ARP-T1-associated Bazex-Dupré-Christol syndrome is an inherited basal cell cancer with ciliary defects characteristic of ciliopathies.

Actin-Related Protein-Testis1 (ARP-T1)/ACTRT1 gene mutations cause the Bazex-Dupré-Christol Syndrome (BDCS) characterized by follicular atrophoderma, hypotrichosis, and basal cell cancer. Here, we report an ARP-T1 interactome (PXD016557) that includes proteins involved in ciliogenesis, endosomal recycling, and septin ring formation. In agreement, ARP-T1 localizes to the midbody during cytokinesis and the basal body of primary cilia in interphase. Tissue samples from ARP-T1-associated BDCS patients have reduced ciliary length. The severity of the shortened cilia significantly correlates with the ARP-T1 levels, which was further validated by ACTRT1 knockdown in culture cells. Thus, we propose that ARP-T1 participates in the regulation of cilia length and that ARP-T1-associated BDCS is a case of skin cancer with ciliopathy characteristics.

Turkish Ectodermal Dysplasia Cohort: From Phenotype to Genotype in 17 Families.

Hypohidrotic or anhidrotic ectodermal dysplasia (HED/EDA) is characterized by impaired development of the hair, teeth, or sweat glands. HED/EDA is inherited in an X-linked, autosomal dominant, or autosomal recessive pattern and caused by the pathogenic variants in 4 genes: EDA, EDAR, EDARADD, and WNT10A. The aim of the present study was to perform molecular screening of these 4 genes in a cohort of Turkish individuals diagnosed with HED/EDA. We screened for pathogenic variants of WNT10A, EDA, EDAR, and EDARADD through Sanger sequencing. We further assessed the clinical profiles of the affected individuals in order to establish phenotype-genotype correlation. In 17 (63%) out of 27 families, 17 pathogenic variants, 8 being novel, were detected in the 4 well-known ectodermal dysplasia genes. EDAR and EDA variants were identified in 6 families each, WNT10A variants in 4, and an EDARADD variant in 1, accounting for 35.3, 35.3, 23.5, and 5.9% of mutation-positive families, respectively. The low mutation detection rate of the cohort and the number of the EDAR pathogenic variants being as high as the EDA ones were the most noteworthy findings which could be attributed to the high consanguinity rate.

Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas.

Basal cell carcinoma (BCC), the most common human cancer, results from aberrant activation of the Hedgehog signaling pathway. Although most cases of BCC are sporadic, some forms are inherited, such as Bazex-Dupré-Christol syndrome (BDCS)-a cancer-prone genodermatosis with an X-linked, dominant inheritance pattern. We have identified mutations in the ACTRT1 gene, which encodes actin-related protein T1 (ARP-T1), in two of the six families with BDCS that were examined in this study. High-throughput sequencing in the four remaining families identified germline mutations in noncoding sequences surrounding ACTRT1. These mutations were located in transcribed sequences encoding enhancer RNAs (eRNAs) and were shown to impair enhancer activity and ACTRT1 expression. ARP-T1 was found to directly bind to the GLI1 promoter, thus inhibiting GLI1 expression, and loss of ARP-T1 led to activation of the Hedgehog pathway in individuals with BDCS. Moreover, exogenous expression of ACTRT1 reduced the in vitro and in vivo proliferation rates of cell lines with aberrant activation of the Hedgehog signaling pathway. In summary, our study identifies a disease mechanism in BCC involving mutations in regulatory noncoding elements and uncovers the tumor-suppressor properties of ACTRT1.

Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti.

The NF-κB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF-κB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-κB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.

