Two novel missense mutations in HAIRY-AND-ENHANCER-OF-SPLIT-7 in a family with spondylocostal dysostosis

Spondylocostal dysostosis (SCD) is an inherited disorder with abnormal vertebral segmentation that results in 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). Four genes causing a subset of autosomal recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300), MESP2 (SCDO2: MIM 608681), LFNG (SCDO3: MIM609813) and HES7 (SCDO4). These genes are all essential components of the Notch signalling pathway, which has multiple roles in development and disease. Previously, only a single SCD-causative missense mutation was described in HES7. In this study, we have identified two new missense mutations in the HES7 gene in a single family, with only individuals carrying both mutant alleles being affected by SCD. In vitro functional analysis revealed that one of the mutant HES7 proteins was unable to repress gene expression by DNA binding or protein heterodimerization.     

Pseudodominant inheritance of spondylocostal dysostosis type 1 caused by two familial delta-like 3 mutations

Spondylocostal dysostoses (SCD) are a heterogeneous group of disorders of axial skeletal malformation characterized by multiple vertebral segmentation defects and rib anomalies. Sporadic cases with diverse phenotypes, sometimes including multiple organ abnormalities, are relatively common, and monogenic forms demonstrating autosomal recessive (AR) and, more rarely, autosomal dominant (AD) inheritance have been reported. We previously showed that mutations in delta-like 3 (DLL3), a somitogenesis gene that encodes a ligand for the notch signaling pathway, cause AR SCD with a consistent pattern of abnormal segmentation. We studied an SCD family previously reported to show AD inheritance, in which the phenotype is similar to that in AR cases. Direct DLL3 sequencing of individuals in two generations identified the affected father as homozygous for a novel frameshift mutation, 1440delG. His two affected children were compound heterozygotes for this mutation and a novel missense mutation, G504D, the first putative missense mutation reported in the transmembrane domain of DLL3. Their two unaffected siblings were heterozygotes for the 1440delG mutation. Pseudodominant inheritance has been confirmed, and the findings raise potential consequences for genetic counseling in relation to the SCD disorders.     

Homozygous DMRT2 variant associates with severe rib malformations in a newborn

Spondylocostal dysostosis (SCD) is a rare disorder characterized by vertebral segmentation defects and malformations of the ribs. SCD patients have some degree of (kypho)scoliosis, short stature and suffer from respiratory impairment due to the reduced size of their thoracic cage. Mutations in DLL3, MESP2, LFNG, HES7, TBX6, and RIPPLY2 are known to cause different subtypes of SCD. Here, we report on a male neonate with an apparent distinct SCD‐like phenotype only partly overlapping the previously described SCD subtypes. The proband presented with severe rib malformations (missing, fused, bifid, and hypoplastic ribs), vertebral malformations (intervertebral fusions of the laminae and irregular ossification of the vertebral bodies), and a mild scoliosis. Clear segmentation defects of the vertebral bodies were lacking. Other dysmorphic features were present as well. Severe respiratory insufficiency was present from birth. Whole exome sequencing identified a homozygous start‐loss variant in DMRT2 (NM_006557.6: c.1A > T p.[Met1?]) being a likely cause of the SCD‐like phenotype in the proband. Mutations in DMRT2 (OMIM#604935) have not been described in relation to SCD‐related phenotypes in humans before. However, Dmrt2 knock‐out mice exhibit severe rib and vertebral defects that strikingly overlap with the radiological phenotype of the proband reported here. Therefore, it seems plausible that mutations in DMRT2 are associated with a different (novel) subtype of SCD mainly characterized by severe rib anomalies but lacking clear segmentation defects of the vertebral bodies.     

