HS is a highly debilitating inflammatory skin disease presenting deep-seated lesions, abscesses, inflamed nodules, pus-discharging tunnels and scars, typically occurring in intertiginous apocrine gland-bearing regions. The pathogenesis of HS is complex and multifactorial in which a strict interplay between genetic, immunologic, infectious and hormonal factors have been identified, though the precise molecular mechanisms underlying these aspects have not yet been fully characterised. A further level of complexity is given by the fact that commonly HS may also occur in combination with other comorbidities involving multiple organ systems giving rise to syndromic variants. In this work the attention was focused on a syndromic variant of HS, PASH syndrome. The aim of this work was to identify genetic changes in HS and PASH patients, trying to understand their role in these diseases. To this aim, in the present study one family with one PASH patient and two families presenting a familiar form of HS were recruited and subjected to genetic analysis. These assays allowed the identification of specific variants in each family that were further characterised in in vitro studies in order to define their effect on PASH and HS phenotype. In a PASH patient a missense variant in DSP gene, encoding for desmoplakin (DSP) that is an obligate and the most abundant component of desmosomes, was identified in homozygosis while the variant was carried in heterozygosis by his healthy parents. Experimental procedures were performed on skin sample biopsies, in silico and in an in vitro genetic model obtained in spontaneously immortalised human keratinocyte cells line (HaCaT cells) engineered with CRISPR-Cas9 technology to carry the mutation of interest. Results confirmed a potential damaging effect of the identified single nucleotide variation (SNV) in the skin since it has been seen to: impact protein structure; potentially contribute to an insufficient recruitment of DSP on desmosomes; determine higher levels of water loss through the stratum corneum of the epidermis; do not influence keratinocytes proliferation and differentiation. This damaging effect has been proposed to alter the mechanical adhesion properties of desmosomes and lead to the disruption of tissue integrity by affecting permeability properties. In the case of the Sardinian family, a rare mutation that has never been previously associated to any disease was found in affected family members in heterozygosis on ZNF318 gene, encoding for a zinc finger protein associated with androgen receptor signalling. An in vitro genetic model was assessed in HaCaT cells through CRISPR-Cas9 technology in order to retrieve cellular clones carrying the mutation of interest. In vitro assays are currently ongoing and are aimed at characterising any variation in androgen receptor signal transduction pathway.In a Friulian family, a frameshift insertion in the DCD gene, encoding for the antimicrobial peptide dermcidin, was found in heterozygosis in affected family members but not in the healthy control. Further studies aimed at assessing the antimicrobial activity of the mutated peptide together with the quantification of its levels in the sweat of all family members will be performed. In this study, new findings on the genetics of HS and PASH syndrome were obtained that collectively allowed to shed light into novel scenarios potentially involved in the onset and progression of these disorders.

L’idrosadenite suppurativa (HS) è una patologia infiammatoria cutanea altamente debilitante caratterizzata dalla presenza di lesioni profonde, ascessi, noduli infiammati, lesioni purulente e cicatrici nelle regioni inverse del corpo ricche di ghiandole apocrine. La patogenesi dell’HS è complessa e multifattoriale che prevede una stretta interazione tra fattori genetici, immunologici, infettivi e ormonali, attraverso meccanismi molecolari non ancora completamente definiti. Un ulteriore livello di complessità si ritrova nel fatto che spesso i casi HS manifestano simultaneamente varie comorbidità che coinvolgono più sistemi d’organi, dando origine a delle varianti sindromiche in cui l’HS costituisce un segno distintivo. In questo lavoro l’attenzione è stata focalizzata su una variante sindromica nota come sindrome PASH. Lo scopo di questo lavoro è stato quello di identificare varianti genetiche in pazienti affetti da HS e PASH, cercando di definire il loro ruolo nell’insorgenza, progressione e severità di queste patologie. A tal fine, nel presente studio sono state reclutate e sottoposte ad analisi genetica una famiglia con un paziente PASH e due famiglie che presentavano una forma famigliare di HS. Queste indagini hanno consentito l’identificazione di varianti geniche specifiche in ciascuna famiglia, che sono state ulteriormente caratterizzate in seguito all’allestimento di modelli in vitro al fine di comprendere il loro effetto sul fenotipo PASH e HS. In un paziente PASH è stata identificata una variante missenso in omozigosi nel gene DSP che codifica per la desmoplachina (DSP), una componente obbligatoria e la più abbondante dei desmosomi, mentre la stessa è presente in eterozigosi nei sani. Sono state eseguite delle procedure sperimentali su biopsie di pelle, in silico e in un modello genetico in vitro ottenuto in una linea cellulare di cheratinociti spontaneamente immortalizzati (HaCaT) ingegnerizzate con la tecnologia di CRISPR-Cas9 per farle esprimere la variante d’interesse. Complessivamente i risultati hanno confermato un potenziale effetto dannoso della variante nella pelle in quanto è stato osservato che essa impatta la struttura della proteina, presumibilmente contribuisce ad un reclutamento insufficiente di DSP sui desmosomi, determina livelli elevati di perdita di acqua attraverso lo strato corneo della pelle e non influenza la proliferazione e il differenziamento dei cheratinociti. È stato proposto che questo effetto dannoso alteri le proprietà meccaniche di adesione dei desmosomi e ad una perdita dell’integrità strutturale del tessuto che sembra influenzare le caratteristiche di permeabilità del distretto cutaneo. Nel caso della famiglia sarda, è stata identificata una rara variante in eterozigosi presente negli individui affetti sul gene ZNF318, che codifica per una proteina zinc-finger associata alla pathway di segnalazione degli androgeni, che non è stata associata a nessuna patologia. È stato sviluppato un modello genetico in vitro sulle HaCaT mediante la tecnologia di CRISPR-Cas9 allo scopo di generare cloni che esprimessero la variante d’interesse. Saggi in vitro che mirano a rilevare cambiamenti nella trasduzione del segnale del recettore degli androgeni sono attualmente in fase di esecuzione. Nella famiglia friulana è stata trovata una frameshift insertion nel gene DCD, che codifica per il peptide antimicrobico dermocidina, in eterozigosi negli individui affetti della famiglia ma assente nei sani. Verranno effettuati ulteriori studi volti a valutare l’attività antimicrobica del peptide mutato unitamente alla quantificazione dei suoi livelli nel sudore di tutti gli individui della famiglia. In questo studio, sono state ottenute nuove informazioni relative alla base genetica di HS e PASH che hanno permesso di mettere in luce nuovi scenari potenzialmente coinvolti nell’insorgenza e nella progressione di tali patologie.

