Whole exome sequencing for gene discovery in lethal fetal disorders

My PhD thesis focuses on the delineation of monogenic causes of lethal fetal anomaly phenotypes, supervised by PD Dr. med. Isabel Filges.
Chromosomal anomalies including submicroscopic copy number variations (CNV) account for a significant fraction of about 10% of fetal structural anomalies. For fetal congenital anomaly syndromes with a normal high-resolution karyotype, the monogenic etiology of the phenotypes is poorly understood. Our objective was to use exome sequencing (ES) to identify genes that were not previously reported in a disease-associated context and novel variants in known disease genes to increase our knowledge on genotype-phenotype correlations in fetal anomalies. Ultimately, we aimed at improving clinical care for affected families.
In the first part of the project we used family-based high throughput Next Generation Sequencing (NGS) of families with two or more fetuses affected by a recurrent rare lethal fetal anomaly pattern or a single fetus with a phenotype pattern indicating a defect in a fundamental human developmental pathway. We sequenced the exome of the affected individuals and parents and analyzed the detected variants, regarding their potential to alter protein function, using databases on variant frequencies in healthy and affected individuals, bioinformatic functional prediction and conservation analysis programs, cross-species phenotyping and pathway analysis as well as current knowledge extracted from medical and biological literature. In total 19 families with 26 fetuses participated in the study. In 12 families (63%) a candidate gene was detected (CENPF, DNHD1, FGFR2, KIF14, KIF4A, MKS1, OTX2, PIGW, PTK7, RYR1, SMAD3 and TTC28) and in 6 (50%) of those a definite diagnosis was achieved (CENPF, FGFR2, KIF14, MKS1, OTX2 and RYR1).
We studied selected candidate genes (RYR1, KIF4A, PTK7 and SMAD3) and variants for their consequences on the functional level. For those four genes I designed and implemented mRNA
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dosage analysis, for PTK7 a cell culture model and for SMAD3 a chicken embryo model in collaboration with the laboratory for regulatory evolution, in order to also elucidate the underlying disease mechanism. Results were returned to the patients within the genetic consultation and counselling of the families regarding recurrence risk for further pregnancies, options for prenatal and preimplantation diagnosis as well as pregnancy management.