[Show abstract][Hide abstract] ABSTRACT: Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure,
syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals
have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity
mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift
mutation, c.571dupA (p.Ile191Asnfs*6), in
, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The func-
tion of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a
pivotal role in cell division of neural progenitors. Sanger sequencing of
in a further eight unrelated individuals with clinical
features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines
(LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle
poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules
owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data
from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function
are a major cause of Filippi syndrome
The American Journal of Human Genetics 11/2014; · 11.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 syndrome.
Human Molecular Genetics 04/2014; · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Ligase IV syndrome is a rare differential diagnosis for Nijmegen Breakage syndrome owing to a shared predisposition to lympho-reticular malignancies, significant microcephaly and radiation hypersensitivity. Only 16 cases with mutations in LIG4 have been described to date with phenotypes varying from malignancy in developmentally normal individuals, to severe combined immunodeficiency and early mortality. Here we report the identification of biallelic truncating LIG4 mutations in 11 patients with microcephalic primordial dwarfism presenting with restricted prenatal growth and extreme postnatal global growth failure (average OFC -10.1 s.d., height -5.1 s.d.). Subsequently most patients developed thrombocytopenia and leucopenia later in childhood and many were found to have previously unrecognised immunodeficiency following molecular diagnosis. None have yet developed malignancy, though all patients tested had cellular radiosensitivity. A genotype:phenotype correlation was also noted with position of truncating mutations corresponding to disease severity. This work extends the phenotypic spectrum associated with LIG4 mutations, establishing that extreme growth retardation with microcephaly is a common presentation of bilallelic truncating mutations. Such growth failure is therefore sufficient to consider a diagnosis of LIG4 deficiency and early recognition of such cases is important as bone marrow failure, immunodeficiency and sometimes malignancy are long term sequelae of this disorder. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Borjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked recessive intellectual disability (ID) disorder caused by mutations in the PHF6 gene and characterised by variable cognitive impairment, a distinct facial gestalt, obesity, and hypogonadism. Female carriers are usually not affected or only mildly affected, and so far only two females with de novo mutations or deletions in PHF6 have been reported.
We performed PHF6 mutational analysis and screening for intragenic deletions and duplications by quantitative real-time PCR and multiplex ligation dependent probe amplification (MLPA) in female patients with variable ID and a distinct appearance of sparse hair, remarkable facial features, hypoplastic nails, and teeth anomalies. We detected two truncating mutations and two duplications of exons 4 and 5. Furthermore, two female patients with PHF6 deletions and a similar phenotype were identified by routine molecular karyotyping. Recently, two patients with a clinical diagnosis of Coffin-Siris syndrome in early infancy had been found to harbour mutations in PHF6, and their phenotype in advanced ages is now described. Further studies revealed skewed X-inactivation in blood lymphocytes, while it was normal in fibroblasts, thus indicating functional mosaicism.
Our findings indicate that de novo defects in PHF6 in females result in a recognisable phenotype which might have been under-recognised so far and which comprises variable ID, a characteristic facial gestalt, hypoplastic nails, brachydactyly, clinodactyly mainly of fingers IV and V, dental anomalies, and linear skin hyperpigmentation. It shows overlap with BFLS but also additional distinct features, thus adding a new facet to this disorder.
Journal of Medical Genetics 10/2013; · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autosomal recessive cutis laxa type 2B (ARCL2B; OMIM # 612940) is a segmental progeroid disorder caused by mutations in PYCR1 encoding pyrroline-5-carboxylate reductase 1, which is part of the conserved proline de novo synthesis pathway. Here we describe 33 patients with PYCR1-related ARCL from 27 families with initial diagnoses varying between wrinkly skin syndrome, gerodermia osteodysplastica, De Barsy syndrome or more severe progeria syndromes. Given the difficult differential diagnosis of ARCL syndromes we performed a systematic comparison of clinical features of PYCR1-related ARCL. Intrauterine growth retardation, a characteristic triangular facial gestalt, psychomotor retardation, and hypotonia were the most relevant distinctive hallmarks of ARCL due to proline de novo synthesis defects. Corneal clouding or cataracts, athetoid movements, and finger contractures were rather rare features, but had a high predictive value. In our cohort we identified 20 different PYCR1 mutations of which seven were novel. Most of the mutations accumulated in exons 4 to 6. Missense alterations of highly conserved residues were most frequent followed by splice site changes and a single nonsense mutation. Analysis of genotype-phenotype correlation revealed that patients with mutations in the first two exons had lower average clinical scores and absent or only mild intellectual disability. Structural analyses predicted interference with PYCR1 multimerization for a subset of missense mutations. These findings have implications for the clinics as well as the pathomechanism of PYCR1-related ARCL.
