Peter M. Steijlen

Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema

We recently reported two common filaggrin (FLG) null mutations that cause ichthyosis vulgaris and predispose to eczema and secondary allergic diseases. We show here that these common European mutations are ancestral variants carried on conserved haplotypes. To facilitate comprehensive analysis of other populations, we report a strategy for full sequencing of this large, highly repetitive gene, and we describe 15 variants, including seven that are prevalent. All the variants are either nonsense or frameshift mutations that, in representative cases, resulted in loss of filaggrin production in the epidermis. In an Irish case-control study, the five most common European mutations showed a strong association with moderate-to-severe childhood eczema (χ2 test: P = 2.12 × 10−51; Fishers exact test: heterozygote odds ratio (OR) = 7.44 (95% confidence interval (c.i.) = 4.9–11.3), and homozygote OR = 151 (95% c.i. = 20–1,136)). We found three additional rare null mutations in this case series, suggesting that the genetic architecture of filaggrin-related atopic dermatitis consists of both prevalent and rare risk alleles.

Mutations in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosis-lymphedema-telangiectasia.

Hereditary lymphedema is a developmental disorder characterized by chronic swelling of the extremities due to dysfunction of the lymphatic vessels. Two responsible genes have been identified: the vascular endothelial growth factor receptor 3 (VEGFR3) gene, implicated in congenital lymphedema, or Milroy disease, and the forkhead-related transcription factor gene FOXC2, causing lymphedema-distichiasis. We describe three families with an unusual association of hypotrichosis, lymphedema, and telangiectasia. Using microsatellite analysis, we first excluded both VEGFR3 and FOXC2 as causative genes; we then considered the murine ragged phenotype, caused by mutations in the Sox18 transcription factor, as a likely counterpart to the human disease, because it presents a combination of hair and cardiovascular anomalies, including symptoms of lymphatic dysfunction. Two of the families were consanguineous; in affected members of these families, we identified homozygous missense mutations in the SOX18 gene, located in 20q13. The two amino acid substitutions, W95R and A104P, affect conserved residues in the first alpha helix of the DNA-binding domain of the transcription factor. In the third family, the parents were nonconsanguineous, and both the affected child and his brother, who died in utero with hydrops fetalis, showed a heterozygous nonsense mutation that truncates the SOX18 protein in its transactivation domain; this substitution was not found in genomic DNA from either parent and hence constitutes a de novo germline mutation. Thus, we show that SOX18 mutations in humans cause both recessive and dominant hypotrichosis-lymphedema-telangiectasia, suggesting that, in addition to its established role in hair and blood vessel development, the SOX18 transcription factor plays a role in the development and/or maintenance of lymphatic vessels.

Haploinsufficiency of TNXB Is Associated with Hypermobility Type of Ehlers-Danlos Syndrome

