MAU2 and NIPBL Variants Impair the Heterodimerization of the Cohesin Loader Subunits and Cause Cornelia de Lange Syndrome.

The NIPBL/MAU2 heterodimer loads cohesin onto chromatin. Mutations in NIPBL account for most cases of the rare developmental disorder Cornelia de Lange syndrome (CdLS). Here we report a MAU2 variant causing CdLS, a deletion of seven amino acids that impairs the interaction between MAU2 and the NIPBL N terminus. Investigating this interaction, we discovered that MAU2 and the NIPBL N terminus are largely dispensable for normal cohesin and NIPBL function in cells with a NIPBL early truncating mutation. Despite a predicted fatal outcome of an out-of-frame single nucleotide duplication in NIPBL, engineered in two different cell lines, alternative translation initiation yields a form of NIPBL missing N-terminal residues. This form cannot interact with MAU2, but binds DNA and mediates cohesin loading. Altogether, our work reveals that cohesin loading can occur independently of functional NIPBL/MAU2 complexes and highlights a novel mechanism protective against out-of-frame mutations that is potentially relevant for other genetic conditions.

Functional Characterization of the GUCY1A3 Coronary Artery Disease Risk Locus.

BACKGROUND: A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3, which encodes the α1 subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide/cGMP signaling pathway. The mechanism linking common variants in this region with coronary risk is not known. METHODS: Gene expression and protein expression were analyzed with quantitative polymerase chain reaction and immunoblotting, respectively. Putative allele-specific transcription factors were identified with in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed with reporter gene assays. To assess the effect of zinc finger E box-binding homeobox 1 transcription factor (ZEB1), siRNA-mediated knockdown and overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes was analyzed with vascular smooth muscle cell migration assays and platelet aggregation analyses. RESULTS: Whole-blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the nonrisk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of nonrisk allele promoter activity and a significant reduction of endogenous GUCY1A3 expression. Ex vivo-studied platelets from homozygous nonrisk allele carriers displayed enhanced inhibition of ADP-induced platelet aggregation by the nitric oxide donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil compared with homozygous risk allele carriers. Moreover, pharmacological stimulation of sGC led to reduced migration only in vascular smooth muscle cells homozygous for the nonrisk allele. In the Hybrid Mouse Diversity Panel, higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta. CONCLUSIONS: Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the nonrisk allele, leading to an increase in GUCY1A3 expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.

In-depth Characterization of the Homodimerization Domain of the Transcription Factor THAP1 and Dystonia-Causing Mutations Therein.

Mutations in the THAP1 gene encoding the transcription factor THAP1 have been shown to cause DYT6 dystonia. THAP1 contains a highly conserved THAP zinc finger at its N-terminal region which allows specific binding to its target sequences as well as a coiled-coil domain (amino acids 139-190) towards its C-terminus postulated as a protein-protein-binding motif. While several DYT6-causing mutations within the THAP domain were shown to decrease THAP1 activity in transcriptional regulation and DNA-binding, the role of mutations within the coiled-coil domain is rather unknown. Therefore, assigning a function to this domain may enable functional testing of mutations in this region. Notably, THAP1 and other THAP proteins form homodimers; however, the responsible domain has not been elucidated in detail. We show that the region of amino acids 139-185 is involved in formation of THAP1 homodimers by using yeast-two-hybrid, GST pull-down, and cross-linking assays. Surprisingly, all nine reported DYT6-causing missense mutations within this region had no effect on dimerization of THAP1 in GST pull-down and formaldehyde cross-linking assays. In conclusion, we demonstrated that a region of 47 amino acids is involved in THAP1 homodimerization but mutations in this region seem not to impair this mechanism.

Identification and Functional Characterization of Two Intronic NIPBL Mutations in Two Patients with Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronic NIPBL mutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13)_(-8) deletion. While the loss of exon 28 retains the reading frame of the NIBPL transcript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the total NIPBL mRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.

