Trichothiodystrophy-associated MPLKIP maintains DBR1 levels for proper lariat debranching and ectodermal differentiation.

The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene-expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.

Ribosomal Dysfunction Is a Common Pathomechanism in Different Forms of Trichothiodystrophy.

Mutations in a broad variety of genes can provoke the severe childhood disorder trichothiodystrophy (TTD) that is classified as a DNA repair disease or a transcription syndrome of RNA polymerase II. In an attempt to identify the common underlying pathomechanism of TTD we performed a knockout/knockdown of the two unrelated TTD factors TTDN1 and RNF113A and investigated the consequences on ribosomal biogenesis and performance. Interestingly, interference with these TTD factors created a nearly uniform impact on RNA polymerase I transcription with downregulation of UBF, disturbed rRNA processing and reduction of the backbone of the small ribosomal subunit rRNA 18S. This was accompanied by a reduced quality of decoding in protein translation and the accumulation of misfolded and carbonylated proteins, indicating a loss of protein homeostasis (proteostasis). As the loss of proteostasis by the ribosome has been identified in the other forms of TTD, here we postulate that ribosomal dysfunction is a common underlying pathomechanism of TTD.

A functional link between lariat debranching enzyme and the intron-binding complex is defective in non-photosensitive trichothiodystrophy.

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.

Severe trichothiodystrophy and cardiac malformation in a newborn carrying a novel GTF2H5 homozygous truncating variant.

We report a newborn patient with trichothiodystrophy-3 (TTD3) caused by a novel homozygous variant in the GTF2H5 gene. His severe phenotype included congenital ichthyosis, complex posterior cranial fossa anomaly, life-threatening infections, bilateral cryptorchidism, and, notably, a complex cardiac malformation, which is unprecedented in TTD3 patients.

Trichothiodystrophy.

This case report describes an infant with frizzy, coarse, and fragile hair and low-set ears, blepharophimosis, and osteopenia.

Facial clues to the photosensitive trichothiodystrophy phenotype in childhood.

Among genodermatoses, trichothiodystrophies (TTDs) are a rare genetically heterogeneous group of syndromic conditions, presenting with skin, hair, and nail abnormalities. An extra-cutaneous involvement (craniofacial district and neurodevelopment) can be also a part of the clinical picture. The presence of photosensitivity describes three forms of TTDs: MIM#601675 (TTD1), MIM#616390 (TTD2) and MIM#616395 (TTD3), that are caused by variants afflicting some components of the DNA Nucleotide Excision Repair (NER) complex and with more marked clinical consequences. In the present research, 24 frontal images of paediatric patients with photosensitive TTDs suitable for facial analysis through the next-generation phenotyping (NGP) technology were obtained from the medical literature. The pictures were compared to age and sex-matched to unaffected controls using 2 distinct deep-learning algorithms: DeepGestalt and GestaltMatcher (Face2Gene, FDNA Inc., USA). To give further support to the observed results, a careful clinical revision was undertaken for each facial feature in paediatric patients with TTD1 or TTD2 or TTD3. Interestingly, a distinctive facial phenotype emerged by the NGP analysis delineating a specific craniofacial dysmorphic spectrum. In addition, we tabulated every single detail within the observed cohort. The novelty of the present research includes the facial characterization in children with the photosensitive types of TTDs through the 2 different algorithms. This result can become additional criteria for early diagnosis, and for subsequent targeted molecular investigations as well as a possible tailored multidisciplinary personalized management.

TFIIH mutations can impact on translational fidelity of the ribosome.

TFIIH is a complex essential for transcription of protein-coding genes by RNA polymerase II, DNA repair of UV-lesions and transcription of rRNA by RNA polymerase I. Mutations in TFIIH cause the cancer prone DNA-repair disorder xeroderma pigmentosum (XP) and the developmental and premature aging disorders trichothiodystrophy (TTD) and Cockayne syndrome. A total of 50% of the TTD cases are caused by TFIIH mutations. Using TFIIH mutant patient cells from TTD and XP subjects we can show that the stress-sensitivity of the proteome is reduced in TTD, but not in XP. Using three different methods to investigate the accuracy of protein synthesis by the ribosome, we demonstrate that translational fidelity of the ribosomes of TTD, but not XP cells, is decreased. The process of ribosomal synthesis and maturation is affected in TTD cells and can lead to instable ribosomes. Isolated ribosomes from TTD patients show an elevated error rate when challenged with oxidized mRNA, explaining the oxidative hypersensitivity of TTD cells. Treatment of TTD cells with N-acetyl cysteine normalized the increased translational error-rate and restored translational fidelity. Here we describe a pathomechanism that might be relevant for our understanding of impaired development and aging-associated neurodegeneration.

