Phd in HUMAN BIOLOGY AND MEDICAL GENETICS

Thesis title: Prenatal Exome Sequencing in Central Nervous System Anomalies: diagnostic yield and challenges

Trio-based prenatal exome sequencing (pES) showed a significant incremental diagnostic yield over karyotype and chromosomal microarray (CMA) in fetuses with structural anomalies. However, optimized indications, detection rates in different categories of fetal anomalies, and interpretation of variants pathogenicity are still under investigation. The aim of this study was to assess the incremental diagnostic yield of trio-based pES after karyotype and CMA inconclusive result in Central Nervous System (CNS) anomalies. Between January 2019 and December 2022, a cohort of 33 fetuses presenting apparently isolated or non-isolated CNS anomalies was enrolled. In all cases karyotype and CMA analyses were performed. In non-conclusive cases, pES was offered. Trio-based pES was performed in 15 cases on genomic DNA extracted from fetal samplings and parental leukocytes. Library preparation and targeted enrichment were performed using the Twist Human Core Exome Kit and sequenced on the Illumina NovaSeq 6000 platform. Then, a systematic review on the published cohorts of fetuses specifically selected for CNS anomalies was performed according to PRISMA guidelines, including n=12 papers. The incremental diagnostic yield of pES over CMA/karyotype was calculated for each study and pooled in a meta‐analysis using a logistic random mixed-effect model. In our cohort in 4/33 cases (12%) standard karyotype was conclusive. CMA was not diagnostic in any case. In 5/15 cases (33%), pES disclosed likely pathogenic (LP) or pathogenic (P) variants in BICD2, NFIA, ARID1A, RPGRIP1L and ZIC2 genes fitting the fetal phenotypes. In 6/15 (40%) cases, multiple Variants of Uncertain Significance (VUSs) were detected. In one case both VUSs and LP variants were detected, partially related to the fetal phenotype. In three cases no variants were disclosed. Systematic literature review showed an incremental yield ranging from 19% to 57% in antenatal cohorts focused on CNS anomalies. The pooled incremental diagnostic yield estimate resulted 36% (95% C.I.: [28%;44%]) including all CNS anomalies, 22% (95% C.I.: [15%;31%] in apparently isolated CNS anomalies, 32% (95% C.I.: [22%;45%]) in CNS-only related anomalies (one or more) and 45% (95% C.I.: [37%;53%]) in non-isolated CNS anomalies (either ≥ 2 anomalies in CNS, or extra-CNS). In conclusion, meta-analysis showed a substantial diagnostic improvement in performing genome wide sequencing analysis over standard and molecular cytogenetics, supporting the proposal of performing pES earlier in the diagnostic route of CNS anomalies.