Stillbirth is the fetal death in utero after 22 weeks of gestation. The etiology of stillbirth is highly complex as it involves placental, fetal and maternal factors, with significant medical, psychosocial and financial implications. Canonical diagnostic workup includes fetal autopsy, placental histopathological evaluation, and assessment of maternal risk factors. Despite such investigations, a large proportion of cases (up to 60%) remain unexplained. Data regarding the contribution of genetic factors to stillbirth are extremely limited and mainly focused on cytogenetic investigations, with only a few sequencing studies performed to date. This research project aims investigating by exome sequencing (ES) the genetic background of a highly, homogeneous, monocentric cohort of 25 stillbirths showing prominent alterations of the brainstem. All but one fetal loss occurred after 28 gestational weeks. Genomic DNA was extracted from post-mortem formalin-fixed paraffin-embedded (FFPE) brain tissues. DNA samples underwent WES by using the Human Core Exome kit with Mitochondrial Panel on a Novaseq6000 platform. The bioinformatic analysis was primarily focused on genetic variants with a minor allele frequency below 5%, located within exonic and splicing regions. Variants were classified according to the ACMG-AMP and ACGS guidelines, and specific ClinGen recommendations. By filtering ES data with virtual gene panels and conducting pathway enrichment analysis, we identified potentially damaging single nucleotide variants in genes associated with central nervous system development and a range of other different conditions. In 20 fetuses (80%) a potentially damaging variant was detected, while in 5 no significant variants were identified, suggesting possible non-genetic factors or variants within unexamined regions. Among the positive cases, 24 variants were identified across 21 different genes. Half of these variants had uncertain significance, while the remaining were classified as pathogenic (46%) or likely pathogenic (4%). The genetic alterations included 5 nonsense, 2 frameshift, 2 in-frame deletions, 1 splice site, and 14 missense substitutions. The majority of the affected genes, including ATP2A2, CFTR, CHD3, CPT2, CUX2, DYNC1H1, FBXO11, GLI2, MED13L, KMT2E, PRRT2, SAMD9L, SCN10A, SCN9A, SHH, SPG7, TCF4, TRIO, and TUBA8, followed an autosomal dominant inheritance pattern. However, in two cases, a single potentially damaging hit was found in the autosomal recessive gene GUCY1A1 and in the X-linked recessive gene HPRT1 suggesting a distinct inheritance pattern for these specific variants. Key genes like SHH, GLI2, TUBA8, TCF4, SCN10A, and CPT2 were highlighted for their potential impact on stillbirths, with implications in neural development, neuronal migration, and metabolic processes. This study investigated by ES a homogeneous cohort of late stillbirths with peculiar neuropathological anomalies. Central nervous system abnormalities represent a relevant cause of stillbirth, especially in the third trimester of pregnancy, being detected in more than 1/3 cases subjected to fetal autopsy and post-mortem, or in utero, brain magnetic resonance imaging (MRI). Specifically, the brainstem nuclei are implicated in the control of crucial physiological functions during fetal life such as swallowing, breathing movements, heart rate, and blood pressure. Accordingly, the ‘brainstem hypothesis’ implies that dysfunction of such anatomical regions may increase vulnerability to sudden infant death syndrome (SIDS)/sudden unexplained death in childhood (SUDC). Although preliminary, our data support the suitability of ES as first-tier genetic tests for stillbirth workup in ‘difficult’ archival FFPE samples and unveil the relevant contribution of SNVs to late stillbirth and its intriguing overlaps with SIDS/SUDC etiology.
La morte endouterina fetale, o MEF, si verifica dopo 22 settimane di gestazione. L'eziologia delle MEF è altamente complessa, coinvolgendo fattori placentari, fetali e materni, e comporta significative implicazioni mediche, psicosociali e finanziarie. Il percorso diagnostico canonico include l'autopsia fetale, la valutazione istopatologica della placenta e l'analisi dei fattori di rischio materni. Nonostante queste indagini, una grande percentuale di casi (fino al 60%) rimane senza una spiegazione chiara. I dati relativi al contributo dei fattori genetici alle MEF sono estremamente limitati e si concentrano principalmente su indagini citogenetiche, con solo pochi studi di sequenziamento condotti finora. Questo progetto di ricerca mira a indagare, attraverso il sequenziamento dell'esoma (ES), il background genetico di una coorte altamente omogenea e monocentrica di 25 casi di MEF caratterizzati da alterazioni prominenti del tronco encefalico. Tutte le perdite fetali, tranne una, si sono verificate dopo 28 settimane di gestazione. Il DNA genomico è stato estratto da tessuti cerebrali fissati in formalina e inclusi in paraffina (FFPE) post mortem. I campioni di DNA sono stati sottoposti a sequenziamento dell'esoma completo (WES). Filtrando i dati di ES con pannelli genici virtuali e conducendo un'analisi di arricchimento dei pathways, abbiamo identificato varianti nucleotidiche singole (SNV) potenzialmente dannose in geni associati allo sviluppo del sistema nervoso centrale e a una serie di altre condizioni. In 20 feti (80%) è stata rilevata una variante potenzialmente dannosa, mentre in 5 non sono state identificate varianti significative. Tra i casi positivi, sono state identificate 24 varianti in 21 geni diversi. La metà di queste varianti aveva un significato incerto, mentre le rimanenti sono state classificate come patogene (46%) o probabilmente patogene (4%). La maggior parte dei geni coinvolti, inclusi ATP2A2, CFTR, CHD3, CPT2, CUX2, DYNC1H1, FBXO11, GLI2, MED13L, KMT2E, PRRT2, SAMD9L, SCN10A, SCN9A, SHH, SPG7, TCF4, TRIO