Dominant mutations in GRHL3 cause Van der Woude Syndrome and disrupt oral periderm development.

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6(+/-);Grhl3(+/-)) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.

Submicroscopic genomic alterations in Silver-Russell syndrome and Silver-Russell-like patients.

BACKGROUND: Silver-Russell syndrome (SRS, OMIM 180860) features fetal and postnatal growth restriction and variable dysmorphisms. Genetic and epigenetic aberrations on chromosomes 7 and 11 are commonly found in SRS. However, a large fraction of SRS cases remain with unknown genetic aetiology. METHODS: 22 patients with a diagnosis of SRS (10 with H19 hypomethylation and 12 of unknown molecular aetiology) and their parents were studied with the Affymetrix 250K Sty microarray. Several analytical approaches were used to identify genomic aberrations such as copy number changes (CNCs), loss of heterozygosity (LOH) and uniparental disomy (UPD). Selected CNCs were verified with quantitative real-time PCR. RESULTS: The largest unambiguous CNCs were found in patients with previously molecularly unexplained SRS with relatively mild phenotypes: a heterozygous deletion of chromosome 15q26.3 including the IGF1R gene (2.6 Mb), an atypical distal 22q11.2 deletion (1.1 Mb), and a pseudoautosomal region duplication (2.7 Mb) in a male patient. LOH regions of potential relevance to the SRS phenotype were also identified. Importantly, no duplications or UPD of chromosomes 7 or 11 were identified. CONCLUSION: Unexpected submicroscopic genomic events with pathogenic potential were found in three patients with molecularly unexplained SRS that was mild. The findings emphasise that SRS is heterogeneous in genetic aetiology beyond the major groups of H19 hypomethylation and maternal UPD7 and that unbiased genome-scale screens may reveal novel genotype-phenotype correlations.

Novel and recurrent STAT3 mutations in hyper-IgE syndrome patients from different ethnic groups.

We performed clinical, immunological and genetic studies of 12 hyper-IgE syndrome (HIES) patients from 4 Hungarian, 2 Lebanese, one Russian, one Polish, and one Swedish families with autosomal dominant (AD) or sporadic forms of the disease to reveal cross-ethnicity of recurrent and novel mutations in the signal transducer and activator of transcription-3 gene (STAT3). Four patients from 3 Hungarian families, and one Russian, and one Swedish patient carried the heterozygous R382W germline mutation at the DNA-binding site of STAT3. The recurrent V637M mutation affecting the SRC homology 2 (SH2) domain was detected in one Lebanese and one Polish family, and the V463del deletion located in the DNA-binding domain was unveiled in another Lebanese family. A novel H332Y mutation affecting the DNA-binding site of STAT3 in three Hungarian patients from a Gypsy family was also found. The segregation of this mutation with HIES, restriction fragment length polymorphism analysis of STAT3 from patients and controls and the negligible production upon IL-6 stimulation of monocyte chemotactic protein-1 by the patient's blood mononuclear cells suggested that the H332Y mutation was disease-causing. These data suggest, that dominant negative mutations of the DNA-binding and SH2 domains of STAT3 cause AD and sporadic cases of HIES in different ethnic groups with R382W as the predominant mutation found in 5 of the 9 families. Functional and genetic data support that the novel H332Y mutation may result in the loss of function of STAT3 and leads to the HIES phenotype.

Novel and de novo mutations of the IRF6 gene detected in patients with Van der Woude or popliteal pterygium syndrome.

The interferon regulatory factor 6 gene (IRF6) has been identified as the major Van der Woude (VWS) syndrome and popliteal pterygium (PPS) syndrome gene with mutations in the majority of the kindreds. We have studied altogether 17 kindreds from Sweden, Finland, Norway, Thailand and Singapore, and report here 10 mutations, six of them previously unseen. In two kindreds, we could document de novo mutations, both of them changing a codon for a glutamine residue to a stop. No mutation could be detected in the four VWS kindreds from Finland, suggesting a founder effect for a mutation in an atypical noncoding position. Our findings demonstrate that several distinct mutations occur in the Swedish population, and confirm the general notion of a broad spectrum of IRF6 mutations underlying the VWS/PPS phenotypes.

Genome-wide analysis of single nucleotide polymorphisms uncovers population structure in Northern Europe.

