Katharina Keupp

Attenuated BMP1 Function Compromises Osteogenesis, Leading to Bone Fragility in Humans and Zebrafish

Bone morphogenetic protein 1 (BMP1) is an astacin metalloprotease with important cellular functions and diverse substrates, including extracellular-matrix proteins and antagonists of some TGFβ superfamily members. Combining whole-exome sequencing and filtering for homozygous stretches of identified variants, we found a homozygous causative BMP1 mutation, c.34G>C, in a consanguineous family affected by increased bone mineral density and multiple recurrent fractures. The mutation is located within the BMP1 signal peptide and leads to impaired secretion and an alteration in posttranslational modification. We also characterize a zebrafish bone mutant harboring lesions in bmp1a, demonstrating conservation of BMP1 function in osteogenesis across species. Genetic, biochemical, and histological analyses of this mutant and a comparison to a second, similar locus reveal that Bmp1a is critically required for mature-collagen generation, downstream of osteoblast maturation, in bone. We thus define the molecular and cellular bases of BMP1-dependent osteogenesis and show the importance of this protein for bone formation and stability.

Recessive TRAPPC11 Mutations Cause a Disease Spectrum of Limb Girdle Muscular Dystrophy and Myopathy with Movement Disorder and Intellectual Disability

Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.

The missing “link”: an autosomal recessive short stature syndrome caused by a hypofunctional XYLT1 mutation

Proteoglycan (PG) synthesis begins with the sequential addition of a “linker chain”, made up of four sugar residues, to a specific region of a core protein. Defects in the enzymes catalyzing steps two to four of the linker chain synthesis have been shown to cause autosomal recessive human phenotypes while no mutation has yet been reported in humans for the xylosyltransferases 1 and 2 (XT1 and XT2), the initiating enzymes in the linker chain formation. Here, we present a consanguineous Turkish family with two affected individuals presenting with short stature, distinct facial features, alterations of fat distribution, and moderate intellectual disability. X-rays showed only mild skeletal changes in the form of a short femoral neck, stocky and plump long bones and thickened ribs. Using a combination of whole-exome sequencing (WES), determination of homozygous stretches by WES variants, and classical linkage analysis, we identified the homozygous missense mutation c.C1441T in XYLT1, encoding XT1, within a large homozygous stretch on chromosome 16p13.12-p12.1. The mutation co-segregated with the phenotype in the family, is not found in over 13,000 alleles in the exome variant server and is predicted to change a highly conserved arginine at position 481 (p.R481W) located in the putative catalytical domain. Immunostaining of primary patient fibroblasts showed a loss of predominance of Golgi localization in mutant cells. Moreover, western blot analysis of decorin in cell culture supernatant demonstrated glycosylation differences between patient and control cells. Our data provide evidence that functional alterations of XT1 cause an autosomal recessive short stature syndrome associated with intellectual disability.

Mutations in the interleukin receptor IL11RA cause autosomal recessive Crouzon-like craniosynostosis

We have characterized a novel autosomal recessive Crouzon‐like craniosynostosis syndrome in a 12‐affected member family from Antakya, Turkey, the presenting features of which include: multiple suture synostosis, midface hypoplasia, variable degree of exophthalmos, relative prognathism, a beaked nose, and conductive hearing loss. Homozygosity mapping followed by targeted next‐generation sequencing identified a c.479+6T>G mutation in the interleukin 11 receptor alpha gene (IL11RA) on chromosome 9p21. This donor splice‐site mutation leads to a high percentage of aberrant IL11RA mRNA transcripts in an affected individual and altered mRNA splicing determined by in vitro exon trapping. An extended IL11RA mutation screen was performed in a cohort of 79 patients with an initial clinical diagnosis of Crouzon syndrome, pansynostosis, or unclassified syndromic craniosynostosis. We identified mutations segregating with the disease in five families: a German patient of Turkish origin and a Turkish family with three affected sibs all of whom were homozygous for the previously identified IL11RA c.479+6T>G mutation; a family with pansynostosis with compound heterozygous missense mutations, p.Pro200Thr and p.Arg237Pro; and two further Turkish families with Crouzon‐like syndrome carrying the homozygous nonsense mutations p.Tyr232* and p.Arg292*. Using transient coexpression in HEK293T and COS7 cells, we demonstrated dramatically reduced IL11‐mediated STAT3 phosphorylation for all mutations. Immunofluorescence analysis of mouse Il11ra demonstrated specific protein expression in cranial mesenchyme which was localized around the coronal suture tips and in the lambdoidal suture. In situ hybridization analysis of adult zebrafish also detected zfil11ra expression in the coronal suture between the overlapping frontal and parietal plates. This study demonstrates that mutations in the IL11RA gene cause an autosomal recessive Crouzon‐like craniosynostosis.

