Early-onset sarcoidosis (EOS) and inheritable Blau syndrome (BS) share characteristic clinical features of juvenile-onset systemic granulomatosis syndrome that mainly affects skin, joints, and eyes. However, no direct evidence has been shown for the possible common origin of these 2 diseases. Recent discovery of CARD15 mutations in BS families encouraged us to investigate similar CARD15 mutations in EOS patients. Among 10 EOS cases retrospectively collected in Japan, heterozygous missense mutations were found in 9 cases; 4 showed a 1000C>T (R334W in amino acid change) that has been reported in BS, 4 showed novel 1487A>T (H496L) , IntroductionSarcoidosis is a multiorganic inflammatory disease with unknown etiology, characterized by the histologic features of noncaseating epithelioid granulomas. In childhood, 2 distinct types of sarcoidosis have been described. 1 Usually the disease is detected in older children by chest radiography and the clinical manifestations are characterized by a classical triad of lung, lymph node, and eye involvement, similar to those in adults. In contrast, early-onset sarcoidosis (EOS), which usually appears in those younger than 4 years of age, is quite rare and has a distinct triad of skin, joint, and eye disorders, without apparent pulmonary involvement. Compared with an asymptomatic and sometimes naturally disappearing course of the disease in older children, EOS is progressive and in many cases causes severe complications, such as blindness, joint destruction, and visceral involvement. 2 Blau syndrome (BS), also showing early-onset granulomatous arthritis, uveitis, and skin rash, is a rare familial disease transmitted in an autosomal dominant manner. 3 By linkage analysis, the responsible locus for BS was mapped to chromosome 16,4 in which CARD15 has recently been identified as the susceptibility gene. 5 CARD15 (NOD2) is a member of the growing family of nucleotide-binding oligomerization domain (NOD) proteins and composed of 2 amino-terminal caspase recruitment domains (CARDs), one NOD, and carboxy-terminal leucinerich repeats (LRRs). 6,7 While mutations in LRRs are reportedly associated with Crohn disease (CD) and psoriatic arthritis, 8-10 3 types of missense point mutations in the NOD, 1000CϾT (R334W in amino acid change), 1001GϾA (R334Q), and 1405CϾT (L469F), have been discovered in BS families. 5,11,12 It has been discussed since the first report of BS whether EOS and BS are the same diseases. 13 However, no direct evidence of their common origin has been shown and confusion still remains. 14 In the first paper describing genetic abnormalities in BS, the authors recognized no CARD15 mutation in 2 EOS patients and therefore proposed a different etiology of BS and EOS. 5 However, we have recently described a sporadic case of systemic granulomatosis syndrome with clinical features of EOS that showed the same CARD15 mutation as detected in BS. 15 In this report, therefore, we retrospectively collected Japanese EOS cases and searched for CARD15 mutations, to further evaluate the re...

The laminin alpha 1 chain carboxyl-terminal globular domain has been identified as a site of multiple biological activities. Using a systematic screening for cell binding sites with 113 overlapping synthetic peptide beads that covered this domain, we found 19 potential active sequences. Corresponding synthetic peptides were evaluated for direct cell attachment, spreading, and inhibition of cell spreading to a laminin-1 substrate using several cell lines. Five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. Cell spreading on AG-10 was inhibited by beta 1 and alpha 6 integrin antibodies and on AG-32 was inhibited by beta 1, alpha 2, and alpha 6 integrin antibodies. In contrast, cell adhesion and spreading on peptide AG-73 were not inhibited by these antibodies. The minimum active sequences of AG-10, AG-32, and AG-73 were determined to be SIYITRF, IAFQRN, and LQVQLSIR, respectively. These sequences are highly conserved among the different species and different laminin alpha chains, suggesting that they play a critical role for biological function and for interaction with cell surface receptors.

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF.