Phenotypic and molecular characterization of a novel case of dyssegmental dysplasia,Silverman-Handmaker type

Dyssegmental dysplasia, Silverman-Handmaker type (DDSH; #MIM 224410) is an autosomal recessive form of lethal dwarfism characterized by a defect in segmentation and fusion of vertebral bodies components (“anisospondyly”) and by severe limb shortening. It is caused by mutations in the perlecan gene (HSPG2), but so far, only three molecularly confirmed cases have been reported. We report a novel case of DDSH in a fetus that presented at 15 weeks gestation with encephalocele, severe micromelic dwarfism and narrow thorax. After termination of pregnancy, radiographs showed short ribs, short and bent long bones and anisospondyly of two vertebral bodies. The fetus was homozygous for a previously undescribed null mutation in HSPG2.     

Notch inhibition by the ligand DELTA-LIKE 3 defines the mechanism of abnormal vertebral segmentation in spondylocostal dysostosis

Mutations in the DELTA-LIKE 3 (DLL3) gene cause the congenital abnormal vertebral segmentation syndrome, spondylocostal dysostosis (SCD). DLL3 is a divergent member of the DSL family of Notch ligands that does not activate signalling in adjacent cells, but instead inhibits signalling when expressed in the same cell as the Notch receptor. Targeted deletion of Dll3 in the mouse causes a developmental defect in somite segmentation, and consequently vertebral formation is severely disrupted, closely resembling human SCD. In contrast to the canonical Notch signalling pathway, very little is known about the mechanism of cis-inhibition by DSL ligands. Here, we report that Dll3 is not presented on the surface of presomitic mesoderm (PSM) cells in vivo, but instead interacts with Notch1 in the late endocytic compartment. This suggests for the first time a mechanism for Dll3-mediated cis-inhibition of Notch signalling, with Dll3 targeting newly synthesized Notch1 for lysosomal degradation prior to post-translational processing and cell surface presentation of the receptor. An inhibitory role for Dll3 in vivo is further supported by the juxtaposition of Dll3 protein and Notch1 signalling in the PSM. Defining a mechanism for cis-inhibition of Notch signalling by Dll3 not only contributes greatly to our understanding of this ligand's function during the formation of the vertebral column, but also provides a paradigm for understanding how other ligands of Notch cis-inhibit signalling.     

Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing

Background

Heterozygous copy-number and missense variants in CNTNAP2 and NRXN1 have repeatedly been associated with a wide spectrum of neuropsychiatric disorders such as developmental language and autism spectrum disorders, epilepsy and schizophrenia. Recently, homozygous or compound heterozygous defects in either gene were reported as causative for severe intellectual disability.

Methods

99 patients with severe intellectual disability and resemblance to Pitt-Hopkins syndrome and/or suspected recessive inheritance were screened for mutations in CNTNAP2 and NRXN1. Molecular karyotyping was performed in 45 patients. In 8 further patients with variable intellectual disability and heterozygous deletions in either CNTNAP2 or NRXN1, the remaining allele was sequenced.

Results

By molecular karyotyping and mutational screening of CNTNAP2 and NRXN1 in a group of severely intellectually disabled patients we identified a heterozygous deletion in NRXN1 in one patient and heterozygous splice-site, frameshift and stop mutations in CNTNAP2 in four patients, respectively. Neither in these patients nor in eight further patients with heterozygous deletions within NRXN1 or CNTNAP2 we could identify a defect on the second allele. One deletion in NRXN1 and one deletion in CNTNAP2 occurred de novo, in another family the deletion was also identified in the mother who had learning difficulties, and in all other tested families one parent was shown to be healthy carrier of the respective deletion or mutation.

Conclusions

We report on patients with heterozygous defects in CNTNAP2 or NRXN1 associated with severe intellectual disability, which has only been reported for recessive defects before. These results expand the spectrum of phenotypic severity in patients with heterozygous defects in either gene. The large variability between severely affected patients and mildly affected or asymptomatic carrier parents might suggest the presence of a second hit, not necessarily located in the same gene.     

