Ingrid van de Laar

Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis

Thoracic aortic aneurysms and dissections are a main feature of connective tissue disorders, such as Marfan syndrome and Loeys-Dietz syndrome. We delineated a new syndrome presenting with aneurysms, dissections and tortuosity throughout the arterial tree in association with mild craniofacial features and skeletal and cutaneous anomalies. In contrast with other aneurysm syndromes, most of these affected individuals presented with early-onset osteoarthritis. We mapped the genetic locus to chromosome 15q22.2–24.2 and show that the disease is caused by mutations in SMAD3. This gene encodes a member of the TGF-β pathway that is essential for TGF-β signal transmission. SMAD3 mutations lead to increased aortic expression of several key players in the TGF-β pathway, including SMAD3. Molecular diagnosis will allow early and reliable identification of cases and relatives at risk for major cardiovascular complications. Our findings endorse the TGF-β pathway as the primary pharmacological target for the development of new treatments for aortic aneurysms and osteoarthritis.

Phenotypic spectrum of the SMAD3-related aneurysms–osteoarthritis syndrome

Background Aneurysms–osteoarthritis syndrome (AOS) is a new autosomal dominant syndromic form of thoracic aortic aneurysms and dissections characterised by the presence of arterial aneurysms and tortuosity, mild craniofacial, skeletal and cutaneous anomalies, and early-onset osteoarthritis. AOS is caused by mutations in the SMAD3 gene. Methods A cohort of 393 patients with aneurysms without mutation in FBN1, TGFBR1 and TGFBR2 was screened for mutations in SMAD3. The patients originated from The Netherlands, Belgium, Switzerland and USA. The clinical phenotype in a total of 45 patients from eight different AOS families with eight different SMAD3 mutations is described. In all patients with a SMAD3 mutation, clinical records were reviewed and extensive genetic, cardiovascular and orthopaedic examinations were performed. Results Five novel SMAD3 mutations (one nonsense, two missense and two frame-shift mutations) were identified in five new AOS families. A follow-up description of the three families with a SMAD3 mutation previously described by the authors was included. In the majority of patients, early-onset joint abnormalities, including osteoarthritis and osteochondritis dissecans, were the initial symptom for which medical advice was sought. Cardiovascular abnormalities were present in almost 90% of patients, and involved mainly aortic aneurysms and dissections. Aneurysms and tortuosity were found in the aorta and other arteries throughout the body, including intracranial arteries. Of the patients who first presented with joint abnormalities, 20% died suddenly from aortic dissection. The presence of mild craniofacial abnormalities including hypertelorism and abnormal uvula may aid the recognition of this syndrome. Conclusion The authors provide further insight into the phenotype of AOS with SMAD3 mutations, and present recommendations for a clinical work-up.

Aggressive Cardiovascular Phenotype of Aneurysms-Osteoarthritis Syndrome Caused by Pathogenic SMAD3 Variants

OBJECTIVES The purpose of this study was describe the cardiovascular phenotype of the aneurysms-osteoarthritis syndrome (AOS) and to provide clinical recommendations. BACKGROUND AOS, caused by pathogenic SMAD3 variants, is a recently described autosomal dominant syndrome characterized by aneurysms and arterial tortuosity in combination with osteoarthritis. METHODS AOS patients in participating centers underwent extensive cardiovascular evaluation, including imaging, arterial stiffness measurements, and biochemical studies. RESULTS We included 44 AOS patients from 7 families with pathogenic SMAD3 variants (mean age: 42 ± 17 years). In 71%, an aortic root aneurysm was found. In 33%, aneurysms in other arteries in the thorax and abdomen were diagnosed, and in 48%, arterial tortuosity was diagnosed. In 16 patients, cerebrovascular imaging was performed, and cerebrovascular abnormalities were detected in 56% of them. Fifteen deaths occurred at a mean age of 54 ± 15 years. The main cause of death was aortic dissection (9 of 15; 60%), which occurred at mildly increased aortic diameters (range: 40 to 63 mm). Furthermore, cardiac abnormalities were diagnosed, such as congenital heart defects (6%), mitral valve abnormalities (51%), left ventricular hypertrophy (19%), and atrial fibrillation (22%). N-terminal brain natriuretic peptide (NT-proBNP) was significantly higher in AOS patients compared with matched controls (p < 0.001). Aortic pulse wave velocity was high-normal (9.2 ± 2.2 m/s), indicating increased aortic stiffness, which strongly correlated with NT-proBNP (r = 0.731, p = 0.005). CONCLUSIONS AOS predisposes patients to aggressive and widespread cardiovascular disease and is associated with high mortality. Dissections can occur at relatively mildly increased aortic diameters; therefore, early elective repair of the ascending aorta should be considered. Moreover, cerebrovascular abnormalities were encountered in most patients.

