X-linked CHARGE-like Abruzzo-Erickson syndrome and classic cleft palate with ankyloglossia result from TBX22 splicing mutations.

X-linked cleft palate (CPX) is caused by mutations in the gene encoding the TBX22 transcription factor and is known to exhibit phenotypic variability, usually involving either a complete, partial or submucous cleft palate, with or without ankyloglossia. This study hypothesized a possible involvement of TBX22 in a family with X-linked, CHARGE-like Abruzzo-Erickson syndrome, of unknown etiology. The phenotype extends to additional features including sensorineural deafness and coloboma, which are suggested by the Tbx22 developmental expression pattern but not previously associated in CPX patients. A novel TBX22 splice acceptor mutation (c.593-5T>A) was identified that tracked with the phenotype in this family. A novel splice donor variant (c.767+5G>A) and a known canonical splice donor mutation (c.767+1G>A) affecting the same exon were identified in patients with classic CPX phenotypes and were comparatively analyzed using both in silico and in vitro splicing studies. All three variants were predicted to abolish normal mRNA splicing and an in vitro assay indicated that use of alternative splice sites was a likely outcome. Collectively, the data showed the functional effect of several novel intronic splice site variants but most importantly confirms that TBX22 is the gene underlying Abruzzo-Erickson syndrome, expanding the phenotypic spectrum of TBX22 mutations.

A functional haplotype variant in the TBX22 promoter is associated with cleft palate and ankyloglossia.

BACKGROUND: Mutations in the T-box transcription factor gene TBX22 are found in patients with X-linked cleft palate and ankyloglossia (CPX), and are reported in approximately 5% of all non-syndromic cleft palate patients. Clinical variability in CPX ranges from a mild or occult submucous cleft palate to a severe, complete cleft of the secondary palate. AIMS: To explore the possibility that mutations lying outside of the TBX22 coding region might contribute to the phenotype, a non-coding upstream exon and its upstream regulatory region were investigated. METHODS AND RESULTS: We sequenced 137 patients with cleft palate without coding region mutations and 295 controls. While no unique mutations were identified, seven single nucleotide polymorphisms (SNPs) were noted. These variants segregate into four distinct haplotypes. Individually, two of the SNPs associate significantly with cleft palate, as does the haplotype containing the rare allele of both SNPs. Analysis of the patient cohorts stratified for the presence of ankyloglossia significantly increases these associations. Reporter assays were used to analyse each of these haplotypes and the impact of individual SNPs. An important functional role for rs41307258 results in a decreased promoter activity of up to 50%. CONCLUSIONS: CPX-like patients harbouring this promoter haplotype are therefore associated with decreased TBX22 transcriptional activity. The risk haplotype, in concert with additional genetic and/or environmental factors, may contribute to the phenotypic variation observed and provide a novel causative mechanism for cleft palate, especially in patients with ankyloglossia.

Disruption of FOXF2 as a Likely Cause of Absent Uvula in an Egyptian Family.

This study investigated the genetic basis of an unusual autosomal dominant phenotype characterized by familial absent uvula, with a short posterior border of the soft palate, abnormal tonsillar pillars, and velopharyngeal insufficiency. Cytogenetic analysis and single-nucleotide polymorphism-based linkage analysis were investigated in a 4-generation family with 8 affected individuals. Whole exome sequencing data were overlaid, and segregation analysis identified a single missense variant, p.Q433P in the FOXF2 transcription factor, that fully segregated with the phenotype. This was found to be in linkage disequilibrium with a small 6p25.3 tandem duplication affecting FOXC1 and GMDS. Notably, the copy number imbalances of this region are commonly associated with pathologies that are not present in this family. Bioinformatic predictions with luciferase reporter studies of the FOXF2 missense variant indicated a negative impact, affecting both protein stability and transcriptional activation. Foxf 2 is expressed in the posterior mouse palate, and knockout animals develop an overt cleft palate. Since mice naturally lack the structural equivalent of the uvula, we demonstrated FOXF2 expression in the developing human uvula. Decipher also records 2 individuals with hypoplastic or bifid uvulae with copy number variants affecting FOXF2. Nevertheless, given cosegregation with the 6p25.3 duplications, we cannot rule out a combined effect of these gains and the missense variant on FOXF2 function, which may account for the rare palate phenotype observed.

