Russo Silvia, Calzari Luciano, Mussa Alessandro, Mainini Ester, Cassina Matteo, Di Candia Stefania, Clementi Maurizio, Guzzetti Sara, Tabano Silvia, Miozzo Monica, Sirchia Silvia, Finelli Palma, Prontera Paolo, Maitz Silvia, Sorge Giovanni, Calcagno Annalisa, Maghnie Mohamad, Divizia Maria Teresa, Melis Daniela, Manfredini Emanuela, Ferrero Giovanni Battista, Pecile Vanna, Larizza Lidia
Human Molecular Genetics Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy.
Department of Pediatric and Public Health Sciences, University of Turin, Torino, Italy.
Clin Epigenetics. 2016 Mar 1;8:23. doi: 10.1186/s13148-016-0183-8. eCollection 2016.
Multiple (epi)genetic defects affecting the expression of the imprinted genes within the 11p15.5 chromosomal region underlie Silver-Russell (SRS) and Beckwith-Wiedemann (BWS) syndromes. The molecular diagnosis of these opposite growth disorders requires a multi-approach flowchart to disclose known primary and secondary (epi)genetic alterations; however, up to 20 and 30 % of clinically diagnosed BWS and SRS cases remain without molecular diagnosis. The complex structure of the 11p15 region with variable CpG methylation and low-rate mosaicism may account for missed diagnoses. Here, we demonstrate the relevance of complementary techniques for the assessment of different CpGs and the importance of testing multiple tissues to increase the SRS and BWS detection rate.
Molecular testing of 147 and 450 clinically diagnosed SRS and BWS cases provided diagnosis in 34 SRS and 185 BWS patients, with 9 SRS and 21 BWS cases remaining undiagnosed and herein referred to as "borderline." A flowchart including complementary techniques and, when applicable, the analysis of buccal swabs, allowed confirmation of the molecular diagnosis in all borderline cases. Comparison of methylation levels by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in borderline and control cases defined an interval of H19/IGF2:IG-DMR loss of methylation that was distinct between "easy to diagnose" and "borderline" cases, which were characterized by values ≤mean -3 standard deviations (SDs) compared to controls. Values ≥mean +1 SD at H19/IGF2: IG-DMR were assigned to borderline hypermethylated BWS cases and those ≤mean -2 SD at KCNQ1OT1: TSS-DMR to hypomethylated BWS cases; these were supported by quantitative pyrosequencing or Southern blot analysis. Six BWS cases suspected to carry mosaic paternal uniparental disomy of chromosome 11 were confirmed by SNP array, which detected mosaicism till 10 %. Regarding the clinical presentation, borderline SRS were representative of the syndromic phenotype, with exception of one patient, whereas BWS cases showed low frequency of the most common features except hemihyperplasia.
A conclusive molecular diagnosis was reached in borderline methylation cases, increasing the detection rate by 6 % for SRS and 5 % for BWS cases. The introduction of complementary techniques and additional tissue analyses into routine diagnostic work-up should facilitate the identification of cases undiagnosed because of mosaicism, a distinctive feature of epigenetic disorders.
影响11p15.5染色体区域印记基因表达的多种（表观）遗传缺陷是Silver-Russell（SRS）综合征和Beckwith-Wiedemann（BWS）综合征的基础。这些相反生长障碍的分子诊断需要一个多方法流程图来揭示已知的原发性和继发性（表观）遗传改变；然而，高达20%至30%临床诊断的BWS和SRS病例仍未得到分子诊断。11p15区域复杂的结构、可变的CpG甲基化和低频率的嵌合现象可能是漏诊的原因。在此，我们证明了互补技术对评估不同CpG的相关性以及检测多个组织以提高SRS和BWS检出率的重要性。
对147例临床诊断的SRS病例和450例临床诊断的BWS病例进行分子检测，为34例SRS患者和185例BWS患者提供了诊断，9例SRS病例和21例BWS病例仍未确诊，在此称为“临界病例”。一个包括互补技术以及在适用时对颊拭子进行分析的流程图，使得所有临界病例的分子诊断得以确认。通过甲基化特异性多重连接依赖性探针扩增（MS-MLPA）对临界病例和对照病例的甲基化水平进行比较，确定了H19/IGF2:IG-DMR甲基化缺失的区间，该区间在“易于诊断”和“临界”病例之间是不同的，“易于诊断”病例与对照相比其值≤平均值-3个标准差（SDs）。H19/IGF2:IG-DMR处值≥平均值+1个SD被归为临界高甲基化BWS病例，KCNQ1OT1:TSS-DMR处值≤平均值-2个SD被归为低甲基化BWS病例；这些通过定量焦磷酸测序或Southern印迹分析得到支持。6例疑似携带11号染色体嵌合型父源单亲二体的BWS病例通过SNP阵列得到证实，该阵列检测到高达10%的嵌合现象。关于临床表现方面，除了1例患者外，临界SRS病例具有该综合征表型的代表性，而BWS病例除半身肥大外，最常见特征的出现频率较低。
临界甲基化病例达成了确定性的分子诊断，使SRS病例的检出率提高了6%，BWS病例的检出率提高了5%。将互补技术和额外的组织分析引入常规诊断检查应有助于识别因嵌合现象（表观遗传疾病的一个显著特征）而未确诊的病例。