Recurrence of perinatal lethal osteogenesis imperfecta in sibships: Parsing the risk between parental mosaicism for dominant mutations and autosomal recessive inheritance

PurposeRecurrence of lethal osteogenesis imperfecta in families results from either dominant (parental mosaicism) or recessive inheritance. The proportion of these two mechanisms is not known, and determination of the contribution of each is important to structure genetic counseling for these families.MethodsWe measured the recurrence rate of lethal osteogenesis imperfecta after the birth of an affected infant. We determined the rate of parental mosaicism in a subset of families in which we had identified dominant mutations. In 37 families in which two or more affected infants were born, we identified mutations and determined the proportion that resulted from recessive inheritance.ResultsThe recurrence rate after the first affected pregnancy was 1.3%. The rate of parental mosaicism in families in which a dominant mutation was identified in a first affected child was 16%. In 37 families with two affected infants, 26 had dominant mutations, seven had recessive mutations, and we failed to find mutations in four. The overall recurrence rate for couples after two or more affected infants was 32%; 27% for families with parental mosaicism, 31% for recessive mutations, and 50% for families with no identified mutation.ConclusionsIn most populations, recurrence of lethal osteogenesis imperfecta usually results from parental mosaicism for dominant mutations, but the carrier frequency of recessive forms of osteogenesis imperfecta will alter that proportion. Mutation identification is an important tool to assess risk and facilitate prenatal or Genet Med 2011:13(2):125–130.     

Novel splice site mutation in CNNM4 gene in a family with Jalili syndrome

Jalili syndrome is a rare autosomal recessive genetic disease characterized by the association of amelogenesis imperfecta and cone-rod retinal dystrophy. This syndrome is caused by mutations in the CNNM4 gene. Different types of CNNM4 mutations have been reported; missense, nonsense, large deletions, single base insertion, and duplication.We used Sanger sequencing to analyze a large consanguineous family with three siblings affected with Jalili syndrome, suspected clinically after dental and ophthalmological examination. These patients are carrying a novel homozygous mutation in the splice site acceptor of intron 3 (c.1682-1G > C) in the CNNM4 gene.We compare the findings of the present family to those from literature, in order to further delineate Jalili syndrome.     

Monozygotic twins discordant for spondylocostal dysostosis

Spondylocostal dysostosis (SCD) is a rare skeletal dysplasia with clinical and radiological manifestations, consisting of short neck and trunk, barrel-shaped chest, protuberant abdomen, scoliosis and abnormalities of vertebral segmentation and of the ribs. Both autosomal recessive and autosomal dominant inheritance have been described. We report on monozygotic twins discordant for the syndrome, either due to a post-zygotic mutation or development of a phenocopy. To our best knowledge, this is the fourth report of SCD in identical twins, and the first one of discordance in monozygotic twins. © 1994 Wiley-Liss, Inc.     

Homozygous deletion of the entire AAAS gene in a triple A syndrome patient

Triple A syndrome, a multisystemic autosomal recessive disease, is characterized by the clinical triad of adrenal insufficiency, alacrima and achalasia in combination with progressive neurological impairments. The disorder is caused by homozygous or compound heterozygous mutations in the AAAS gene. Here we present the clinical and molecular data of a ten year old patient with triple A syndrome. Array CGH analysis confirmed the PCR-based assumption of a homozygous deletion of the entire AAAS gene in the patient and a heterozygous deletion in both parents. We demonstrate that the patient carries a 15 kb deletion and identified the 5′ and 3’ breakpoints outside the AAAS gene. This is the first report of a triple A syndrome patient with a homozygous deletion of the entire AAAS gene.     