Hidden mutations in Cornelia de Lange syndrome limitations of sanger sequencing in molecular diagnostics.

Cornelia de Lange syndrome (CdLS) is a well-characterized developmental disorder. The genetic cause of CdLS is a mutation in one of five associated genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) accounting for about 70% of cases. To improve our current molecular diagnostic and to analyze some of CdLS candidate genes, we developed and established a gene panel approach. Because recent data indicate a high frequency of mosaic NIPBL mutations that were not detected by conventional sequencing approaches of blood DNA, we started to collect buccal mucosa (BM) samples of our patients that were negative for mutations in the known CdLS genes. Here, we report the identification of three mosaic NIPBL mutations by our high-coverage gene panel sequencing approach that were undetected by classical Sanger sequencing analysis of BM DNA. All mutations were confirmed by the use of highly sensitive SNaPshot fragment analysis using DNA from BM, urine, and fibroblast samples. In blood samples, we could not detect the respective mutation. Finally, in fibroblast samples from all three patients, Sanger sequencing could identify all the mutations. Thus, our study highlights the need for highly sensitive technologies in molecular diagnostic of CdLS to improve genetic diagnosis and counseling of patients and their families.

THAP1, the gene mutated in DYT6 dystonia, autoregulates its own expression.

THAP1 encodes a transcription factor but its regulation is largely elusive. TOR1A was shown to be repressed by THAP1 in vitro. Notably, mutations in both of these genes lead to dystonia (DYT6 or DYT1). Surprisingly, expressional changes of TOR1A in THAP1 mutation carriers have not been detected indicating additional levels of regulation. Here, we investigated whether THAP1 is able to autoregulate its own expression. Using in-silico prediction, luciferase reporter gene assays, and (quantitative) chromatin immunoprecipitation (ChIP), we defined the THAP1 minimal promoter to a 480bp-fragment and demonstrated specific binding of THAP1 to this region which resulted in repression of the THAP1 promoter. This autoregulation was disturbed by different DYT6-causing mutations. Two mutants (Ser6Phe, Arg13His) were shown to be less stable than wildtype THAP1 adding to the effect of reduced binding to the THAP1 promoter. Overexpressed THAP1 is preferably degraded through the proteasome. Notably, endogenous THAP1 expression was significantly reduced in cells overexpressing wildtype THAP1 as demonstrated by quantitative PCR. In contrast, higher THAP1 levels were detected in induced pluripotent stem cell (iPS)-derived neurons from THAP1 mutation carriers. Thus, we identified a feedback-loop in the regulation of THAP1 expression and demonstrated that mutant THAP1 leads to higher THAP1 expression levels. This compensatory autoregulation may contribute to the mean age at onset in the late teen years or even reduced penetrance in some THAP1 mutation carriers.

Functional characterization of NIPBL physiological splice variants and eight splicing mutations in patients with Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B'. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Functional interaction of osteogenic transcription factors Runx2 and Vdr in transcriptional regulation of Opn during soft tissue calcification.

Loss of Abcc6 gene expression was identified to be responsible for dystrophic calcification of the heart (DCC) or vessels after acute injury in several strains of laboratory mice. This calcification shares features with osteogenesis and may involve osteogenic factors. Tissue expression of osteopontin (Opn) and 11 osteogenic transcription factors were studied in vivo in mouse models for DCC and in vitro using luciferase reporter gene assays. Compared with DCC-resistant C57BL/6 mice, a significant increase in Opn transcription was demonstrated in necrotic lesions of both DCC-susceptible C3H/He and B6.C3H(Dyscalc1) congenic mice at day 3 after injury. Significant increases in gene expression were also demonstrated for the transcription factors runt domain-containing transcription factor 2 (Runx2), vitamin D receptor (Vdr), SRY (sex-determining region Y)-box 9 protein, and Nfkb1 in C3H/He mice versus C57BL/6 controls. However, only Runx2 remained significantly increased in the B6.C3H(Dyscalc1) congenic mice, which carry only the Dyscalc1 locus with functional Abcc6 deletion on a C57BL/6 genetic background. Luciferase assay use increased Opn promoter activity, which was demonstrated after overexpression of Runx2. A poly-T stretch insertion was identified to stabilize the binding of Runx2, thus significantly enhancing Opn promoter activity. This Runx2-mediated activation was further enhanced by cotransfection with Vdr. Our data suggest a key role of Runx2 in the regulation of Opn in a model of cardiovascular calcification and demonstrate a synergistic cooperation of Runx2 and Vdr.

