Search

Contains fulltext : 125776.pdf (publisher's version ) (Closed access), The Forkhead box G1 (FOXG1) gene encodes a transcriptional repressor essential for early development of the telencephalon. Intragenic mutations and gene deletions leading to haploinsufficiency cause the congenital variant of Rett syndrome. We here describe Rett syndrome-like patients, three of them carrying a balanced translocation with breakpoint in the chromosome 14q12 region, and one patient having a 14q12 microdeletion excluding the FOXG1 gene. The hypothesis of long-range FOXG1-regulatory elements in this region was supported by our finding of reduced FOXG1 mRNA and protein levels in platelets and skin fibroblasts from these cases. Given that FOXG1 is not only expressed in brain but also in platelets, we have studied platelet morphology in these patients and two additional patients with FOXG1 mutations. Electron microscopy of their platelets showed some enlarged, rounder platelets with often abnormal alpha, and fewer dense granules. Platelet function studies were possible in one 14q12 translocation patient with a prolonged Ivy bleeding time and a patient with a heterozygous FOXG1 c.1248C>G mutation (p.Tyr416X). Both have a prolonged PFA-100 occlusion time with collagen and epinephrine and reduced aggregation responses to low dose of ADP and epinephrine. Dense granule ATP secretion was normal for strong agonists but absent for epinephrine. In conclusion, our study shows that by using platelets functional evidence of cis-regulatory elements in the 14q12 region result in reduced FOXG1 levels in patients' platelets having translocations or deletions in that region. These platelet functional abnormalities deserve further investigation regarding a non-transcriptional regulatory role for FOXG1 in these anucleated cells.

BACKGROUND: Jeune asphyxiating thoracic dystrophy (JATD) is a rare, often lethal, recessively inherited chondrodysplasia characterised by shortened ribs and long bones, sometimes accompanied by polydactyly, and renal, liver and retinal disease. Mutations in intraflagellar transport (IFT) genes cause JATD, including the IFT dynein-2 motor subunit gene DYNC2H1. Genetic heterogeneity and the large DYNC2H1 gene size have hindered JATD genetic diagnosis. AIMS AND METHODS: To determine the contribution to JATD we screened DYNC2H1 in 71 JATD patients JATD patients combining SNP mapping, Sanger sequencing and exome sequencing. RESULTS AND CONCLUSIONS: We detected 34 DYNC2H1 mutations in 29/71 (41%) patients from 19/57 families (33%), showing it as a major cause of JATD especially in Northern European patients. This included 13 early protein termination mutations (nonsense/frameshift, deletion, splice site) but no patients carried these in combination, suggesting the human phenotype is at least partly hypomorphic. In addition, 21 missense mutations were distributed across DYNC2H1 and these showed some clustering to functional domains, especially the ATP motor domain. DYNC2H1 patients largely lacked significant extra-skeletal involvement, demonstrating an important genotype-phenotype correlation in JATD. Significant variability exists in the course and severity of the thoracic phenotype, both between affected siblings with identical DYNC2H1 alleles and among individuals with different alleles, which suggests the DYNC2H1 phenotype might be subject to modifier alleles, non-genetic or epigenetic factors. Assessment of fibroblasts from patients showed accumulation of anterograde IFT proteins in the ciliary tips, confirming defects similar to patients with other retrograde IFT machinery mutations, which may be of undervalued potential for diagnostic purposes.

Item does not contain fulltext, Intellectual disability (ID) disorders are genetically and phenotypically highly heterogeneous and present a major challenge in clinical genetics and medicine. Although many genes involved in ID have been identified, the etiology is unknown in most affected individuals. Moreover, the function of most genes associated with ID remains poorly characterized. Evidence is accumulating that the control of gene transcription through epigenetic modification of chromatin structure in neurons has an important role in cognitive processes and in the etiology of ID. However, our understanding of the key molecular players and mechanisms in this process is highly fragmentary. Here, we identify a chromatin-modification module that underlies a recognizable form of ID, the Kleefstra syndrome phenotypic spectrum (KSS). In a cohort of KSS individuals without mutations in EHMT1 (the only gene known to be disrupted in KSS until now), we identified de novo mutations in four genes, MBD5, MLL3, SMARCB1, and NR1I3, all of which encode epigenetic regulators. Using Drosophila, we demonstrate that MBD5, MLL3, and NR1I3 cooperate with EHMT1, whereas SMARCB1 is known to directly interact with MLL3. We propose a highly conserved epigenetic network that underlies cognition in health and disease. This network should allow the design of strategies to treat the growing group of ID pathologies that are caused by epigenetic defects.

Item does not contain fulltext, We studied two unrelated boys with intellectual disability (ID) and a striking facial resemblance suggestive of a hitherto unappreciated syndrome. Exome sequencing in both families identified identical de novo mutations in PACS1, suggestive of causality. To support these genetic findings and to understand the pathomechanism of the mutation, we studied the protein in vitro and in vivo. Altered PACS1 forms cytoplasmic aggregates in vitro with concomitant increased protein stability and shows impaired binding to an isoform-specific variant of TRPV4, but not the full-length protein. Furthermore, consistent with the human pathology, expression of mutant PACS1 mRNA in zebrafish embryos induces craniofacial defects most likely in a dominant-negative fashion. This phenotype is driven by aberrant specification and migration of SOX10-positive cranial, but not enteric, neural-crest cells. Our findings suggest that PACS1 is necessary for the formation of craniofacial structures and that perturbation of its functions results in a specific syndromic ID phenotype.

Item does not contain fulltext, RATIONALE: Congenital heart malformations are a major cause of morbidity and mortality, especially in young children. Failure to establish normal left-right (L-R) asymmetry often results in cardiovascular malformations and other laterality defects of visceral organs. OBJECTIVE: To identify genetic mutations causing cardiac laterality defects. METHODS AND RESULTS: We performed a genome-wide linkage analysis in patients with cardiac laterality defects from a consanguineous family. The patients had combinations of defects that included dextrocardia, transposition of great arteries, double-outlet right ventricle, atrioventricular septal defects, and caval vein abnormalities. Sequencing of positional candidate genes identified mutations in NPHP4. We performed mutation analysis of NPHP4 in 146 unrelated patients with similar cardiac laterality defects. Forty-one percent of these patients also had laterality defects of the abdominal organs. We identified 8 additional missense variants that were absent or very rare in control subjects. To study the role of nphp4 in establishing L-R asymmetry, we used antisense morpholinos to knockdown nphp4 expression in zebrafish. Depletion of nphp4 disrupted L-R patterning as well as cardiac and gut laterality. Cardiac laterality defects were partially rescued by human NPHP4 mRNA, whereas mutant NPHP4 containing genetic variants found in patients failed to rescue. We show that nphp4 is involved in the formation of motile cilia in Kupffer's vesicle, which generate asymmetrical fluid flow necessary for normal L-R asymmetry. CONCLUSIONS: NPHP4 mutations are associated with cardiac laterality defects and heterotaxy. In zebrafish, nphp4 is essential for the development and function of Kupffer's vesicle cilia and is required for global L-R patterning.

Contains fulltext : 108772.pdf (publisher's version ) (Closed access), Walker-Warburg syndrome (WWS) is an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy (CMD) and aberrant a-dystroglycan glycosylation. Here we report mutations in the ISPD gene (encoding isoprenoid synthase domain containing) as the second most common cause of WWS. Bacterial IspD is a nucleotidyl transferase belonging to a large glycosyltransferase family, but the role of the orthologous protein in chordates is obscure to date, as this phylum does not have the corresponding non-mevalonate isoprenoid biosynthesis pathway. Knockdown of ispd in zebrafish recapitulates the human WWS phenotype with hydrocephalus, reduced eye size, muscle degeneration and hypoglycosylated a-dystroglycan. These results implicate ISPD in a-dystroglycan glycosylation in maintaining sarcolemma integrity in vertebrates.

Rationale: Congenital heart malformations are a major cause of morbidity and mortality, especially in young children. Failure to establish normal left-right (L-R) asymmetry often results in cardiovascular malformations and other laterality defects of visceral organs. Objective: To identify genetic mutations causing cardiac laterality defects. Methods and Results: We performed a genome-wide linkage analysis in patients with cardiac laterality defects from a consanguineous family. The patients had combinations of defects that included dextrocardia, transposition of great arteries, double-outlet right ventricle, atrioventricular septal defects, and caval vein abnormalities. Sequencing of positional candidate genes identified mutations in NPHP4. We performed mutation analysis of NPHP4 in 146 unrelated patients with similar cardiac laterality defects. Forty-one percent of these patients also had laterality defects of the abdominal organs. We identified 8 additional missense variants that were absent or very rare in control subjects. To study the role of nphp4 in establishing L-R asymmetry, we used antisense morpholinos to knockdown nphp4 expression in zebrafish. Depletion of nphp4 disrupted L-R patterning as well as cardiac and gut laterality. Cardiac laterality defects were partially rescued by human NPHP4 mRNA, whereas mutant NPHP4 containing genetic variants found in patients failed to rescue. We show that nphp4 is involved in the formation of motile cilia in Kupffer's vesicle, which generate asymmetrical fluid flow necessary for normal L-R asymmetry. Conclusions: NPHP4 mutations are associated with cardiac laterality defects and heterotaxy. In zebrafish, nphp4 is essential for the development and function of Kupffer's vesicle cilia and is required for global L-R patterning.

Chromosome 8p23.1 is a common hotspot associated with major congenital malformations, including congenital diaphragmatic hernia (CDH) and cardiac defects. We present findings from high-resolution arrays in patients who carry a loss (n = 18) or a gain (n = 1) of sub-band 8p23.1. We confirm a region involved in both diaphragmatic and heart malformations. Results from a novel CNVConnect algorithm, prioritizing protein–protein interactions between products of genes in the 8p23.1 hotspot and products of previously known CDH causing genes, implicated GATA4, NEIL2, and SOX7 in diaphragmatic defects. Sequence analysis of these genes in 226 chromosomally normal CDH patients, as well as in a small number of deletion 8p23.1 patients, showed rare unreported variants in the coding region; these may be contributing to the diaphragmatic phenotype. We also demonstrated that two of these three genes were expressed in the E11.5–12.5 primordial mouse diaphragm, the developmental stage at which CDH is thought to occur. This combination of bioinformatics and expression studies can be applied to other chromosomal hotspots, as well as private microdeletions or microduplications, to identify causative genes and their interaction networks.

Item does not contain fulltext

Pierpont syndrome is a multiple congenital anomaly syndrome with learning disability first described in 1998. There are only three patients with Pierpont syndrome who have previously been published in the literature. Details of a series of patients with features of this condition were therefore obtained retrospectively to better characterize its key features. These patients were noted to have distinctive shared facial characteristics, in addition to plantar fat pads and other limb abnormalities. Further individuals with equally striking hand and foot findings were identified whose facies were less characteristic, and hence we considered them unlikely to be affected with the same condition. Despite several patients with possible Pierpont syndrome having had high-resolution array CGH or SNP array, the etiology of this phenotype remains unknown. Whilst it is as yet unclear whether it is a single entity, there appears to be a group of patients in whom Pierpont syndrome may be a recognizable condition, with typical facies, particularly when smiling, and characteristic hand and foot findings.

01 juni 2010, Contains fulltext : 88081.pdf (publisher's version ) (Closed access), Schinzel-Giedion syndrome is characterized by severe mental retardation, distinctive facial features and multiple congenital malformations; most affected individuals die before the age of ten. We sequenced the exomes of four affected individuals (cases) and found heterozygous de novo variants in SETBP1 in all four. We also identified SETBP1 mutations in eight additional cases using Sanger sequencing. All mutations clustered to a highly conserved 11-bp exonic region, suggesting a dominant-negative or gain-of-function effect.

Context: Short stature has an incidence of three in 100 in children. Reliable molecular genetic testing may be crucial in the context of beneficial disease management. Deletions spanning or surrounding the SHOX gene account for a significant proportion of patients with idiopathic short stature (ISS) and allied disorders, such as Leri-Weill dyschondrosteosis. Objective: Several shortcomings of current strategies for copy number profiling of the SHOX region prompted us to develop an improved test for molecular diagnostics of the SHOX region. Design and Results:Weintroduced a quantitative PCR (qPCR)-based copy number profiling test, consisting of 11 amplicons targeting clinically relevant regions, i.e. the SHOX gene and regulatory regions. To ensure an optimal sensitivity and specificity, this test was validated in 32 controls and 18 probands with previously identified copy number changes. In addition, 152 probands with SHOX-associated phenotypes were screened, revealing 10 novel copy number changes. Conclusion: This highly validated qPCR test supersedes other approaches for copy number screening of the SHOX region in terms of reliability, accuracy, and cost efficiency. In addition, another strong point is the fact that it can be easily implemented in any standard equipped molecular laboratory. Our qPCR-based test is highly recommended for molecular diagnostics of idiopathic short stature and allied disorders. Copyright

Contains fulltext : 69152.pdf (publisher's version ) (Closed access), BACKGROUND: Recent molecular studies of breakpoints of recurrent chromosome rearrangements revealed the role of genomic architecture in their formation. In particular, segmental duplications representing blocks of >1 kb with >90% sequence homology were shown to mediate non-allelic homologous recombination (NAHR). However, the occurrence of the majority of newly detected submicroscopic imbalances cannot be explained by the presence of segmental duplications. Therefore, further studies are needed to investigate whether architectural features other than segmental duplications mediate these rearrangements. METHODS: We analysed a series of patients with breakpoints clustering within chromosome band 5q35. Using high density arrays and subsequent quantitative polymerase chain reaction (qPCR), we characterised the breakpoints of four interstitial deletions (including one associated with an unbalanced paracentric inversion), a duplication and a familial reciprocal t(5;18)(q35;q22) translocation. Results and CONCLUSION: Five of the breakpoints were located within an interval of approximately 265 kb encompassing the RANBP17 and TLX3 genes. This region is also targeted by the recurrent cryptic t(5;14)(q35;q32) translocation, which occurs in approximately 20% of childhood T cell acute lymphoblastic leukaemia (T-ALL). In silico analysis indicated the architectural features most likely to contribute to the genomic instability of this region, which was supported by our molecular data. Of further interest, in two patients and the familial translocation, the delineated breakpoint regions encompassed highly homologous LINEs (long interspersed nuclear elements), suggesting that NAHR between these LINEs may have mediated these rearrangements.

Contains fulltext : 70803.pdf (publisher's version ) (Closed access), Oral-facial-digital type I (OFDI) syndrome is a male-lethal X-linked dominant developmental disorder belonging to the heterogeneous group of oral-facial-digital syndromes (OFDS). OFDI is characterized by malformations of the face, oral cavity, and digits. Central nervous system (CNS) abnormalities and cystic kidney disease can also be part of this condition. This rare genetic disorder is due to mutations in the OFD1 gene that encodes a centrosome/basal body protein necessary for primary cilium assembly and for left-right axis determination, thus ascribing OFDI to the growing number of disorders associated to ciliary dysfunction. We now report a mutation analysis study in a cohort of 100 unrelated affected individuals collected worldwide. Putative disease-causing mutations were identified in 81 patients (81%). We describe 67 different mutations, 64 of which represent novel mutations, including 36 frameshift, nine missense, 11 splice-site, and 11 nonsense mutations. Most of them concentrate in exons 3, 8, 9, 12, 13, and 16, suggesting that these exons may represent mutational hotspots. Phenotypic characterization of the patients provided a better definition of the clinical features of OFDI syndrome. Our results indicate that renal cystic disease is present in 60% of cases >18 years of age. Genotype-phenotype correlation did not reveal significant associations apart for the high-arched/cleft palate most frequently associated to missense and splice-site mutations. Our results contribute to further expand our knowledge on the molecular basis of OFDI syndrome.

Contains fulltext : 70830.pdf (publisher's version ) (Closed access), Fundamental processes influencing human growth can be revealed by studying extreme short stature. Using genetic linkage analysis, we find that biallelic loss-of-function mutations in the centrosomal pericentrin (PCNT) gene on chromosome 21q22.3 cause microcephalic osteodysplastic primordial dwarfism type II (MOPD II) in 25 patients. Adults with this rare inherited condition have an average height of 100 centimeters and a brain size comparable to that of a 3-month-old baby, but are of near-normal intelligence. Absence of PCNT results in disorganized mitotic spindles and missegregation of chromosomes. Mutations in related genes are known to cause primary microcephaly (MCPH1, CDK5RAP2, ASPM, and CENPJ).

Contains fulltext : 71235.pdf (publisher's version ) (Open Access), BACKGROUND: Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS: We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS: We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS: We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Contains fulltext : 51769.pdf (publisher's version ) (Closed access), CHARGE syndrome is characterized by a wide clinical variability. During the past years the phenotypic spectrum was markedly expanded. Limb anomalies were initially not recognized as part of the phenotype but more recently mild limb anomalies were described in approximately 30% of the patients. We report on three patients with several major features of CHARGE syndrome who, in addition, presented severe limb anomalies including monodactyly, tibia aplasia, and bifid femora. Three different heterozygous truncating mutations in the CHD7 gene were detected. It has been hypothesized before that the CHARGE syndrome is caused by a disruption of mesenchymal-epithelial interaction. Given the expression of the CHD7 gene in the developing limb bud, it was anticipated that limb defects would belong to the spectrum of manifestations of CHARGE syndrome. The present observations provide further support to this hypothesis.

Item does not contain fulltext, Array-based whole genome investigation or molecular karyotyping enables the genome-wide detection of submicroscopic imbalances. Proof-of-principle experiments have demonstrated that molecular karyotyping outperforms conventional karyotyping with regard to detection of chromosomal imbalances. This article identifies areas for which the technology seems matured and areas that require more investigations. Molecular karyotyping should be part of the genetic diagnostic work-up of patients with developmental disorders. For the implementation of the technique for other constitutional indications and in prenatal diagnosis, more research is appropriate. Also, the article aims to provide best practice guidelines for the application of array comparative genomic hybridisation to ensure both technical and clinical quality criteria that will optimise and standardise results and reports in diagnostic laboratories. In short, both the specificity and the sensitivity of the arrays should be evaluated in every laboratory offering the diagnostic test. Internal and external quality control programmes are urgently needed to evaluate and standardise the test results between laboratories.

Contains fulltext : 50277.pdf (publisher's version ) (Closed access), We present three unrelated children with distinctive congenital facial skin lesions. All three children had two to three well-circumscribed, round or oval vesicular lesions, 1/2-1 cm in diameter on each cheek at birth. The lesions were located along an arc from the top of the ear to the corner of the mouth. Patient 1 was born with a unilateral cleft lip and palate, and a cutaneous hemangioma in the right palm. She is developing normally. Patient 2 has neurological sequelae after suffering an unexplained large left-sided intracerebral hemorrhage perinatally. Patient 3 has a small chin, somewhat cupped ears and a nevus on the left foot. He is developing normally. This condition has been described in the dermatological literature as focal facial dermal hypoplasia with preauricular localization. No cases with associated anomalies have been published previously. Most cases have been sporadic but familial occurrence compatible with autosomal dominant and autosomal recessive inheritance has been documented. If an embryonic fusion defect of the mandibular and maxillary prominences underlies the anomaly, the cleft lip and palate seen in one of our patients may be non-coincidental. No mutations in the TWIST2 gene were found in DNA extracted from peripheral leukocytes in the two children who were investigated.

Contains fulltext : 49619.pdf (publisher's version ) (Closed access), The prevalence of congenital heart defects is approximately 1% of all live births. Identifying the genes responsible for cardiac malformation is the first step to understand pathogenesis. Heterozygous mutations in the CSX/NKX2-5 (NKX2E) gene have been identified to cause atrial septal defect (ASD) and/or atrioventricular (AV) conduction disturbance in some families. However, there is great variability in expressivity of the phenotype between the patients with a CSX/NKX2-5 mutation.We screened four sporadic patients and three index cases of families with ASD and/or conduction defects. In one of them, a CSX/NKX2-5 mutation was identified. This novel mutation (p.Tyr256X) was inherited in a three-generation family causing five individuals to have cardiac anomalies ranging from ASD to arrhythmias. Interestingly, all the observed AV conduction disturbances were at the nodal level, manifesting first as an AV block of the first degree and evolving toward a second-degree block. Atrial fibrillation, previously reported in three individuals with CSX/NKX2-5 mutations, was observed in three patients.

Contains fulltext : 48815.pdf (publisher's version ) (Closed access), Opitz syndrome (OS; MIM 145410 and MIM 300000) is a congenital midline malformation syndrome characterized by hypertelorism, hypospadias, cleft lip/palate, laryngotracheoesophageal (LTE) abnormalities, imperforate anus, developmental delay, and cardiac defects. The X-linked form (XLOS) is caused by mutations in the MID1 gene, which encodes a microtubule-associated RBCC protein. In this study, phenotypic manifestations of patients with and without MID1 mutations were compared to determine genotype-phenotype correlations. We detected 10 novel mutations, 5 in familial cases, 2 in sporadic cases, and 3 in families for whom it was not clear if they were familial or sporadic. The genotype and phenotype was compared for these 10 families, clinically diagnosed OS patients found not to have MID1 mutations, and 4 families in whom we have previously reported MID1 mutations. This combined data set includes clinical and mutation data on 70 patients. The XLOS patients with MID1 mutations were less severely affected than patients with MID1 mutations reported in previous studies, particularly in functionally significant neurologic, LTE, anal, and cardiac abnormalities. Minor anomalies were more prevalent in patients with MID1 mutations compared to those without mutations in this study. Female MID1 mutation carriers had milder phenotypes compared to male MID1 mutation carriers, with the most common manifestation being hypertelorism in both sexes. Most of the anomalies found in the patients of the present study do not correlate with the MID1 mutation type, with the possible exception of LTE malformations. This study demonstrates the wide spectrum of severity and manifestations of OS. It also shows that XLOS patients with MID1 mutations may be less severely affected than indicated in prior reports.