) و(ELISA. الاعتمادية والراحة والفعالية من حيث التكلفة لهذة الطريقة جعلها قابلة للتطبيق على التنميط الجيني HLA - B27 في الممارسات الروتينيه.• اعطت MLCT نتائج افضل من ELISA حيث استخدم الاخير للكشف عن sHLA - B27. كلا التقنيتين تحمل نتائج ايجابية كاذبة وسلبية كاذبة عاليه.• في المختبرات التي لاتتوفر فيها التشخيصات الجزيئيه، يمكن استخدام طريقة MLCT مع استخدام مضخمات صفيحة كبيرة (large panel antisera) لتقليل النتائج الكاذبه. • لم يؤدي الكشف عن ELISA لـsHLA - B27 في المصل الى الحساسيه او التحديد المطلوب. لتعزيز الايجابية، يوصى استخدام عينات البلازما ومحلول الاستعزاز(enhancing solution) لمنع فقدان sHLA - B27 اثناء التخثر وكذلك لمنع التفاعل المتصالب (cross - reaction) مع HLA - B7.• هناك ارتباط قوي بين HLA - B27 والتهاب الفقار المقسط بين المرضى العراقيين. • هناك ميل للمرض للتاثير على الرجال الايجابي HLA - B27 اكثر من النساء الايجابية HLA - B27.• تظهر اعراض AS في وقت اسبق في وجود جين HLA - B27 ومدة المرض كانت اطول في مرضى HLA - B27 الايجابيين من المرضى السلبيين، ولا يوجد اي ارتباطات مهمة لـ HLA - B27 مع المتغييرات المدروسة الاخرى.• هناك غلبة الذكور في الحالات AS وتبدا الاعراض عادة في الشباب | Ankylosing spondylitis (AS) is a chronic inflammatory disease affecting the sacroiliac joints, spine and not infrequently peripheral joints. AS affects 0.1 - 1% of general population. The exact cause of AS is still unknown but a strong genetic predisposition for AS was determined with a strong association with HLA - B27 positivity. HLA - B27 is present in >90% of white AS patients and 50 - 80% of non - white AS patients. There are several methods for the detection of HLA - B27 as serological methods (microlymphocytotoxicity (MLCT) and enzyme linked immunosorbent assay (ELISA)) and molecular techniques (DNA - based e.g. PCR). Serological methods were at first the standard tests for detection of HLA - B27 antigens but were replaced by DNA - based HLA typing because serologic methods have several disadvantages e.g. being sensitive to down - regulation or conformational changes of the antigens, cannot detect the protein structure differences caused by single or limited nucleotide polymorphism, have false - negative or false - positive results for several causes such as cross reactivity to other antibodies with different HLA class I antigens due to the extensive similarity within the class, altered antigenic epitopes, platelet or erythrocyte contamination. These changes to the protein (antigen) structure occur frequently in response to concurrent infection, drug therapy and health state of the patient.PCR - based techniques are not affected by these protein changes and are thus more representative to the state of the gene product (present / absent); in addition, it can detect allelic differences at the nucleotide level. It is a rapid and a selective method for the detection of HLA - B2701 to HLA - B2728 subtypes; it is capable of detecting a single base difference in DNA sequence between the two alleles and can be applied on any source of good quality DNA regardless of cell viability.Aims of the study : • To detect HLA - B27 status among patients with ankylosing spondylitis using real - time PCR technique.• To compare serologic and molecular techniques in verifying HLA - B27 status, and• To assess the correlation of HLA - B27 status with some clinical variables.Patients, Materials and Methods : A cross - sectional study was conducted on 83 adult Iraqi patients with AS diagnosed at the Rheumatology and Rehabilitation Consultation Clinic at the Medical City - Baghdad / Iraq. Patients’ data reporting and subsequent testing was performed following patients’ consents. Demographic and clinical data of all patients were reported. The frequency of HLA - B27 was determined in all enrolled patients by real - time polymerase chain reaction (PCR) using HLA - B27 RealFast™ kit as well as by ELISA method using (human leukocyte antigen) ELISA kit. Three to five ml of peripheral blood sample was aspirated from each and every enrolled patient. It was divided into two samples : 1.5 - 2 ml was