IRF6 mutation screening in non-syndromic orofacial clefting: analysis of 1521 families.

Van der Woude syndrome (VWS) is an autosomal dominant malformation syndrome characterized by orofacial clefting (OFC) and lower lip pits. The clinical presentation of VWS is variable and can present as an isolated OFC, making it difficult to distinguish VWS cases from individuals with non-syndromic OFCs. About 70% of causal VWS mutations occur in IRF6, a gene that is also associated with non-syndromic OFCs. Screening for IRF6 mutations in apparently non-syndromic cases has been performed in several modestly sized cohorts with mixed results. In this study, we screened 1521 trios with presumed non-syndromic OFCs to determine the frequency of causal IRF6 mutations. We identified seven likely causal IRF6 mutations, although a posteriori review identified two misdiagnosed VWS families based on the presence of lip pits. We found no evidence for association between rare IRF6 polymorphisms and non-syndromic OFCs. We combined our results with other similar studies (totaling 2472 families) and conclude that causal IRF6 mutations are found in 0.24-0.44% of apparently non-syndromic OFC families. We suggest that clinical mutation screening for IRF6 be considered for certain family patterns such as families with mixed types of OFCs and/or autosomal dominant transmission.

TLR4 mutations are associated with endotoxin hyporesponsiveness in humans.

There is much variability between individuals in the response to inhaled toxins, but it is not known why certain people develop disease when challenged with environmental agents and others remain healthy. To address this, we investigated whether TLR4 (encoding the toll-like receptor-4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signalling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress.

Identification of novel candidate genes associated with cleft lip and palate using array comparative genomic hybridisation.

AIM AND METHOD: We analysed DNA samples isolated from individuals born with cleft lip and cleft palate to identify deletions and duplications of candidate gene loci using array comparative genomic hybridisation (array-CGH). RESULTS: Of 83 syndromic cases analysed we identified one subject with a previously unknown 2.7 Mb deletion at 22q11.21 coinciding with the DiGeorge syndrome region. Eighteen of the syndromic cases had clinical features of Van der Woude syndrome and deletions were identified in five of these, all of which encompassed the interferon regulatory factor 6 (IRF6) gene. In a series of 104 non-syndromic cases we found one subject with a 3.2 Mb deletion at chromosome 6q25.1-25.2 and another with a 2.2 Mb deletion at 10q26.11-26.13. Analyses of parental DNA demonstrated that the two deletion cases at 22q11.21 and 6q25.1-25.2 were de novo, while the deletion of 10q26.11-26.13 was inherited from the mother, who also has a cleft lip. These deletions appear likely to be causally associated with the phenotypes of the subjects. Estrogen receptor 1 (ESR1) and fibroblast growth factor receptor 2 (FGFR2) genes from the 6q25.1-25.2 and 10q26.11-26.13, respectively, were identified as likely causative genes using a gene prioritization software. CONCLUSION: We have shown that array-CGH analysis of DNA samples derived from cleft lip and palate subjects is an efficient and productive method for identifying candidate chromosomal loci and genes, complementing traditional genetic mapping strategies.

Defective expression of p56lck in an infant with severe combined immunodeficiency.

Severe combined immune deficiency (SCID) is a heterogeneous disorder characterized by profound defects in cellular and humoral immunity. We report here an infant with clinical and laboratory features of SCID and selective CD4 lymphopenia and lack of CD28 expression on CD8(+) T cells. T cells from this patient showed poor blastogenic responses to various mitogens and IL-2. Other T cell antigen receptor- induced responses, including upregulation of CD69, were similarly inhibited. However, more proximal T cell antigen receptor signaling events, such as anti-CD3 induced protein tyrosine phosphorylation, phosphorylation of mitogen-associated protein kinase, and calcium mobilization were intact. Although p59fyn and ZAP-70 protein tyrosine kinases were expressed at normal levels, a marked decrease in the level of p56lck was noted. Furthermore, this decrease was associated with the presence of an alternatively spliced lck transcript lacking the exon 7 kinase encoding domain. These data suggest that a deficiency in p56lck expression can produce a SCID phenotype in humans.

IRF6 and SPRY4 Signaling Interact in Periderm Development.