RAP1-mediated MEK/ERK pathway defects in Kabuki syndrome

The genetic disorder Kabuki syndrome (KS) is characterized by developmental delay and congenital anomalies. Dominant mutations in the chromatin regulators lysine (K)-specific methyltransferase 2D (KMT2D) (also known as MLL2) and lysine (K)-specific demethylase 6A (KDM6A) underlie the majority of cases. Although the functions of these chromatin-modifying proteins have been studied extensively, the physiological systems regulated by them are largely unknown. Using whole-exome sequencing, we identified a mutation in RAP1A that was converted to homozygosity as the result of uniparental isodisomy (UPD) in a patient with KS and a de novo, dominant mutation in RAP1B in a second individual with a KS-like phenotype. We elucidated a genetic and functional interaction between the respective KS-associated genes and their products in zebrafish models and patient cell lines. Specifically, we determined that dysfunction of known KS genes and the genes identified in this study results in aberrant MEK/ERK signaling as well as disruption of F-actin polymerization and cell intercalation. Moreover, these phenotypes could be rescued in zebrafish models by rebalancing MEK/ERK signaling via administration of small molecule inhibitors of MEK. Taken together, our studies suggest that the KS pathophysiology overlaps with the RASopathies and provide a potential direction for treatment design.

Gene panel testing of 5589 BRCA1/2-negative index patients with breast cancer in a routine diagnostic setting: results of the German Consortium for Hereditary Breast and Ovarian Cancer

The prevalence of germ line mutations in non‐BRCA1/2 genes associated with hereditary breast cancer (BC) is low, and the role of some of these genes in BC predisposition and pathogenesis is conflicting. In this study, 5589 consecutive BC index patients negative for pathogenic BRCA1/2 mutations and 2189 female controls were screened for germ line mutations in eight cancer predisposition genes (ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D, and TP53). All patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germ line testing. The highest mutation prevalence was observed in the CHEK2 gene (2.5%), followed by ATM (1.5%) and PALB2 (1.2%). The mutation prevalence in each of the remaining genes was 0.3% or lower. Using Exome Aggregation Consortium control data, we confirm significant associations of heterozygous germ line mutations with BC for ATM (OR: 3.63, 95%CI: 2.67–4.94), CDH1 (OR: 17.04, 95%CI: 3.54–82), CHEK2 (OR: 2.93, 95%CI: 2.29–3.75), PALB2 (OR: 9.53, 95%CI: 6.25–14.51), and TP53 (OR: 7.30, 95%CI: 1.22–43.68). NBN germ line mutations were not significantly associated with BC risk (OR:1.39, 95%CI: 0.73–2.64). Due to their low mutation prevalence, the RAD51C and RAD51D genes require further investigation. Compared with control datasets, predicted damaging rare missense variants were significantly more prevalent in CHEK2 and TP53 in BC index patients. Compared with the overall sample, only TP53 mutation carriers show a significantly younger age at first BC diagnosis. We demonstrate a significant association of deleterious variants in the CHEK2, PALB2, and TP53 genes with bilateral BC. Both, ATM and CHEK2, were negatively associated with triple‐negative breast cancer (TNBC) and estrogen receptor (ER)‐negative tumor phenotypes. A particularly high CHEK2 mutation prevalence (5.2%) was observed in patients with human epidermal growth factor receptor 2 (HER2)‐positive tumors.

Non-small cell neuroendocrine carcinoma of the ovary in a BRCA2-germline mutation carrier: A case report and brief review of the literature

Non-small cell neuroendocrine carcinomas (NSCNEC) account for 2% of gynecological cancer cases and are associated with a poor prognosis due to delayed diagnosis and aggressive tumor behavior. BRCA2-associated ovarian carcinomas predominantly possess a high-grade serous phenotype, which respond to platinum and targeted therapy with PARP inhibitors. Presented here is the case of an adult patient with NSCNEC of the ovaries associated with a deleterious BRCA2 germline mutation. The pathogenic mutation was also confirmed on the somatic level, while the wild-type allele had a high variant fraction, suggesting loss of heterozygosity. To the best of our knowledge, this is the first report of an adult BRCA2 germline mutation carrier with the rare NSCNEC of the ovary phenotype. Therefore, ovarian cancer patients with histological subtypes other than high-grade serous carcinomas should be tested for BRCA1/2 mutations, as they may benefit from targeted therapy with poly (ADP-ribose) polymerase inhibitors.

Erratum to: A hypofunctional PAX1 mutation causes autosomal recessively inherited otofaciocervical syndrome

Otofaciocervical syndrome (OFCS) is an autosomal recessively inherited disorder characterized by facial dysmorphism, external ear anomalies with preauricular pits and hearing impairment, branchial cysts or fistulas, anomalies of the vertebrae and the shoulder girdle, and mild intellectual disability. In a large consanguineous family with OFCS from Turkey, we performed whole-exome sequencing (WES) of a single pooled DNA sample of four affected individuals. Filtering for variants with a percentage of alternate reads C90 % and a coverage of at least five reads identified only a single novel homozygous variant, c.497G[T, located in PAX1 that co-segregated with the disease in the family. PAX1 encodes a transcription factor with a critical role in pattern formation during embryogenesis in vertebrates. The mutation is predicted to substitute the glycine at position 166 to valine (p.G166V) within the highly conserved paired-box domain of the PAX1 protein. We performed a dual luciferase reporter assay to examine the transactivation of a regulatory sequence in the Nkx3-2 promoter region, which is a direct target of mouse Pax1 transcriptional regulation. We observed a significantly reduced transactivation in HEK293T cells overexpressing Pax1 in comparison to Pax1 expressing cells, indicating a reduced DNAbinding affinity of the mutant protein. Taken together, our results show that the strategy of pooling DNA is a powerful, cost-effective application for WES in consanguineous families and establish PAX1 as a new disease-causing gene for OFCS and as part of the EYA-DACH-SIX-PAX network, important in early embryogenesis.