Classical and next generation sequencing approaches applied to the molecular diagnosis of congenital hypothyroidism

BERNAL BARQUERO, CE; MARTIN, M; GEYSELS, RC; PEYRET, V; PAPENDIECK, P; TESTA, G; MIRAS, M; CHIESA, AE; CITTERIO, CE; NICOLA, JP

Congreso; XVIII Latin American Thyroid Congress; 2021

Latin American Thyroid Society

Congenital hypothyroidism (CH) is the most frequent endocrine disorder in pediatric patients with an incidence of
1:2,000-4,000 newborns. To date, over thirty monogenic forms of CH have been identified, highlighting the genetic heterogeneity of
the disease. A meta-analysis demonstrated that only 5%-10% of patients with thyroid dysgenesis and 45%-88% of patients with thyroid
dyshormonogenesis are diagnosed using single-gene sequencing. Objective: To apply classical and next generation sequencing (NGS)
approaches to investigate the etiology of CH. Methods: A cohort of 16 patients with permanent CH due to thyroid dysgenesis (TD,
n = 2), dyshormonogenesis (TDH, n = 14), or thyroid hormone resistance (THR, n = 1) was enrolled in the study. Most patients
with TDH showed defective iodide accumulation as revealed by thyroid scintigraphy. 2 TDH patients showed a syndromic disease
(hypoacusia or epilepsy). The etiology of the disease was explored by SLC5A5 gene sequencing (n = 5), 17 candidate gene sequencing
panel (n = 11), or whole-exome sequencing (WES, n = 3). NGS variants were further validated using Sanger sequencing. In silico and
functional in vitro analyzes were used to investigate the pathogenicity of the variants identified. Results: SLC5A5 gene sequencing
revealed compound heterozygous variants (p.D396V; c.970-3A>C) in 1 out of 5 TDH patients analyzed. Gene sequencing panel
showed simple or compound heterozygous variants in genes involved in thyroid organogenesis or hormonogenesis. One TD patient
showed a heterozygous FOXE1 variant (p.P203R). One TDH patient showed compound heterozygous TG variants (p.Q29*; c.177-
2A>C). The remaining TDH patients showed heterozygous TG (p.F1542Vfs*20; p.T2563C; p.S523P) or DUOX2 (p.E1496Dfs*51)
variants. Duo WES uncovered a candidate heterozygous BCOR variant (p.S1431Y) in THR patient, and a candidate heterozygous
TUBB3 (p.Q23*) along with a TG variant (p.G653D) in a TDH patient with epilepsy. In silico and/or in vitro functional analyzes
classified 2 variants as benign and 10 as pathogenic (4 missense, 4 nonsense, 2 splice disruptive). Conclusions: NGS has emerged as
an attractive technology to explore the genetic basis of CH, particularly when the clinical presentation is not clear to guide candidate
gene analysis. Gene sequencing panel revealed variants in genes that would have been a priori excluded based on the phenotype of the
patient. WES may provide novel insights into the pathogenesis of the disease.