Rare mutations in IRF6 and GRHL3 cause Van der Woude syndrome, an autosomal dominant orofacial clefting disorder. Common variants in IRF6 and GRHL3 also contribute risk for isolated orofacial clefting. Similarly, variants within genes that encode receptor tyrosine kinase (RTK) signaling components, including members of the FGF pathway, EPHA3 and SPRY2, also contribute risk for isolated orofacial clefting. In the mouse, loss of Irf6 or perturbation of Fgf signaling leads to abnormal oral epithelial adhesions and cleft palate. Oral adhesions can result from a disruption of periderm formation. Here, we find that IRF6 and SPRY4 signaling interact in periderm function. We crossed Irf6 heterozygous ( Irf6+/-) mice with transgenic mice that express Spry4 in the basal epithelial layer ( TgKRT14::Spry4). While embryos with either of these mutations can have abnormal oral adhesions, using a new quantitative assay, we observed a nonadditive effect of abnormal oral epithelial adhesions in the most severely affected double mutant embryos ( Irf6+/-;TgKRT14::Spry4). At the molecular level, the sites of abnormal oral adhesions maintained periderm-like cells that express keratin 6, but we observed abnormal expression of GRHL3. Together, these data suggest that Irf6 and RTK signaling interact in regulating periderm differentiation and function, as well as provide a rationale to screen for epistatic interactions between variants in IRF6 and RTK signaling pathway genes in human orofacial clefting populations.

Full Spectrum of Postnatal Tooth Phenotypes in a Novel Irf6 Cleft Lip Model.

Clefting of the lip, with or without palatal involvement (CLP), is associated with a higher incidence of developmental tooth abnormalities, including hypodontia and supernumerary teeth, aberrant crown and root morphologies, and enamel defects, although the underlying mechanistic link is poorly understood. As most CLP genes are expressed throughout the oral epithelium, the authors hypothesized that the expression of CLP genes may persist in the dental epithelium and thus, in addition to their earlier role in labiopalatine development, may play an important functional role in subsequent tooth patterning and amelogenesis. To address this, the authors generated a unique conditional knockout model involving the major CLP gene, Irf6, that overcomes the previously reported perinatal lethality to enable assessment of any posteruption dental phenotypes. A dental epithelium-specific Irf6 conditional knockout (Irf6-cKO) mouse was generated via a Pitx2-Cre driver line. Dental development was analyzed by microcomputed tomography, scanning electron microscopy, histology, immunohistochemistry, and quantitative polymerase chain reaction. Irf6-cKO mice displayed variable hypodontia, occasional supernumerary incisors and molars, as well as crown and root patterning anomalies, including peg-shaped first molars and taurodontic and C-shaped mandibular second molars. Enamel density was reduced in preeruption Irf6-cKO mice, and some shearing of enamel rods was noted in posteruption incisors. There was also rapid attrition of Irf6-cKO molars following eruption. Histologically, Irf6-cKO ameloblasts exhibited disturbances in adhesion and polarity, and delayed enamel formation was confirmed immunohistochemically. Altered structure of Hertwig's epithelial root sheath was also observed. These data support a role for IRF6 in tooth number, crown and root morphology and amelogenesis that is likely due to a functional role of Irf6 in organization and polarity of epithelial cell types. This data reinforce the notion that various isolated tooth defects could be considered part of the CLP spectrum in relatives of an affected individual.

A novel epithelial-expressed ETS gene, ELF3: human and murine cDNA sequences, murine genomic organization, human mapping to 1q32.2 and expression in tissues and cancer.

The ETS family of genes are implicated in cancers such as Ewings sarcoma, acute myeloid leukemia and chronic myelomonocytic leukemia. Further, they have important functions in embryonic development. Hence, identification and characterization of members of this family are important. We identify a novel ETS family member, ELF3, and report its human and murine cDNA sequences. The mouse cDNA has an alternatively spliced transcript with an extra 60 bp inserted. Hence we present the organization of the murine Elf3 gene together with its exon/intron structure. This gene consists of 9 exons and 8 introns spanning 4.8 kb. ELF3 binds and transactivates ETS sequences and interestingly also shows the ability to bind a GGAT-like purine core, a preferential ETS1/ETS2 type binding site. The expression of ELF3, unlike most other ETS family members, is absent in hematopoietic cells and hematopoietic organs in humans and mice. Intriguingly, the gene is specifically expressed in cell lines of epithelial origin and in organs such as lung, stomach, intestine, kidney that have specialized epithelial cells. We localize the human gene to 1q32.2, a region that is amplified in epithelial tumors of the breast, lung and prostate. Finally, we show that ELF3 expression is increased in a lung carcinoma and adenocarcinoma, as compared to normal tissue. ELF3 is also expressed in cell lines derived from lung cancers. These results suggest that this novel ETS gene may be involved in lung tumorigenesis.

Extent of duplex DNA underwinding induced by RecA protein binding in the presence of ATP.

