Encarna Guillén-Navarro

Mutation of HAIRY-AND-ENHANCER-OF-SPLIT-7 in humans causes spondylocostal dysostosis

Spondylocostal dysostosis (SCD) is an inherited disorder that is characterized by the presence of extensive hemivertebrae, truncal shortening and abnormally aligned ribs. It arises during embryonic development by a disruption of formation of somites (the precursor tissue of the vertebrae, ribs and associated tendons and muscles). Previously, three genes causing a subset of autosomal recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300), MESP2 (SCDO2: MIM 608681) and LFNG (SCDO3: MIM609813). These genes are all important components of the Notch signaling pathway, which has multiple roles in development and disease. Here we have used autozygosity mapping to identify a mutation in a fourth Notch pathway gene, Hairy-and-Enhancer-of-Split-7 (HES7), in an autosomal recessive SCD family. HES7 encodes a bHLH-Orange domain transcriptional repressor protein that is both a direct target of the Notch signaling pathway, and part of a negative feedback mechanism required to attenuate Notch signaling. A missense mutation was identified in the DNA-binding domain of the HES7 protein. Functional analysis revealed that the mutant HES7 was not able to repress gene expression by DNA binding or protein heterodimerization. This is the first report of mutation in the human HES7 gene, and provides further evidence for the importance of the Notch signaling pathway in the correct patterning of the axial skeleton.

Mutations in PLOD2 cause autosomal-recessive connective tissue disorders within the Bruck syndrome—Osteogenesis imperfecta phenotypic spectrum†

PLOD2 and FKBP10 are genes mutated in Bruck syndrome (BS), a condition resembling osteogenesis imperfecta (OI), but that is also typically associated with congenital joint contractures. Herein, we sought mutations in six consanguineous BS families and detected changes in either PLOD2 or FKBP10 in all cases. Two probands were found with a homozygous frameshift mutation in the alternative exon 13a of PLOD2, indicating that specific inactivation of the longer protein isoform encoded by this gene is sufficient to cause BS. In addition, by homozygosity mapping, followed by a candidate gene approach, we identified a homozygous donor splice site mutation in PLOD2 in a patient with autosomal‐recessive OI (AR‐OI). Screening of additional samples also revealed compound heterozygous mutations in PLOD2 in two brothers, one affected with mild AR‐OI and the other with mild BS. Thus, PLOD2 in addition to causing BS is also associated with AR‐OI phenotypes of variable severity. Hum Mutat 33:1444–1449, 2012.

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.

CDKN1C (p57Kip2) analysis in Beckwith–Wiedemann syndrome (BWS) patients: Genotype–phenotype correlations, novel mutations, and polymorphisms

Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by macroglossia, macrosomia, and abdominal wall defects. It is a multigenic disorder caused in most patients by alterations in growth regulatory genes. A small number of individuals with BWS (5–10%) have mutations in CDKN1C, a cyclin‐dependent kinase inhibitor of G1 cyclin complexes that functions as a negative regulator of cellular growth and proliferation. Here, we report on eight patients with BWS and CDKN1C mutations and review previous reported cases. We analyzed 72 patients (50 BWS, 17 with isolated hemihyperplasia (IH), three with omphalocele, and two with macroglossia) for CDKN1C defects with the aim to search for new mutations and to define genotype–phenotype correlations. Our findings suggest that BWS patients with CDKN1C mutations have a different pattern of clinical malformations than those with other molecular defects. Polydactyly, genital abnormalities, extra nipple, and cleft palate are more frequently observed in BWS with mutations in CDKN1C. The clinical observation of these malformations may help to decide which genetic characterization should be undertaken (i.e., CDKN1C screening), thus optimizing the laboratory evaluation for BWS.

