Thyroid nodules in xeroderma pigmentosum patients: a feature of premature aging.

PURPOSE: Xeroderma pigmentosum (XP) is an autosomal recessive disease with defective DNA repair, a markedly increased risk of skin cancer, and premature aging. Reports from North Africa have described thyroid nodules in XP patients, but thyroid nodule prevalence has never been determined in XP patients enrolled in our natural history study at the National Institutes of Health (NIH). METHODS: We performed thyroid ultrasound examinations on all 29 XP patients examined from 2011 to 2019 and assessed nodule malignancy using the Thyroid Imaging Reporting and Data System. Thyroid nodule prevalence was also obtained from comparison cohorts. DNA sequencing was performed on thyroid tissue from XP patients who had surgery for thyroid cancer. RESULTS: Thyroid nodules were identified in 18/29 XP patients (62%). The median age of patients with thyroid nodules in our XP cohort (20 years) was younger than that of three comparison groups: 36 years (California study-208 subjects), 48 years (Korean study-24,757 subjects), and 52 years (NIH-682 research subjects). Multiple (2-4) thyroid nodules were found in 12/18 (67%) of the patients with nodules. Autopsy examination revealed follicular adenomas in 4/8 (50%) additional XP patients. DNA sequencing revealed rare mutations in two other XP patients with papillary thyroid cancer. CONCLUSIONS: XP patients have an increased incidence of thyroid nodules at an early age in comparison to the general population. These finding confirm another premature aging feature of XP.

Activated STING in a vascular and pulmonary syndrome.

BACKGROUND: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-ß, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. CONCLUSIONS: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyosis.

We recently mapped the disease locus for severe autosomal recessive lamellar ichthyosis (LI) to chromosome 14q11 and showed complete linkage with TGM1, the gene encoding transglutaminase 1. We have now identified point mutations in TGM1 in two of the multiplex LI families used in the linkage study. Each nucleotide change causes a non-conservative amino acid substitution of histidine for one of two adjacent arginine residues in exon 3 of the gene (Arg141His, Arg142His). Within the transglutaminase family, these arginines are invariant within a conserved region, distant from the catalytic site of the enzyme. We hypothesize that these mutations adversely affect formation of crosslinks essential in production of cornified cell envelopes and a normal stratum corneum layer of the skin.

Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis.

Erythrokeratodermia variabilis (EKV, OMIM 133200) is an autosomal dominant genodermatosis with considerable intra- and interfamilial variability. It has a disfiguring phenotype characterized by the independent occurrence of two morphologic features: transient figurate red patches and localized or generalized hyperkeratosis. Both features can be triggered by external factors such as trauma to the skin. After initial linkage to the RH locus on 1p, EKV was mapped to an interval of 2.6 cM on 1p34-p35, and a candidate gene (GJA4) encoding the gap junction protein alpha-4 (connexin 31, Cx31) was excluded by sequence analysis. Evidence in mouse suggesting that the EKV region harbours a cluster of epidermally expressed connexin genes led us to characterize the human homologues of GJB3 (encoding Cx31) and GJB5 (encoding Cx31.1). GJB3, GJB5 and GJA4 were localized to a 1.1-Mb YAC in the candidate interval. We detected heterozygous missense mutations in GJB3 in four EKV families leading to substitution of a conserved glycine by charged residues (G12R and G12D), or change of a cysteine (C86S). These mutations are predicted to interfere with normal Cx31 structure and function, possibly due to a dominant inhibitory effect. Our results implicate Cx31 in the pathogenesis of EKV, and provide evidence that intercellular communication mediated by Cx31 is crucial for epidermal differentiation and response to external factors.

Linkage of epidermolytic hyperkeratosis to the type II keratin gene cluster on chromosome 12q.

We investigated the molecular genetics of epidermolytic hyperkeratosis (EHK), a dominant disorder characterized by epidermal blistering, hyperkeratosis, vacuolar degeneration and clumping of keratin filaments. Based on this pathology, we have excluded by linkage analysis several candidate genes for the disease; in contrast, complete linkage was obtained with the type II keratin, K1, on 12q11-q13. Linkage in this region of chromosome 12 was confirmed using several other markers, and multi-locus linkage analyses further supported this location. Keratins are excellent EHK gene candidates since their expression is specific to the suprabasal epidermal layers. In the pedigree studied here, a type II keratin gene, very probably K1, is implicated as the site of the molecular defect causing EHK.