A novel missense mutation in the gene EDARADD associated with an unusual phenotype of hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia (HED) is a rare disorder characterized by deficient development of structures derived from the ectoderm including hair, nails, eccrine glands, and teeth. HED forms that are caused by mutations in the genes EDA, EDAR, or EDARADD may show almost identical phenotypes, explained by a common signaling pathway. Proper interaction of the proteins encoded by these three genes is important for the activation of the NF-κB signaling pathway and subsequent transcription of the target genes. Mutations in the gene EDARADD are most rarely implicated in HED. Here we describe a novel missense mutation, c.367G>A (p.Asp123Asn), in this gene which did not appear to influence the interaction between EDAR and EDARADD proteins, but led to an impaired ability to activate NF-κB signaling. Female members of the affected family showed either unilateral or bilateral amazia. In addition, an affected girl developed bilateral ovarian teratomas, possibly associated with her genetic condition.

Genetic characterization of congenital tufting enteropathy: epcam associated phenotype and involvement of SPINT2 in the syndromic form.

Congenital tufting enteropathy (CTE) is a rare and severe enteropathy recently ascribed to mutations in the epcam gene. Here we establish SPINT2, previously ascribed to congenital sodium diarrhea, as a second gene associated with CTE and report molecular and immunohistochemistry data in 57 CTE patients. Inclusion criteria were early onset diarrhea and intestinal insufficiency with the typical histological CTE abnormalities. The clinical phenotype was registered, the entire coding regions of epcam and SPINT2 sequenced, and immunostaining of EpCAM and SPINT2 performed on intestinal biopsies. An epcam mutation was involved in 41 patients (73 %) who mainly displayed isolated digestive symptoms. Mutations severely affected gene expression since the EpCAM signal on intestinal tissues was either undetectable or low and irregular. Twelve other patients (21 %) carried mutations in SPINT2, and were phenotypically characterized by systematic association with keratitis (p < 10(-4)) and, for half of them, with choanal atresia (p < 10(-4)). Dependency on parenteral nutrition (PN) was comparable in patients with epcam or SPINT2 mutations, but the frequent epcam mutation c.556-14A>G (abnormal splicing) was significantly associated with a better outcome (p = 0.032) with milder PN dependency to weaning in some cases. Finally, four patients (7 %) with isolated digestive symptoms had no detectable epcam or SPINT2 mutation. Two candidate genes, Elf3 and Claudin7, were excluded from this population. Our study allows us to separate CTE patients into at least three genetic classes, each with specific phenotypes. The genetics approach raises the question of the distinction between two congenital enteropathies. Our findings should help improve the diagnosis of CTE, guide toward strategies of long-term PN management, and limit indications for intestinal transplantation to life-threatening PN complications.

First clinical description of an infant with interleukin-36-receptor antagonist deficiency successfully treated with anakinra.

YM is the first son of Tunisian consanguineous parents who developed, at 2 weeks of life, an erythematous and scaly eruption, with subsequent rapid evolution toward generalized pustular psoriasis. Afterward, cutaneous flares of diffuse erythematous rash and pustules involving the whole body appeared, with a once weekly periodicity. Intense irritability was present during flares without fever. Moreover, since 1 month of age the infant presented with diarrhea, dysphagia, and reduced feeding rate, with failure to thrive. Laboratory tests during acute flares showed marked leukocytosis, thrombocytosis, and anemia without C-reactive protein elevation. Skin biopsy and clinical presentation were consistent with pustular psoriasis; nevertheless, the patient did not respond to high-potency topical corticosteroids and retinoid acid. As the patient presented with repeated skin flares early after birth, as well as serious constitutional distress with failure to thrive, an autoinflammatory syndrome like interleukine-1-receptor antagonist deficiency or interleukin-36-receptor antagonist deficiency (DITRA) was considered. The hypothesis was reinforced by parental consanguinity, and absence of skin lesion improvement under standard topical treatment. Genetic analyses showed a homozygous mutation in the IL36RN gene (L27P), which represents the same mutation recently described in DITRA patients. At age 6 months we started treatment with the recombinant interleukin-1 receptor antagonist anakinra with efficacy both on constitutional symptoms and skin involvement. DITRA is a recently described autoinflammatory disease characterized by repeated flares of generalized pustular psoriasis, high fever, asthenia, and systemic inflammation. We report herein the first exhaustive clinical description of an infant with DITRA who was successfully treated with anakinra.