Hidradenitis suppurativa: identification of the main cellular and molecular pathways involved in immune and cutaneous cell biology / Gratton, Rossella. - (2021 Oct 15).

Hidradenitis suppurativa: identification of the main cellular and molecular pathways involved in immune and cutaneous cell biology.

GRATTON, ROSSELLA
2021-10-15

Abstract

HS is a highly debilitating inflammatory skin disease presenting deep-seated lesions, abscesses, inflamed nodules, pus-discharging tunnels and scars, typically occurring in intertiginous apocrine gland-bearing regions. The pathogenesis of HS is complex and multifactorial in which a strict interplay between genetic, immunologic, infectious and hormonal factors have been identified, though the precise molecular mechanisms underlying these aspects have not yet been fully characterised. A further level of complexity is given by the fact that commonly HS may also occur in combination with other comorbidities involving multiple organ systems giving rise to syndromic variants. In this work the attention was focused on a syndromic variant of HS, PASH syndrome. The aim of this work was to identify genetic changes in HS and PASH patients, trying to understand their role in these diseases. To this aim, in the present study one family with one PASH patient and two families presenting a familiar form of HS were recruited and subjected to genetic analysis. These assays allowed the identification of specific variants in each family that were further characterised in in vitro studies in order to define their effect on PASH and HS phenotype. In a PASH patient a missense variant in DSP gene, encoding for desmoplakin (DSP) that is an obligate and the most abundant component of desmosomes, was identified in homozygosis while the variant was carried in heterozygosis by his healthy parents. Experimental procedures were performed on skin sample biopsies, in silico and in an in vitro genetic model obtained in spontaneously immortalised human keratinocyte cells line (HaCaT cells) engineered with CRISPR-Cas9 technology to carry the mutation of interest. Results confirmed a potential damaging effect of the identified single nucleotide variation (SNV) in the skin since it has been seen to: impact protein structure; potentially contribute to an insufficient recruitment of DSP on desmosomes; determine higher levels of water loss through the stratum corneum of the epidermis; do not influence keratinocytes proliferation and differentiation. This damaging effect has been proposed to alter the mechanical adhesion properties of desmosomes and lead to the disruption of tissue integrity by affecting permeability properties. In the case of the Sardinian family, a rare mutation that has never been previously associated to any disease was found in affected family members in heterozygosis on ZNF318 gene, encoding for a zinc finger protein associated with androgen receptor signalling. An in vitro genetic model was assessed in HaCaT cells through CRISPR-Cas9 technology in order to retrieve cellular clones carrying the mutation of interest. In vitro assays are currently ongoing and are aimed at characterising any variation in androgen receptor signal transduction pathway.In a Friulian family, a frameshift insertion in the DCD gene, encoding for the antimicrobial peptide dermcidin, was found in heterozygosis in affected family members but not in the healthy control. Further studies aimed at assessing the antimicrobial activity of the mutated peptide together with the quantification of its levels in the sweat of all family members will be performed. In this study, new findings on the genetics of HS and PASH syndrome were obtained that collectively allowed to shed light into novel scenarios potentially involved in the onset and progression of these disorders.
15-ott-2021
CROVELLA, SERGIO
33
2019/2020
Settore MED/05 - Patologia Clinica
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2997561
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