Isolated NIBPL missense mutations that cause Cornelia de Lange syndrome alter MAU2 interaction.

Cornelia de Lange syndrome (CdLS; or Brachmann-de Lange syndrome) is a dominantly inherited congenital malformation disorder with features that include characteristic facies, cognitive delays, growth retardation and limb anomalies. Mutations in nearly 60% of CdLS patients have been identified in NIPBL, which encodes a regulator of the sister chromatid cohesion complex. NIPBL, also known as delangin, is a homolog of yeast and amphibian Scc2 and C. elegans PQN-85. Although the exact mechanism of NIPBL function in sister chromatid cohesion is unclear, in vivo yeast and C. elegans experiments and in vitro vertebrate cell experiments have demonstrated that NIPBL/Scc2 functionally interacts with the MAU2/Scc4 protein to initiate loading of cohesin onto chromatin. To test the significance of this model in the clinical setting of CdLS, we fine-mapped the NIBPL-MAU2 interaction domain and tested the functional significance of missense mutations and variants in NIPBL and MAU2 identified in these minimal domains in a cohort of patients with CdLS. We demonstrate that specific novel mutations at the N-terminus of the MAU2-interacting domain of NIBPL result in markedly reduced MAU2 binding, although we appreciate no consistent clinical difference in the small group of patients with these mutations. These data suggest that factors in addition to MAU2 are essential in determining the clinical features and severity of CdLS.

First pregnancy and life after preimplantation genetic diagnosis by polar body analysis for mucopolysaccharidosis type I.

Preimplantation genetic diagnosis (PGD) may help couples at risk to avoid pregnancies with known genetic diseases. In Germany, the only option to perform PGD is the analysis of polar bodies (PB). Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder. Q70X is one of the frequent diseases causing mutations of alpha-L-iduronidase (IDUA), leading to a severe phenotype with mental retardation and various somatic abnormalities, and making a request for PGD is understandable. Using five polymorphic DNA markers from the vicinity of IDUA, PGD on first PB was performed for a consanguineous couple, both heterozygotes of the Q70X mutation of IDUA. Sixteen first PB were obtained by laser assisted hatching of the zona pellucida. Genotyping led to the conclusion that 3/16 oocytes carried wild-type IDUA alleles. Only one of these oocytes showed pronucleus formation after intracytoplasmic sperm injection and was transferred on day 2 after oocyte retrieval. A singleton pregnancy was established. Prenatal diagnosis showed a fetus heterozygous for Q70X. For MPS I, PB analysis is a feasible way to perform PGD and it may be an acceptable alternative for couples with moral objections to embryo selection, or for countries in which genetic testing of the embryo is prohibited.

Polar body diagnosis for hemophilia a using multiplex PCR for linked polymorphic markers.

Preimplantation genetic diagnosis (PGD) is usually performed on blastomeres. In Germany, the only possibility to perform PGD is by analysis of polar bodies. We performed PGD using polar bodies in a woman who is a carrier of hemophilia A. Multiplex PCR followed by nested fluorescent PCR for five linked polymorphic markers was established. From 11 analyzed polar bodies, only 1 showed alleles linked to the mutation. The corresponding oocyte was transferred and no pregnancy was established. As seen in other investigations, the rate of heterozygous first polar bodies is surprisingly high.