Combination of lipid metabolism alterations and their sensitivity to inflammatory cytokines in human lipin-1-deficient myoblasts.

Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha+Interleukin-1beta(TNF1α+IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines.

A founder effect at the EPCAM locus in Congenital Tufting Enteropathy in the Arabic Gulf.

Mutations of the EPCAM gene have been recently identified in Congenital Tufting Enteropathy (CTE), a severe autosomal recessive gastrointestinal insufficiency of childhood requiring parenteral nutrition and occasionally intestinal transplantation. Studying seven multiplex consanguineous families from the Arabic peninsula (Kuwait and Qatar) we found that most patients were homozygote for a c.498insC mutation in exon 5. The others carried a novel mutation IVS4-2A→G. Both mutations were predicted to truncate the C-terminal domain necessary to anchorage of EPCAM at the intercellular membrane. Consistently, immunohistochemistry of intestinal biopsies failed to detect the EPCAM protein at the intercellular membrane level. The c.498insC mutation was found on the background of a minimal common haplotype of 473kb suggesting a very old founder effect (5000-6000 yrs).

Clinical and histologic features of incontinentia pigmenti in adults with nuclear factor-κB essential modulator gene mutations.

BACKGROUND: Incontinentia pigmenti (IP) is a multisystem disorder, in which cutaneous symptoms can be accompanied by dental, ocular, and central nervous system defects. In adults, the clinical diagnosis of IP is based principally on the late onset of stage 4 lesions and their association with dental, nail, ocular, or central nervous system anomalies. Nevertheless, these lesions are often unrecognized. OBJECTIVES: Our aim was assessment of IP manifestations in adults to clarify diagnostic criteria for mild forms of the disease, to help physicians detect adult IP in the presence of subtle lesions and avoid misdiagnosis. METHOD: We conducted clinical and histologic examination of 25 adults with IP and nuclear factor-κB essential modulator gene rearrangement or mutations. RESULTS: Linear atrophic, hypopigmented, and hairless lesions (stage 4) are constant in adults. Apoptotic keratinocytes in the epidermis or dermis and atrophic hair follicles, with absence of arrector pili muscles, are frequently observed. In contrast, nipple anomalies are rare. LIMITATIONS: We were unable to determine the age of the onset of IP stage 4 lesions. CONCLUSION: Skin manifestations are constant in adult patients with IP. Histology is characteristic and could be considered as a minor diagnostic criterion of IP. Nipple anomalies also may be considered as a minor criterion. Detection of such subtle manifestations can evoke IP in patients with repeated miscarriages or unexplained neurologic manifestations.

Only four genes (EDA1, EDAR, EDARADD, and WNT10A) account for 90% of hypohidrotic/anhidrotic ectodermal dysplasia cases.

Hypohidrotic and anhidrotic ectodermal dysplasia (HED/EDA) is a rare genodermatosis characterized by abnormal development of sweat glands, teeth, and hair. Three disease-causing genes have been hitherto identified, namely, (1) EDA1 accounting for X-linked forms, (2) EDAR, and (3) EDARADD, causing both autosomal dominant and recessive forms. Recently, WNT10A gene was identified as responsible for various autosomal recessive forms of ectodermal dysplasias, including onycho-odonto-dermal dysplasia (OODD) and Schöpf-Schulz-Passarge syndrome. We systematically studied EDA1, EDAR, EDARADD, and WNT10A genes in a large cohort of 65 unrelated patients, of which 61 presented with HED/EDA. A total of 50 mutations (including 32 novel mutations) accounted for 60/65 cases in our series. These four genes accounted for 92% (56/61 patients) of HED/EDA cases: (1) the EDA1 gene was the most common disease-causing gene (58% of cases), (2)WNT10A and EDAR were each responsible for 16% of cases. Moreover, a novel disease locus for dominant HED/EDA mapped to chromosome 14q12-q13.1. Although no clinical differences between patients carrying EDA1, EDAR, or EDARADD mutations could be identified, patients harboring WNT10A mutations displayed distinctive clinical features (marked dental phenotype, no facial dysmorphism), helping to decide which gene should be first investigated in HED/EDA.