To the Editor: Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable connective-tissue disorders, generally affecting skin, joints, and blood vessels. The most recent classification recognizes six subtypes (Beighton et al. 1998), of which the hypermobility type (HT-EDS [formerly EDS type III] [MIM 130020]) is the most common. This type of EDS is similar to benign joint hypermobility syndrome (BJHS), and both are often considered to represent the same hyperlaxity syndrome, since no clear clinical distinction can be made (Grahame 1999). Although various causative genes have been found in all other types of EDS, the genetic basis of HT-EDS or BJHS remains unexplained (Steinmann et al. 2002). One family has been described that has a missense mutation in COL3A1 (Narcisi et al. 1994), resulting in a phenotype that resembles HT-EDS, without obvious vascular complications. Mutations in COL3A1 generally result in the severe vascular type of EDS (MIM 130050). To our knowledge, no other cases of COL3A1 mutations in HT-EDS have been reported. Recently, we showed that deficiency of the extracellular-matrix protein tenascin-X (TNX), encoded by the TNXB gene, causes a new type of recessively inherited EDS (Schalkwijk et al. 2001). Patients with complete deficiency of TNX showed marked joint hypermobility, skin hyperextensibility, and easy bruising. The absence of atrophic scars and recessive inheritance distinguishes TNX deficiency from the classical type of EDS. In our initial report (Schalkwijk et al. 2001), only a few heterozygous family members were available for examination. Here, we have examined all 20 heterozygous family members (individuals from families A–D in table 1) who were available for further study, regardless of clinical symptoms; in all of these individuals, we have found significantly reduced serum TNX levels (56% ± 6% vs. 100% ± 14% in the control population; P<.001, by Students t test) (fig. 1f), and, in 17 of them, we have confirmed heterozygosity for a truncating TNXB mutation (table 1). Clinical examination revealed generalized joint hypermobility in nine family members (45%), using the Beighton score (Beighton et al. 1973), for HT-EDS, or the Brighton criteria (Grahame et al. 2000), for BJHS (table 1 and fig. 1e). Skin hyperextensibility and easy bruising, frequently seen in the individuals with complete TNX deficiency, were absent. A number of patients with haploinsufficiency had recurring joint dislocations and chronic joint pain, as are seen in HT-EDS and BJHS. Only four family members carrying two normal TNXB alleles were available for study, of whom none had hypermobility. The local medical ethics committee (CMO Regio Arnhem-Nijmegen) approved the study protocol, and informed consent was obtained from all patients. Figure  1 TNXB haploinsufficiency and generalized joint hypermobility. a–d, Pedigrees of families A–D (also see table 1). e, Joint hypermobility in individual III9 from family A. f, Distribution of serum TNX levels in control population, population ... Table 1 Clinical and Molecular Findings in Individuals with TNXB Haploinsufficiency/Reduced Serum TNX Levels A striking finding is that 0 of the 6 males with haploinsufficiency fulfilled the clinical criteria for HT-EDS or BJHS, whereas 9 of 14 (64%) females were positive. This finding is in accordance with previous population-based studies that show a female preponderance in joint hypermobility syndromes (Larsson et al. 1987; Rikken-Bultman et al. 1997). In a control group of 30 unaffected females of the same age as the females with haploinsufficiency in the present study, we found no individuals with a Beighton score >4. This indicates that the prevalence of generalized joint hypermobility in a population of females with haploinsufficiency is significantly higher than in a control population (P<.001, by χ2 test). No sex differences in serum TNX levels in unaffected individuals and individuals with haploinsufficiency were found (not shown). Because our observations in families carrying previously described TNXB mutations suggested an association between TNXB haploinsufficiency and joint hypermobility, we wondered about the prevalence of TNXB haploinsufficiency in patients with HT-EDS. We measured serum TNX levels (by ELISA) in an unselected cohort of 80 patients with HT-EDS who were recruited through the Dutch organization for patients with EDS. All patients were diagnosed with HT-EDS by a medical specialist, and ∼90% were female. Although the mean serum TNX level was not different in the cohort with HT-EDS overall (99.4%±19.7%) (fig. 1f), six of these patients (7.5% [all female]) had serum TNX levels >2.5 SDs (65%) below the mean for unaffected individuals. On the basis of the normal distribution of serum TNX levels, only 0.6% of individuals would be expected to have such low serum TNX levels, which is significantly less than the frequency found in the population with HT-EDS described in the present study (P<.001, by Fishers exact test). Clinically, patients with reduced TNX levels showed hypermobile joints, often associated with joint subluxations and chronic musculoskeletal pain (table 1). The clinical findings in these patients differ from those with complete TNX deficiency. Patients with haploinsufficiency do not have skin hyperextensibility and lack the easy bruising seen in patients with TNX deficiency. In addition, TNXB haploinsufficiency is expected to be an autosomal dominant trait, which is in accordance with the observed mode of inheritance of HT-EDS and BJHS. On screening for the presence of a 30-kb deletion described previously (Burch et al. 1997; Schalkwijk et al. 2001), we found that this deletion was present in one of these six patients. The 30-kb deletion creates a fusion gene of TNXB and XA, a partial duplicate of TNXB. The XA gene has an internal deletion that truncates its ORF, rendering XA and the fusion gene nonfunctional (Gitelman et al. 1992). The deleted allele also lacks CYP21, so this individual is also a carrier for congenital adrenal hyperplasia. Subsequently, we PCR amplified and directly sequenced the coding regions and the intron-exon boundaries of TNXB in the other five patients presumed to have haploinsufficiency (for primers used, see Schalkwijk et al. 2001). One patient (individual F in table 1) was heterozygous for a 2-bp deletion, [AA56063] del, in exon 8, resulting in a premature stop codon at the position of amino acid 1231. In the other four patients, we were unable to identify mutations in TNXB. These patients may have mutations, in regulatory sequences or in exons of the TNXB gene, that have not yet been identified, or they may represent the extreme in normal variation of TNX expression. In conclusion, in the present study, we have reported a genetic defect associated with HT-EDS or BJHS. On the basis of the observed phenotype in patients with complete TNX deficiency and the high prevalence of generalized joint hypermobility in heterozygous females, this is likely to be a causative relationship. Reduced TNX expression could disturb deposition of collagen (Mao et al. 2002) and the elastic fiber network (Burch et al. 1997), as has been shown for complete TNX deficiency, resulting in increased laxity of ligaments and tendons. TNXB haploinsufficiency is dominantly inherited and appears to produce clinical findings primarily in women, consistent with clinical descriptions of HT-EDS. Although we identified inactivating TNX mutations in only 2.5% of this cohort with HT-EDS, 7.5% had serum TNX levels low enough to affect collagen metabolism. The present study demonstrates that TNXB haploinsufficiency is associated with HT-EDS and suggests that locus heterogeneity exists for this disorder, as it does for other types of EDS (Byers 1994).

Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis

Marie Unna hereditary hypotrichosis (MUHH) is an autosomal dominant form of genetic hair loss. In a large Chinese family carrying MUHH, we identified a pathogenic initiation codon mutation in U2HR, an inhibitory upstream ORF in the 5′ UTR of the gene encoding the human hairless homolog (HR). U2HR is predicted to encode a 34–amino acid peptide that is highly conserved among mammals. In 18 more families from different ancestral groups, we identified a range of defects in U2HR, including loss of initiation, delayed termination codon and nonsense and missense mutations. Functional analysis showed that these classes of mutations all resulted in increased translation of the main HR physiological ORF. Our results establish the link between MUHH and U2HR, show that fine-tuning of HR protein levels is important in control of hair growth, and identify a potential mechanism for preventing hair loss or promoting hair removal.

Photodynamic therapy versus topical imiquimod versus topical fluorouracil for treatment of superficial basal-cell carcinoma: a single blind, non-inferiority, randomised controlled trial

BACKGROUND Superficial basal-cell carcinoma is most commonly treated with topical non-surgical treatments, such as photodynamic therapy or topical creams. Photodynamic therapy is considered the preferable treatment, although this has not been previously tested in a randomised control trial. We assessed the effectiveness of photodynamic therapy compared with imiquimod or fluorouracil in patients with superficial basal-cell carcinoma. METHODS In this single blind, non-inferiority, randomised controlled multicentre trial, we enrolled patients with a histologically proven superficial basal-cell carcinoma at seven hospitals in the Netherlands. Patients were randomly assigned to receive treatment with methylaminolevulinate photodynamic therapy (MAL-PDT; two sessions with an interval of 1 week), imiquimod cream (once daily, five times a week for 6 weeks), or fluorouracil cream (twice daily for 4 weeks). Follow-up was at 3 and 12 months post-treatment. Data were collected by one observer who was blinded to the assigned treatment. The primary outcome was the proportion of patients free of tumour at both 3 and 12 month follow up. A pre-specified non-inferiority margin of 10% was used and modified intention-to-treat analyses were done. This trial is registered as an International Standard Randomised controlled trial (ISRCTN 79701845). FINDINGS 601 patients were randomised: 202 to receive MAL-PDT, 198 to receive imiquimod, and 201 to receive fluorouracil. A year after treatment, 52 of 196 patients treated with MAL-PDT, 31 of 189 treated with imiquimod, and 39 of 198 treated with fluorouracil had tumour residue or recurrence. The proportion of patients tumour-free at both 3 and 12 month follow-up was 72.8% (95% CI 66.8-79.4) for MAL-PDT, 83.4% (78.2-88.9) for imiquimod cream, and 80.1% (74.7-85.9) for fluorouracil cream. The difference between imiquimod and MAL-PDT was 10.6% (95% CI 1.5-19.5; p=0.021) and 7.3% (-1.9 to 16.5; p=0.120) between fluorouracil and MAL-PDT, and between fluorouracil and imiquimod was -3.3% (-11.6 to 5.0; p=0.435. For patients treated with MAL-PDT, moderate to severe pain and burning sensation were reported most often during the actual MAL-PDT session. For other local adverse reactions, local skin redness was most often reported as moderate or severe in all treatment groups. Patients treated with creams more often reported moderate to severe local swelling, erosion, crust formation, and itching of the skin than patients treated with MAL-PDT. In the MAL-PDT group no serious adverse events were reported. One patient treated with imiquimod and two patients treated with fluorouracil developed a local wound infection and needed additional treatment in the outpatient setting. INTERPRETATION Topical fluorouracil was non-inferior and imiquimod was superior to MAL-PDT for treatment of superficial basal-cell carcinoma. On the basis of these findings, imiquimod can be considered the preferred treatment, but all aspects affecting treatment choice should be weighted to select the best treatment for patients. FUNDING Grant of the Netherlands Organization for Scientific Research ZONMW (08-82310-98-08626).

