The complexity of disease spectra in the vast and multifaceted field of paediatric surgery requires ever-changing strategies for diagnosis, establishment of prognosis and for therapeutic strategies. Notably, the combination of clinical skills and basic science research has proven invaluable in the development of new standards for the management of paediatric surgical diseases.
In this context, the advent of techniques allowing the study of genes and proteins, such as polymerase chain reaction (PCR), DNA sequencing, Western Blotting and chromosome imaging have immensely broaden the comprehension of such diseases, to the point that a molecular genetic investigation has become standard for the establishment of the diagnosis and/or prognosis in such diseases. Examples abound, ranging from the identification of protein targets in tumoral diseases, the analysis of gene variants responsible for the aetiology of several surgical pathologies or the investigation of chromosomal rearrangements which are linked to the prognosis of tumours, only naming a few.
The aim of the current “Professorial dissertation” (Habilitationsschrift) is to illustrate the importance of such molecular genetic studies for the future development of the paediatric surgery. One of the main areas in which these techniques have encountered ample resonance is the field of paediatric oncology. Accordingly, we have investigated cytogenetic and molecular genetic abnormalities in an infantile mucoepidermoid carcinoma (MEC) of the lung, and describe in chapter 2 the newly reported fusion transcript (and potential oncogene), MECT1-MAML2, which has been shown to encode a protein chimera that acts as a co-activator of the cAMP signalling pathway and whose expression has been linked to a better prognosis of MEC in children [19, 47, 96]. Furthermore, we attempted to understand the possible genetic mechanisms underlying one of the most common solid tumours in childhood, neuroblastomas (NB), which are neural-crest derived tumours whose clinical presentation, response to therapy and outcome of the treatment varies greatly [55]. To ascertain whether PHOX2b is indeed involved in the development of sporadic NB, we analyzed the complete PHOX2b coding region in 69 tumour DNA samples from NB patients through direct DNA sequencing, hypothesizing that PHOX2b mutations and/or polymorphisms were frequent in NB patients and thus directly associated to NB development. The results are shown in Chapter 3.
However, genetic abnormalities are not only responsible for tumoral diseases in paediatric patients, but also play a crucial role in diseases affecting the development and the functionality of different organs and systems. An example of such is Hirschsprung disease (HSCR), a congenital disorder characterized by the absence of enteric ganglia in a variable extent in the intestine of affected children. HSCR is transmitted in a complex pattern of inheritance, and the principal gene involved is located at 10q11.2, the RET proto-oncogene [3, 23, 30, 36, 72]. Although RET mutations resulting in either RET-protein truncation or functional inactivation have been associated with the HSCR phenotype, these are only identified in 15-20% of sporadic HSCR cases, and in only 50% of the familial cases [5, 15]. Therefore, in Chapter 4, we aimed to identify genomic rearrangements in the coding sequence of the RET proto-oncogene as well as in three HSCR-associated genes (ZEB2, EDN3 and GDNF) in 80 HSCR patients by using Multiplex Ligation-dependent Probe Amplification (MLPA), assessing if further variants of these genes might account for the unexplained cases.
Similarly, the central congenital hypoventilation syndrome (CCHS) is an autonomous control disease resulting in hypoventilation, hypercapnia and hypoxia during quiet sleep. The main genetic variants detected in CCHS patients have been mutations (deletions or expansions) in the PHOX2b gene, which in some series are present in up to 97% of isolated cases [103]. However, this disease is often associated to other neural crest pathologies, such as HSCR, which is linked to RET variants as mentioned above. Hence, it is feasible to infer that microdeletions or amplifications in sensitive areas of RET or in any of the other aforementioned associated genes may account for some of the CCHS/HSCR cases in which RET germline variants have not been identified. We have attempted in Chapter 5 to identify genomic rearrangements in the coding sequence of the RET, ZEB2, EDN3 and GDNF in 38 CCHS or CCHS/HSCR patients by MLPA.
Finally, genetic techniques are not solely useful in rare, seldom syndromes as depicted above. Another disease, the infantile hypertrophic pyloric stenosis (IHPS) is the most common indication for laparotomy in the first year of life, and still fatal if left untreated. A genetic background for the aetiology of IHPS can be inferred from the frequent familial occurrence of the disease and from recent research findings. Due to the essential role of nitric oxide (NO.) in the relaxation of the smooth muscle cells, one of the established candidate genes for the aetiology of IHPS is the neuronal Nitric Oxide Synthase (NOS1 or nNOS). Suitably, we attempted to further elucidate the association of NOS1 with IHPS by sequencing of the complete coding region of the gene in IHPS patients, hypothesizing that NOS1 genomic variants are indeed present and more frequent in pyloric stenosis affected children compared to normal controls. The results of this study are shown in Chapter 6.
In summary, in the following pages the following aims and specific hypotheses were tested:
Aim: to demonstrate that the application of molecular genetic techniques and the development of a “genetically oriented” thinking for diagnostic and therapy strategies may greatly improve the breadth of expertise of paediatric surgeons.
Hypothesis I: The translocation t(11;19)(q14~21;p12~13), and the consequent expression of the MECT1-MAML2 fusion chimera serve as a diagnostic and prognostic marker for low- to intermediate-grade lung mucoepidermoid carcinoma.
Hypothesis II: PHOX2b mutations and/or polymorphisms are present in neuroblastoma patients and thus directly associated to NB development.
Hypothesis III: Genomic rearrangements in RET are responsible for the Hirschsprung disease (HSCR) phenotype in patients without identifiable germline RET variants.
Hypothesis IV: genomic rearrangements in RET are responsible for the HSCR phenotype or for a “HSCR trait” in individuals without identifiable germline RET variants in patients with central congenital hypoventilation syndrome and HSCR.
Hypothesis V: NOS1 genomic variants are present and more frequent in pyloric stenosis affected children compared to normal controls.