Jutta Becker

Exome sequencing identifies truncating mutations in human SERPINF1 in autosomal-recessive osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. After mutations in all known OI genes had been excluded by Sanger sequencing, we applied next-generation sequencing to analyze the exome of a single individual who has a severe form of the disease and whose parents are second cousins. A total of 26,922 variations from the human reference genome sequence were subjected to several filtering steps. In addition, we extracted the genotypes of all dbSNP130-annotated SNPs from the exome sequencing data and used these 299,494 genotypes as markers for the genome-wide identification of homozygous regions. A single homozygous truncating mutation, affecting SERPINF1 on chromosome 17p13.3, that was embedded into a homozygous stretch of 2.99 Mb remained. The mutation was also homozygous in the affected brother of the index patient. Subsequently, we identified homozygosity for two different truncating SERPINF1 mutations in two unrelated patients with OI and parental consanguinity. All four individuals with SERPINF1 mutations have severe OI. Fractures of long bones and severe vertebral compression fractures with resulting deformities were observed as early as the first year of life in these individuals. Collagen analyses with cultured dermal fibroblasts displayed no evidence for impaired collagen folding, posttranslational modification, or secretion. SERPINF1 encodes pigment epithelium-derived factor (PEDF), a secreted glycoprotein of the serpin superfamily. PEDF is a multifunctional protein and one of the strongest inhibitors of angiogenesis currently known in humans. Our data provide genetic evidence for PEDF involvement in human bone homeostasis.

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.

Acetylcholine Receptor Pathway Mutations Explain Various Fetal Akinesia Deformation Sequence Disorders

Impaired fetal movement causes malformations, summarized as fetal akinesia deformation sequence (FADS), and is triggered by environmental and genetic factors. Acetylcholine receptor (AChR) components are suspects because mutations in the fetally expressed gamma subunit (CHRNG) of AChR were found in two FADS disorders, lethal multiple pterygium syndrome (LMPS) and Escobar syndrome. Other AChR subunits alpha1, beta1, and delta (CHRNA1, CHRNB1, CHRND) as well as receptor-associated protein of the synapse (RAPSN) previously revealed missense or compound nonsense-missense mutations in viable congenital myasthenic syndrome; lethality of homozygous null mutations was predicted but never shown. We provide the first report to our knowledge of homozygous nonsense mutations in CHRNA1 and CHRND and show that they were lethal, whereas novel recessive missense mutations in RAPSN caused a severe but not necessarily lethal phenotype. To elucidate disease-associated malformations such as frequent abortions, fetal edema, cystic hygroma, or cardiac defects, we studied Chrna1, Chrnb1, Chrnd, Chrng, and Rapsn in mouse embryos and found expression in skeletal muscles but also in early somite development. This indicates that early developmental defects might be due to somite expression in addition to solely muscle-specific effects. We conclude that complete or severe functional disruption of fetal AChR causes lethal multiple pterygium syndrome whereas milder alterations result in fetal hypokinesia with inborn contractures or a myasthenic syndrome later in life.

Mutations in SEC24D, Encoding a Component of the COPII Machinery, Cause a Syndromic Form of Osteogenesis Imperfecta

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205(∗)) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish).

CHARGE and Kabuki syndromes: A phenotypic and molecular link

CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding gene CHD7. Kabuki syndrome, another developmental disorder, is characterized by typical facial features in combination with developmental delay, short stature, prominent digit pads and visceral abnormalities. Mutations in the KMT2D gene, which encodes a H3K4 histone methyltransferase, are the major cause of Kabuki syndrome. Here, we report a patient, who was initially diagnosed with CHARGE syndrome based on the spectrum of inner organ malformations like choanal hypoplasia, heart defect, anal atresia, vision problems and conductive hearing impairment. While sequencing and MLPA analysis of all coding exons of CHD7 revealed no pathogenic mutation, sequence analysis of the KMT2D gene identified the heterozygous de novo nonsense mutation c.5263C > T (p.Gln1755*). Thus, our patient was diagnosed with Kabuki syndrome. By using co-immunoprecipitation, immunohistochemistry and direct yeast two hybrid assays, we could show that, like KMT2D, CHD7 interacts with members of the WAR complex, namely WDR5, ASH2L and RbBP5. We therefore propose that CHD7 and KMT2D function in the same chromatin modification machinery, thus pointing out a mechanistic connection, and presenting a probable explanation for the phenotypic overlap between Kabuki and CHARGE syndromes.

Mutation Update for Kabuki Syndrome Genes KMT2D and KDM6A and Further Delineation of X-Linked Kabuki Syndrome Subtype 2.

Kabuki syndrome (KS) is a rare but recognizable condition that consists of a characteristic face, short stature, various organ malformations, and a variable degree of intellectual disability. Mutations in KMT2D have been identified as the main cause for KS, whereas mutations in KDM6A are a much less frequent cause. Here, we report a mutation screening in a case series of 347 unpublished patients, in which we identified 12 novel KDM6A mutations (KS type 2) and 208 mutations in KMT2D (KS type 1), 132 of them novel. Two of the KDM6A mutations were maternally inherited and nine were shown to be de novo. We give an up‐to‐date overview of all published mutations for the two KS genes and point out possible mutation hot spots and strategies for molecular genetic testing. We also report the clinical details for 11 patients with KS type 2, summarize the published clinical information, specifically with a focus on the less well‐defined X‐linked KS type 2, and comment on phenotype–genotype correlations as well as sex‐specific phenotypic differences. Finally, we also discuss a possible role of KDM6A in Kabuki‐like Turner syndrome and report a mutation screening of KDM6C (UTY) in male KS patients.

