Arie van Haeringen

Heterozygous Germline Mutations in the p53 Homolog p63 Are the Cause of EEC Syndrome

EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63alpha, but not p63beta and p63gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.

Mutations in SWI/SNF chromatin remodeling complex gene ARID1B cause Coffin-Siris syndrome

We identified de novo truncating mutations in ARID1B in three individuals with Coffin-Siris syndrome (CSS) by exome sequencing. Array-based copy-number variation (CNV) analysis in 2,000 individuals with intellectual disability revealed deletions encompassing ARID1B in 3 subjects with phenotypes partially overlapping that of CSS. Taken together with published data, these results indicate that haploinsufficiency of the ARID1B gene, which encodes an epigenetic modifier of chromatin structure, is an important cause of CSS and is potentially a common cause of intellectual disability and speech impairment.

Germline deletion of the miR-17 ∼ 92 cluster causes skeletal and growth defects in humans

MicroRNAs (miRNAs) are key regulators of gene expression in animals and plants. Studies in a variety of model organisms show that miRNAs modulate developmental processes. To our knowledge, the only hereditary condition known to be caused by a miRNA is a form of adult-onset non-syndromic deafness, and no miRNA mutation has yet been found to be responsible for any developmental defect in humans. Here we report the identification of germline hemizygous deletions of MIR17HG, encoding the miR-17∼92 polycistronic miRNA cluster, in individuals with microcephaly, short stature and digital abnormalities. We demonstrate that haploinsufficiency of miR-17∼92 is responsible for these developmental abnormalities by showing that mice harboring targeted deletion of the miR-17∼92 cluster phenocopy several key features of the affected humans. These findings identify a regulatory function for miR-17∼92 in growth and skeletal development and represent the first example of an miRNA gene responsible for a syndromic developmental defect in humans.

Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome.

We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both alleles of POLR1C in three individuals with TCS. These findings identify two additional genes involved in TCS, confirm the genetic heterogeneity of TCS and support the hypothesis that TCS is a ribosomopathy.

MLL2 mutation spectrum in 45 patients with Kabuki syndrome

Kabuki Syndrome (KS) is a rare syndrome characterized by intellectual disability and multiple congenital abnormalities, in particular a distinct dysmorphic facial appearance. KS is caused by mutations in the MLL2 gene, encoding an H3K4 histone methyl transferase which acts as an epigenetic transcriptional activator during growth and development. Direct sequencing of all 54 exons of the MLL2 gene in 45 clinically well‐defined KS patients identified 34 (75.6%) different mutations. One mutation has been described previously, all others are novel. Clinically, all KS patients were sporadic, and mutations were de novo for all 27 families for which both parents were available. We detected nonsense (n=11), frameshift (n=17), splice site (n=4) and missense (n=2) mutations, predicting a high frequency of absent or non‐functional MLL2 protein. Interestingly, both missense mutations located in the C‐terminal conserved functional domains of the protein. Phenotypically our study indicated a statistically significant difference in the presence of a distinct facial appearance (p=0.0143) and growth retardation (p=0.0040) when comparing KS patients with an MLL2 mutation compared to patients without a mutation. Our data double the number of MLL2 mutations in KS reported so far and widen the spectrum of MLL2 mutations and disease mechanisms in KS.

Craniosynostosis and multiple skeletal anomalies in humans and zebrafish result from a defect in the localized degradation of retinoic acid.

Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency.

Distinct Effects of Allelic NFIX Mutations on Nonsense-Mediated mRNA Decay Engender Either a Sotos-like or a Marshall-Smith Syndrome

