Katarina Dathe

Duplications Involving a Conserved Regulatory Element Downstream of BMP2 Are Associated with Brachydactyly Type A2

Autosomal-dominant brachydactyly type A2 (BDA2), a limb malformation characterized by hypoplastic middle phalanges of the second and fifth fingers, has been shown to be due to mutations in the Bone morphogenetic protein receptor 1B (BMPR1B) or in its ligand Growth and differentiation factor 5 (GDF5). A linkage analysis performed in a mutation-negative family identified a novel locus for BDA2 on chromosome 20p12.3 that incorporates the gene for Bone morphogenetic protein 2 (BMP2). No point mutation was identified in BMP2, so a high-density array CGH analysis covering the critical interval of approximately 1.3 Mb was performed. A microduplication of approximately 5.5 kb in a noncoding sequence approximately 110 kb downstream of BMP2 was detected. Screening of other patients by qPCR revealed a similar duplication in a second family. The duplicated region contains evolutionary highly conserved sequences suggestive of a long-range regulator. By using a transgenic mouse model we can show that this sequence is able to drive expression of a X-Gal reporter construct in the limbs. The almost complete overlap with endogenous Bmp2 expression indicates that a limb-specific enhancer of Bmp2 is located within the identified duplication. Our results reveal an additional functional mechanism for the pathogenesis of BDA2, which is duplication of a regulatory element that affects the expression of BMP2 in the developing limb.

Deletion and Point Mutations of PTHLH Cause Brachydactyly Type E

Autosomal-dominant brachydactyly type E (BDE) is a congenital limb malformation characterized by small hands and feet predominantly as a result of shortened metacarpals and metatarsals. In a large pedigree with BDE, short stature, and learning disabilities, we detected a microdeletion of approximately 900 kb encompassing PTHLH, the gene coding for parathyroid hormone related protein (PTHRP). PTHRP is known to regulate the balance between chondrocyte proliferation and the onset of hypertrophic differentiation during endochondral bone development. Inactivation of Pthrp in mice results in short-limbed dwarfism because of premature differentiation of chondrocyte. On the basis of our initial finding, we tested further individuals with BDE and short stature for mutations in PTHLH. We identified two missense (L44P and L60P), a nonstop (X178WextX( *)54), and a nonsense (K120X) mutation. The missense mutation L60P was tested in chicken micromass culture with the replication-competent avian sarcoma leukosis virus retroviral expression system and was shown to result in a loss of function. Thus, loss-of-function mutations in PTHLH cause BDE with short stature.

Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5W414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.

Molecular analysis of two novel missense mutations in the GDF5 proregion that reduce protein activity and are associated with brachydactyly type C

Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5(T201P) and proGDF5(L263P) revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFβ1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.

Drug safety in pregnancy: the German Embryotox institute

Since 1988, the German Embryotox institute combines individual counselling of pregnant women and their health care providers (HCP) with research on drug safety in pregnancy. In addition, Embryotox offers web-based information which covers the most important and most frequently requested pharmaceutical substances. In contrast to ready-made drug risk information in package leaflets and other product information, individual counselling considers different clinical settings such as (1) looking for a drug of choice or planning pregnancy under medication, (2) risk assessment of a particular drug that has already been taken during an (unplanned) pregnancy and (3) evaluation of an adverse pregnancy outcome in association with a particular medication. Using the three established developmental toxicants valproic acid, isotretinoin, and renin-angiotensin-aldosterone system (RAAS) inhibitors as an example, the need of detailed information is illustrated. Through the risk communication process, pregnancy outcome data are routinely collected by Embryotox. This approach uses the advantages of a pre-existing communication structure and of dealing with motivated responders. Engagement in the treatment plan facilitates receiving reliable data on drug exposure as well as detailed follow-up data. Based on these patient records, prospective datasets are evaluated in observational cohort studies in comparison to non-exposed control cohorts. In addition, retrospective datasets received as suspected adverse drug reactions from multiple German sources allow a screening for signals of teratogenicity and distinct patterns of developmental toxicity. Clinical expertise in specialties such as teratology, paediatrics, embryology, obstetrics and human genetics are required to ensure high-quality assessment of drug safety in pregnancy.