Adverse effects of trichothiodystrophy DNA repair and transcription gene disorder on human fetal development.

The effects of DNA repair and transcription gene abnormalities in human pre-natal life have never been studied. Trichothiodystrophy (TTD) is a rare (affected frequency of 10(-6)) recessive disorder caused by mutations in genes involved in nucleotide excision repair (NER) pathway and in transcription. Based on our novel clinical observations, we conducted a genetic epidemiologic study to investigate gestational outcomes associated with TTD. We compared pregnancies resulting in TTD-affected offspring (n = 24) with respect to abnormalities during their antenatal and neonatal periods to pregnancies resulting in their unaffected siblings (n = 18), accounting for correlation, and to population reference values. Significantly higher incidence of several severe gestational complications was noted in TTD-affected pregnancies. Small for gestational age (SGA) <10th percentile [Relative risk (RR ) = 9.3, 95% CI = 1.4-60.5, p = 0.02], SGA <3rd percentile (RR = 7.2, 95% CI = 1.1-48.1, p = 0.04), and neonatal intensive care unit (NICU) hospitalization (RR = 6.4, 95% CI = 1.4-29.5, p = 0.02) occurred more frequently among TTD-affected neonates compared with their unaffected siblings. Compared with reference values from general obstetrical population, pregnancies that resulted in TTD-affected infants were significantly more likely to be complicated by hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome (RR = 35.7, 95% CI = 7.6-92.5, p = 0.0002), elevated mid-trimester maternal serum human chorionic gonadotropin (hCG) levels (RR = 14.3, 95% CI = 7.0-16.6, p < 0.0001), SGA <3rd percentile (RR = 13.9, 95% CI = 7.4-21.1, p < 0.0001), pre-term delivery (<32 weeks) (RR = 12.0, 95% CI = 4.9-21.6, p < 0.0001), pre-eclampsia (RR = 4.0, 95% CI = 1.6-7.4, p = 0.006), and decreased fetal movement (RR = 3.3, 95% CI = 1.6-5.2, p = 0.0018). Abnormal placental development is an underlying mechanism that may explain the constellation of observed complications in our study. Thus, we hypothesize that TTD DNA repair and transcription genes play an important role in normal human placental development.

Trichothiodystrophy: a systematic review of 112 published cases characterises a wide spectrum of clinical manifestations.

Trichothiodystrophy (TTD) is a rare, autosomal recessive disease, characterised by brittle, sulfur deficient hair and multisystem abnormalities. A systematic literature review identified 112 patients ranging from 12 weeks to 47 years of age (median 6 years). In addition to hair abnormalities, common features reported were developmental delay/intellectual impairment (86%), short stature (73%), ichthyosis (65%), abnormal characteristics at birth (55%), ocular abnormalities (51%), infections (46%), photosensitivity (42%), maternal pregnancy complications (28%) and defective DNA repair (37%). There was high mortality, with 19 deaths under the age of 10 years (13 infection related), which is 20-fold higher compared to the US population. The spectrum of clinical features varied from mild disease with only hair involvement to severe disease with profound developmental defects, recurrent infections and a high mortality at a young age. Abnormal characteristics at birth and pregnancy complications, unrecognised but common features of TTD, suggest a role for DNA repair genes in normal fetal development.

Xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome: a complex genotype-phenotype relationship.

Patients with the rare genetic disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS) have defects in DNA nucleotide excision repair (NER). The NER pathway involves at least 28 genes. Three NER genes are also part of the basal transcription factor, TFIIH. Mutations in 11 NER genes have been associated with clinical diseases with at least eight overlapping phenotypes. The clinical features of these patients have some similarities but also have marked differences. NER is involved in protection against sunlight-induced DNA damage. While XP patients have 1000-fold increase in susceptibility to skin cancer, TTD and CS patients have normal skin cancer risk. Several of the genes involved in NER also affect somatic growth and development. Some patients have short stature and immature sexual development. TTD patients have sulfur deficient brittle hair. Progressive sensorineural deafness is an early feature of XP and CS. Many of these clinical diseases are associated with developmental delay and progressive neurological degeneration. The main neuropathology of XP is a primary neuronal degeneration. In contrast, CS and TTD patients have reduced myelination of the brain. These complex neurological abnormalities are not related to sunlight exposure but may be caused by developmental defects as well as faulty repair of DNA damage to neuronal cells induced by oxidative metabolism or other endogenous processes.