Familial CHARGE syndrome and the CHD7 gene: a recurrent missense mutation, intrafamilial recurrence and variability.

CHARGE syndrome is an autosomal dominant condition that is caused by mutations in the CHD7 gene. Few familial cases of this syndrome have been reported and these were characterized by a wide clinical variability. We here report on five CHD7 mutation positive families and comment on their clinical features. We observed somatic and germline mosaicism as well as parent‐to‐child transmission of non‐mosaic CHD7 mutations as causes of familial CHARGE syndrome. In one family with two affected sibs a somatic mutation was identified in lymphocytes of a clinically unaffected parent (2520G > A in exon 8). This is the second report of somatic CHD7 mosaicism in an unaffected parent. In two further families with affected siblings, we could not detect the mutation in parental lymphocytes suggesting germline mosaicism. The previously reported clinical variability was strikingly present in all five families. We find that alterations in CHD7 can result in a very mild phenotype, characterized by only a few minor symptoms of the CHARGE syndrome clinical spectrum. Such a mild phenotype was present in two families that shared the same 6322G > A missense mutation. These two families showed parent‐to‐child transmission. Phenotypically milder forms of CHARGE syndrome have a higher risk of transmission to multiple family members.

Genomic SNP array as a gold standard for prenatal diagnosis of foetal ultrasound abnormalities

BackgroundWe have investigated whether replacing conventional karyotyping by SNP array analysis in cases of foetal ultrasound abnormalities would increase the diagnostic yield and speed of prenatal diagnosis in clinical practice.Findings/resultsFrom May 2009 till June 2011 we performed HumanCytoSNP-12 array (HCS) (http://www.Illumina.com) analysis in 207 cases of foetal structural abnormalities. HCS allows detecting unbalanced genomic abnormalities with a resolution of about 150/200 kb. All cases were selected by a clinical geneticist after excluding the most common aneuploidies by RAD (rapid aneuploidy detection). Pre-test genetic counselling was offered in all cases.In 24/207 (11,6%) foetuses a clinically relevant genetic abnormality was detected. Only 8/24 abnormalities would have been detected if only routine karyotyping was performed. Submicroscopic abnormalities were found in 16/207 (7,7%) cases. The array results were achieved within 1-2 weeks after amniocentesis.ConclusionsPrenatal SNP array testing is faster than karyotyping and allows detecting much smaller aberrations (~0.15 Mb) in addition to the microscopic unbalanced chromosome abnormalities detectable with karyotyping (~ > 5 Mb). Since karyotyping would have missed 66% (16/24) of genomic abnormalities in our cohort, we propose to perform genomic high resolution array testing assisted by pre-test counselling as a primary prenatal diagnostic test in cases of foetal ultrasound abnormalities.

PRRT2 phenotypes and penetrance of paroxysmal kinesigenic dyskinesia and infantile convulsions

Objective: To describe the phenotypes and penetrance of paroxysmal kinesigenic dyskinesia (PKD), a movement disorder characterized by attacks of involuntary movements occurring after sudden movements, infantile convulsion and choreoathetosis (ICCA) syndrome, and benign familial infantile convulsions (BFIC), caused by PRRT2 mutations. Methods: We performed clinical and genetic studies in 3 large families with ICCA, 2 smaller families with PKD, and 4 individuals with sporadic PKD. Migraine was also present in several individuals. Results: We detected 3 different PRRT2 heterozygous mutations: the recurrent p.Arg217Profs*8 mutation, previously reported, was identified in 2 families with ICCA, 2 families with PKD, and one individual with sporadic PKD; one novel missense mutation (p.Ser275Phe) was detected in the remaining family with ICCA; and one novel truncating mutation (p.Arg217*) was found in one individual with sporadic PKD. In the 2 remaining individuals with sporadic PKD, PRRT2 mutations were not detected. Importantly, PRRT2 mutations did not cosegregate with febrile convulsions or with migraine. The estimated penetrance of PRRT2 mutations was 61%, if only the PKD phenotype was considered; however, if infantile convulsions were also taken into account, the penetrance was nearly complete. Considering our findings and those reported in literature, 23 PRRT2 mutations explain ∼56% of the families analyzed. Conclusions: PRRT2 mutations are the major cause of PKD or ICCA, but they do not seem to be involved in the etiology of febrile convulsions and migraine. The identification of PRRT2 as a major gene for the PKD-ICCA-BFIC spectrum allows better disease classification, molecular confirmation of the clinical diagnosis, and genetic testing and counseling.

Cardiovascular malformations caused by NOTCH1 mutations do not keep left: data on 428 probands with left-sided CHD and their families

Purpose:We aimed to determine the prevalence and phenotypic spectrum of NOTCH1 mutations in left-sided congenital heart disease (LS-CHD). LS-CHD includes aortic valve stenosis, a bicuspid aortic valve, coarctation of the aorta, and hypoplastic left heart syndrome.Methods:NOTCH1 was screened for mutations in 428 nonsyndromic probands with LS-CHD, and family histories were obtained for all. When a mutation was detected, relatives were also tested.Results:In 148/428 patients (35%), LS-CHD was familial. Fourteen mutations (3%; 5 RNA splicing mutations, 8 truncating mutations, 1 whole-gene deletion) were detected, 11 in familial disease (11/148 (7%)) and 3 in sporadic disease (3/280 (1%)). Forty-nine additional mutation carriers were identified among the 14 families, of whom 12 (25%) were asymptomatic. Most of these mutation carriers had LS-CHD, but 9 (18%) had right-sided congenital heart disease (RS-CHD) or conotruncal heart disease (CTD). Thoracic aortic aneurysms (TAAs) occurred in 6 mutation carriers (probands included 6/63 (10%)).Conclusion:Pathogenic mutations in NOTCH1 were identified in 7% of familial LS-CHD and in 1% of sporadic LS-CHD. The penetrance is high; a cardiovascular malformation was found in 75% of NOTCH1 mutation carriers. The phenotypic spectrum includes LS-CHD, RS-CHD, CTD, and TAA. Testing NOTCH1 for an early diagnosis in LS-CHD/RS-CHD/CTD/TAA is warranted.Genet Med 18 9, 914–923.

Functional analysis of novel TBX5 T-box mutations associated with Holt–Oram syndrome

AIMS Holt-Oram syndrome (HOS) is a heart/hand syndrome clinically characterized by upper limb and cardiac malformations. Mutations in T-box transcription factor 5 (TBX5) underlie this syndrome, the majority of which lead to premature stops. In this study, we present our functional analyses of five (novel) missense TBX5 mutations identified in HOS patients, most of whom presented with severe cardiac malformations. METHODS AND RESULTS Functional characterization of mutant proteins shows a dramatic loss of DNA-binding capacity, as well as diminished binding to known cardiac interaction partners NKX2-5 and GATA4. The disturbance of these interactions leads to a loss of function, as measured by the reduced activation of Nppa and FGF10 in rat heart derived cells, although with variable severity. Two out of the five mutations are peculiar: one, p.H220del, is associated with additional extra-cardiac defects, perhaps by interfering with other T-box dependant pathways, and another, p.I106V, leads to limb defects only, which is supported by its normal interaction with cardiac-specific interaction partners. CONCLUSION Overall, our data are consistent with the hypothesis that these novel missense mutations in TBX5 lead to functional haploinsufficiency and result in a reduced transcriptional activation of target genes, which is likely central to the pathogenesis of HOS.

NPHP4 Variants are Associated with Pleiotropic Heart Malformations

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 Kupffers 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 Kupffers vesicle cilia and is required for global L-R patterning.

Limb anomalies in patients with CHARGE syndrome: an expansion of the phenotype.

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.