Deletions of the RUNX2 gene are present in about 10% of individuals with cleidocranial dysplasia

Cleidocranial Dysplasia (CCD) is an autosomal dominant skeletal disorder characterized by hypoplastic or absent clavicles, increased head circumference, large fontanels, dental anomalies, and short stature. Hand malformations are also common. Mutations in RUNX2 cause CCD, but are not identified in all CCD patients. In this study we screened 135 unrelated patients with the clinical diagnosis of CCD for RUNX2 mutations by sequencing analysis and demonstrated 82 mutations 48 of which were novel. By quantitative PCR we screened the remaining 53 unrelated patients for copy number variations in the RUNX2 gene. Heterozygous deletions of different size were identified in 13 patients, and a duplication of the exons 1 to 4 of the RUNX2 gene in one patient. Thus, heterozygous deletions or duplications affecting the RUNX2 gene may be present in about 10% of all patients with a clinical diagnosis of CCD which corresponds to 26% of individuals with normal results on sequencing analysis. We therefore suggest that screening for intragenic deletions and duplications by qPCR or MLPA should be considered for patients with CCD phenotype in whom DNA sequencing does not reveal a causative RUNX2 mutation.

Mutations in WNT10A are frequently involved in oligodontia associated with minor signs of ectodermal dysplasia

Ectodermal dysplasias (ED) are a clinically and genetically heterogeneous group of hereditary disorders that have in common abnormal development of ectodermal derivatives. Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of eccrine sweat glands, hair, and teeth. The X‐linked form of the disease, caused by mutations in the EDA gene, represents the majority of patients with the hypohidrotic form. Autosomal dominant and autosomal recessive forms are occasionally seen, and result from mutations in at least three genes (WNT10A, EDAR, or more rarely EDARADD). We have screened for mutations in EDAR (commonly involved in the hypohidrotic form) and WNT10A (involved in a wide spectrum of ED and in isolated hypodontia) in a cohort of 36 patients referred for EDA molecular screening, which failed to identify any mutation. We identified eight EDAR mutations in five patients (two with homozygous mutations, one with compound heterozygous mutations, and two with heterozygous mutation), four of which were novel variants. We identified 28 WNT10A mutations in 16 patients (5 with homozygous mutations, 7 with compound heterozygous mutations, and 4 with heterozygous mutations), seven of which were novel variants. Our study allows a more precise definition of the phenotypic spectrum associated with EDAR and WNT10A mutations and underlines the importance of the implication of WNT10A among patients with ED.

Exome sequencing unravels unexpected differential diagnoses in individuals with the tentative diagnosis of Coffin–Siris and Nicolaides–Baraitser syndromes

Coffin–Siris syndrome (CSS) and Nicolaides–Baraitser syndrome (NCBRS) are rare intellectual disability/congenital malformation syndromes that represent distinct entities but show considerable clinical overlap. They are caused by mutations in genes encoding members of the BRG1- and BRM-associated factor (BAF) complex. However, there are a number of patients with the clinical diagnosis of CSS or NCBRS in whom the causative mutation has not been identified. In this study, we performed trio-based whole-exome sequencing (WES) in ten previously described but unsolved individuals with the tentative diagnosis of CSS or NCBRS and found causative mutations in nine out of ten individuals. Interestingly, our WES analysis disclosed overlapping differential diagnoses including Wiedemann–Steiner, Kabuki, and Adams–Oliver syndromes. In addition, most likely causative de novo mutations were identified in GRIN2A and SHANK3. Moreover, trio-based WES detected SMARCA2 and SMARCA4 deletions, which had not been annotated in a previous Haloplex target enrichment and next-generation sequencing of known CSS/NCBRS genes emphasizing the advantages of WES as a diagnostic tool. In summary, we discuss the phenotypic and diagnostic challenges in clinical genetics, establish important differential diagnoses, and emphasize the cardinal features and the broad clinical spectrum of BAF complex disorders and other disorders caused by mutations in epigenetic landscapers.