Mutations in the human homologue of the Drosophila patched gene in Caucasian and African-American nevoid basal cell carcinoma syndrome patients.

The nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin syndrome, is a multisystem autosomal dominant disorder. The salient features of this syndrome include multiple basal cell carcinomas, palmar and/or plantar pits, odontogenic keratocysts, skeletal and developmental anomalies, and ectopic calcification. Other features include such tumors as ovarian fibromas and medulloblastomas. There is extensive interfamilial as well as intrafamilial variability with respect to the manifestation and severity of the phenotype. Alterations in the human homologue (PTCH) of the Drosophila segment polarity gene patched have been identified in NBCCS patients as well as tumors associated with this syndrome. We report several mutations in this gene in NBCCS patients and present the clinical phenotypes of the individuals in whom these mutations were identified.

Protein tyrosine kinase and protein phosphotyrosine phosphatase in normal and psoriatic skin.

Protein tyrosine kinase and protein phosphotyrosine phosphatase activities were measured in extracts of skin samples from patients with psoriasis. Both kinase and phosphatase activities were significantly greater in samples taken from an involved area, characterized by epidermal hyperproliferation, than from adjacent skin of normal appearance. Samples from skin of non-psoriatic individuals were indistinguishable from the normal-appearing skin of psoriatic patients. There was no detectable change in the apparent Km for either ATP or casein of the protein tyrosine activity in plaques compared with controls. Phosphorylation of endogenous proteins was also increased about 2-fold in plaque extracts compared with controls. Both epidermal growth factor and platelet-derived growth factor stimulated endogenous protein tyrosine phosphorylation in particulate fractions of plaque biopsies but not in solubilized extracts nor in any control fractions. Our data suggest that increased protein tyrosine phosphorylation and dephosphorylation activity and growth factor sensitivity are important factors in non-malignant hyperplastic cell growth.

Epidermolytic hyperkeratosis: applied molecular genetics.

Epidermolytic hyperkeratosis is an autosomal dominant ichthyosis characterized by blistering, especially at birth and during childhood, and hyperkeratosis. Epidermolytic hyperkeratosis presents striking clinical heterogeneity, particularly between families. Several avenues of research have implicated an abnormality of epidermal differentiation in the pathogenesis of this disease. In a three-generation family with 20 affected individuals, we tested a variety of candidate loci and identified linkage to the type II keratin region on chromosome 12. Further investigation revealed a mutation in the H1 subdomain of the keratin 1 gene as the cause of EHK in this family. Because keratin 10 is the co-expressed partner of keratin 1, it was not surprising when abnormalities in keratin 10 were found in other families with EHK. We have examined 52 patients from 21 families and have identified at least six clinical phenotypes. The most useful distinguishing feature was the presence or absence of severe hyperkeratosis of the palms and soles. We and others are continuing to search for and characterize mutations in keratin 1 and 10 in patients with epidermolytic hyperkeratosis. Correlation of the clinical disease types with the specific mutations should lead to a better understanding of the relationship between keratin structure and function in normal and diseased epidermis.

Inhibition of epidermal adenyl cyclase by lithium carbonate.

An in vitro floating system was used to investigate the effect of lithium carbonate (Li2CO3) on the activity of adenyl cyclase in normal pig epidermis. Li2CO3 decreased the responsiveness of adenyl cyclase to stimulation by histamine, adenosine monophosphate (AMP) and epinephrine. This abnormality is similar but not identical to the previously described impaired responsiveness of adenyl cyclase to epinephrine and PGE2 in psoriatic plaques compared to normal adjacent skin. Involved and uninvolved skin from a psoriatic on lithium therapy demonstrated decreased responsiveness to in vitro stimulation by epinephrine, histamine and adenosine when compared to skin from psoriatics who were not on lithium therapy. These results are consistent with the observation that lithium therapy worsens psoriatic lesions.

