Clinical and molecular genetic characterization of congenital malformations

Sammanfattning: Congenital malformations are important causes of perinatal mortality and morbidity, and around 4% of children are diagnosed with a malformation during their first year of life. Despite improved surgical treatment, several malformations are associated with lifelong sequelae requiring specialized health care. Important issues for these families are the etiology, prognosis and recurrence risk of the malformation in future pregnancies. Nowadays, around 50% of patients with malformations in combination with cognitive impairment receive an etiologic diagnosis after genetic evaluation. The aims of this thesis were to increase the knowledge of genetic causes behind congenital malformations, to improve clinical genetic investigations of these patients and at the same time to identify genes involved in normal and impaired organ development. Study I Whole-body human chimerism is the result of two zygotes giving rise to one individual, and is a rarely detected condition that can cause congenital anomalies. We have studied the molecular background of a 46,XX/47,XY,+14 karyotype identified in clinical genetic investigation in a boy with disorder of sex development (DSD). Based on molecular findings, we suggest that the chimerism in our patient is the result of dispermic fertilization of a parthenogenetically activated oocyte. This study highlights chimerism as an underlying cause of distinct cell populations in an individual, and shows the difficulty of predicting the severity of associated phenotypes in mosaic or chimeric forms of genetic aberrations. Study II Tetrasomy 14 is a rare condition associated with multiple malformations, cognitive impairment and mortality when present in non-mosaic form. We report on molecular genetic and mitochondrial studies in an 8-year-old girl with a marker chromosome 14. We showed that the marker chromosome originated from maternal meiosis and was present in all cells analyzed, providing evidence that survival beyond infancy is possible in non-mosaic forms of this condition. The results emphasize importance of updating existing data on clinical outcomes of patients with severe diseases to correspond to high standard pediatric care. Study III VACTERL association is a condition with multiple malformations including vertebral (V) anorectal (A) cardiac (C) tracheoesophageal (TE) renal (L) and limb (L) anomalies, without a known common cause. We performed array comparative genomic hybridization (array CGH) and DNA sequencing of the candidate genes PCSK5, HOXD13 and CHD7 to investigate the role of copy number variants (CNV) and single gene defects in 39 patients and fetal cases with VACTERL association or a VACTERL-like phenotype. We identified pathogenic gene dose alterations in 2/39 patients (5%) and a pathogenic mutation in CHD7 in one patient, while single nucleotide variants of unclear significance were detected in PCSK5. We concluded that copy number variants are not common causes of VACTERL association and that CHARGE and VACTERL syndromes represent important differential diagnoses. Study IV In this study, we have investigated the hypothesis that genetic mosaicism in malformed organs could be an underlying cause of congenital malformations. Array CGH analyses using DNA derived from cardiac tissue in 23 patients with congenital heart malformations were performed, and findings of pathogenic or unclear variants were compared with presence in blood in the same individuals. We identified pathogenic gene dose alterations in 2/23 patients (9%) and did not find evidence for mosaicism. We concluded that identification of copy number variants are important in individual cases of congenital malformations and that genetic mosaicism warrants further study using other molecular genetic technologies. Study V We have studied the presence of copy number variants in 25 patients with congenital anorectal malformations, esophageal atresia and hydronephrosis using array CGH analysis and identified pathogenic variants in 2/25 cases (8%). We describe a mosaic structural variant of tetrasomy 15 identified in a patient with syndromic esophageal atresia, and report a novel putative susceptibility region for esophageal atresia. Mosaicism for pathogenic or unclear variants was investigated in both tissue and blood in eight cases and did not reveal discrepancies. The study shows importance of copy number analysis in individual patients with gastrointestinal malformations. General conclusions from our studies are that tissue-specific genetic mosaicism for copy number variants does not appear to be a common cause of congenital malformations and that CNV analysis is important in patients with congenital malformations as identification of high penetrance variants in some families markedly improves accuracy of recurrence risk estimations in these families.