Pregnancy outcome after chelation therapy in Wilson disease. Evaluation of the German Embryotox Database.

Continuation of treatment is recommended for pregnant women with Wilson disease. Therapy options include the copper chelating agents d-penicillamine and trientine. However, there are still uncertainties concerning a possible teratogenic risk. In this case series, we report on the outcome of 20 pregnancies with maternal chelator exposure at least during the first trimester. Of these 20 pregnancies documented by the German Embryotox Project, 14 were prospectively ascertained and 6 were retrospective. No major birth defects were observed. Three of the 14 prospective cases resulted in a spontaneous abortion, and one pregnancy was electively terminated. Our results do not support the hypothesis of teratogenicity based on earlier case reports of congenital anomalies. Therefore our study may contribute to reassure women needing chelation therapy during pregnancy. However, it must be taken into account that the sample size of this case series is too limited to make final conclusions on teratogenic effects.

Brachydactyly Type C patient with compound heterozygosity for p.Gly319Val and p.Ile358Thr variants in the GDF5 proregion: benign variants or mutations?

We report on a Brachydactyly Type C (BDC) patient with clinically inconspicuous parents. Molecular genetic analyses revealed compound heterozygosity for two GDF5 variants. The variant c.956G>T (p.Gly319Val) was inherited from her mother and has been reported in exome sequencing projects, whereas c.1073T>C (p.Ile358Thr) has never been reported so far. In silico, both variants were predicted to be ‘disease-causing’, but the fact that p.Ile358Thr was predicted by SIFT to be ‘tolerated’ raised our suspicion. Therefore, we performed in vitro assays. To our surprise, GDF5G319V showed pronounced loss of function in luciferase reporter assays and in vitro chondrogenesis, whereas GDF5I358T and GDF5WT had comparable biological activities. Western blot analyses revealed decreased protein levels after overexpression of GDF5G319V. In absence of linkage or de novo mutation, several scenarios could explain the underlying mechanism of the patient’s phenotype. Owing to reduced activity of GDF5G319V in our functional assays, p.Gly319Val might be causative for BDC, but typically evoke an unrecognizably mild phenotype or even nonpenetrance. Another possibility is that our assays failed to pinpoint the disease-causing mechanism of the p.Ile358Thr allele. A final possibility is that compound heterozygosity for p.Ile358Thr and p.Gly319Val is more deleterious to GDF5 activity than either variant alone. Until all possible explanations can be rigorously tested experimentally, a precise recurrence risk counseling for the parents and the affected child is not possible.

Autosomal dominant Robinow syndrome associated with a novel DVL3 splice mutation

Robinow syndrome is a clinically and genetically heterogeneous disorder characterized by mesomelic limb shortening, distinctive facial features, and variable oral, cardiac, vertebral, and urogenital malformations. We identified the novel de novo splice acceptor mutation c.1715‐2A > C in DVL3 in a 15‐year‐old female patient with typical features of Robinow syndrome. By studying DVL3 transcripts in this patient, we confirmed expression of both wild‐type and mutant alleles. Mutant DVL3 mRNAs were found to harbor a deletion of four nucleotides at the beginning of exon 15 and encode a protein product with a distinct −1 reading‐frame C‐terminus. The data demonstrate that mutant DVL3 proteins associated with Robinow syndrome show truncation of the C‐terminus and share 83 novel amino acid residues before the stop codon confirming highly specific DVL3 alterations to be associated with this syndrome. The phenotype of the Robinow syndrome‐affected female reported here is typical as she shows mesomelia and mild hand anomalies as well as characteristic facial anomalies. She also exhibited a supraumbilical midline abdominal raphe which has not been observed in other patients with Robinow syndrome. In contrast to the clinical data of four previously reported individuals with DVL3‐related Robinow syndrome, short stature was not present in this individual at the age of 15 years. These findings expand the clinical spectrum of Robinow syndrome associated with DVL3 mutations. To date, comparison of clinical data of DVL3 mutation‐positive individuals with those of patients with genetically different forms did not allow delineation of gene‐specific phenotypes.