Combination of linkage mapping and microarray-expression analysis identifies NF-kappaB signaling defect as a cause of autosomal-recessive mental retardation.

Autosomal-recessive inheritance accounts for nearly 25% of nonsyndromic mental retardation (MR), but the extreme heterogeneity of such conditions markedly hampers gene identification. Combining autozygosity mapping and RNA expression profiling in a consanguineous Tunisian family of three MR children with mild microcephaly and white-matter abnormalities identified the TRAPPC9 gene, which encodes a NF-kappaB-inducing kinase (NIK) and IkappaB kinase complex beta (IKK-beta) binding protein, as a likely candidate. Sequencing analysis revealed a nonsense variant (c.1708C>T [p.R570X]) within exon 9 of this gene that is responsible for an undetectable level of TRAPPC9 protein in patient skin fibroblasts. Moreover, TNF-alpha stimulation assays showed a defect in IkBalpha degradation, suggesting impaired NF-kappaB signaling in patient cells. This study provides evidence of an NF-kappaB signaling defect in isolated MR.

Unusual presentation of a severe autosomal recessive anhydrotic ectodermal dysplasia with a novel mutation in the EDAR gene.

We report on an 18-year-old woman, born to first-cousin parents, presenting with a severe form of anhydrotic ectodermal dysplasia (EDA/HED). She had sparse hair, absent limb hair, absent sweating, episodes of hyperpyrexia, important hypodontia, and hyperconvex nails. She also showed unusual clinical manifestations such as an absence of breasts, a rudimentary extranumerary areola and nipple on the left side, and marked palmo-plantar hyperkeratosis. Light microscopy of skin biopsies showed orthokeratotic hyperkeratosis and absence of sweat glands. A novel homozygous mutation (IVS9 + 1G > A) in the EDAR gene was identified. This mutation results in a total absence of EDAR transcripts and consequently of the EDAR protein, which likely results in abolition of all ectodysplasin-mediated NF-kappaB signaling. This is the first complete loss-of-function mutation in the EDAR gene reported to date, which may explain the unusual presentation of HED in this patient, enlarging the clinical spectrum linked to the dysfunction of the ectodysplasin mediated NF-kappaB signaling.

Alterations of the IKBKG locus and diseases: an update and a report of 13 novel mutations.

Mutations in the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), also called nuclear factor-kappaB (NF-kB) essential modulator (NEMO), gene are the most common single cause of incontinentia pigmenti (IP) in females and anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID) in males. The IKBKG gene, located in the Xq28 chromosomal region, encodes for the regulatory subunit of the inhibitor of kappaB (IkB) kinase (IKK) complex required for the activation of the NF-kB pathway. Therefore, the remarkably heterogeneous and often severe clinical presentation reported in IP is due to the pleiotropic role of this signaling transcription pathway. A recurrent exon 4_10 genomic rearrangement in the IKBKG gene accounts for 60 to 80% of IP-causing mutations. Besides the IKBKG rearrangement found in IP females (which is lethal in males), a total of 69 different small mutations (missense, frameshift, nonsense, and splice-site mutations) have been reported, including 13 novel ones in this work. The updated distribution of all the IP- and EDA-ID-causing mutations along the IKBKG gene highlights a secondary hotspot mutation in exon 10, which contains only 11% of the protein. Furthermore, familial inheritance analysis revealed an unexpectedly high incidence of sporadic cases (>65%). The sum of the observations can aid both in determining the molecular basis of IP and EDA-ID allelic diseases, and in genetic counseling in affected families.

The NEMO mutation creating the most-upstream premature stop codon is hypomorphic because of a reinitiation of translation.