e TUBA8, seguivano un modello di eredità autosomica dominante. Tuttavia, in due casi, è stato identificato un singolo hit potenzialmente dannoso nel gene autosomico recessivo GUCY1A1 e nel gene X-linked recessivo HPRT1, suggerendo un modello di eredità distinto per queste varianti specifiche. Geni chiave come SHH, GLI2, TUBA8, TCF4, SCN10A e CPT2 sono stati evidenziati per il loro potenziale impatto sulle MEF, con implicazioni nello sviluppo neurale, nella migrazione neuronale e nei processi metabolici. Questo studio ha indagato, tramite ES, una coorte omogenea di MEF tardive con peculiari anomalie neuropatologiche. Le anomalie del sistema nervoso centrale rappresentano una causa rilevante di morte endouterina fetale, soprattutto nel terzo trimestre di gravidanza, essendo rilevate in più di 1/3 dei casi sottoposti ad autopsia fetale e risonanza magnetica cerebrale post mortem o in utero. In particolare, i nuclei del tronco encefalico sono implicati nel controllo di funzioni fisiologiche cruciali durante la vita fetale come la deglutizione, i movimenti respiratori, la frequenza cardiaca e la pressione sanguigna. Di conseguenza, l'ipotesi del "tronco encefalico" implica che la disfunzione di tali regioni anatomiche possa aumentare la vulnerabilità alla sudden infant death syndrome (SIDS) o alla sudden unexplained death in childhood (SUDC). Sebbene preliminari, i nostri dati supportano l'adeguatezza del sequenziamento dell'esoma come test genetico di prima linea per l'analisi della MEF in campioni archivistici FFPE 'difficili' e rivelano il rilevante contributo delle SNV alla natimortalità tardiva e i suoi intriganti collegamenti con l'eziologia della SIDS/SUDC.
Esplorazione dell'architettura genetica attraverso il sequenziamento dell'esoma in una coorte di stillbirths tardive con peculiari anomalie del tronco encefalico
SPENNACCHIO, AGNESE
2023/2024
Abstract
Stillbirth is the fetal death in utero after 22 weeks of gestation. The etiology of stillbirth is highly complex as it involves placental, fetal and maternal factors, with significant medical, psychosocial and financial implications. Canonical diagnostic workup includes fetal autopsy, placental histopathological evaluation, and assessment of maternal risk factors. Despite such investigations, a large proportion of cases (up to 60%) remain unexplained. Data regarding the contribution of genetic factors to stillbirth are extremely limited and mainly focused on cytogenetic investigations, with only a few sequencing studies performed to date. This research project aims investigating by exome sequencing (ES) the genetic background of a highly, homogeneous, monocentric cohort of 25 stillbirths showing prominent alterations of the brainstem. All but one fetal loss occurred after 28 gestational weeks. Genomic DNA was extracted from post-mortem formalin-fixed paraffin-embedded (FFPE) brain tissues. DNA samples underwent WES by using the Human Core Exome kit with Mitochondrial Panel on a Novaseq6000 platform. The bioinformatic analysis was primarily focused on genetic variants with a minor allele frequency below 5%, located within exonic and splicing regions. Variants were classified according to the ACMG-AMP and ACGS guidelines, and specific ClinGen recommendations. By filtering ES data with virtual gene panels and conducting pathway enrichment analysis, we identified potentially damaging single nucleotide variants in genes associated with central nervous system development and a range of other different conditions. In 20 fetuses (80%) a potentially damaging variant was detected, while in 5 no significant variants were identified, suggesting possible non-genetic factors or variants within unexamined regions. Among the positive cases, 24 variants were identified across 21 different genes. Half of these variants had uncertain significance, while the remaining were classified as pathogenic (46%) or likely pathogenic (4%). The genetic alterations included 5 nonsense, 2 frameshift, 2 in-frame deletions, 1 splice site, and 14 missense substitutions. The majority of the affected genes, including ATP2A2, CFTR, CHD3, CPT2, CUX2, DYNC1H1, FBXO11, GLI2, MED13L, KMT2E, PRRT2, SAMD9L, SCN10A, SCN9A, SHH, SPG7, TCF4, TRIO, and TUBA8, followed an autosomal dominant inheritance pattern. However, in two cases, a single potentially damaging hit was found in the autosomal recessive gene GUCY1A1 and in the X-linked recessive gene HPRT1 suggesting a distinct inheritance pattern for these specific variants. Key genes like SHH, GLI2, TUBA8, TCF4, SCN10A, and CPT2 were highlighted for their potential impact on stillbirths, with implications in neural development, neuronal migration, and metabolic processes. This study investigated by ES a homogeneous cohort of late stillbirths with peculiar neuropathological anomalies. Central nervous system abnormalities represent a relevant cause of stillbirth, especially in the third trimester of pregnancy, being detected in more than 1/3 cases subjected to fetal autopsy and post-mortem, or in utero, brain magnetic resonance imaging (MRI). Specifically, the brainstem nuclei are implicated in the control of crucial physiological functions during fetal life such as swallowing, breathing movements, heart rate, and blood pressure. Accordingly, the ‘brainstem hypothesis’ implies that dysfunction of such anatomical regions may increase vulnerability to sudden infant death syndrome (SIDS)/sudden unexplained death in childhood (SUDC). Although preliminary, our data support the suitability of ES as first-tier genetic tests for stillbirth workup in ‘difficult’ archival FFPE samples and unveil the relevant contribution of SNVs to late stillbirth and its intriguing overlaps with SIDS/SUDC etiology.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28369