Extracraniofacial anomalies in craniofacial microsomia: retrospective analysis of 991 patients.

Craniofacial microsomia (CFM) is characterized by unilateral or bilateral underdevelopment of the facial structures arising from the first and second pharyngeal arches, but extracraniofacial anomalies may also be present. This retrospective study provides an overview of the prevalence, types, and characteristics of extracraniofacial anomalies in patients with CFM. All patients diagnosed with CFM seen at four craniofacial centres were included. The patient charts were reviewed and data on patient characteristics and extracraniofacial anomalies were extracted. Of the 991 patients included, 462 (47%) had extracraniofacial anomalies. The prevalence of extracraniofacial anomalies in the various tracts was as follows: vertebral 28%, central nervous system 11%, circulatory system 21%, respiratory tract 3%, gastrointestinal tract 9%, and urogenital tract 11%. Compared to patients without extracraniofacial anomalies, those with an extracraniofacial anomaly were at higher risk of having additional extracraniofacial anomalies in other tracts. The prevalence of extracraniofacial anomalies was greater in patients with bilateral CFM, a more severe mandibular deformity, or facial nerve or soft tissue deformity. Patients with CFM should be screened for extracraniofacial anomalies by physical examination with specific attention to the circulatory, renal, and neurological tracts. Diagnostically, electrocardiography, echocardiography, spine radiography, and renal ultrasound should be performed for patients at risk of extracraniofacial anomalies.

Characterizing the skull base in craniofacial microsomia using principal component analysis.

The aim of this study was to compare the anatomical differences in the skull base between the affected and non-affected side in patients with craniofacial microsomia (CFM), and to compare the affected and non-affected sides with measurements from a normal population. Three-dimensional computed tomography scans of 13 patients with unilateral CFM and 19 normal patients (age range 7-12 years) were marked manually with reliable homologous landmarks. Principal component analysis (PCA), as part of a point distribution model (PDM), was used to analyse the variability within the normal and preoperative CFM patient groups. Through analysis of the differences in the principal components calculated for the two groups, a model was created to describe the differences between CFM patients and normal age-matched controls. The PDMs were also used to describe the shape changes in the skull base between the cohorts and validated this model. Using thin-plate splines as a means of interpolation, videos were created to visualize the transformation from CFM skull to normal skull, and to display the variability in shape changes within the groups themselves. In CFM cases, the skull base showed significant asymmetry. Anatomical areas around the glenoid fossa and mastoid process showed the most asymmetry and restriction of growth, suggesting a pathology involving the first and second pharyngeal arches.

Heterogeneity in polyadenylation cleavage sites in mammalian mRNA sequences: implications for SAGE analysis.

The analysis of a human thyroid serial analysis of gene expression (SAGE) library shows the presence of an abundant SAGE tag corresponding to the mRNA of thyroglobulin (TG). Additional, less abundant tags are present that can not be linked to any other known gene, but show considerable homology to the wild-type TG tag. To determine whether these tags represent TG mRNA molecules with alternative cleavage, 3'-RACE clones were sequenced. The results show that the three putative TG SAGE tags can be attributed to TG transcripts and reflect the use of alternative polyadenylation cleavage sites downstream of a single polyadenylation signal in vivo. By screening more than 300 000 sequences corresponding to human, mouse and rat transcripts for this phenomenon we show that a considerable percentage of mRNA transcripts (44% human, 22% mouse and 22% rat) show cleavage site heterogeneity. When analyzing SAGE-generated expression data, this phenomenon should be considered, since, according to our calculations, 2.8% of human transcripts show two or more different SAGE tags corresponding to a single gene because of alternative cleavage site selection. Both experimental and in silico data show that the selection of the specific cleavage site for poly(A) addition using a given polyadenylation signal is more variable than was previously thought.

Genes differentially expressed in medulloblastoma and fetal brain.

Serial analysis of gene expression (SAGE) was used to identify genes that might be involved in the development or growth of medulloblastoma, a childhood brain tumor. Sequence tags from medulloblastoma (10229) and fetal brain (10692) were determined. The distributions of sequence tags in each population were compared, and for each sequence tag, pairwise chi2 test statistics were calculated. Northern blot was used to confirm some of the results obtained by SAGE. For 16 tags, the chi2 test statistic was associated with a P value < 10(-4). Among those transcripts with a higher expression in medulloblastoma were the genes for ZIC1 protein and the OTX2 gene, both of which are expressed in the cerebellar germinal layers. The high expression of these two genes strongly supports the hypothesis that medulloblastoma arises from the germinal layer of the cerebellum. This analysis shows that SAGE can be used as a rapid differential screening procedure.

