Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

In papillary renal neoplasm with reverse polarity (PRNRP), the status of chromosomal copy number alterations, especially chromosomes 7/17 gain and chromosome Y loss, has remained controversial. In the literatures, there is a discrepancy among the results of chromosomal alteration in PRNRP depending on the analytical methods. Here, we comprehensively analyzed the status of chromosomal abnormalities in PRNRP. Nineteen PRNRP cases were analyzed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), five of which were additionally subjected to array-based comparative genomic hybridization (aCGH) analysis. Fifteen cases of PRCC were used as controls. From the aCGH results, no genome copy number abnormalities were found in the five PRNRP cases. By FISH, numbers of nuclei with abnormal chromosomal signals in PRNRP (centromere 7 gain: 11-21% of nuclei, centromere 17 gain: 11% of nuclei, centromere Y loss: 14-31% of nuclei) were similar to those in non-neoplastic tubular cells (centromere 7 gain: 11-15% of nuclei, centromere 17 gain: 12-15% of nuclei, centromere Y loss: 13-45% of nuclei). c-MET immunohistochemical overexpression, a substitute marker for chromosome 7 trisomy, was observed in 0 of 19 PRNRP cases, consistent with the analyses by aCGH and NGS regarding chromosome 7 gain. Taken together, the frequency of chromosomal alterations in PRNRP is similar to that in non-neoplastic tubular cells, and lower than that in PRCC. Our data suggest that PRNRP has a different tumorigenesis and is a distinct entity from PRCC.

Endometriosis is a complex disease that affects 10-15% of women of reproductive age. Familial studies show that relatives of affected patients have a higher risk of developing the disease, implicating a genetic role for this disorder. Little is known about the impact of germline genomic copy number variant (CNV) polymorphisms on the heredity of the disease. In this study, we describe a rare CNV identified in two sisters with familial endometriosis, which contain genes that may increase the susceptibility and progression of this disease. We investigated the presence of CNVs from the endometrium and blood of the sisters with endometriosis and normal endometrium of five women as controls without the disease using array-CGH through the Agilent 2x400K platform. We excluded common CNVs that were present in the database of genomic variation. We identified, in both sisters, a rare CNV gain affecting 113kb at band 3q12.2 involving two candidate genes: ADGRG7 and TFG. The CNV gain was validated by qPCR. ADGRG7 is located at 3q12.2 and encodes a G protein-coupled receptor influencing the NF-kappaβ pathway. TFG participates in chromosomal translocations associated with hematologic tumor and soft tissue sarcomas, and is also involved in the NF-kappa B pathway. The CNV gain in this family provides a new candidate genetic marker for future familial endometriosis studies. Additional longitudinal studies of affected families must confirm any associations between this rare CNV gain and genes involved in the NF-kappaβ pathway in predisposition to endometriosis.

Neurodevelopmental disorders are a group of complex multifactorial disorders characterized by cognitive impairment, communication deficits, abnormal behaviour, and/or motor skills resulting from abnormal neural development. Copy number variants (CNVs) are genetic alterations often associated with neurodevelopmental disorders. We evaluated the diagnostic efficacy of the array-comparative genomic hybridization (a-CGH) method and its relevance as a routine diagnostic test in patients with neurodevelopmental disorders for the identification of the molecular alterations underlying or contributing to the clinical manifestations. In the present study, we analysed 1800 subjects with neurodevelopmental disorders using a CGH microarray. We identified 208 (7%) pathogenetic CNVs, 2202 (78%) variants of uncertain significance (VOUS), and 504 (18%) benign CNVs in the 1800 patients analysed. Some alterations contain genes potentially related to neurodevelopmental disorders including CHRNA7, ANKS1B, ANKRD11, RBFOX1, ASTN2, GABRG3, SHANK2, KIF1A SETBP1, SNTG2, CTNNA2, TOP3B, CNTN4, CNTN5, and CNTN6. The identification of interesting significant genes related to neurological disorders with a-CGH is therefore an essential step in the diagnostic procedure, allowing a better understanding of both the pathophysiology of these disorders and the mechanisms underlying their clinical manifestations.

Mitochondrial disorders are characterized by a huge clinical, biochemical, and genetic heterogeneity, which poses significant diagnostic challenges. Several studies report that more than 50% of patients with suspected mitochondrial disease could have a non-mitochondrial disorder. Thus, only the identification of the causative pathogenic variant can confirm the diagnosis. Herein, we describe the diagnostic journey of a family suspected of having a mitochondrial disorder who were referred to our Genetics Department. The proband presented with the association of cerebellar ataxia, COX-negative fibers on muscle histology, and mtDNA deletions. Whole exome sequencing (WES), supplemented by a high-resolution array, comparative genomic hybridization (array-CGH), allowed us to identify two pathogenic variants in the non-mitochondrial SYNE1 gene. The proband and her affected sister were found to be compound heterozygous for a known nonsense variant (c.13258C>T, p.(Arg4420Ter)), and a large intragenic deletion that was predicted to result in a loss of function. To our knowledge, this is the first report of a large intragenic deletion of SYNE1 in patients with cerebellar ataxia (ARCA1). This report highlights the interest in a pangenomic approach to identify the genetic basis in heterogeneous neuromuscular patients with the possible cause of mitochondrial disease. Moreover, even rare copy number variations should be considered in patients with a phenotype suggestive of SYNE1 deficiency.

