Genetic investigation of syndromic obesity and development of biological models to study the Xia-Gibbs syndrome

Author(s)

Laura Machado Lara Carvalho

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2023-01-17

Examining board members

Carla Rosenberg; Maria Rita dos Santos e Passos Bueno; Alexander Augusto de Lima Jorge; Juliana Ferreira de Oliveira

• Advisor: Carla Rosenberg; Celia Priszkulnik Koiffmann; Ana Cristina Victorino Krepischi; Tiago Antônio de Oliveira Mendes

Abstract

INTRODUCTION: Syndromic obesity is defined by the association of obesity with one or more of the following features: developmental delay, intellectual disability, dysmorphic traits, and congenital malformations. Most cases of syndromic obesity do not have their genetic origin elucidated, which impairs genetic counseling of the families and precludes the development of therapeutic methods; therefore, further clinical and genetic studies are required. In this thesis, syndromic obesity was the topic of study, with the initial genetic investigation of a cohort with this condition. We identify in this cohort a case of Xia-Gibbs syndrome, a syndromic form of intellectual disability caused by AHDC1 heterozygous mutations, that has poorly understood pathophysiological mechanisms, and for which there is still no recognized association with obesity; we then decided to develop biological models for its study. OBJECTIVES: (1) To identify causal genetic variants in a cohort of 20 patients with syndromic obesity who were negative for Prader-Willi, Fragile X syndromes, and MLPA SALSA P064, P036 and P070 tests; (2) to develop biological models (cellular and animal) to study the Xia-Gibbs syndrome. METHODS: (1) For those cases with a previous negative result in chromosome microarray analyses (CMA), we performed exome sequencing (Agilent V5) of the patients and their parents (five trios). For the others, exome sequencing of the trios (total of 15) was performed with probe capture for coding regions and for an optimized backbone for evaluation of copy number variation (CNVs; OneSeq Agilent). (2) In addition to the patient with the loss-of-function AHDC1 variant identified in the syndromic obesity cohort, two other Xia-Gibbs patients were recruited. For the development of induced pluripotent stem cells (iPSC) lines, we used the reprogramming method of cells from the three patients. To develop the animal model (zebrafish), we edited the ahdc1 gene using CRISPR-Cas9. RESULTS: (1) In the group in which we analyzed only SNVs and indels (because they were already negative for CMA), we had a diagnostic rate of 40% (2/5 MYT1L and GRIA3 genes); in the group in which we combined the analysis of CNVs, SNVs and indels, the diagnostic rate was 47% (7/15 MED13L, AHDC1, EHTM1, STAG2, KIAA0442, MEIS2 and SETD1A genes). Two of these patients (with MYT1L and MED13L causal variants) were also described as case reports, in addition to the paper referring to the complete cohort. (2) We obtained three iPSC lines with different loss-of-function AHDC1 variants. These cells have a human iPSC typical morphology, show expression of pluripotency factors (SOX2, SSEA-4, OCT3/4 and NANOG) and have the ability to differentiate into the three germ layers which is evidenced by the expression of PAX6, HAND1 and SOX17 by the in vitro derived embryoid bodies. The zebrafish line obtained has an insertion of four base pairs in the ahdc1 coding region, it is fertile, and the genotypic frequencies of its offspring follow Mendelian proportions. CONCLUSIONS: (1) The nine causal variants identified involve known syndromic intellectual disability genes; only two of these genes (MYT1L and EHMT1) are related to syndromes in which the association with obesity is well defined. In syndromic obesity investigations, the possibility that the etiology of the syndromic condition may be independent of obesity should not be ruled out. Genome-wide investigation approaches (such as microarray and exome/genome sequencing) should be prioritized rather than gene panels focused on syndromic obesity. (2) We have developed the first biological models for Xia-Gibbs syndrome, which we will use to study its yet poorly understood pathophysiology, as well as to evaluate the hypothesis that the neural mechanisms for hunger and satiety are altered in patients. CONTRIBUTIONS OF THIS THESIS: The genetic study of a syndromic obesity cohort, the presentation of case reports and a comprehensive review about this topic presented in this thesis contributed to the diagnosis and genetic counseling of patients and their families. The development of biological models of Xia-Gibbs syndrome, should lead to the understanding of the mechanisms underlying AHDC1 mutations and emerging therapeutic targets. Finally, this thesis also comprises articles meant to support genetic education, helping to take science beyond the walls of our University. (AU)

• FAPESP's process: 18/08486-3 - Genetic bases of Syndromic Obesity and molecular mechanisms involved in its physiopathology
• Grantee: Laura Machado Lara Carvalho
• Support Opportunities: Scholarships in Brazil - Doctorate