BACKGROUND: Genome-wide data provide a powerful tool for inferring patterns of genetic variation and structure of human populations. PRINCIPAL FINDINGS: In this study, we analysed almost 250,000 SNPs from a total of 945 samples from Eastern and Western Finland, Sweden, Northern Germany and Great Britain complemented with HapMap data. Small but statistically significant differences were observed between the European populations (F(ST) = 0.0040, p<10(-4)), also between Eastern and Western Finland (F(ST) = 0.0032, p<10(-3)). The latter indicated the existence of a relatively strong autosomal substructure within the country, similar to that observed earlier with smaller numbers of markers. The Germans and British were less differentiated than the Swedes, Western Finns and especially the Eastern Finns who also showed other signs of genetic drift. This is likely caused by the later founding of the northern populations, together with subsequent founder and bottleneck effects, and a smaller population size. Furthermore, our data suggest a small eastern contribution among the Finns, consistent with the historical and linguistic background of the population. SIGNIFICANCE: Our results warn against a priori assumptions of homogeneity among Finns and other seemingly isolated populations. Thus, in association studies in such populations, additional caution for population structure may be necessary. Our results illustrate that population history is often important for patterns of genetic variation, and that the analysis of hundreds of thousands of SNPs provides high resolution also for population genetics.

A locus on 2p12 containing the co-regulated MRPL19 and C2ORF3 genes is associated to dyslexia.

DYX3, a locus for dyslexia, resides on chromosome 2p11-p15. We have refined its location on 2p12 to a 157 kb region in two rounds of linkage disequilibrium (LD) mapping in a set of Finnish families. The observed association was replicated in an independent set of 251 German families. Two overlapping risk haplotypes spanning 16 kb were identified in both sample sets separately as well as in a joint analysis. In the German sample set, the odds ratio for the most significantly associated haplotype increased with dyslexia severity from 2.2 to 5.2. The risk haplotypes are located in an intergenic region between FLJ13391 and MRPL19/C2ORF3. As no novel genes could be cloned from this region, we hypothesized that the risk haplotypes might affect long-distance regulatory elements and characterized the three known genes. MRPL19 and C2ORF3 are in strong LD and were highly co-expressed across a panel of tissues from regions of adult human brain. The expression of MRPL19 and C2ORF3, but not FLJ13391, were also correlated with the four dyslexia candidate genes identified so far (DYX1C1, ROBO1, DCDC2 and KIAA0319). Although several non-synonymous changes were identified in MRPL19 and C2ORF3, none of them significantly associated with dyslexia. However, heterozygous carriers of the risk haplotype showed significantly attenuated expression of both MRPL19 and C2ORF3, as compared with non-carriers. Analysis of C2ORF3 orthologues in four non-human primates suggested different evolutionary rates for primates when compared with the out-group. In conclusion, our data support MRPL19 and C2ORF3 as candidate susceptibility genes for DYX3.

Strong conservation of the human NF2 locus based on sequence comparison in five species.

We analyzed 137 kb covering human neurofibromatosis 2 ( NF2) tumor suppressor locus and orthologous loci from baboon, mouse, rat, and pufferfish Takifugu rubripes. A predominant feature of human-rodent conservation is a very similar distribution of conserved islands, regarding length, position, and degree of identity. By use of a threshold of 75% identity over > or =100 bp of gap-free alignment, comparisons of human-mouse sequences resulted in 3.58% for extra-exonic conservation, which can be compared to 4.5% of exonic sequence content within the human locus. We identified a duplication of neurofibromin 2 in pufferfish, which resulted in two putative proteins with 74% and 76% identity to the human protein. One distinct island (called inter 1), conserved between all analyzed species, was located between promoters of the NIPSNAP1 and NF2 genes. Inter 1 might represent a novel regulatory element, important for the function of this locus. The high level of intronic conservation in the NF2 locus suggests that a number of unknown regulatory elements might exist within this gene. These elements could be affected by disease-causing mutations in NF2 patients and NF2-associated tumors. Alternatively, this conservation might be explained by presence of not yet characterized transcriptional unit(s) within this locus.

A full-coverage, high-resolution human chromosome 22 genomic microarray for clinical and research applications.

We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.