A method is described for the accurate determination of the superhelical density (omega) of highly underwound circular DNA molecules. Using this method, duplex DNA bound by RecA protein in the presence of ATP at pH 7.5 is found to be underwound by 39.6% (omega = -0.396), corresponding to a helical periodicity of 17.4 base-pairs per turn. The underwinding is increased to 41% (17.9 base-pairs per turn) in the presence of low levels of ATP gamma S, in good agreement with the 18.6 base-pairs per turn reported previously. In spite of the extensive underwinding, the distribution of DNA topoisomers produced by RecA protein binding is small. This indicates a high degree of structural uniformity among RecA-double-stranded DNA complexes in the presence of ATP.

Matroshka and ectopic polymorphisms: Two new classes of DNA sequence variation identified at the Van der Woude syndrome locus on 1q32-q41.

Van der Woude syndrome (VWS) is an orofacial clefting disorder with an autosomal dominant pattern of inheritance. In our efforts to clone the VWS gene, 900 kb of genomic sequence from the VWS candidate region at chromosome 1q32-q41 was analyzed for new DNA sequence variants. We observed that in clone CTA-321i20 a 7922 bp sequence is absent relative to the sequence present in PAC clone RP4-782d21 at positions 1669-9590, suggesting the presence of a deletion/insertion (del/ins) polymorphism. Embedded in this 7922 bp region was a TTCC short tandem repeat (STR). Genotype analysis showed that both the internal STR and the (del/ins) mutation were true polymorphisms. This is a novel example of intraallelic variation, a polymorphism within a polymorphism, and we suggest that it be termed a "Matroshka" polymorphism. Further genetic and DNA sequence analysis indicated that the ancestral state of the 1669-9590 del/ins polymorphism was the insertion allele and that the original deletion mutation probably occurred only once. A second class of novel DNA sequence variation was discovered on chromosome 5 that shared a 328 bp identical sequence with this region on chromosome 1. A single nucleotide polymorphism (SNP) was detected by SSCP using a pair of primers derived from the chromosome 1 sequence. Surprisingly, these primers also amplified the identical locus on chromosome 5, and the SNP was only located on chromosome 5. Since the probe unexpectedly detected alleles from another locus, we suggest that this type of sequence variant be termed an "ectopic" polymorphism. These two novel classes of DNA sequence polymorphisms have the potential to confound genetic and DNA sequence analysis and may also contribute to variation in disease phenotypes.

Complex RNA processing of TDRKH, a novel gene encoding the putative RNA-binding tudor and KH domains.

The sequence from a human EST (IMAGE:259322) with homology to the nucleotide-sensitive chloride conductance regulator (ICln) was used to screen a human aortic cDNA library. The probe sequence was from a region of the EST lacking homology to ICln, and the goal was to isolate an ICln-like gene. A 2843bp cDNA clone with an open reading frame coding for a 561 amino acid protein was isolated. This clone had no homology to ICln. PROSITE analysis of the putative protein sequence reveals one tudor and two K homology (KH) domains. The gene has therefore been named TDRKH. Both KH and tudor motifs are involved in binding to RNA or single-strand DNA. PCR analysis demonstrated that TDRKH is alternatively spliced in several ways and alternatively polyadenylated at multiple sites. Northern analysis confirmed the presence of messages of multiple lengths with predominant bands at 2.8 and 4.0 kb and also demonstrated that TDRKH is widely expressed in human tissues. Within an intron of TDRKH, there is a region with 90% homology to ICln. This sequence, which is incorporated into the alternatively spliced message represented by IMAGE:259322, contains a 2 bp deletion that disrupts the ICln reading frame and therefore represents an ICln pseudogene. The TDRKH gene was mapped to the Epidermal Differentiation Complex (EDC) at chromosome 1q21 by radiation hybrid mapping and STS content of genomic clones from that region. The EDC contains a large cluster of related genes involved in terminal differentiation of the epidermis. It remains to be determined whether TDRKH has a specific role in epithelial function.

Structure and mapping of the human beta-defensin HBD-2 gene and its expression at sites of inflammation.

We cloned a second human beta-defensin gene, HBD-2, and determined its gene structure and expression in inflamed tissue sections. The entire gene spanned about 2 kb with two small exons and one intron. Radiation hybrid studies confirmed the location on chromosome 8p, were consistent with the order HNP-1, HBD-1 and HBD-2, and located HBD-2 as the most centromeric of the genes. By three-color fluorescence in situ hybridization on both free chromatin fiber mapping and interphase mapping, HBD-1, HBD-2 and HNP-1 were mapped to chromosome 8p23. HBD-1 was within 40-100kb of HNP-1, while HBD-2 was about 500-600 kb from HBD-1, with the most likely order HNP-1, HBD-1, HBD-2. The expression of HBD-2 was locally regulated by inflammation. HBD-2 mRNA was markedly increased in the epidermis surrounding inflamed regions, but not detectable in adjacent non-inflamed areas, a distribution that was confirmed at the peptide level by immunostaining with HBD-2 antibody. The HBD-2 gene is the first member of the human defensin family that is locally inducible by inflammation.