We initially performed exome-sequencing 11 of the two UV S S-A patients, Kps3 and XP24KO (details described in Methods, Supplementary Note, Table 2c). The patients were homozygous for c.367A>T mutation in UVSSA, which led to a premature termination, p.Lys123* (Fig. 1a, b). We identified the same homozygous mutation in Kps2 (sib. of Kps3), and a homozygous c.87delG, causing a frameshift p.Ile31Phefs*9, in an Israeli patient UV S S24TA (Fig. 1b, c, Supplementary Note, Supplementary Fig. 1). The identified mutations are summarized in Fig. 1d. We did not detect the 80kDa UVSSA protein in any of the UV S S-A patients (Fig. 1e). We additionally examined several mild xeroderma pigmentosum (XP) cases; in one such case, XP70TO 12 (Supplementary Table 1), we identified a homozygous p.Cys32Arg, in the UVSSA (Fig. 1c, d), implying that XP70TO is also in the UV S S-A group. The mutant protein was stably expressed in XP70TO cells (Fig. 1f, Supplementary Fig. 2a-d). 4Allele frequencies of the identified mutations in a control population were examined (Supplementary Note, Supplementary Fig. 3a). Haploinsufficiency for UVSSA is negligible as the parents of Kps2/Kps3 showed no symptoms 4 . In parallel with exome-sequencing, we performed whole-genome SNP-genotyping to identify runs-of-homozygosity (ROH) shared among the patients. We identified three overlapping-ROHs (> 1Mbps) on autosomes, one of which encompasses the UVSSA locus (Fig. 1g, Supplementary Table 3a, b, Supplementary Fig. 3b, c). No chromosome copy number variation was detected (Supplementary Fig. 3d).The above findings strongly suggest that the mutations in UVSSA in the UV S S-A patients are causal for the disease; we therefore, next examined the NER-activities in the UV S S-A cells (Fig. 2). Unscheduled-DNA-synthesis (UDS 13 , defective in XP) was nearly normal; however, RNA-synthesis-recovery (RRS 14 , defective in UV S S and in CS) was reduced in all cell-strains mutated in UVSSA ( Fig. 2a, b; UDS and RRS were measured using a recently-developed rapid non-radioactive system 15,16 ). Similarly, siRNA-based depletion of the UVSSA gene (Fig. 2c) caused a drastic reduction of RRS (Fig. 2d, Supplementary Fig. 4), whereas UDS was unaffected (Fig. 2e). Ectopic-expression of the wild-type UVSSA cDNA in UV S S-A cells restored normal RRS ( Fig. 2f; V5-tagged-UVSSA immunofluorescent-staining shown in Fig. 2g), while it did not affect RRS-level in normal, CS-A, or CS-B cells; neither ERCC8 nor ERCC6 cDNA expression in UV S S-A cells restored the RRS-level.We conclude that KIAA1530/UVSSA is the causal gene for UV S S-A.ERCC8 and ERCC6 genes are responsible for both CS and UV S S 7,8 . To evaluate whether UVSSA mutations may also result in CS-phenotypes, we sequenced 5 the UVSSA gene of 61 CS-patients whose genetic defects had not yet been determined (Supplementary Table 4). We found no obvious mutations except for four novel heterozygous changes. These changes as well as the SNPs, also found in control and UV S S-A individuals, do not affect the RRS-activity (Suppleme...

Laminin-5, consisting of the ␣3, ␤3, and ␥2 chains, is localized in the skin basement membrane and supports the structural stability of the epidermo-dermal linkage and regulates various cellular functions. The ␣ chains of laminins have been shown to have various biological activities. In this study, we identified a sequence of the ␣3 chain C-terminal globular domain (LG1-LG5 modules) required for both heparin binding and cell adhesion using recombinant proteins and synthetic peptides. We found that the LG3 and LG4 modules have activity for heparin binding and that LG4 has activity for cell adhesion. Studies with synthetic peptides delineated the A3G75aR sequence (NSFMALYLSKGR, residues 1412-1423) within LG4 as a major site for both heparin and cell binding. Substitution mutations in LG4 and A3G75aR identified the Lys and Arg of the A3G75aR sequence as critical for these activities. Cell adhesion to LG4 and A3G75aR was inhibited by heparitinase I treatment of cells, suggesting that cell binding to the A3G75aR site was mediated by cell surface heparan sulfate proteoglycans. We showed by affinity chromatography that syndecan-2 from fibroblasts bound to LG4. Solid-phase assays confirmed that syndecan-2 interacted with the A3G75aR peptide sequence. Stably transfected 293T cells with expression vectors for syndecan-2 and -4, but not glypican-1, specifically adhered to LG4 and A3G75aR. These results indicate that the A3G75aR sequence within the laminin ␣3 LG4 module is responsible for cell adhesion and suggest that syndecan-2 and -4 mediate this activity.Laminins are extracellular proteins primarily present at the basement membrane, where they provide structural stability and exert many biological functions, including cell adhesion, migration, proliferation, and differentiation, and they are also involved in angiogenesis and tumor invasion (for reviews, see Refs. 1 and 2). Laminins are a large family of glycoproteins, consisting of at least 12 isoforms (laminin-1 to -12) (3-5). Each laminin is composed of three different chains, ␣, ␤, and ␥. There are 11 laminin chains, five ␣ chains (␣1-␣5), three ␤ chains (␤1-␤3), and three ␥ chains (␥1-␥3), whose expression is regulated temporarily and specially during development and tissue repair. Three chains are assembled into a cross-shaped heterotrimer (␣␤␥) by forming a triple-stranded coiled-coil structure through the ␣-helical domain of each chain (6, 7).Laminin-5 (␣3␤3␥2) is a component of anchoring fibrils and forms a complex with the hemidesmosome apparatus and stabilizes the basement membrane structure by forming supramolecular complexes with laminin-6 and -7, collagen VII (8, 9), and fibulin-2 (10). Mutations in laminin-5 (11, 12) cause congenital skin blister diseases junctional epidermolysis bullosa. Disruption of the laminin ␣3 gene in mice resulted in abnormal hemidesmosomes and blister formation in the skin (13). Laminin-5 is also shown to be an adhesive substrate for keratinocytes (14). In these processes, the ␣ 3 ␤ 1 integrin was identified as a cellular recepto...