DLL3 as a candidate gene for vertebral malformations

Investigations have not identified a major locus for congenital vertebral malformations. Based on observations in mice, we hypothesized that mutations in DLL3, a member of the notch-signaling pathway, might contribute to human vertebral malformations. We sequenced the DLL3 gene in 50 patients with congenital vertebral malformations. A Caucasian male patient with VACTERL manifestations including a T5-T6 block vertebrae was heterozygous for a "G" to "A" missense mutation changing glycine to arginine at codon 269. This residue is conserved in mammals, including chimpanzee, mouse, dog, and rat. Additional testing in the patient did not show evidence of chromosome abnormalities. The patient's asymptomatic mother was also heterozygous for the missense mutation. Since this mutation was not observed in a control population and leads to an amino acid change, it may be clinically significant. The mutation was not found in a control population of 87 anonymous individuals. Several established mechanisms could explain the mutation in both the patient and his asymptomatic mother (susceptibility allele requiring additional environmental factors, somatic mosaicism, multigenic inheritance). Documenting the absence of the mutation in a larger control population or the presence of the mutation in additional affected patients, or documenting a functional difference in DLL3 would provide further evidence supporting its causal role.     

A cluster of autosomal recessive spondylocostal dysostosis caused by three newly identified DLL3 mutations segregating in a small village

In 1982, one of us reported a cluster of eight individuals affected by spondylocostal dysostosis (SD, MIM 277300) in four nuclear families indigenous to a village from eastern Switzerland. We tested the hypothesis that the molecular basis for this cluster was segregation of a single mutation in the DLL3 gene, recently linked to SD. Marker haplotypes around the DLL3 locus contradicted this hypothesis as three different haplotypes were seen in affected individuals, but sequence analysis showed that three unreported DLL3 mutations were segregating: a duplication of 17 bp in exon 8 (c.1285-1301dup), a single-nucleotide deletion in exon 5 (c.615delC), and a R238X nonsense mutation in exon 6. Contrary to our initial assumption of a single allele segregating in this small community, three different pathogenic alleles were observed, with a putative founder mutation occurring at the homozygous state but also compounding with, and thus revealing, two other independent mutations. As all three mutations predict truncation of the DLL3 protein and loss of the membrane-attaching domain, the results confirm that autosomal recessive spondylocostal dysostosis represents the null phenotype of DLL3, with remarkable phenotypic consistency across families.     

A tale of 2 cousins: An atypical and a typical case of abetalipoproteinemia

Abetalipoproteinemia (ABL) is a rare recessive genetic disease caused by mutations of the MTTP gene. This disease is characterised by a defect in the lipidation of APO B and the absence of VLDL and chylomicron production. Patients affected by ABL present neurological, hemalogical and gastro-intestinal symptoms due to deficiency in lipophilic vitamins and fat malabsorption. We herein report the case of two cousins, one presenting classical symptoms of abetalipoproteinemia and one presenting a much attenuated phenotype. The proband carried a novel combination of MTTP mutations, the 1867+1G>A and the R540C mutations. This patient never received any vitamin supplements and was relatively free of symptoms despite an undetectable APO B concentration. Her cousin was homozygous for 1867+1G>A MTTP mutation and presented most of the classical symptoms of ABL. In conclusion we report a very unusual kindred where on affected member is strongly symptomatic of ABL whereas the other presents very mostly asymptomatic disease suggesting that ABL can present itself with a very incomplete clinical penetrance.     

Identification of bi-allelic LFNG variants in three patients and further clinical and molecular refinement of spondylocostal dysostosis 3

Spondylocostal dysostosis (SCD), a condition characterized by multiple segmentation defects of the vertebrae and rib malformations, is caused by bi-allelic variants in one of the genes involved in the Notch signaling pathway that tunes the “segmentation clock” of somitogenesis: DLL3, HES7, LFNG, MESP2, RIPPLY2, and TBX6. To date, seven individuals with LFNG variants have been reported in the literature. In this study we describe two newborns and one fetus with SCD, who were found by trio-based exome sequencing (trio-ES) to carry homozygous (c.822-5C>T) or compound heterozygous (c.[863dup];[1063G>A]) and (c.[521G>T];[890T>G]) variants in LFNG. Notably, the c.822-5C>T change, affecting the polypyrimidine tract of intron 5, is the first non-coding variant reported in LFNG. This study further refines the clinical and molecular features of spondylocostal dysostosis 3 and adds to the numerous investigations supporting the usefulness of trio-ES approach in prenatal and neonatal settings.     

