1. Χριστίνα Κανακά-Gantenbein, Καθηγήτρια Παιδιατρικής – Παιδιατρικής Ενδοκρινολογίας, Διευθύντρια της Α’ Παιδιατρικής Κλινικής της Ιατρικής Σχολής του Πανεπιστημίου Αθηνών στο Νοσοκομείο Παίδων «Η Αγία Σοφία» (Επιβλέπουσα Καθηγήτρια)
2. Ανδρέας Σκορίλας, Καθηγητής Κλινικής Βιοχημείας, Τμήμα Βιολογίας, Σχολή Θετικών Επιστημών, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
3. Γεώργιος Χρούσος, Ομότιμος Καθηγητής Παιδιατρικής και Διευθυντής Ερευνητικού Πανεπιστημιακού Ινστιτούτου Υγείας Μητέρας, Παιδιού και Ιατρικής Ακρίβειας του Πανεπιστημίου Αθηνών
4. Νικόλαος Τεντολούρης, Καθηγητής παθολογίας και Υπεύθυνος της Διαβητολογικής Μονάδας και του Ερευνητικού Διαβητολογικού Εργαστηρίου, Α' Προπαιδευτικής Παθολογικής Κλινικής Πανεπιστημίου Αθηνών, Γενικό Νοσοκομείο Αθηνών «Λαϊκό»
5. Iωάννα-Pαχήλ Traeger-Συνοδινού, Καθηγήτρια Γενετικής στο Εργαστήριο Ιατρικής Γενετικής του Πανεπιστημίου Αθηνών
6. Εμμανουήλ Ζουμάκης, Επίκουρος Καθηγητής Μοριακής Βιοχημείας, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
7. Χρήστος Κοντός, Επίκουρος Καθηγητής Μοριακής Βιολογίας Τμήμα Βιολογίας, Σχολή Θετικών Επιστημών, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Introduction: A large number of endocrinological disorders have genetic etiology. Τhe application of hitherto widely used conventional techniques of Molecular Biology (e.g. Sanger sequencing) fails to clarify in many cases the genetic defect. This weakness is covered by Next Generation Sequencing (NGS), which allows the parallel and massive sequencing of many genes in many patients simultaneously in a single assay. Typical examples of endocrinological diseases whose identification of molecular disorder is not always feasible by conventional methods are the Monogenic Diabetes Mellitus MODY, the Congenital Hyperinsulinism (CHI), the Combined Pituitary Hormone Deficiency (CPHD), and the Disorders of Sex Development (DSD). Patients with MODY and CHI have impaired insulin secretion from the beta cells of the islets of Langerhans of the pancreas. In particular, patients with MODY have reduced insulin secretion and hence hyperglycemia, whereas patients with CHI have increased insulin secretion, disproportionate to circulating blood glucose levels, and consequently hypoglycemia. Patients with CPHD may have anatomical pituitary abnormalities and facial malformations, as well as at least two pituitary hormone deficiencies, while patients with DSD may have abnormalities in sex determination and differentiation, resulting in newborns with ambiguous genitalia. The identification of molecular defect is of outmost importance, not only for the identification of the pathogenic variants of the responsible for the above disorders genes, but also for the phenotype-genotype correlation, for the treatment, the prognosis and the recognition of the accompanying features as well as providing genetic counseling to the patient and his/her family. In addition, the identification of molecular defect is important for the treatment of the newborn with DSD, as it can determine the decision on the sex of the child, a decision with significant impact on psychosocial adjustment and future quality of his/her life.
Objective: The aim of the present thesis was to identify the molecular defects by employing the methodology of next generation sequencing (Targeted Gene Panel, TGP or Whole Exome Sequencing, WES) in patients with genetic endocrinological disorders, in which the used techniques of molecular analysis have failed to detect the molecular defect. Specifically, the genetic defect of 50 patients with MODY, 17 patients with CHI, 2 patients with CPHD and 2 patients with DSD was investigated.