Discovery of new human beta-defensins using a genomics-based approach.

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.

Breakpoint of a balanced translocation (X:14) (q27.1;q32.3) in a girl with severe hemophilia B maps proximal to the factor IX gene.

Hemophilia B is an X-linked bleeding disorder caused by the deficiency of coagulation factor (F)IX, with an estimated prevalence of 1 in 30 000 male births. It is almost exclusively seen in males with rare exceptions. We report a girl who was diagnosed with severe (<1%) FIX deficiency at 4 months of age. Cytogenetic studies in the patient showed a balanced translocation between one of the X-chromosomes and chromosome 14, with breakpoints at bands Xq27.1 and 14q32.3. Both parents were found to have normal chromosomes. Late replication studies by incorporation of 5-bromodeoxyuridine showed non-random inactivation of the normal X-chromosome, a phenomenon frequently seen in balanced X/autosome translocations. To map the breakpoint, fluorescent in-situ hybridization was performed. A PAC DNA probe, RP6-88D7 (which contains the FIX gene) hybridized only on the normal chromosome X as well as onto the derivative 14. Using a PAC DNA probe, RP11-963P9 that is located proximal to the FIX gene, we obtained signals on the normal and derivative X and also on the derivative 14. We conclude that the breakpoint is located within the DNA sequence of this clone mapping proximal to the FIX gene. Since the FIX gene seems to be intact in the derivative 14, the breakpoint may affect an upstream regulatory sequence that subjects the gene to position effect variegation (PEV).

Microdeletions at chromosome bands 1q32-q41 as a cause of Van der Woude syndrome.

Van der Woude syndrome (VWS) is an autosomal dominant disorder comprising cleft lip and/or cleft palate and lip pits. We reported previously a family whose underlying mutation is a 500-800 kb deletion localized to chromosome bands 1q32-q41 [Sander et al., 1994: Hum Mol Genet 3:576-578]. Along with cleft lip/palate and lip pits, affected relatives exhibit developmental delays, suggesting that the function of a gene nearby may also be disrupted. To further localize the VWS gene we searched for other deletions that cause VWS. An allele loss assay was performed using a novel highly polymorphic marker, D1S3753. From a panel of 37 unrelated individuals, we detected an allele loss in one family, indicating the presence of a deletion. In this family, the phenotype in three generations of affected individuals was confined to the cardinal signs of VWS. Surprisingly, mapping of the new deletion showed that it extended 0.2-1 Mb beyond the proximal breakpoint for the deletion described previously. No deletions were detected in seven cases of popliteal pterygia syndrome, 76 cases of mixed syndromic forms of cleft lip and palate, and 178 cases of nonsyndromic cleft lip and palate. These observations suggest that genetic searches for microdeletions should be routine in screening patients for causes of VWS and may facilitate the positional cloning efforts of the VWS gene and of a nearby gene or genes that may be involved in brain development.

Improved cytotoxic T-lymphocyte immune responses to a tumor antigen by vaccines co-expressing the SLAM-associated adaptor EAT-2.

The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.

[Van-der-Woude Syndrome]. / Van-der-Woude-Syndrom.

We report on two families with different expression of a Van-der-Woude-Syndrome (VWS) and with proven mutation of the IRF6- gene. The Van-der-Woude syndrome is a rare disease, typically consisting of congenital pits of the lower lip in combination with cleft lip or cleft palate or both. The Van-der-Woude syndrome is an autosomal dominant syndrome with variable expression. The penetrance is between 0,89 and 0,99. It is important to establish the correct diagnosis by careful investigation of patients with cleft lip or cleft palate and their parents. Genetic counselling is recommended in such cases.

Homology-dependent underwinding of duplex DNA in recA protein generated paranemic complexes.

RecA protein promoted formation of paranemic joints, in which a recA-ssDNA complex and a nicked circular dsDNA molecule are homologously aligned without net cross-strand interwinding, is accompanied by extensive underwinding of the dsDNA molecule. When the nick is sealed by DNA ligase, a highly negatively superhelical DNA molecule is formed. This underwinding has the following properties: (a) it occurs within 2 min; (b) it is completely homology dependent; (c) it does not require a homologous free DNA end. The resulting underwound DNA species is comprised of a heterogeneous population of topoisomers. The degree of unwinding exhibits a strong dependence on the fractional length of homology in the dsDNA molecule and indicates that paranemic joints can extend for at least 2900 base pairs. The rapid underwinding associated with paranemic joint formation is followed by a longer phase in which the dsDNA molecule is more extensively underwound.