UNASSIGNED: utism spectrum disorder (ASD) is a heterogeneous clinical condition, and its genetic basis is widely confirmed. The chromosomal microarray analysis (CMA) is a first-line diagnostic test that identifies copy number variants (CNVs). Some of these genomic rearrangements are associated with ASD, but the meaning of most of them is still unknown.
UNASSIGNED: We performed a comparative genome hybridization (array-CGH) analysis in 130 children with confirmed ASD. Genetic results were analyzed and compared to clinical phenotype.
UNASSIGNED: 61/130 children carry CNVs, 44 presenting variants of unknown significance (u-CNVs), and 17 with susceptibility-CNVs (c-CNVs). Clinical evaluation showed no differences in cognitive abilities, language and EEG abnormalities, ASD symptoms among CNVs group and other patients. Finally, we highlight the role of GPHN, IMMP2L and ZMYND11, as ASD susceptibility genes.
UNASSIGNED: Our findings underscore the importance of array-CGH in ASD children since new CNVs and emerging genes appear to be associated with different clinical pictures.

Cat Eye Syndrome (CES) is a rare genetic disease caused by the presence of a small supernumerary marker chromosome derived from chromosome 22, which results in a partial tetrasomy of 22p-22q11.21. CES is classically defined by association of iris coloboma, anal atresia, and preauricular tags or pits, with high clinical and genetic heterogeneity. We conducted an international retrospective study of patients carrying genomic gain in the 22q11.21 chromosomal region upstream from LCR22-A identified using FISH, MLPA, and/or array-CGH. We report a cohort of 43 CES cases. We highlight that the clinical triad represents no more than 50% of cases. However, only 16% of CES patients presented with the three signs of the triad and 9% not present any of these three signs. We also highlight the importance of other impairments: cardiac anomalies are one of the major signs of CES (51% of cases), and high frequency of intellectual disability (47%). Ocular motility defects (45%), abdominal malformations (44%), ophthalmologic malformations (35%), and genitourinary tract defects (32%) are other frequent clinical features. We observed that sSMC is the most frequent chromosomal anomaly (91%) and we highlight the high prevalence of mosaic cases (40%) and the unexpectedly high prevalence of parental transmission of sSMC (23%). Most often, the transmitting parent has mild or absent features and carries the mosaic marker at a very low rate (<10%). These data allow us to better delineate the clinical phenotype associated with CES, which must be taken into account in the cytogenetic testing for this syndrome. These findings draw attention to the need for genetic counseling and the risk of recurrence.

Complex chromosomal rearrangements are rare events compatible with survival, consisting of an imbalance and/or position effect of one or more genes, that contribute to a range of clinical presentations. The investigation and diagnosis of these cases are often difficult. The interpretation of the pattern of pairing and segregation of these chromosomes during meiosis is important for the assessment of the risk and the type of imbalance in the offspring. Here, we investigated two unrelated pediatric carriers of complex rearrangements of chromosome 7. The first case was a 2-year-old girl with a severe phenotype. Conventional cytogenetics evidenced a duplication of part of the short arm of chromosome 7. By array-CGH analysis, we found a complex rearrangement with three discontinuous trisomy regions (7p22.1p21.3, 7p21.3, and 7p21.3p15.3). The second case was a newborn investigated for hypodevelopment and dimorphisms. The karyotype analysis promptly revealed a structurally altered chromosome 7. The array-CGH analysis identified an even more complex rearrangement consisting of a trisomic region at 7q11.23q22 and a tetrasomic region of 4.5 Mb spanning 7q21.3 to q22.1. The mother\'s karyotype examination revealed a complex rearrangement of chromosome 7: the 7q11.23q22 region was inserted in the short arm at 7p15.3. Finally, array-CGH analysis showed a trisomic region that corresponds to the tetrasomic region of the son. Our work proved that the integration of several technical solutions is often required to appropriately analyze complex chromosomal rearrangements in order to understand their implications and offer appropriate genetic counseling.

The lack of effective screening and successful treatment contributes to high ovarian cancer mortality, making it the second most common cause of gynecologic cancer death. Development of chemoresistance in up to 75% of patients is the cause of a poor treatment response and reduced survival. Therefore, identifying potential and effective biomarkers for its diagnosis and prognosis is a strong critical need. Copy number alterations are frequent in cancer, and relevant for molecular tumor stratification and patients\' prognoses. In this study, array-CGH analysis was performed in three cell lines and derived cancer stem cells (CSCs) to identify genes potentially predictive for ovarian cancer patients\' prognoses. Bioinformatic analyses of genes involved in copy number gains revealed that AhRR and PPP1R3C expression negatively correlated with ovarian cancer patients\' overall and progression-free survival. These results, together with a significant association between AhRR and PPP1R3C expression and ovarian cancer stemness markers, suggested their potential role in CSCs. Furthermore, AhRR and PPP1R3C\'s increased expression was maintained in some CSC subpopulations, reinforcing their potential role in ovarian cancer. In conclusion, we reported for the first time, to the best of our knowledge, a prognostic role of AhRR and PPP1R3C expression in serous ovarian cancer.

The 5q deletion syndrome is a relatively rare condition caused by the monoallelic interstitial deletion of the long arm of chromosome 5. Patients described in literature usually present variable dysmorphic features, behavioral disturbance, and intellectual disability (ID); moreover, the involvement of the APC gene (5q22.2) in the deletion predisposes them to tumoral syndromes (Familial Adenomatous Polyposis and Gardner syndrome). Although the development of gastrointestinal tract malignancies has been extensively described, the genetic causes underlying neurologic manifestations have never been investigated. In this study, we described a new patient with a 19.85 Mb interstitial deletion identified by array-CGH and compared the deletions and the phenotypes reported in other patients already described in the literature and the Decipher database. Overlapping deletions allowed us to highlight a common region in 5q22.1q23.1, identifying KCNN2 (5q22.3) as the most likely candidate gene contributing to the neurologic phenotype.