By using a combination of array comparative genomic hybridization and a candidate gene approach, we identified nuclear factor I/X (NFIX) deletions or nonsense mutation in three sporadic cases of a Sotos-like overgrowth syndrome with advanced bone age, macrocephaly, developmental delay, scoliosis, and unusual facies. Unlike the aforementioned human syndrome, Nfix-deficient mice are unable to gain weight and die in the first 3 postnatal weeks, while they also present with a spinal deformation and decreased bone mineralization. These features prompted us to consider NFIX as a candidate gene for Marshall-Smith syndrome (MSS), a severe malformation syndrome characterized by failure to thrive, respiratory insufficiency, accelerated osseous maturation, kyphoscoliosis, osteopenia, and unusual facies. Distinct frameshift and splice NFIX mutations that escaped nonsense-mediated mRNA decay (NMD) were identified in nine MSS subjects. NFIX belongs to the Nuclear factor one (NFI) family of transcription factors, but its specific function is presently unknown. We demonstrate that NFIX is normally expressed prenatally during human brain development and skeletogenesis. These findings demonstrate that allelic NFIX mutations trigger distinct phenotypes, depending specifically on their impact on NMD.

Coffin-siris syndrome and the BAF complex: Genotype-phenotype study in 63 patients

De novo germline variants in several components of the SWI/SNF‐like BAF complex can cause Coffin–Siris syndrome (CSS), Nicolaides–Baraitser syndrome (NCBRS), and nonsyndromic intellectual disability. We screened 63 patients with a clinical diagnosis of CSS for these genes (ARID1A, ARID1B, SMARCA2, SMARCA4, SMARCB1, and SMARCE1) and identified pathogenic variants in 45 (71%) patients. We found a high proportion of variants in ARID1B (68%). All four pathogenic variants in ARID1A appeared to be mosaic. By using all variants from the Exome Variant Server as test data, we were able to classify variants in ARID1A, ARID1B, and SMARCB1 reliably as being pathogenic or nonpathogenic. For SMARCA2, SMARCA4, and SMARCE1 several variants in the EVS remained unclassified, underlining the importance of parental testing. We have entered all variant and clinical information in LOVD‐powered databases to facilitate further genotype–phenotype correlations, as these will become increasingly important because of the uptake of targeted and untargeted next generation sequencing in diagnostics. The emerging phenotype–genotype correlation is that SMARCB1 patients have the most marked physical phenotype and severe cognitive and growth delay. The variability in phenotype seems most marked in ARID1A and ARID1B patients. Distal limbs anomalies are most marked in ARID1A patients and least in SMARCB1 patients. Numbers are small however, and larger series are needed to confirm this correlation.

A new diagnostic workflow for patients with mental retardation and/or multiple congenital abnormalities: test arrays first

High-density single-nucleotide polymorphism (SNP) genotyping technology enables extensive genotyping as well as the detection of increasingly smaller chromosomal aberrations. In this study, we assess molecular karyotyping as first-round analysis of patients with mental retardation and/or multiple congenital abnormalities (MR/MCA). We used different commercially available SNP array platforms, the Affymetrix GeneChip 262K NspI, the Genechip 238K StyI, the Illumina HumanHap 300 and HumanCNV 370 BeadChip, to detect copy number variants (CNVs) in 318 patients with unexplained MR/MCA. We found abnormalities in 22.6% of the patients, including six CNVs that overlap known microdeletion/duplication syndromes, eight CNVs that overlap recently described syndromes, 63 potentially pathogenic CNVs (in 52 patients), four large segments of homozygosity and two mosaic trisomies for an entire chromosome. This study shows that high-density SNP array analysis reveals a much higher diagnostic yield as that of conventional karyotyping. SNP arrays have the potential to detect CNVs, mosaics, uniparental disomies and loss of heterozygosity in one experiment. We, therefore, propose a novel diagnostic approach to all MR/MCA patients by first analyzing every patient with an SNP array instead of conventional karyotyping.

Exclusion of the dysplastic nevus syndrome (DNS) locus from the short arm of chromosome 1 by linkage studies in Dutch families.

Familial dysplastic nevus syndrome (DNS) is an autosomal dominant premalignant condition characterized by multiple large moles of variable size and color and a strongly increased risk for cutaneous malignant melanoma. In order to determine the chromosomal localization of the DNS gene, linkage studies were initiated in six large Dutch families. No support was obtained for linkage between the loci for DNS and the rhesus blood group on chromosome 1. Data from additional markers (DNF15S1, D1Z2, FUCA1, D1S17, D1S57, and PGM1) make it possible to exclude the DNS gene from the short arm of chromosome 1 in these Dutch families.