Molecular Genetics and Metabolism 08/2013; · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure, and variable intellectual disability. MCPH is caused by mutations of eleven different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis, however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.
Human Molecular Genetics 08/2013; · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromatin remodeling complexes are known to modify chemical marks on histones or to induce conformational changes of the chromatin in order to regulate transcription. De novo dominant mutations in different members of the SWI/SNF chromatin remodeling complex have recently been described in individuals with Coffin-Siris (CSS) and Nicolaides-Baraitser (NCBRS) syndromes. Using a combination of whole-exome sequencing, NGS-based sequencing of 23 SWI/SNF complex genes, and molecular karyotyping in 46 previously undescribed individuals with CSS and NCBRS, we identified a de novo 1-bp deletion (c.677delG, p.Gly226Glufs*53) and a de novo missense mutation (c.914G>T, p.Cys305Phe) in PHF6 in two individuals diagnosed with CSS. PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex implicating dysfunction of a second chromatin remodeling complex in the pathogenesis of CSS-like phenotypes. Altogether, we identified mutations in 60% of the studied individuals (28/46), located in the genes ARID1A, ARID1B, SMARCB1, SMARCE1, SMARCA2, and PHF6. We show that mutations in ARID1B are the main cause of CSS, accounting for 76% of identified mutations. ARID1B and SMARCB1 mutations were also found in individuals with the initial diagnosis of NCBRS. These individuals apparently belong to a small subset who display an intermediate CSS/NCBRS phenotype. Our proposed genotype-phenotype correlations are important for molecular screening strategies.
Human Molecular Genetics 08/2013; · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 ≥90 % 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(G157V) in comparison to Pax1(WT) expressing cells, indicating a reduced DNA-binding 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.
[Show abstract][Hide abstract] ABSTRACT: 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 American Journal of Human Genetics 07/2013; · 11.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nager syndrome (MIM #154400) is the best-known preaxial acrofacial dysostosis, mainly characterized by craniofacial and preaxial limb anomalies. The craniofacial abnormalities mainly consist of downslanting palpebral fissures, malar hypoplasia, micrognathia, external ear anomalies, and cleft palate. The preaxial limb defects are characterized by radial and thumb hypoplasia or aplasia, duplication of thumbs and proximal radioulnar synostosis. Haploinsufficiency of SF3B4 (MIM *605593), which encodes SAP49, a component of the pre-mRNA spliceosomal complex, has recently been identified as the underlying cause of Nager syndrome. In our study, we performed exome sequencing in two and Sanger sequencing of SF3B4 in further ten previously unreported patients with the clinical diagnosis of Nager syndrome, including one familial case. We identified heterozygous SF3B4 mutations in seven out of twelve patients. Four of the seven mutations were shown to be de novo; in three individuals, DNA of both parents was not available. No familial mutations were discovered. Three mutations were nonsense, three were frameshift mutations and one T > C transition destroyed the translation start signal. In three of four SF3B4 negative families, EFTUD2 was analyzed, but no pathogenic variants were identified. Our results indicate that the SF3B4 gene is mutated in about half of the patients with the clinical diagnosis of Nager syndrome and further support genetic heterogeneity for this condition.
[Show abstract][Hide abstract] ABSTRACT: We report that hypofunctional alleles of WNT1 cause autosomal-recessive osteogenesis imperfecta, a congenital disorder characterized by reduced bone mass and recurrent fractures. In consanguineous families, we identified five homozygous mutations in WNT1: one frameshift mutation, two missense mutations, one splice-site mutation, and one nonsense mutation. In addition, in a family affected by dominantly inherited early-onset osteoporosis, a heterozygous WNT1 missense mutation was identified in affected individuals. Initial functional analysis revealed that altered WNT1 proteins fail to activate canonical LRP5-mediated WNT-regulated β-catenin signaling. Furthermore, osteoblasts cultured in vitro showed enhanced Wnt1 expression with advancing differentiation, indicating a role of WNT1 in osteoblast function and bone development. Our finding that homozygous and heterozygous variants in WNT1 predispose to low-bone-mass phenotypes might advance the development of more effective therapeutic strategies for congenital forms of bone fragility, as well as for common forms of age-related osteoporosis.
