Lipa Gene Mutation in PED-LIPIGEN (Pediatric FH Subjects)
关键词
抽象
描述
Lysosomal acid lipase (LAL) is encoded by LIPA gene located on chromosome 10q23.3-q23 and consists of 10 exons. LIPA mRNA (messenger RiboNucleic Acid) (GenBank accession number NM_000235) is 2782 bp long and encodes a mature protein of 375 residues (GenBank accession number NP_000226). The sequencing of all 10 exons of LIPA gene will consist of 10 PCR (Polymerase Chain Reaction) amplification reactions (for the 10 exons and the proximal promoter) followed by 20 sequence reactions (forward and reverse sequencing) with appropriate primers designed to include the intron-exon boundaries. This analysis will be performed in about 250 FH pediatric subjects as specified in project description.
The sequencing work will be performed taking advantage of 2 automated 8 capillaries automated DNA Sequencer (3500 Genetic Analyzer, Thermo Fisher Scientific, Monza, Italy) currently available in the laboratory of the Units involved in the project.
In case of identification of unreported sequence variants, the presence of these mutations will be assessed in a sample of at least 100 normolipidemic subjects of the population, in order to define whether the nucleotide changes are rare sequence variations (with a putative functional effect) or represent common polymorphisms. In case of finding of rare variants in the coding regions, an in silico analysis will be performed by using two different softwares (Polyphen, http://genetics.bwh.harvard.edu/pph/ and Panther, http://www.pantherdb.org/) to predict the putative damaging role of the mutations on the protein. In case of intronic variants, the specifically designed software Automated Splice Site Analysis will be applied (https://www.splice.uwo.ca/).
Putative causal mutations will be also tested for co-segregation in available families in affected and unaffected members.
In order to test the effect of variants on enzyme activity LAL-activity will be assayed with dried blood spot (DBS) technique using the inhibitors Lalistat 2 in carriers and non carriers of these mutations belonging to available kindred.
日期
| 最后验证: | 06/30/2020 |
| 首次提交: | 05/20/2019 |
| 提交的预估入学人数: | 06/10/2019 |
| 首次发布: | 06/11/2019 |
| 上次提交的更新: | 07/21/2020 |
| 最近更新发布: | 07/22/2020 |
| 实际学习开始日期: | 08/31/2017 |
| 预计主要完成日期: | 06/30/2021 |
| 预计完成日期: | 06/30/2021 |
状况或疾病
干预/治疗
Other: FH pediatric patients
相
手臂组
| 臂 | 干预/治疗 |
|---|---|
| FH pediatric patients 1000 clinically diagnosed FH pediatric patients (age <18 years) included in the LIPIGEN (Lipid TransPort Disorders italian Genetic Network) database | Other: FH pediatric patients Observational study: There is no intervention. |
资格标准
| 有资格学习的性别 | All |
| 取样方式 | Non-Probability Sample |
| 接受健康志愿者 | 是 |
| 标准 | Inclusion Criteria: - Pediatric subjects (<18 years old) with a clinical diagnosis of FH and without identified pathogenic mutations in the known candidate genes. Exclusion Criteria: - Subjects with a clinical diagnosis of FH with identified pathogenic mutations in the known candidate genes. |
结果
主要结果指标
1. Prevalence of patients with mutations of LIPA gene among clinically diagnosed FH subjects [2 years from start of the study]
次要成果指标
1. Frequency of specific mutations of LIPA gene among clinically diagnosed FH subjects [2 years from start of the study]