Amorphic mutations in the NF- kappa B essential modulator (NEMO) cause X-dominant incontinentia pigmenti, which is lethal in males in utero, whereas hypomorphic mutations cause X-recessive anhidrotic ectodermal dysplasia with immunodeficiency, a complex developmental disorder and life-threatening primary immunodeficiency. We characterized the NEMO mutation 110_111insC, which creates the most-upstream premature translation termination codon (at codon position 49) of any known NEMO mutation. Surprisingly, this mutation is associated with a pure immunodeficiency. We solve this paradox by showing that a Kozakian methionine codon located immediately downstream from the insertion allows the reinitiation of translation. The residual production of an NH(2)-truncated NEMO protein was sufficient for normal fetal development and for the subsequent normal development of skin appendages but was insufficient for the development of protective immune responses.

Novel splicing mutation in the NEMO (IKK-gamma) gene with severe immunodeficiency and heterogeneity of X-chromosome inactivation.

We report on a family with three stillborn males, three affected males who were small for gestational age and died within 8 months, and one male who died at age 5 years. This boy had cone-shaped teeth and oligoodontia. He had serious bacterial infections and inflammatory bowel disease. Mutations in the NF-kappaB essential modulator (NEMO) gene have recently been shown to be the cause of a group of ectodermal dysplasia and immunodeficiency disorders (EDA-ID). Analysis of the NEMO gene revealed a nucleotide change in the consensus sequence of the splicing donor site of exon 6 IVS6 + 5G --> A(1027 + 5G --> A), which has not previously been described in EDA-ID. RT-PCR analysis of fibroblast RNA from an aborted affected male fetus demonstrated a skipping of exons 4, 5, and 6 which resulted in a truncated protein of about 35 kDa revealed by NEMO antibody. The skipping of exons 4, 5, and 6 did not affect the ORF of the C-terminal of NEMO encoded by exons 7, 8, 9, and 10, which contains a coiled-coil motif (CC2), a leucin-zipper (LZ), and a zinc finger motif (ZF) sub-domains of NEMO. IkappaBalpha degradation was strongly impaired in the fetal fibroblasts, suggesting an impaired NF-kappaB signaling. One healthy carrier had a completely skewed X-inactivation pattern with the normal X active, whereas the two other carriers had a random X-inactivation pattern. This family may represent a new phenotype within the EDA-ID disorders. From the heterogeneity in X-inactivation phenotype, we conclude that this mutation is not deleterious enough to be lethal for peripheral blood cells.

TAB2, TRAF6 and TAK1 are involved in NF-kappaB activation induced by the TNF-receptor, Edar and its adaptator Edaradd.

Activation of the NF-kappaB pathway by the TNF-receptor Edar (Ectodysplasin receptor) and its downstream adaptator Edaradd (Edar-associated death domain) is essential for the development of hair follicles, teeth, exocrine glands and other ectodermal derivatives. Dysfunction of Edar signalling causes hypohidrotic/anhidrotic ectodermal dysplasia (ED), a disorder characterized by sparse hair, lack of sweat glands and malformation of teeth. The Edar signalling pathway stimulates NF-kappaB transcription factors via an activation of the IkappaB kinase (IKK) complex. To gain further insight into the mechanism of IKK activation by Edar and Edaradd, we performed a yeast two-hybrid screen and isolated TAB2 (TAK1-binding protein 2) as a binding partner of Edaradd. TAB2 is an adaptator protein that brigdes TRAF6 (TNF-receptor-associated factor 6) to TAK1 (TGFbeta-activated kinase 1), allowing TAK1 activation and subsequent IKK activation. Here, we show that endogenous and overexpressed TAB2, TRAF6 and TAK1 co-immunoprecipitated with Edaradd in 293 cells. Moreover, we show that dominant negative forms of TAB2, TRAF6 and TAK1 blocked the NF-kappaB activation induced by Edaradd. These results support the involvement of the TAB2/TRAF6/TAK1 signalling complex in the Edar signal transduction pathway and have important implications for our understanding of NF-kappaB activation and EDs in human.