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201904151249411

H19 RT-PCR Real Time cod. BM-030 -Quantification assay of H19           

 

Principle of the test : Quantitative analysis of H19 mRNA expression

 

Technology : Relatitive Quantitative Real Time PCR

Gene Target : H19

Internal Control: GAPDH

Specimen : cDNA

Results : ΔΔCt method  

Reporting Units : Arbitrary Units (AU)

Number of tests : 25 tests BM-030

Kit storage : -20°C

Necessary equipment : 7500 Real Time PCR System

Status: Ready to use

 

 

H19 RT-PCR Real Time cod. BM-030
Quantitative Analysis of H19 mRNA expression

 

Imprinting disorders are a group of congenital diseases caused by dysregulation of genomic imprinting, affecting prenatal and postnatal growth, neurocognitive development, metabolism and cancer predisposition. Aberrant expression of imprinted genes can be achieved through different mechanisms, classified into epigenetic - if not involving DNA sequence change - or genetic in the case of altered genomic sequence. Despite the underlying mechanism, the phenotype depends on the parental allele affected and opposite phenotypes may result depending on the involvement of the maternal or the paternal chromosome(1).

Beckwith-Wiedemann syndrome (BWS) (OMIM #130650) is the most common congenital overgrowth condition (1:10.500 live births)(2) and represents the paradigm of genetic imprinting disorders and cancer predisposition syndromes. BWS is caused by several epigenetic and genetic defects. In approximately 85% of patients disturbed expression of imprinted genes located into two separate domains on chromosome 11p15.5 is found. In this chromosomal region, two differentially methylated imprinting centers (IC) ( H19/IGF2:IG-DMR and KCNQ1OT1:TSS-DMR, commonly referred to as IC1 and IC2, respectively) control the expression of genes involved in cell cycle progression and somatic growth control.

SRS (OMIM #180860) is the phenotypic and genetic opposite disorder of BWS, has an estimate incidence of 1:30,000 to 1:100,0003 and represents the paradigm of genetic restricted growth imprinting disorders and poor feeding predisposition. The most common mechanisms is LoM at IC1 on the paternal chromosome 11p15 (IC1-LoM), which is detected in 40-60% of patients. IC1-LoM results in reduced  IGF2  expression and increased  H19  expression(3).

The insulin-like growth factor-2 (Igf2)-H19 locus encodes important paternally imprinted genes that govern normal embryonic development. While Igf-2 encodes IGF2, which is an autocrine/paracrine mitogen,  transcription of H19 gives rise to non-coding mRNA that is a precursor of several microRNAs (miRNAs) that negatively affect cell proliferation. The proper imprinting of a differentially methylated region (DMR) within this locus, with methylation of the paternal chromosome and a lack of methylation on the maternal chromosome, regulates expression of both of these genes so that Igf2 is transcribed only from the paternal chromosome and H19 only from the maternal chromosome. Erasure of imprinting (hypomethylation) of the Igf2-H19 locus on both chromosomes, leads to downregulation of Igf2 and upregulation of H19 expression. Hypermethylation of this locus on both chromosomes (loss of imprinting) results in Igf2 overexpression(4).

Imprinting disorders are largely underdiagnosed because of the broad range of clinical signs, the overlap of presentation among different disorders, the presence of mild phenotypes, the mitigation of the phenotype with age and the limited availability of molecular techniques employed for diagnosis(1).

 

BioMole has developed and validated a novel quantitative analysis of H19 mRNA expression. This method is based on real-time PCR. Relative quantification of mRNA expression of H19 was achieved by normalization to the reference gene GAPDH. Relative quantification of target gene expression in patients was performed with the ΔΔCt method and the relative H19 fold changes were determined. Results are expressed in corresponding arbitrary units (AU) User friendly and complete, the H19 RT-PCR Real Time kit is suitable for any laboratory.

 

 

Reference:

1.       Syndromic Disorders Caused by Disturbed Human Imprinting

Diana Carli, Evelise Riberi, Giovanni Battista Ferrero, and Alessandro Mussa.

J.Clin Res Pediatr Endocrinol 2020;12(1):1-16.

2.       Prevalence of Beckwith-Wiedemann syndrome in North West of Italy

Mussa A, Russo S, De Crescenzo A, Chiesa N, Molinatto C, Selicorni A, Richiardi L, Larizza L, Silengo MC, Riccio A, Ferrero GB. Am J Med Genet A 2013;161:2481-2486. Epub 2013 Aug 5.

3.       Diagnosis and management of Silver-Russell syndrome: first international consensus statement.

Wakeling EL, Brioude F, Lokulo-Sodipe O, O’Connell SM, Salem J, Bliek J, Canton AP, Chrzanowska KH, Davies JH, Dias RP, Dubern B, Elbracht M, Giabicani E, Grimberg A, Grønskov K, Hokken- Koelega AC, Jorge AA, Kagami M, Linglart A, Maghnie M, Mohnike K, Monk D, Moore GE, Murray PG, Ogata T, Petit IO, Russo S, Said E, Toumba M, Tümer Z, Binder G, Eggermann T, Harbison MD, Temple IK, Mackay DJ, Netchine I. Nat Rev Endocrinol 2016;13:105-124. Epub 2016 Sep 2.

4.       Igf2-H19, an imprinted tandem gene, is an important regulator of embryonic development, a guardian of proliferation of adult pluripotent stem cells,a regulator of longevity, and a ‘passkey’ to cancerogenesis.

Mariusz Z. Ratajczak  Folia Histochem. Cytobiol. 2012;50(2):171-179.