A new seipin-associated neurodegenerative syndrome

Background Seipin/BSCL2 mutations can cause type 2 congenital generalised lipodystrophy (BSCL) or dominant motor neurone diseases. Type 2 BSCL is frequently associated with some degree of intellectual impairment, but not to fatal neurodegeneration. In order to unveil the aetiology and pathogenetic mechanisms of a new neurodegenerative syndrome associated with a novel BSCL2 mutation, six children, four of them showing the BSCL features, were studied. Methods Mutational and splicing analyses of BSCL2 were performed. The brain of two of these children was examined postmortem. Relative expression of BSCL2 transcripts was analysed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in different tissues of the index case and controls. Overexpressed mutated seipin in HeLa cells was analysed by immunofluorescence and western blotting. Results Two patients carried a novel homozygous c.985C>T mutation, which appeared in the other four patients in compound heterozygosity. Splicing analysis showed that the c.985C>T mutation causes an aberrant splicing site leading to skipping of exon 7. Expression of exon 7-skipping transcripts was very high with respect to that of the non-skipped transcripts in all the analysed tissues of the index case. Neuropathological studies showed severe neurone loss, astrogliosis and intranuclear ubiquitin(+) aggregates in neurones from multiple cortical regions and in the caudate nucleus. Conclusions Our results suggest that exon 7 skipping in the BSCL2 gene due to the c.985C>T mutation is responsible for a novel early onset, fatal neurodegenerative syndrome involving cerebral cortex and basal ganglia.

Autosomal Dominant Oculoauriculovertebral Spectrum and 14q23.1 Microduplication

Oculoauriculovertebral spectrum (OAVS; OMIM 164210) is characterized by anomalies derived from an abnormal development of the first and second branchial arches, with marked inter and intra‐familial phenotypic variability. Main clinical features are defects on aural, oral, mandibular, and vertebral development. Cardiac, pulmonary, renal, skeletal, and central nervous system anomalies have also been described. Most affected individuals are isolated cases in otherwise normal families. Autosomal dominant inheritance has been observed in about 2–10% of cases and linkage analysis as well as array‐CGH analysis have detected candidate loci for OAVS offering new insights into the understanding of pathogenesis of this entity. We describe a family with clinical diagnosis of OAVS, autosomal dominant inheritance pattern, and detection of a 14q23.1 duplication of 1.34u2009Mb in size which segregates with the phenotype. This region contains OTX2, which is involved in the development of the forebrain, eyes, and ears, and appears to be a good candidate gene for OAVS.

Expanding the mutation spectrum in 182 Spanish probands with craniosynostosis: identification and characterization of novel TCF12 variants

Craniosynostosis, caused by the premature fusion of one or more of the cranial sutures, can be classified into non-syndromic or syndromic and by which sutures are affected. Clinical assignment is a difficult challenge due to the high phenotypic variability observed between syndromes. During routine diagnostics, we screened 182 Spanish craniosynostosis probands, implementing a four-tiered cascade screening of FGFR2, FGFR3, FGFR1, TWIST1 and EFNB1. A total of 43 variants, eight novel, were identified in 113 (62%) patients: 104 (92%) detected in level 1; eight (7%) in level 2 and one (1%) in level 3. We subsequently screened additional genes in the probands with no detected mutation: one duplication of the IHH regulatory region was identified in a patient with craniosynostosis Philadelphia type and five variants, four novel, were identified in the recently described TCF12, in probands with coronal or multisuture affectation. In the 19 Saethre–Chotzen syndrome (SCS) individuals in whom a variant was detected, 15 (79%) carried a TWIST1 variant, whereas four (21%) had a TCF12 variant. Thus, we propose that TCF12 screening should be included for TWIST1 negative SCS patients and in patients where the coronal suture is affected. In summary, a molecular diagnosis was obtained in a total of 119/182 patients (65%), allowing the correct craniosynostosis syndrome classification, aiding genetic counselling and in some cases provided a better planning on how and when surgical intervention should take place and, subsequently the appropriate clinical follow up.