Qualitative and quantitative analysis of cytosol retinoid binding proteins in human skin.

The distribution of the cytosol retinol and retinoic acid binding proteins are known to vary greatly within the different layers of the eye, a retinoid target organ. We have analyzed the cytosol retinoid binding from adult human skin, another retinoid target organ, and examined the relative contribution of the epidermis and dermis to the total retinoid binding. The mean specific activity of [3H]retinol (0.52 +/- 0.06 pmol/mg protein) and [3H]retinoic acid (3.20 +/- 0.45 pmol/mg protein) binding to cytosol preparations from different specimens of adult human skin was determined. On the average these skins bound 7-fold more retinoic acid than retinol. When skin was treated with EDTA and separated into epidermal and dermal fractions, [3H]retinol and [3H]retinoic acid binding was found in the cytosol derived from epidermis (0.36 +/- 0.03 pmol/mg protein, 3.69 +/- 0.13 pmol/mg protein, respectively) but not from dermis. To confirm that the absence of dermal binding was not due to loss during the EDTA separation, we assayed skin keratomed at 0.1, 0.2, and 0.3 mm. The skin obtained at 0.1 mm was upper epidermis and exhibited binding for both retinol and retinoid acid. The 0.2 mm skin, which added lower epidermis but little dermal contamination, had higher specific activities for both retinol and retinoic acid binding. The 0.3 mm skin which added primarily dermis, had lower specific activities for binding both retinoids. This is consistent with the concept that the epidermis is responsible for the majority of retinoid binding in adult human skin obtained from the lower limb.

Congenital recessive ichthyosis unlinked to loci for epidermal transglutaminases.

Congenital recessive ichthyosis has a broad range of clinical presentations, which may be considered a spectrum of phenotypes with classic lamellar ichthyosis at one pole and classic congenital ichthyosiform erythroderma at the other. The identification of mutations in the transglutaminase-1 gene as a cause of lamellar ichthyosis implicates transglutaminases in other congenital recessive ichthyoses. We investigated two multiplex families with clinical manifestations between the two poles for linkage to the transglutaminase-1 locus on chromosome 14. Strongly negative lod scores prompted a search for linkage to two other epidermally expressed transglutaminases, transglutaminase-2 and transglutaminase-3, on chromosome 20. No evidence for linkage was found. These data confirm the hypothesis that the congenital recessive ichthyoses are genetically heterogeneous and in two families exclude two other transglutaminases that could be considered as candidate loci for at least some of the nonlamellar recessive ichthyoses.

A mutation in the V1 end domain of keratin 1 in non-epidermolytic palmar-plantar keratoderma.

Mutations in keratin 9 have been found in families with an epidermolytic form of palmar-plantar keratoderma (PPK). In another form of PPK (Unna-Thost type), epidermolysis is not observed histologically. We studied a pedigree with this non-epidermolytic form of PPK. By gene linkage analysis, the type I keratin locus could be excluded but complete linkage with the type II keratin region was found. Sequence analysis identified a single base change in the amino-terminal V1 variable subdomain of keratin 1, which caused a lysine to isoleucine substitution. This non-conservative mutation completely cosegregated with the disease and was not observed in 50 unrelated unaffected individuals. An examination of keratin amino-terminal sequences revealed a previously unreported 22-residue window in the V1 subdomain that is conserved among most type II keratins. The altered lysine is an invariant residue in this conserved sequence. Previously described keratin mutations affect the central regions important for filament assembly and stability, and cause diseases characterized by cellular degeneration or disruption. This is the first disease mutation in a keratin chain variable end region. The observation that it is not associated with epidermolysis supports the concept that the amino-terminal domain of keratins may be involved in supramolecular interactions of keratin filaments rather than stability. Therefore, hyperkeratosis associated with this mutation may be due to perturbations in the interactions of the keratin end domain with other cellular components.

Mutations in the H1 and 1A domains in the keratin 1 gene in epidermolytic hyperkeratosis.

