Division of Pediatric Gastroenterology.
Stanley Division of Neurovirology.
J Pediatr Gastroenterol Nutr. 2021 Mar 1;72(3):436-441. doi: 10.1097/MPG.0000000000003012.
Genetic susceptibility has been proposed as etiopathogenic in several pediatric liver diseases including autoimmune hepatitis (AIH). High throughput sequencing (HTPS) has been applied to archived needle liver biopsies obtained from adults but rarely to pediatric biopsies. For conclusive diagnosis of AIH, most subjects have an initial formalin-fixed paraffin embedded (FFPE) needle liver biopsy that is eventually archived and may be stored for decades.
Our goal was to develop methods to utilize tissue from archived needle liver biopsies for extraction of RNA sufficient to produce HTPS data.
We extracted total RNA from 45 FFPE needle liver biopsy samples (24 AIH type 1 patients and 21 controls [ages 15_11 and 19_10]; biopsy storage time 0.5-20 years) and constructed cDNA libraries that were then sequenced on an Illumina HiSeq2000 platform.
Forty (89%) of the libraries produced high-quality sequences for further analyses. The average number of sequences obtained per library from HTPS was 55,136,519 (range 14,914,291-184,027,499). There was a significant inverse relationship between the number of human reads obtained and the age of the specimen (P < 2_10_7). It was possible to classify more than 90% of the reads as known genes in samples that had been stored for less than 10 years.
Archived needle liver biopsies can be used for sequence based interrogation of the etiologic origins of complex liver diseases of young subjects, such as AIH.
遗传易感性已被提出是几种儿科肝病（包括自身免疫性肝炎（AIH））的病因学发病机制。高通量测序（HTPS）已应用于成人的存档针肝活检，但很少应用于儿科活检。为了明确诊断 AIH，大多数患者最初进行福尔马林固定石蜡包埋（FFPE）的针肝活检，最终进行存档，可能会储存数十年。
我们的目标是开发利用存档针肝活检组织提取 RNA 的方法，以获得足够进行 HTPS 数据的 RNA。
我们从 45 个 FFPE 针肝活检样本（24 例 AIH 1 型患者和 21 例对照[年龄 15_11 和 19_10]；活检储存时间 0.5-20 年）中提取总 RNA，并构建 cDNA 文库，然后在 Illumina HiSeq2000 平台上进行测序。
40（89%）个文库产生了可用于进一步分析的高质量序列。从 HTPS 获得的每个文库的平均序列数为 55,136,519（范围 14,914,291-184,027,499）。从 HTPS 获得的人类读数数量与标本的年龄呈显著负相关（P<2_10_7）。在储存时间少于 10 年的样本中，超过 90%的读数可以归类为已知基因。
存档针肝活检可用于对年轻患者（如 AIH）的复杂肝脏疾病的病因学进行基于序列的探究。