A novel 22 bp deletion in a Tunisian phenylketonuria family

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase. To date, more than 530 mutations in the PAH gene have been reported. In Tunisia, this disease seems to be the result of point mutations, few studies have been published about molecular defects of PKU in our country. In this study, we report a novel deletion in exon 6 of two brothers in a Tunisian family after DHPLC analysis and sequencing of the exon 6 of the PAH gene.     

Autosomal recessive otofaciocervical syndrome type 2 with novel homozygous small insertion in PAX1 gene

Otofaciocervical syndrome (OTFCS) is described as a single gene disorder of both autosomal dominant and autosomal recessive inheritance. The major clinical features of OTFCS include ear malformations (external/middle/inner ear), facial dysmorphism, shoulder girdle abnormalities, vertebral anomalies, and mild intellectual disability. The autosomal recessive form of OTFCS syndrome (OTFCS2) has been recently reported to be caused due to homozygous mutations in PAX1 gene. Here we report a third family of OTFCS2 phenotype wherein whole exome sequencing identified a novel homozygous small insertion in PAX1 as the underlying genetic cause.     

Spondylocostal dysostosis in a pregnancy complicated by confined placental mosaicism for tetrasomy 9p

The spondylocostal dysostoses (SCD) are a clinically and genetically heterogeneous group of disorders characterized by defects of vertebral segmentation and rib abnormalities. We report on the diagnosis of two siblings with SCD. Diagnosis was first made in a female infant following a pregnancy that was complicated by early fetal hydrops and a nuchal translucency of 8.2 mm in the first trimester. The clinical picture was complicated by the co-existent diagnosis of confined placental mosaicism (CPM) for tetrasomy 9p. To our knowledge, this is the first report of CPM for tetrasomy 9p. Postnatally the diagnosis of SCD was made on the basis of radiographic findings comprising multiple anomalies of the cervical and thoracic vertebrae and multiple fused and dysplastic ribs. Radiographic investigation of other family members showed that the infant's 4-year-old sibling had fusion of four ribs on the right side, indicating a less severe form of SCD. Testing of the genes DLL3, MESP2, and LFNG did not identify a mutation, suggesting that the siblings may have a new molecular subtype of SCD.     

CRTAP mutations in lethal and severe osteogenesis imperfecta: the importance of combining biochemical and molecular genetic analysis

Autosomal recessive lethal and severe osteogenesis imperfecta (OI) is caused by the deficiency of cartilage-associated protein (CRTAP) and prolyl-3-hydroxylase 1 (P3H1) because of CRTAP and LEPRE1 mutations. We analyzed five families in which 10 individuals had a clinical diagnosis of lethal and severe OI with an overmodification of collagen type I on biochemical testing and without a mutation in the collagen type I genes. CRTAP mutations not described earlier were identified in the affected individuals. Although it seems that one important feature of autosomal recessive OI due to CRTAP mutations is the higher consistency of radiological features with OI type II-B/III, differentiation between autosomal dominant and autosomal recessive OI on the basis of clinical, radiological and biochemical investigations proves difficult in the affected individuals reported here. These observations confirm that once a clinical diagnosis of OI has been made in an affected individual, biochemical testing for overmodification of collagen type I should always be combined with molecular genetic analysis of the collagen type I genes. If no mutations in the collagen type I genes are found, additional molecular genetic analysis of the CRTAP and LEPRE1 genes should follow. This approach will allow proper identification of the genetic cause of lethal or severe OI, which is important in providing prenatal diagnosis, preimplantation genetic diagnosis and estimating recurrence risk.