TFIIH stabilization recovers the DNA repair and transcription dysfunctions in thermo-sensitive trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. The photosensitive form of TTD (PS-TTD) is most commonly caused by mutations in the ERCC2/XPD gene encoding a subunit of the transcription/DNA repair complex TFIIH. Here we report novel ERCC2/XPD mutations affecting proper protein folding, which generate thermo-labile forms of XPD associated with thermo-sensitive phenotypes characterized by reversible aggravation of TTD clinical signs during episodes of fever. In patient cells, the newly identified XPD variants result in thermo-instability of the whole TFIIH complex and consequent temperature-dependent defects in DNA repair and transcription. Improving the protein folding process by exposing patient cells to low temperature or to the chemical chaperone glycerol allowed rescue of TFIIH thermo-instability and a concomitant recovery of the complex activities. Besides providing a rationale for the peculiar thermo-sensitive clinical features of these new cases, the present findings demonstrate how variations in the cellular concentration of mutated TFIIH impact the cellular functions of the complex and underlie how both quantitative and qualitative TFIIH alterations contribute to TTD clinical features.

Debilitating hip degeneration in trichothiodystrophy: Association with ERCC2/XPD mutations, osteosclerosis, osteopenia, coxa valga, contractures, and osteonecrosis.

Trichothiodystrophy (TTD) is a rare, autosomal recessive, multisystem disorder of DNA repair and transcription with developmental delay and abnormalities in brain, eye, skin, nervous, and musculoskeletal systems. We followed a cohort of 37 patients with TTD at the National Institutes of Health (NIH) from 2001 to 2019 with a median age at last observation of 12 years (range 2-36). Some children with TTD developed rapidly debilitating hip degeneration (DHD): a distinctive pattern of hip pain, inability to walk, and avascular necrosis on imaging. Ten (27%) of the 37 patients had DHD at median age 8 years (range 5-12), followed by onset of imaging findings at median age 9 years (range 5-13). All 10 had mutations in the ERCC2/XPD gene. In 7 of the 10 affected patients, DHD rapidly became bilateral. DHD was associated with coxa valga, central osteosclerosis with peripheral osteopenia of the skeleton, and contractures/tightness of the lower limbs. Except for one patient, surgical interventions were generally not effective at preventing DHD. Four patients with DHD died at a median age of 11 years (range 9-15). TTD patients with ERCC2/XPD gene mutations have a high risk of musculoskeletal abnormalities and DHD leading to poor outcomes. Monitoring by history, physical examination, imaging, and by physical medicine and rehabilitation specialists may be warranted.

Establishment of a human induced pluripotent stem cell line, KMUGMCi003-A, from a patient with trichothiodystrophy 1 (TTD1) bearing compound heterozygous missense mutations in the ERCC2 gene.

Trichothiodystrophy 1 (TTD1) is a rare, autosomal recessive, multisystem disorder characterized by the sulfur-deficient brittle hair, cutaneous photosensitivity, high risk of skin cancer, psychomotor retardation. TTD1 is caused by homozygous or compound heterozygous mutation in ERCC2 gene. The peripheral blood mononuclear cells (PBMCs) from a patient carrying two heterozygous missense mutations of the ERCC2 gene were reprogrammed using the CytoTune-iPS2.0 Sendai Reprogramming Kit. The putative compound heterozygous mutation in ERCC2 will cause the abnormal protein, which is known to associated with TTD1. The established human induced pluripotent cell (hiPSC) line will enable proper in vitro disease modelling of TTD1.

Trichothiodystrophy hair shafts display distinct ultrastructural features.

Hair shafts from three trichothiodystrophy (TTD) patients with mutations in the ERCC2 (XPD) gene were examined by transmission electron microscopy. TTD is a rare, recessive disorder with mutations in several genes in the DNA repair/transcription pathway, including ERCC2. Unlike previous studies, the hair shafts were examined after relaxation of their structure by partial disulphide bond reduction in the presence of sodium dodecyl sulphate, permitting improved visualization. Compared with hair shafts of normal phenotype, TTD cuticle cells displayed aberrant marginal bands and exocuticle layers. Clusters of cells stained differently (light versus dark) in the cortex of aberrant shafts, and the keratin macrofibrils appeared much shorter in the cytoplasm. Considerable heterogeneity in these properties was evident among samples and even along the length of single hair shafts. The results are consistent with not only a paucity of high sulphur components, such as keratin-associated proteins, but also a profound imbalance in protein content and organization.