collected in a plain tube and left to clot for serological (ELISA) test which was performed at The Teaching Laboratories / Medical City Campus and another 2.5 - 3 ml sample was collected in K2 EDTA tube and was kept frozen for DNA analysis which was performed at the Central Public Health Laboratory.Results : The mean age of patients (± SD) was (38.4±9.8) years (range 16 - 62 years). Male to female ratio was (9 : 1), male patients were 76 and female patients were 6. The mean age of disease onset of patients was 32.13±9.08 years and the mean age of disease duration of patients was 6.5±5.8 years. Enthesitis was observed in 44 (53%), peripheral arthritis was present in 14 (16.9%), eye involvement was present in 12 (14.5%), cardiovascular disease and psoriasis were in 1 (1.2%) each.HLA - B27 positivity was detected in 55 (66.3%) patients by real - time PCR and remaining 28 (33.7%) cases were negative HLA - B27. The disease onset was earlier and disease duration was longer in HLA - B27 - positive patients (p - value<0.05 for both). There were statistically significant correlation between HLA - B27 positivity with gender and uveitis, (p= 0.002, 0.032, respectively) and there were no statistically significant associations of HLA - B27 positivity with age at diagnosis, family history, enthesitis, peripheral arthritis, ESR, CRP, radiological findings and treatment types. The sHLA - B27 status by ELISA test for 83 cases showed 32 (38.6%) positive cases. Comparing the results of both tests using real - time PCR results as a control, 11 HLA - B27 cases were false positive and 34 patients were false negative results. Thus, the sensitivity of ELISA was 38.18% and specificity was 60.71%, this method detects soluble HLA - B27, so it can be affected by several factors including ongoing patient’s treatment and patient’s health. Out of 83 cases, 28 (33.7%) cases have initial serological test for detection of HLA - B27 by microlymphocytotoxicity (MLCT) test; the comparison of ELISA and MLCT results with real - time PCR (used as a control) revealed that ELISA method showed 13 (46.4%) false negative results and 3 (10.7%) false positive results, (sensitivity=60.6%, specificity=72.72%, positive predictive value (PPV)=86.95%) while MLCT showed 9 (32.1%) false negative results and 2 (7.1%) false positive, positive (sensitivity =68.96% and specificity =80%, PPV=90.9%).Conclusions : • Real - time PCR detection of HLA - B27 status is superior to serological techniques (ELISA and MLCT). The reliability, convenience and cost effectiveness of this method make it applicable to HLA - B27 genotyping in routine practices.• MLCT yielded better results than ELISA when the latter was used for detection of sHLA - B27. Both serologic techniques carry high false positive and negative results.• In labs where molecular diagnostics are not available, MLCT test can be used instead adding large panel antisera to reduce false results.• ELISA detection of sHLA - B27 in serum did not give the required sensitivity or specificity. To enhance positivity, the use of plasma samples and an enhancing solution are recommended to prevent the loss of sHLA - B27 during clotting and to prevent cross reaction with HLA - B7.• There is a strong association between HLA - B27 and ankylosing spondylitis among Iraqi patients.• There is a tendency of the disease to affect HLA - B27 positive men more than HLA - B27 positive women.• Symptoms of AS appear earlier in the presence of HLA - B27 gene and the duration of the disease was longer in HLA - B27 positive than negative patients. Uveitis is more likely to occur in HLA - B27 positive than negative patients, and there are no significant associations of HLA - B27 with the other studied variables. • There is a male predominance in AS cases and the symptoms start usually in young adults.
طيف وتردد طفرات الجين K - ras عند المرضى العراقيين المصابين بسرطان القولون والمستقيم المفرق == Spectrum and Frequency of K - ras Mutations among Iraqi Patients with Sporadic Colorectal Cancer