Serial analysis of gene expression as a tool to assess the human thyroid expression profile and to identify novel thyroidal genes.

The assessment of the expression profile of normal human thyroid tissue using serial analysis of gene expression (SAGE) generated a collection of 10,994 sequence transcripts (tags). Each tag represented a messenger RNA transcript, and, in total, 6099 different tags could be distinguished. The presence and abundance of thyroid-specific transcripts showed the overall expression profile to be from a normal thyroid cell. The expression level of several transcripts was confirmed on Northern blot. Seventy percent of tags could not be attributed to a known human gene and, therefore, possibly correspond to novel genes putatively involved in thyroid function. The tag sequence generated by the SAGE technique can be used to further characterize these novel genes. In this way, application of the SAGE technique to thyroid tissue gives insight in the expression profile of a normal thyroid gland and provides the information to characterize novel genes involved in thyroid pathology, such as congenital hypothyroidism and thyroid neoplasia.

Up to date with human thyroglobulin.

The coding region of the human thyroglobulin (TG) mRNA has been resequenced, and comparison with the TG sequence originally published in 1987 showed many variations. All of the variations were validated in 20--40 other alleles, and this resulted in the revision of 41 nucleotide positions. This review presents the revised wild-type human TG sequence, including all known exon/exon boundaries and additional data on the TG mRNA population, concerning alternative splicing and variability of the polyadenylation cleavage site. The amino acid sequence derived shows one additional, 12 changed, and 10 polymorphic residues. Protein characteristics, such as acceptor and donor tyrosine residues, N-glycosylation sites, cysteine-rich repeats, the proposed receptor domain, and antigenic epitopes, are included, and their relationship to the revised sequence is discussed. Furthermore, all reported TG mutations causing dyshormonogenesis in humans and animals are designated in the nucleotide and amino acid sequences. This up-to-date profile of the human TG molecule presents the features of importance for its complex role in thyroid hormonogenesis, and is the basis for future studies on the structure--function relationship.

The revised 8307 base pair coding sequence of human thyroglobulin transiently expressed in eukaryotic cells.

We developed a transient transfection system for human thyroglobulin (TG) cDNA in both human thyroid cells and in COS-1 cells. Four overlapping TG cDNA fragments were amplified by reverse transcription-PCR from RNA of normal thyroid tissue. The most 5' fragment includes the natural translation initiation site and the sequence encoding the signal peptide (SP). After subcloning, the nucleotide sequence was determined and compared with the published human sequence, resulting in the detection of 30 nucleotide variations. For validation purposes, all variations were screened in 6-12 normal human alleles. Twenty-one were present in all screened alleles and have to be revised in the published nucleotide sequence. Since one variation concerns a triplet insertion, the coding sequence of the mature human thyroglobulin is 8307 nucleotides encoding 2750 amino acids. The TG cDNA constructs were transiently transfected in HTori 3 and COS-1 cells and protein expression was detected using a polyclonal anti-human-TG on fixed cells and after SDS-PAGE. In both cell-lines all four TG protein fragments were expressed. The mannose structures detected on the proteins by lectins and localization after expression in the cells suggest that only the N-terminal TG fragment (containing the SP) is directed to the endoplasmatic reticulum but is unable to reach the Golgi complex. The described expression system in human thyrocytes will be a helpful tool in studying the structure-function relationship of human TG in thyroid hormonogenesis.

Cleft lip with cleft palate, ankyloglossia, and hypodontia are associated with TBX22 mutations.

X-linked cleft palate and ankyloglossia (CPX) are caused by mutations in the TBX22 transcription factor. To investigate whether patients with ankyloglossia alone or in the presence of other craniofacial features including hypodontia or CLP might be caused by TBX22 mutations, we analyzed 45 Thai patients with isolated ankyloglossia, 2 unusual CPA families, and 282 non-syndromic Thai and UK patients with CLP. Five putative missense mutations were identified, including 3 located in the T-box binding domain (R120Q, R126W, and R151L) that affects DNA binding and/or transcriptional repression. The 2 novel C-terminal mutations, P389Q and S400Y, did not affect TBX22 activity. Mutations R120Q and P389Q were identified in patients with ankyloglossia only, while R126W and R151L were present in families that included CLP. Several individuals in these families were also found to have micro/hypodontia. This study has expanded the phenotypic spectrum of TBX22-related mutations to include dental anomalies and cleft lip.