Patients and Methods: Fifty patients with MODY were screened for the detection of pathogenic variants in a group of 7 MODY genes (GCK, HNF1A, HNF4A, HNF1B, INS, ABCC8 and KCNJ11). Seventeen patients with CHI were screened for the detection of pathogenic variants in the above gene group, many of which have been linked also to CHI, and additionally in 5 CHI genes (GLUD1, HADH, INSR, SLC16A1, TRMT10A). Patients with MODY or CHI, in whom did not detect pathogenic variants employing NGS methodology, were screened for deletions and duplications of GCK, HNF1A, HNF4A, HNF1B and ABCC8 genes employing MLPA (Multiplex Ligation-dependent Probe). In addition, the genetically undiagnosed patients with CHI were also tested for the pathogenic intronic variants c.1333-1013A>G of the ABCC8 gene and c.636+471G>T of the HADH gene employing Sanger sequencing. In the present thesis, the frequency of the p.S385C variant of the KCNJ11 gene in the Greek population was also investigated. In order to detect pathogenic variants responsible for the phenotype characteristics of 2 patients with CPHD and 2 patients with DSD were used WES. All pathogenic variants detected employing the NGS methodology were confirmed by Sanger sequencing.
Results: The 28% (14/50) of MODY patients carried pathogenic variants of the ABCC8 (8%, 4/50), GCK (8%, 4/50), HNF1A (6%, 3/50), HNF1B (4%, 2/50) and HNF4A (2%, 1/50) genes, while the 6% (3/50) of the patients carried variants of uncertain significance of the ABCC8 gene. No pathogenic variants were detected in the KCNJ11 and INS genes. Five novel pathogenic variants were identified: p.C371X of the GCK gene, p.N402Y of the HNF1A gene, p.E285K of the HNF4A gene, p.M1514T and p.S1386F of the ABCC8 gene. In addition, 2 unrelated patients with MODY carried de novo heterozygous deletion of the whole HNF1B gene. The 23.5% (4/17) of patients with CHI carried pathogenic variants of the ABCC8 (11.8%, 2/17), GCK (5.9%, 1/17) and HNF4A (5.9%, 1/17) genes, whereas the 11.8% (2/17) of the patients with CHI carried variants of uncertain significance of the KCNJ11, INSR and HNF4A genes. Three novel pathogenic variants were detected: p.V71A of the GCK gene, p.R333P of the HNF4A gene and p.I445Sfs * 5 of the ABCC8 gene. The frequency of p.S385C variant of the KCNJ11 gene in the Greek population was found to be polymorphism (1.35% > 1%). The application of WES in 2 patients with CPHD revealed pathogenic variants of the HS6ST1, IL17RD and SOX9 (novel variant) genes in the first patient and pathogenic variants of the BMP4, GNRH1 and SRA1 genes in the second patient, which are probably responsible for some of their clinical characteristics. Moreover, the application of WES in 2 patients with DSD unveiled pathogenic variants of the TACR3, WNT7A genes and SAMD9 (de novo variant), PMM2, ACTN4 genes respectively, which are probably responsible for their clinical characteristics.
Conclusions: The application of the NGS methodology provided genetic diagnosis in 28% of patients with MODY and 23.5% of patients with CHI, allowed the identification of 5 novel pathogenic variants in patients with MODY and 3 in patients with CHI. It has also revealed that pathogenic variants of the ABCC8 gene detected in patients with MODY were equal to the pathogenic variants of the GCK gene detected in the same MODY cohort. In addition, the application of WES in previously undiagnosed patients with CPHD and patients with DSD, unveiled several variants which are probably responsible for their clinical features. It is concluded that the application of the NGS methodology provides rapid results, increases diagnostic accuracy and is more cost-effective than Sanger sequencing. Therefore, the NGS methodology constitues a useful and powerful tool for the diagnosis of genetic endocrinological disorders.