背景:Prader-Willi综合征(PWS,MIM176,270)和Angelman综合征(AS,MIM105,830)是由染色体15q11-13的印记缺陷引起的,母体基因表达缺失导致AS,父系基因表达导致PWS。诊断,一旦在大多数情况下通过使用甲基化特异性PCR测试确定,能够进行适当的治疗干预,并避免了进一步调查的需要。在斯里兰卡(以及其他低收入和中等收入国家),PWS/AS的基因检测有限,主要是因为父母无法支付测试费用,因为这些测试不是由卫生服务机构资助的。
方法:90例(46例女性),临床特征提示PWS(n=37)和AS(n=53),由儿科内分泌学家和儿科神经科医生推荐,被招募。在知情同意后获得临床信息和血液样本。在使用内部方法和试剂盒对DNA进行亚硫酸氢盐修饰后,提取DNA并进行甲基化特异性PCR(MS-PCR)。使用已知的阳性对照验证结果。在确认PWS和AS的情况下,使用亲子三人DNA样本来确定该疾病是否归因于缺失或单亲二体。确定了两种修饰方法的MS-PCR测试和微卫星分析的成本。
结果:在怀疑的PWS病例中,19/37为阳性,而5/53的疑似AS病例为阳性。AS的较低识别率可能与该疾病的临床特征与其他疾病的重叠有关。基于试剂盒的修饰方法更可靠,耗时少,并且在我们的实验室中具有成本效益。
结论:本研究中描述的基于试剂盒的修饰,然后进行MS-PCR,可以对可疑的PWS/AS病例进行更实惠的基因检测,这可能会通过针对受影响病例的适当治疗来改善患者护理。关于低复发风险的父母遗传咨询是可能的,尤其是在确认缺失或单亲二分法的情况下。在MS-PCR中,具有强烈临床怀疑AS的阴性病例,需要进行UBE3A突变测试。此外,在强烈临床怀疑的情况下,也可能需要进行印迹中心突变/缺失测试,MS-PCR阴性PWS和AS病例。
BACKGROUND: Prader-Willi syndrome (PWS, MIM 176,270) and Angelman syndrome (AS, MIM 105,830) are caused by imprinting defects of chromosome 15q11-13, with loss of maternal gene expression causing AS and paternal gene expression causing PWS. The diagnosis, once established in most cases by using a methylation-specific PCR test, enables appropriate therapeutic interventions and avoids the need for further investigations. Genetic testing for PWS/AS is limited in Sri Lanka (and in other low- and middle-income countries), mainly because parents are unable to pay for testing as these are not funded by the health service.
METHODS: Ninety cases (46 female) with clinical features suggesting PWS (n = 37) and AS (n = 53), referred by a pediatric endocrinologist and a pediatric neurologist, were recruited. Clinical information and blood samples were obtained following informed consent. DNA was extracted and methylation-specific PCR (MS-PCR) was performed following bisulfite modification of DNA by using an in-house method and a kit. Results were validated using known positive controls. Parent-child trio DNA samples were used in cases with confirmed PWS and AS to determine if the disease was due to a deletion or uniparental disomy. The cost of the MS-PCR testing of the two modification methods and the microsatellite analysis was determined.
RESULTS: Among the suspected PWS cases, 19/37 were positive, while 5/53 of the suspected AS cases were positive. The lower identification rate of AS is probably related to the overlap of clinical features of this condition with other disorders. The kit-based modification method was more reliable, less time-consuming, and cost-effective in our laboratory.
CONCLUSIONS: The kit-based modification followed by MS-PCR described in this study enables more affordable genetic testing of suspected PWS/AS cases, and this is likely to improve patient care by targeting appropriate therapy for the affected cases. Parental genetic counselling is made possible regarding the low recurrence risk, especially where a deletion or uniparental disomy is confirmed. In MS-PCR, negative cases with a strong clinical suspicion of AS, UBE3A mutation testing is required. In addition, imprinting center mutation/deletion testing may also be needed in strongly clinically suspected, MS-PCR negative PWS and AS cases.