ESPE Abstracts (2015) 84 P-1-131

Increased Detection Rate of Paired Box Domain Gene Mutations by Application of Multiplex Ligation-Dependent Probe Amplification Analysis in Patients with Primary Congenital Hypothyroidism and Thyroid Dysgenesis

Malgorzata Kumorowicz-Czocha,b, Pia Hermannsc, Anna Madetko-Talowskad & Joahim Pohlenzc


aPrivate Pediatrics and Pediatric Endocrinology Practice, Cracow, Poland; bDepartment of Pediatric and Adolescent Endocrinology, Chair of Pediatrics, Polish-American Institute of Pediatrics, Jagiellonian University Medical College (by 2014), Cracow, Poland; cDepartment of Pediatrics, Johannes Gutenberg University Medical School, Mainz, Germany; dDivision of Medical Genetics, Chair of Pediatrics, Polish-American Institute of Pediatrics, Medical College, Jagiellonian University, Cracow, Poland


Background: The contribution of mutations in paired box domain (PAX8) gene in children with congenital hypothyroidism (CH) and thyroid dysgenesis (TD) still remains a subject of interest of researchers. While quantitative PCR and direct sequencing concentrate on single gene fragment analysis and identification of point mutations, multiplex ligation-dependent probe amplification (MLPA) analysis might improve the detection rate of PAX8 mutations in patients with congenital CH caused by TD.

Objective and hypotheses: To determine if MLPA could improve the detection rate of PAX8 gene mutations in patients with CH and TD.

Method: The study included 45 children from south-eastern Poland selected via already established neonatal screening for primary CH. DNA was extracted from peripheral blood samples with the use of Master Pure DNA Purification Kit (Epicentre Biotechnologies). DNA samples were used in two types of genetic analysis of PAX8 gene: Sanger sequencing method (promoter region and 12 exons with their exon-intron boundaries were sequenced) and MLPA technique (SALSA MLPA kit P319-A1 THYROID).

Results: Sanger sequencing method revealed PAX8 mutations in 5 out of 45 (11.1%) patients with CH and TD. In two of them heterozygous substitutions with the amino acid change in the coding sequence (c.700G>A, p.E234K and c.1225C>T, p.P409S respectively) were detected. In remaining three children missense variant within the promoter sequence of PAX8 gene (456C>T) was revealed. Application of MLPA analysis allowed identification of heterozygous deletion of exon 7 of PAX8 gene in two more (4.4%) patients in the examined group. In total, heterozygous PAX8 mutations were detected in seven out of 45 patients (15.5%).

Conclusion: In the study MLPA analysis increased PAX8 mutation rate from 11.1 to 15.5%. Application of MLPA analysis, in addition to direct sequencing, both improves and expands genetic analysis for CH and TD.

Funding: The work was funded in part by the Fritz-Thyssen-Stiftung and MAIFOR.

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