Trichothiodystrophy (TTD) is a rare hereditary disorder characterized by a hair dysplasia and associated with numerous symptoms affecting mainly organs of neuroectodermal origin. About half of the reported TTD patients are photosensitive, with a persistency of UV-induced DNA lesions. The photosensitive TTD patients are defective in either of three genes (XPB, XPD or TTDA) encoding distinct subunits of the TFIIH complex, a DNA repair factor that is also required for transcription by RNA polymerase II. Although photosensitive TTD was originally associated with defects in DNA repair, recent data are highlighting subtle transcription deficiencies. In contrast, very little is known on non-photosensitive TTD that is characterized by normal DNA repair. To date, only two genes (MPLKIP/TTDN1 and RNF113A) of unknown function have been identified as disease genes and they account for less than 20% of the cases. Based on the observation that TTD phenotype is linked to transcription alterations, we investigated the cellular levels of different RNA polymerase II- basal transcription factors in primary skin fibroblasts from the fifteen unsolved non-photosensitive TTD cases available in the laboratory. TBP, TFIIB, TFIIE and the photosensitive TTD factor TFIIH were analyzed by immunoblotting. Normal cellular amounts of all the different proteins were observed in fourteen out of the fifteen analyzed patients. In contrast, fibroblasts from TTD28PV were characterized by marked reductions (to about 40% of normal) in the levels of both and subunits of TFIIE. Reduced levels of TFIIE complex were also confirmed by immunofluorescence staining of the patient cells, pointing to the involvement of the TFIIE complex in the pathological phenotype of TTD28PV. Targeted Sanger sequencing of the genes encoding the two TFIIE subunits (GTF2E1 and GTF2E2), revealed the presence of a homozygous missense mutation (p.Asp187Tyr) in the GTF2E2 gene which encodes the TFIIE protein. Subsequent quantitative RT-PCR assays in TTD28PV cells demonstrated that the GTF2E2 mutation does not affect the gene transcription. Thus, the mutation directly impacts on the stability of the TFIIE protein. As suggested by the finding that both subunits are equally reduced in the patient cells and confirmed by GTF2E2 silencing, the instability of TFIIE affects the stability of the whole TFIIE complex. Given the strong functional relationship between TFIIE and TFIIH (which is central to the onset of photosensitive TTD), it is likely that the mutation found in GTF2E2 is pathogenic. Thus, this work identifies a potential new locus for non-photosensitive TTD.
La tricotiodistrofia (TTD) è una malattia ereditaria caratterizzata da anomalie dei capelli alle quali si associano sintomi a carico di organi di origine neuroectodermica. La metà circa dei casi TTD presenta fotosensibilità dovuta a incapacità di rimuovere in modo efficiente le lesioni prodotte dalla luce UV sul DNA. I pazienti TTD fotosensibili sono mutati in tre geni (XPB, XPD o TTDA) che codificano per subunità di TFIIH, un complesso coinvolto nella riparazione dei danni al DNA e nella trascrizione mediata dalla RNA polimerasi II. La forma fotosensibile della TTD è stata inizialmente associata a difetti nella riparazione del DNA ma le recenti ricerche stanno rivelando la presenza anche di lievi alterazioni trascrizionali. Molto limitate sono invece le conoscenze sulla TTD non-fotosensibile caratterizzata da una normale capacità riparativa. Ad oggi, solo due geni di funzione ignota (MPLKIP/TTDN1 e RNF113A) sono stati identificati come responsabili della malattia in circa il 20% dei casi. Partendo dall’osservazione che il fenotipo TTD è associato a difetti nella trascrizione, ci siamo proposti di valutare i livelli cellulari di diversi fattori generali della trascrizione mediata dalla RNA polimerasi II nei quindici pazienti TTD non-fotosensibili irrisolti i cui fibroblasti primari della cute sono disponibili presso il laboratorio. Mediante immunoblotting sono stati analizzati TBP, TFIIB, TFIIE ed il complesso TFIIH coinvolto nella TTD fotosensibile. Normali livelli di tutte le proteine analizzate sono