In the autosomal dominant disorder epidermolytic hyperkeratosis, the structural integrity of the keratin intermediate filaments is altered in the suprabasal layers of the epidermis. We and others have used genetic linkage studies and mutation analysis to establish that single amino acid substitutions in either the keratin 1 or keratin 10 chains can cause epidermolytic hyperkeratosis. However, a larger database of mutations is required to better understand the relationship between specific mutations in these keratin chains and their effect on keratin filament structure. A larger database will also provide a catalog that may be useful for genetic counseling purposes. In this paper, we report the identification of three new mutations of the keratin 1 chain of epidermolytic hyperkeratosis probands in highly conserved residues in the H1 or beginning of the 1A rod domain segments. These correspond to regions involved in molecular overlaps between neighboring molecules in keratin filaments. Using an in vitro assay, synthetic peptides bearing these substitutions show diminished capacity to disassemble preformed filaments in vitro in comparison to the wild type peptides. Moreover, analyses of all mutations in epidermolytic hyperkeratosis known to date demonstrate remarkable clustering in the molecular overlap region. We conclude that non-conservative substitutions in the overlap region are likely to interfere with normal keratin filament structure and function, leading to pathology.

Etretinate: effect of milk intake on absorption.

Since etretinate, an aromatic retinoid useful in the treatment of psoriasis and other skin disorders is lipid-soluble, it may be poorly absorbed in the absence of a fat load. We therefore studied serum concentrations of etretinate and its major metabolite (Ro 10-1670) after the controlled administration of etretinate. After an overnight fast, 6 Darier's disease and 4 psoriatic patients received a 1 mg/kg morning dose of etretinate with water or 1 pint of whole milk (fat load). There was a 260% increase (p less than 0.0005) in the mean of each patient's increase in the baseline-corrected peak serum concentration of etretinate after administration with milk (115 +/- 15 micrograms/dl) compared to after administration with water (32 +/- 4 micrograms/dl). Over a 24-h period there was an overall 296 +/- 26% (p less than 0.0005) increase in serum etretinate after administration with milk compared to water in 5 patients with Darier's disease. In contrast to the serum etretinate, there was a 17% mean decrease (p less than 0.025) in the corrected peak serum concentration of Ro 10-1670 in all 10 patients after administration of etretinate with milk compared to water. The net result of these alterations is that the mean corrected serum concentration of etretinate is higher than Ro 10-1670 at all time points measured after milk administration. In contrast, after administration of etretinate with water the major retinoid in the serum is Ro 10-1670. Establishing the clinical significance of these alterations may require controlled clinical trials.

Linkage studies in erythrokeratodermias: fine mapping, genetic heterogeneity and analysis of candidate genes.

Erythrokeratodermias are a clinically heterogeneous group of rare autosomal dominant disorders of cornification with overlapping features including hyperkeratosis and erythema. We ascertained five extended pedigrees with different phenotypes for a linkage study. Three families presented with localized erythrokeratodermia variabilis, and one with erythrokeratodermia and ataxia. Another family had Greither disease associated with variable hyperkeratotic plaques. Despite their phenotypic differences, both erythrokeratodermia variabilis and erythrokeratodermia with ataxia map to a common region in 1p34-p35. Multipoint linkage and haplotype analyses place erythrokeratodermia variabilis between the marker D1S496 and D1S186 with a maximum LOD score of 12.88. Our linkage results provide compelling evidence for genetic homogeneity among families of mixed European and French-Canadian origin. In contrast, results excluded Greither's disease from the established erythrokeratodermia variabilis gene region indicating genetic heterogeneity of erythrokeratodermias. Based on recombinations, two genes assigned to 1p34-p35 were excluded: cartilage matrix protein and avian myelocytosis viral oncogene. Connexin-37 (GJA4), a member of the connexin gene family, maps within the erythrokeratodermia variabilis region and is an attractive candidate gene. Direct sequencing of the coding region of GJA4 in four patients revealed several variations, including a novel polymorphism within the 5' cytoplasmic domain, but no pathogenic mutations were found, thus excluding Connexin-37 as a candidate. There is evidence, however, that other epidermally expressed connexins cluster in this region, and one may yet be determined to play a role in the pathogenesis of erythrokeratodermia variabilis.