Metronidazole-Induced Hepatitis in a Teenager With Xeroderma Pigmentosum and Trichothiodystrophy Overlap.

A teenage girl had the rare combined phenotype of xeroderma pigmentosum and trichothiodystrophy, resulting from mutations in the XPD (ERCC2) gene involved in nucleotide excision repair (NER). After treatment with antibiotics, including metronidazole for recurrent infections, she showed signs of acute and severe hepatotoxicity, which gradually resolved after withdrawal of the treatment. Cultured skin fibroblasts from the patient revealed cellular sensitivity to killing by metronidazole compared with cells from a range of other donors. This reveals that the metronidazole sensitivity was an intrinsic property of her cells. It is well recognized that patients with Cockayne syndrome, another NER disorder, are at high risk of metronidazole-induced hepatotoxicity, but this had not been reported in individuals with other NER disorders. We would urge extreme caution in the use of metronidazole in the management of individuals with the xeroderma pigmentosum and trichothiodystrophy overlap or trichothiodystrophy phenotypes.

Nucleolar TFIIE plays a role in ribosomal biogenesis and performance.

Ribosome biogenesis is a highly energy-demanding process in eukaryotes which requires the concerted action of all three RNA polymerases. In RNA polymerase II transcription, the general transcription factor TFIIH is recruited by TFIIE to the initiation site of protein-coding genes. Distinct mutations in TFIIH and TFIIE give rise to the degenerative disorder trichothiodystrophy (TTD). Here, we uncovered an unexpected role of TFIIE in ribosomal RNA synthesis by RNA polymerase I. With high resolution microscopy we detected TFIIE in the nucleolus where TFIIE binds to actively transcribed rDNA. Mutations in TFIIE affects gene-occupancy of RNA polymerase I, rRNA maturation, ribosomal assembly and performance. In consequence, the elevated translational error rate with imbalanced protein synthesis and turnover results in an increase in heat-sensitive proteins. Collectively, mutations in TFIIE-due to impaired ribosomal biogenesis and translational accuracy-lead to a loss of protein homeostasis (proteostasis) which can partly explain the clinical phenotype in TTD.

Reduced levels of prostaglandin I2 synthase: a distinctive feature of the cancer-free trichothiodystrophy.

The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I2 synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.

Protein instability associated with AARS1 and MARS1 mutations causes trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable.

Novel ERCC2 variant in trichothiodystrophy infant: the first case report in China.

BACKGROUND: Trichothiodystrophy (TTD) is a rare, autosomal recessive, multisystem disorder most commonly caused by variants in ERCC2. CASE PRESENTATION: Here, we describe the first Chinese patient with a novel variant in ERCC2. A male infant, who was born to a healthy non-consanguineous couple, exhibited brittle hair, hair loss ichthyosis, eczema, retinal pigmentation and hypospadias. He carried a novel heterozygous ERCC2 variant. The maternal variant (c.2191-18_2213del) is a previous described genomic deletion that affects the splicing of intron 22. The paternal variant (c.1666-1G > A), that occurs in the splice site of intron 17 and likely alters ERCC2 gene function through aberrant splicing, has not been reported previously. CONCLUSIONS: Our case reported a novel pathogenic variant in ERCC2, which expanded the known genetic variants associated with TTD.

A novel MPLKIP-variant in three Finnish patients with non-photosensitive trichothiodystrophy type 4.

Trichothiodystrophy is a group of multisystem neuroectodermal disorders with dysplastic hair as the cardinal symptom. We describe three patients from two Finnish families in whom whole-exome sequencing revealed a novel homozygous variant, c.26del, p.(Pro9Glnfs*144) in the MPLKIP-gene, confirming the diagnosis of non-photosensitive trichothiodystrophy type 4 (TTD4). The variant was confirmed by Sanger sequencing and inherited from unaffected carrier parents. This report adds to the literature by expanding the genetic and phenotypic spectra of MPLKIP-related trichothiodystrophy. We describe dysmorphic features in the patients and provide a comparison of clinical characteristics in patients with TTD4 reported to date.