The American Journal of Human Genetics 03/2013; · 11.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.
PLoS ONE 01/2013; 8(11):e78496. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kraniosynostosen gehören mit einer Inzidenz von 1:2000 bis 1:3000 Geburten zu den häufigsten kraniofazialen Anomalien. Die durch die vorzeitige Verknöcherung einer oder mehrerer Schädelnähte verursachte Wachstumshemmung kann zu schweren Deformitäten des Schädel- und Gesichtsskeletts führen. Dies sorgt nicht nur für eine große ästhetische Beeinträchtigung, sondern hat auch funktionelle Auswirkungen für die Patienten. Hierzu können u. a. gehören: intrakranielle Drucksteigerung, Atrophie des N. opticus, Atem-, Hör- und Entwicklungsstörungen. Trotz großer Anstrengungen konnten bisher nur für einen Teil der autosomal-dominanten syndromalen Kraniosynostosen die ursächlichen Gene, z. B ,,fibroblast growth factor receptor 1-3“ (FGFR1-3), ,,twist basic helix-loop-helix transcription factor 1“ (TWIST1) etc., gefunden werden. Die Ätiologie der nichtsyndromalen Kraniosynostosen bleibt weiterhin ungeklärt. Aufgrund der verbreiteten Anwendung neuer Sequenziertechnologien zur Identifizierung neuer kausaler Gene bei Patienten mit Kraniosynostose kann in den nächsten Jahren mit der Entschlüsselung vieler weiterer krankheitsverursachender Gene gerechnet werden. Insbesondere die syndromalen Formen der Kraniosynostose bedürfen aufgrund ihrer klinischen Komplexität einer interdisziplinären Betreuung. Die einzige Therapieoption besteht derzeit in der kraniofazialen Chirurgie, welche aber die genetisch determinierten pathologischen Wachstumsmuster der komplexen syndromalen Kraniosynostosen langfristig oft nicht beheben kann.
Medizinische Genetik 01/2013; 25(3). · 0.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is known that FGFR2 gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5-10% of breast tumors. In our study, we sequenced the most important coding regions of FGFR2 in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in FGFR2 and FGF10. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer.
PLoS ONE 01/2013; 8(3):e60264. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetische Nierenerkrankungen haben an den sich im Kindes- bis Jugendalter manifestierenden Nierenerkrankungen einen bedeutenden Anteil, gewinnen aber auch zunehmend an Bedeutung für nur langsam progrediente oder spät im Erwachsenenalter auftretende Erkrankungen. Ein erheblicher Teil insbesondere kindlicher Nierenerkrankungen ist mit terminaler Niereninsuffizienz und/oder weiterer relevanter Morbidität verbunden. Eine frühzeitige (molekulare) Diagnosestellung kann für eine bessere prognostische Einschätzung Voraussetzung sein und bietet Möglichkeiten hinsichtlich einer optimierten symptomatischen Therapie.Mechanistisch betrachtet stellen mit Mutationen in Ionenkanälen assoziierte Nephropathien – neben strukturellen Defekten des glomerulären Filters (z. B. COL4A3, LAMB2, Nephrin) und Störungen der für die Entwicklung des Urogenitaltrakts relevanter Signalwege (z. B. HNF1B, WT1) – eine prototypische und zahlenmäßig wesentliche Gruppe dar.Die molekulargenetische Aufklärung der (hypokaliämischen) Salzverlusttubulopathien hat ganz wesentlich zu unserem Verständnis der zentralen Rolle der Niere im Salzhaushalt beigetragen. Das Spektrum renaler Ionenkanalerkrankungen soll an den Beispielen der klassischen Salzverlusttubulopathien (Bartter- und Gitelman-Syndrom), der Transient-receptor-potential-(TRP)-Kanalgruppe sowie der Rolle von Kanalveränderungen bei Aldosteronismus und angeborenen Hypertonusformen dargestellt werden.
Medizinische Genetik 01/2013; 25(4). · 0.09 Impact Factor