Absence of activating mutations in ras and gsp oncogenes in a cohort of nine patients with sporadic pediatric thyroid tumors.

BACKGROUND: Characterization of the genetic background of pediatric thyroid carcinomas could aid in distinguishing between differently staged tumors with respect to treatment and prognosis. Two known genetic factors associated with thyroid carcinoma, the proto-oncogenes gsp and ras were investigated. PROCEDURE: DNA was extracted from paraffin sections from both tumor and normal thyroid tissue of nine patients (ages 9-16 years). Of these patients, eight were diagnosed with papillary carcinoma and one with follicular adenoma. The coding exons of gsp and the three known ras genes (H, K, and N-ras) were screened for mutations using SSCP-analysis. RESULTS: There were no mutations present in the ras and gsp proto-oncogenes hot spots, however, LOH of H-ras (chromosome location 11p15.5) was found in tumor tissue from one patient and a homozygous mutation in exon 12 of gsp causing a Pro-->Ser conversion was present in the thyroid tumor tissue from another patient. Two silent polymorphisms were detected, H-ras exon1, 86T-->C and gsp exon 5, 81T-->C. CONCLUSIONS: Our results indicate that the ras/gsp mutations found are probably late events in the tumorigenesis representing general oncogenic stress. In conclusion, it seems that ras/gsp activation is not a factor in the mechanism causing sporadic thyroid carcinoma in children.

Cloning of tissue-specific genes using serial analysis of gene expression and a novel computational substraction approach.

A paradigm of molecular medicine is the identification of functionally specialized genes in the search of defects responsible for human disease. To identify novel genes relevant for thyroid physiology, we applied serial analysis of gene expression (SAGE) and identified 4260 tag sequences that did not match any known gene present in the GenBank database ("no-match" tags). These no-match tags represent still uncharacterized transcripts. Most of them are expected to correspond to housekeeping genes and only a few to genes with a tissue-restricted pattern of expression. To pinpoint the best candidates for tissue-specificity in a large series of tags, we used a computer-based approach. We compared the relative abundance of 80 no match tags in our thyroid SAGE library with the expression level in 14 other SAGE libraries derived from 9 different human tissues. Based on the expression data, we developed the "tissue preferential expression" (TPE) algorithm to discriminate tags expressed specifically in the thyroid. We then selected four tags as preferentially expressed in thyroid. Results were validated by RT-PCR and northern blot on multiple-tissue RNA samples. Finally, the screening of a thyroid cDNA library with expressed sequence tag (EST) sequences related to the selected tags allowed the isolation of four novel thyroid-specific cDNAs. We demonstrate that the computational substraction of SAGE tags by the proposed TPE algorithm is a rapid and reliable way to expedite the cloning of tissue-specific genes through the combined use of SAGE and EST databases.

USAGE: a web-based approach towards the analysis of SAGE data. Serial Analysis of Gene Expression.

MOTIVATION: SAGE enables the determination of genome-wide mRNA expression profiles. A comprehensive analysis of SAGE data requires software, which integrates (statistical) data analysis methods with a database system. Furthermore, to facilitate data sharing between users, the application should reside on a central server and be accessed via the internet. Since such an application was not available we developed the USAGE package. RESULTS: USAGE is a web-based application that comprises an integrated set of tools, which offers many functions for analysing and comparing SAGE data. Additionally, USAGE includes a statistical method for the planning of new SAGE experiments. USAGE is available in a multi-user environment giving users the option of sharing data. USAGE is interfaced to a relational database to store data and analysis results. The USAGE query editor allows the composition of queries for searching this database. Several database functions have been included which enable the selection and combination of data. USAGE provides the biologist increased functionality and flexibility for analysing SAGE data. AVAILABILITY: USAGE is freely accessible for academic institutions at http://www.cmbi.kun.nl/usage/. The source code of USAGE is freely available for academic institutions on request from the first author.