IMPE2023 Poster Presentations Growth and Syndromes (15 abstracts)
Next generation sequencing implementation for the diagnosis of endocrine disorders: 4 years-experience in a public paediatric hospital
Paula Scaglia 1,2 , María Esnaola Azcoiti 1,2 , Agustín Izquierdo 1,2 , Lourdes Correa Brito 1,2 , Marianela Maier 1,2 , Gabriela Sanso 1,2 , Bárbara Casali 1,2 , Florencia Villegas 3 , Romina Armando 3 , Nora Sanguineti 1 , Franco Brunello 4 , Sandra Rozental 1 , Diego Raffo 1 , Solange Rosenbrock 1 , María Gabriela Ballerini 1 , Adriana Boywitt 1 , Sebastián Castro 1 , Débora Braslavsky 1 , Analía Freire 1 , Rosa Enacan 1 , Florencia Clement 1 , Romina Grinspon 1 , Ana Keselman 1 , Mirta Gryngarten 1 , Andrea Arcari 1 , Ana Vieites 1 , Ana Chiesa 1 , Patricia Papendieck 1 , María del Carmen Fernández 3 , Marcelo Martí 4 , Claudia Arberas 3 , Ignacio Bergadá 1 , Rodolfo Rey 1,2 & María Gabriela Ropelato 1,2
1Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE) CONICET – FEI – División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina. 2Unidad de Medicina Traslacional, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina. 3Sección Genética Medica, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina. 4Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET, Universidad de Buenos Aires., Buenos Aires, Argentina
Introduction: Most paediatric rare diseases have an underlying genetic cause but making a molecular diagnosis is often still a challenge. The incorporation of Next Generation Sequencing (NGS) technologies into research and clinical workflows has improved the diagnosis of these disorders.
Objective: To evaluate the process of NGS implementation for the diagnosis of genetic endocrine diseases in a tertiary public paediatric Hospital.
Material and methods: DNA was obtained from patients with suspicion of genetic endocrine diseases and their parents. Genetic diagnosis and counselling process involved 3 steps: 1-Pre-test: clinical evaluation by paediatric endocrinologists and geneticists, phenotype description (HPO), selection of adequate genetic testing approach, informed consent. 2-Genetic testing: performed by NGS in a NextSeq500 (Illumina) using targeted capture custom gene panels (Agilent or Twist), clinical exome (TrusightOne, Illumina or Inherited Disease Panel, Agilent) and/or WES (3Billion). Data were analysed by own designed bioinformatic pipelines (GATK). DECoN algorithm was used to predict Copy Number Variants (CNVs). Variants were prioritised using B-platform (Bitgenia) and classified according to ACMG criteria. Sequence variants were confirmed by Sanger sequencing, while GAP-PCR, MLPA or CGH-array (180K, Agilent) were used for CNVs confirmation. Segregation analysis was performed. 3-Post-test: Report and genetic counselling.
Results: From March 2018 to July 2022, we included 140 probands (52 girls) with gonadal axis disorders (n=41), growth disorders (n=40, including 13 rasopathies), thyroid disorders (n=24), DSD (n=22), neuroendocrine hereditary tumours (n=5), and other endocrine disorders (n=8). We found 91 variants (29 novel) in 55 different genes: 58.2% missense, 15.4% frameshift, 9.9% splicing, 8.8% nonsense, 1.1% inframe duplication and 6.6% CNVs (5 deletions, 1 duplication). Sixty-five relevant variants (40 pathogenic, 22 likely pathogenic, 3 VUS) in genes related to phenotypes with appropriate familial segregation study were found in 59 positive cases, yielding a global diagnostic efficiency of 42.1% (59/140). In 61 negative cases, no variants associated to phenotype could be prioritised. Twenty cases remained as inconclusive for one of the following reasons: only VUS found in a gene related to phenotype, only one pathogenic/likely pathogenic variant in a recessive gene, incomplete or inconclusive segregation study, predicted CNV still requires confirmation by appropriate method.
Conclusions: The successful implementation of NGS methodology allowed the diagnosis of 42% of the cases of our cohort. This approach was flexible enough to uncover the molecular bases underlying diverse endocrine genetic disorders in a paediatric centre.
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