A nonclassical IFITM5 mutation located in the coding region causes severe osteogenesis imperfecta with prenatal onset.

Osteogenesis imperfecta (OI) is a hereditary connective tissue disorder characterized by a wide range of skeletal symptoms. Most patients have dominantly inherited or de novo mutations in COL1A1 or COL1A2. Up to 5% of patients have OI type V, characterized by hyperplastic callus formation after fractures, calcification of the interosseous membrane of the forearm, and a mesh‐like lamellation pattern observed in bone histology. Recently, a heterozygous mutation in the 5′‐untranslated region (UTR) of IFITM5 (c.–14C > T) was identified as the underlying cause of OI type V, and only this specific mutation was subsequently identified in all patient cohorts with this OI subtype. We now present a case of a heterozygous mutation within the coding region of IFITM5 (c.119C > T; p.S40L). The mutation occurred de novo in the patient and resulted in severe OI with prenatal onset and extreme short stature. At the age of 19 months, the typical clinical hallmarks of OI type V were not present. Our finding has important consequences for the genetic “work‐up” of patients suspected to have OI, both in prenatal and in postnatal settings: The entire gene—not only the 5′‐UTR harboring the “classical” OI type V mutation—has to be analyzed to exclude a causal role of IFITM5. We propose that this should be part of the initial diagnostic steps for genetic laboratories performing SANGER sequencing in OI patients.

Cognitive training with and without additional physical activity in healthy older adults: cognitive effects, neurobiological mechanisms, and prediction of training success

Data is inconsistent concerning the question whether cognitive-physical training (CPT) yields stronger cognitive gains than cognitive training (CT). Effects of additional counseling, neurobiological mechanisms, and predictors have scarcely been studied. Healthy older adults were trained with CT (n = 20), CPT (n = 25), or CPT with counseling (CPT+C; n = 23). Cognition, physical fitness, BDNF, IGF-1, and VEGF were assessed at pre- and post-test. No interaction effects were found except for one effect showing that CPT+C led to stronger gains in verbal fluency than CPT (p = 0.03). However, this superiority could not be assigned to additional physical training gains. Low baseline cognitive performance and BDNF, not carrying apoE4, gains in physical fitness and the moderation of gains in physical fitness × gains in BDNF predicted training success. Although all types of interventions seem successful to enhance cognition, our data do not support the hypotheses that CPT shows superior CT gains compared to CT or that CPT+C adds merit to CPT. However, as CPT leads to additional gains in physical fitness which in turn is known to have positive impact on cognition in the long-term, CPT seems more beneficial. Training success can partly be predicted by neuropsychological, neurobiological, and genetic parameters. Unique Identifier: WHO ICTRP (http://www.who.int/ictrp); ID: DRKS00005194.

Systematic assessment of urinary hydroxy-oxo-glutarate for diagnosis and follow-up of primary hyperoxaluria type III

BackgroundThere are currently three distinct autosomal recessive inherited types of primary hyperoxaluria (PH: PHI, PHII, and PHIII), all characterized by the endogenous overproduction of oxalate. The PH type is difficult to differentiate by clinical features alone. In addition to universal general characteristics to all hyperoxaluria subtypes, specific urinary metabolites can be detected: glycolate in PHI, L-glyceric acid in PHII, and hydroxy-oxo-glutarate (HOG) in PHIII. PHIII is considered to be the most benign form and is characterized by severe recurrent urolithiasis in early life, followed by clinical remission in many, but not all patients. We examined urinary HOG (UHOG) excretion as a diagnostic marker and its correlation to progression of the clinical course of PHIII.MethodsUHOG was analyzed by combined ion chromatography/mass spectrometry (IC/MS) in urine samples from 30 PHIII and 68 PHI/II patients and 79 non-PH hyperoxaluria patients.ResultsMean UHOG excretion was significantly higher in patients with PHIII than in those with PHI/II and in non-PH patients(51.6 vs. 6.61 vs. 8.36 μmol/1.73 m2/24 h, respectively; p<0.01).ConclusionsSignificantly elevated UHOG excretion was exclusively seen in PHIII patients and showed a 100 % consensus with the results of hydroxy-oxo-glutarate aldolase (HOGA1) mutational analysis in newly diagnosed patients. However, UHOG excretion did not correlate with clinical course on follow-up and could not be used to discriminate between active stone formers and patients with a clinically uneventful follow-up.

CRTAP variants in early-onset osteoporosis and recurrent fractures.

CRTAP Variants in Early-Onset Osteoporosis and Recurrent Fractures Alice Costantini,* Ilkka Vuorimies, Riikka M€akitie, Mervi K. M€ayr€anp€a€a, Jutta Becker, Minna Pekkinen, Helena Valta, Christian Netzer, Anders K€ampe, Fulya Taylan, Hong Jiao, and Outi M€akitie Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Folkh€alsan Institute of Genetics, University of Helsinki, Helsinki, Finland Children’s Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Institute of Human Genetics, University of Cologne, Cologne, Germany Department of Biosciences and Nutrition, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden Clinical Research Centre, Karolinska University Hospital, Huddinge, Sweden Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden