Disorders of sex development (DSD) refers to a heterogeneous group of congenital conditions of the urogenital tract and the reproductive system affecting human sex determination and/or differentiation, thus representing a condition in which the development of chromosomal, gonadal, or anatomical sex is atypical. The current classification of DSD includes 4 main groups: 46 XY, 46 XX, sex chromosome DSD, and syndromic DSD, where the main pathological conditions are hypospadias, cryptorchidism, and micropenis in affected males, whereas in affected females ovotestis, gonadoblastoma, and ovarian dysgenesis are the main symptoms. In this study, 25 patients (displaying either syndromic or non-syndromic phenotype) were examined through exome analysis (Whole-Exome Sequencing, WES), in both single patient (singleton-WES) or trio (proband and relatives), followed by bioinformatic data analysis. This thesis work aims to evaluate the detection rate of the WES approach in our cohort of DSD patients, compared to the conventional panel-based diagnosis of DSDs along with estimating the utility of WES approach in the discovery of novel genes associated with DSD. In our workflow, variants were filtered according to population frequency (lower than 5% in GnomAD), as wells as type of variant, predicted functional effects, segregation, and NGS quality metrics. To better address the bioinformatic analysis, a specific virtual panel of 305 genes related to DSD was curated. Moreover, other “sub-panels” were created and used for patients showing complementary phenotypes (e.g., azoospermia, ovarian cancer, West syndrome, intellectual disability, autism spectrum disorder, epilepsy, and short stature). WES data analysis allowed us to identify a putative causative variant in 20/25 patients (80%), whereas 5/25 (20%) remained inconclusive. The identified variants were subdivided into two main classes: “Diagnostic” and “Suspected candidate”. “Diagnostic” variants, defined as those predicted as “pathogenic/likely pathogenic” or VUS (variants of uncertain significance) temperature scale from “Hot” to “Tepid” affecting genes already associated with conditions superimposable with the patient phenotype, were highlighted in 5/25 patients (20%). On the other hand, “suspected candidate” variants were considered not clinically conclusive and were classified into the following sub-groups: 1) monoallelic variants in recessive genes; 2) variants with only partial segregation and inheritance overlap; 3) variants with only partial phenotype overlap; 4) variants in “novel” genes; 5) and “miscellaneous”, for example including 3’UTR variants. Suspected candidate variants were evidenced in 15/25 patients (60%). Globally, the 80% diagnostic yield achieved in our cohort is a relevant result, not only compared with other WES-based DSD cohort studies diagnostic rate (66.7%), but especially compared with panel-based DSD cohort studies, in which the detection rate ranges between 20% and 40%. Moreover, the choice of performing WES enabled to identify variants in 9 genes not commonly included in most used commercial DSD gene panels. These findings demonstrate how WES overcomes the panel-based limitations of the subjective genes’ selection. Beyond all the advantages, the use of WES in DSD diagnosis shows some limitations, such as the higher occurrence of VUS compared with other fully penetrant Mendelian conditions, making the molecular diagnosis more challenging. Nonetheless, these preliminary results confirm the key role that WES could have as first-line approach in the study of both syndromic and non-syndromic DSD cases.
I disordini dello sviluppo sessuale (DSD) si riferiscono ad un gruppo eterogeneo di condizioni congenite del tratto urogenitale e del sistema riproduttivo che influenzano la determinazione e/o la differenziazione del sesso umano, rappresentando quindi una condizione in cui lo sviluppo del sesso cromosomico, gonadico o anatomico è atipico. L'attuale classificazione dei DSD comprende quattro categorie principali: 46 XY, 46 XX, DSD da aneuploidie dei cromosomi sessuali e DSD sindromico, in cui le principali condizioni patologiche sono l'ipospadia, il criptorchidismo e il micropene nei maschi affetti, mentre nelle femmine affette i sintomi principali sono l'ovotestis, il gonadoblastoma e la disgenesia ovarica. In questo studio sono stati esaminati 25 pazienti (con fenotipo sindromico o non-sindromico), sui quali abbiamo eseguito l'analisi dell'esoma (Whole-Exome sequencing, WES), in particolare sia come trio- (probando + parenti) sia come singleton-WES (solo probando), e la successiva analisi bioinformatica dei dati. Questo lavoro di tesi mira a valutare la resa diagnostica dell'approccio WES nella nostra coorte di pazienti DSD, rispetto alla diagnosi convenzionale basata sul pannello targettato DSD, e a stimare l'utilità dell'approccio WES nella scoperta di "nuovi" geni associati a DSD. Nelle nostre analisi trio- e singleton-WES, le varianti sono state filtrate in base ad una frequenza di popolazione inferiore al 5% in GnomAD (v2.1.1) e ad altri parametri (tipo di variante, effetti, segregazione, copertura). Le varianti filtrate sono state poi analizzate utilizzando una lista di geni specifica: in particolare, i dati della nostra coorte DSD sono stati filtrati utilizzando un elenco di 305 geni DSD, che comprende quelli associati ai principali fenotipi clinici DSD. Inoltre, sono stati creati e utilizzati altre liste di geni per i pazienti che presentavano un fenotipo più ampio oltre a quello genitourinario. L'analisi dei dati WES sui nostri 25 casi ci ha permesso di identificare una probabile variante causale in 20/25 (80%) casi, mentre 5/25 (20%) casi sono rimasti senza alcuna possibile spiegazione genetica. Tutte le varianti sono state controllate per frequenza nel database GnomAD (v3.1.2) e classificate secondo le linee guida ACMG. Le diverse varianti, le abbiamo suddivise in due classi principali: varianti "diagnostiche" e "candidate sospette". Le varianti "diagnostiche", considerate rare e predette come "probabilmente patogenetiche" o con una scala di temperatura VUS da "Hot" a "Tepid", ricadenti in geni già associati a condizioni sovrapponibili al fenotipo del paziente, sono state evidenziate in 5/25 (20%) pazienti. Le varianti "candidate sospette" sono state suddivise in cinque sottogruppi che ne spiegano il ruolo: varianti monoalleliche in geni recessivi, varianti con parziale sovrapposizione della segregazione e dell’ereditarietà, varianti con parziale sovrapposizione fenotipica, varianti in geni "nuovi", varianti miscellanee come quelle ricadenti in regioni extra-esoniche). Le varianti "candidate sospette" sono state evidenziate in 15/25 (60%) casi. Nella nostra coorte DSD, la resa diagnostica raggiunta è dell'80%, risultato significativo non solo rispetto a quella ottenuta in altri studi su coorti DSD con approccio WES (66,7%), ma soprattutto rispetto a studi di coorte DSD basati su pannelli genici, in cui la resa diagnostica varia tra il 20% e il 40%. Inoltre, la scelta di eseguire il WES ha permesso di identificare 9 geni non riportati nei pannelli genici DSD più comunemente utilizzati per questo tipo di analisi. Questi risultati dimostrano come il WES consenta di superare le limitazioni dell’approccio tramite pannelli targettati, confermando il ruolo chiave che il WES potrebbe ricoprire se usato come approccio di prima linea nello studio di casi DSD, sia sindromici che non sindromici.
Genetic study in an Italian cohort of patients with disorders of sex development (DSD) and assessment of the usefulness of Whole-Exome Sequencing (WES) as first-tier diagnostic test
DI BELLA, CLAUDIA
2021/2022
Abstract
Disorders of sex development (DSD) refers to a heterogeneous group of congenital conditions of the urogenital tract and the reproductive system affecting human sex determination and/or differentiation, thus representing a condition in which the development of chromosomal, gonadal, or anatomical sex is atypical. The current classification of DSD includes 4 main groups: 46 XY, 46 XX, sex chromosome DSD, and syndromic DSD, where the main pathological conditions are hypospadias, cryptorchidism, and micropenis in affected males, whereas in affected females ovotestis, gonadoblastoma, and ovarian dysgenesis are the main symptoms. In this study, 25 patients (displaying either syndromic or non-syndromic phenotype) were examined through exome analysis (Whole-Exome Sequencing, WES), in both single patient (singleton-WES) or trio (proband and relatives), followed by bioinformatic data analysis. This thesis work aims to evaluate the detection rate of the WES approach in our cohort of DSD patients, compared to the conventional panel-based diagnosis of DSDs along with estimating the utility of WES approach in the discovery of novel genes associated with DSD. In our workflow, variants were filtered according to population frequency (lower than 5% in GnomAD), as wells as type of variant, predicted functional effects, segregation, and NGS quality metrics. To better address the bioinformatic analysis, a specific virtual panel of 305 genes related to DSD was curated. Moreover, other “sub-panels” were created and used for patients showing complementary phenotypes (e.g., azoospermia, ovarian cancer, West syndrome, intellectual disability, autism spectrum disorder, epilepsy, and short stature). WES data analysis allowed us to identify a putative causative variant in 20/25 patients (80%), whereas 5/25 (20%) remained inconclusive. The identified variants were subdivided into two main classes: “Diagnostic” and “Suspected candidate”. “Diagnostic” variants, defined as those predicted as “pathogenic/likely pathogenic” or VUS (variants of uncertain significance) temperature scale from “Hot” to “Tepid” affecting genes already associated with conditions superimposable with the patient phenotype, were highlighted in 5/25 patients (20%). On the other hand, “suspected candidate” variants were considered not clinically conclusive and were classified into the following sub-groups: 1) monoallelic variants in recessive genes; 2) variants with only partial segregation and inheritance overlap; 3) variants with only partial phenotype overlap; 4) variants in “novel” genes; 5) and “miscellaneous”, for example including 3’UTR variants. Suspected candidate variants were evidenced in 15/25 patients (60%). Globally, the 80% diagnostic yield achieved in our cohort is a relevant result, not only compared with other WES-based DSD cohort studies diagnostic rate (66.7%), but especially compared with panel-based DSD cohort studies, in which the detection rate ranges between 20% and 40%. Moreover, the choice of performing WES enabled to identify variants in 9 genes not commonly included in most used commercial DSD gene panels. These findings demonstrate how WES overcomes the panel-based limitations of the subjective genes’ selection. Beyond all the advantages, the use of WES in DSD diagnosis shows some limitations, such as the higher occurrence of VUS compared with other fully penetrant Mendelian conditions, making the molecular diagnosis more challenging. Nonetheless, these preliminary results confirm the key role that WES could have as first-line approach in the study of both syndromic and non-syndromic DSD cases.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14895