The hereditary palmoplantar keratoses: an updated review and classification

The palmoplantar keratoses (PPKs) comprise a heterogeneous group of disorders of keratinization, which can be subdivided into hereditary and acquired forms. Many authors have attempted to classify the hereditary forms,1–5 and most classifications have been based on the morphology, distribution, associated symptoms and mode of inheritance. Subsequently, many new forms have been recognized, and what were previously considered to be distinct types have been shown to be variants of a single type, both of which limit the usefulness of previous classifications. Hence, we propose a new, updated classification, which enables accurate diagnosis of these disorders.

A Homozygous Missense Mutation in TGM5 Abolishes Epidermal Transglutaminase 5 Activity and Causes Acral Peeling Skin Syndrome

Peeling skin syndrome is an autosomal recessive genodermatosis characterized by the shedding of the outer epidermis. In the acral form, the dorsa of the hands and feet are predominantly affected. Ultrastructural analysis has revealed tissue separation at the junction between the granular cells and the stratum corneum in the outer epidermis. Genomewide linkage analysis in a consanguineous Dutch kindred mapped the gene to 15q15.2 in the interval between markers D15S1040 and D15S1016. Two homozygous missense mutations, T109M and G113C, were found in TGM5, which encodes transglutaminase 5 (TG5), in all affected persons in two unrelated families. The mutation was present on the same haplotype in both kindreds, indicating a probable ancestral mutation. TG5 is strongly expressed in the epidermal granular cells, where it cross-links a variety of structural proteins in the terminal differentiation of the epidermis to form the cornified cell envelope. An established, in vitro, biochemical cross-linking assay revealed that, although T109M is not pathogenic, G113C completely abolishes TG5 activity. Three-dimensional modeling of TG5 showed that G113C lies close to the catalytic domain, and, furthermore, that this glycine residue is conserved in all known transglutaminases, which is consistent with pathogenicity. Other families with more-widespread peeling skin phenotypes lacked TGM5 mutations. This study identifies the first causative gene in this heterogeneous group of skin disorders and demonstrates that the protein cross-linking function performed by TG5 is vital for maintaining cell-cell adhesion between the outermost layers of the epidermis.

Aplasia cutis congenita and associated disorders: an update.

We present an update of disorders in which aplasia cutis congenita is a feature. Localization of the lesion, important other features, and possible etiology are tabulated. Disorders are classified as chromosomal, monogenic, teratological/exogenous, and unknown. Points of particular interest in history taking and examination of patients with aplasia cutis congenita are presented.

Prevalence of Symptomatic Intracranial Aneurysm and Ischaemic Stroke in Pseudoxanthoma Elasticum

Background: Pseudoxanthoma elasticum (PXE) is an heritable connective tissue disorder with clinical manifestations of the ocular, dermal, and cardiovascular system. The purpose of this study was to investigate the prevalence of symptomatic intracranial aneurysms (IAs) and ischaemic stroke (IS) in PXE. Methods: The records of 100 patients with PXE were retrieved. All patients were contacted and data on complications were collected. The literature was reviewed regarding PXE, ISs, and IAs. Results: No patient with PXE had a symptomatic IA as presenting symptom. One patient presented with an IS. During follow-up of 94 of the 100 patients (mean follow-up 17.1 years, range 1–49 years), none presented a symptomatic IA (3,168 retrospective patient observation years and 1,602 prospective patient observation years). Upper gastrointestinal haemorrhage during follow-up occurred in 17 patients, in 1 patient during aspirin use. One patient has IS as presenting symptom and a recurrence during follow-up, and 7 patients had IS during follow-up. All were caused by small-vessel disease. The relative risk of IS in PXE under 65 years compared with the general population was 3.6 (95% confidence interval 3.3–4.0). Conclusions: On the basis of the currently available data, an association between symptomatic IAs and PXE is unlikely. However, the incidence of IS, due to small-vessel disease, was increased. Antiplatelet therapy in patients with PXE may lead to a high incidence of upper gastrointestinal haemorrhages.

Fractionated 5-aminolaevulinic acid-photodynamic therapy vs. surgical excision in the treatment of nodular basal cell carcinoma: Results of a randomized controlled trial

Background  Skin cancer incidence rates have been increasing for decades and this increase is expected to continue. Surgical excision (SE) is the treatment of first choice for nodular basal cell carcinoma (nBCC). Photodynamic therapy (PDT) has proven to be an effective treatment for superficial basal cell carcinoma. Its long‐term efficacy in nBCC has not yet been established.