stati osservati in quattordici dei quindici pazienti. I fibroblasti della paziente TTD28PV hanno invece mostrato livelli notevolmente ridotti (il 40% circa del normale) delle subunità e di TFIIE. La riduzione dei livelli di complesso TFIIE sono stati confermati anche mediante immunofluorescenza, suggerendo il coinvolgimento di TFIIE nel fenotipo patologico di TTD28PV. Mediante sequenziamento dei due geni che codificano per le subunità di TFIIE (GTF2E1 e GTF2E2) è stata identificata una mutazione missenso (p.Asp187Tyr) in omozigosi in GTF2E2 codificante per la proteina TFIIE. Successive analisi di RT-PCR quantitativa nei fibroblasti della paziente TTD28PV hanno dimostrato che la mutazione in GTF2E2 non altera la trascrizione del gene. La mutazione ha quindi un effetto diretto sulla stabilità della proteina TFIIE. L’osservazione di riduzioni simili nei livelli di entrambe le subunità di TFIIE nelle cellule della paziente ed esperimenti di silenziamento di GTF2E2 hanno confermato che l’instabilità di TFIIE altera la stabilità dell’intero complesso TFIIE. Considerando la stretta relazione funzionale tra TFIIE e TFIIH (fattore centrale nella TTD fotosensibile) è molto probabile che la mutazione identificata in GTF2E2 sia patogenetica. Il lavoro di questa tesi identifica quindi un nuovo potenziale locus per la forma non-fotosensibile della TTD.
Un nuovo gene responsabile della tricotiodistrofia non-fotosensibile
TOLVE, LAURA
2014/2015
Abstract
Trichothiodystrophy (TTD) is a rare hereditary disorder characterized by a hair dysplasia and associated with numerous symptoms affecting mainly organs of neuroectodermal origin. About half of the reported TTD patients are photosensitive, with a persistency of UV-induced DNA lesions. The photosensitive TTD patients are defective in either of three genes (XPB, XPD or TTDA) encoding distinct subunits of the TFIIH complex, a DNA repair factor that is also required for transcription by RNA polymerase II. Although photosensitive TTD was originally associated with defects in DNA repair, recent data are highlighting subtle transcription deficiencies. In contrast, very little is known on non-photosensitive TTD that is characterized by normal DNA repair. To date, only two genes (MPLKIP/TTDN1 and RNF113A) of unknown function have been identified as disease genes and they account for less than 20% of the cases. Based on the observation that TTD phenotype is linked to transcription alterations, we investigated the cellular levels of different RNA polymerase II- basal transcription factors in primary skin fibroblasts from the fifteen unsolved non-photosensitive TTD cases available in the laboratory. TBP, TFIIB, TFIIE and the photosensitive TTD factor TFIIH were analyzed by immunoblotting. Normal cellular amounts of all the different proteins were observed in fourteen out of the fifteen analyzed patients. In contrast, fibroblasts from TTD28PV were characterized by marked reductions (to about 40% of normal) in the levels of both and subunits of TFIIE. Reduced levels of TFIIE complex were also confirmed by immunofluorescence staining of the patient cells, pointing to the involvement of the TFIIE complex in the pathological phenotype of TTD28PV. Targeted Sanger sequencing of the genes encoding the two TFIIE subunits (GTF2E1 and GTF2E2), revealed the presence of a homozygous missense mutation (p.Asp187Tyr) in the GTF2E2 gene which encodes the TFIIE protein. Subsequent quantitative RT-PCR assays in TTD28PV cells demonstrated that the GTF2E2 mutation does not affect the gene transcription. Thus, the mutation directly impacts on the stability of the TFIIE protein. As suggested by the finding that both subunits are equally reduced in the patient cells and confirmed by GTF2E2 silencing, the instability of TFIIE affects the stability of the whole TFIIE complex. Given the strong functional relationship between TFIIE and TFIIH (which is central to the onset of photosensitive TTD), it is likely that the mutation found in GTF2E2 is pathogenic. Thus, this work identifies a potential new locus for non-photosensitive TTD.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/20.500.14239/22464