Nils Peeters

Performant Mutation Identification Using Targeted Next‐Generation Sequencing of 14 Thoracic Aortic Aneurysm Genes

At least 14 causative genes have been identified for both syndromic and nonsyndromic forms of thoracic aortic aneurysm/dissection (TAA), an important cause of death in the industrialized world. Molecular confirmation of the diagnosis is increasingly important for gene‐tailored patient management but consecutive, conventional molecular TAA gene screening is expensive and labor‐intensive. To circumvent these problems, we developed a TAA gene panel for next‐generation sequencing of 14 TAA genes. After validation, we applied the assay to 100 Marfan patients. We identified 90 FBN1 mutations, 44 of which were novel. In addition, Multiplex ligation‐dependent probe amplification identified large deletions in six of the remaining samples, whereas false‐negative results were excluded by Sanger sequencing of FBN1, TGFBR1, and TGFBR2 in the last four samples. Subsequently, we screened 55 syndromic and nonsyndromic TAA patients. We identified causal mutations in 15 patients (27%), one in each of the six following genes: ACTA2, COL3A1, TGFBR1, MYLK, SMAD3, SLC2A10 (homozygous), two in NOTCH1, and seven in FBN1. We conclude that our approach for TAA genetic testing overcomes the intrinsic hurdles of consecutive Sanger sequencing of all candidate genes and provides a powerful tool for the elaboration of clinical phenotypes assigned to different genes.

A New Neurological Syndrome with Mental Retardation, Choreoathetosis, and Abnormal Behavior Maps to Chromosome Xp11

Choreoathetosis is a major clinical feature in only a small number of hereditary neurological disorders. We define a new X-linked syndrome with a unique clinical picture characterized by mild mental retardation, choreoathetosis, and abnormal behavior. We mapped the disease in a four-generation pedigree to chromosome Xp11 by linkage analysis and defined a candidate region containing a number of genes possibly involved in neuronal signaling, including a potassium channel gene and a neuronal G protein-coupled receptor.

Mutation analysis of mitochondrial DNA 12SrRNA and tRNASer(UCN) genes in non-syndromic hearing loss patients

Specific mitochondrial DNA (mtDNA) mutations in 12SrRNA and tRNASer(UCN) cause non-syndromic hearing loss (NSHL). In this study, we screened 466 hearing loss (HL) patients, negative for GJB2 mutations, for mutations in the two mtDNA genes and flanking regions. In total, 43 different variants were identified, 31 of which were polymorphisms, one was a mutation (m.1555A-->G), two were known variants of controversial pathological nature (m.827A-->G and m.961delTinsC(n)) and nine were newly identified variants. The frequency of m.1555A-->G in this set of HL patients was 0.3%, which was lower than expected. To assess the putative causative nature of controversial or newly identified variants, the frequencies of these variants were determined in 400 Belgian control subjects, and their effect on the secondary structure and their conservation among different species was determined. Our data provide further support for a polymorphic nature of the controversial m.961delTinsC(n) variant. In addition, two of the newly identified variants, m.636A-->G in the 12SrRNA flanking tRNA(Phe) and m.990T-->C in 12SrRNA, may be new candidates for pathogenic HL variants. If the pathogenic nature of m.636A-->G can be confirmed, this would be the first NSHL mutation in tRNA(Phe).

An interstitial deletion of chromosome 7 at band q21: A case report and review

We report on a girl with moderate developmental delay and mild dysmorphic features. Cytogenetic investigations revealed a de novo interstitial deletion at the proximal dark band on the long arm of chromosome 7 (7q21.1‐q21.3) in all analyzed G‐banded metaphases of lymphocytes and fibroblasts. Fluorescence in situ hybridization (FISH) and molecular studies defined the breakpoints at 7q21.11 and 7q21.3 on the paternal chromosome 7, with the proximal deletion breakpoint between the elastin gene (localized at 7q11.23) and D7S2517, and the distal breakpoint between D7S652 and the COL1A2 gene (localized at 7q21.3‐q22.1). Deletions of interstitial segments at the proximal long arm of chromosome 7 at q21 are relatively rare. The karyotype–phenotype correlation of these patients is reviewed and discussed. The clinical findings of patients with a deletion at 7q21 significantly overlap with those of patients with maternal uniparental disomy of chromosome 7 (matUPD(7)) and Silver–Russell syndrome (SRS, OMIM 180860). Therefore, 7q21 might be considered a candidate chromosomal region for matUPD(7) and SRS.

KID Syndrome: report of a Scandinavian patient with connexin-26 gene mutation.

Keratitis-ichthyosis-deafness syndrome is a rare genodermatosis, which has recently been connected with mutations in the connexin-26 gene, GJB2. We present a 15-year-old boy with erythroderma, hyperkeratotic plaques and deafness. Sequencing analysis showed a heterozygous missense mutation D50N (148G>A) in GJB2. The boy has not yet manifested characteristic eye lesions but his case shows that tardy development of eye signs should not preclude a clinical diagnosis of keratitis-ichthyosis-deafness syndrome. Besides the typical clinical features, the patients height was above the 98th percentile and he displayed a delayed bone age in his hands. Additionally, he suffered from migrainoid headaches and the results of a magnetic resonance scan of the cerebrum showed he had a large cisterna magna which probably occurred independently from the syndrome. This patient is the first Danish patient in whom the keratitis-ichthyosis-deafness syndrome has been verified by mutation analysis.

Two novel MYLK nonsense mutations causing thoracic aortic aneurysms/dissections in patients without apparent family history

To the Editor: Only 20% of all familial thoracic aortic aneurysms/dissections (FTAAD) cases are explained, of which <1% are caused by mutations in the MYLK gene. So far, 3 heterozygous MYLK mutations have been reported (Figure 1). We identified 2 novel disease-causing MYLK mutations (c.4372C>T, p.Arg1458* & c.4459C>T, p.Arg1487*) (Figure 1) using TAAD gene panel sequencing, explaining the cardiovascular phenotype in 0.6% of the cases (n = 358). Both heterozygous point mutations result in a pre-mature termination codon, probably leading to nonsense-mediated mRNA decay and, hence, haploinsufficiency. This suggests that a loss-of-function mechanism underlies MYLK-related TAAD, fitting prior observations. Screening of first-degree relatives of the proband of family A revealed the mutation only in the father. In family B, the mutation was found in the paternal half-brother of the proband, suggesting either transmission through the father or germline mosaicism. All available siblings of the father (n = 4) tested negative. The probands did not harbor mutations in other known TAAD genes. The study protocol was approved by the ethics committee. In family A (Figure 1), the proband (IV:1) presented with a type B aortic dissection from the left subclavian to the left common iliac artery (diameter descending aorta 30 mm) with a saccular aortic arch aneurysm between the left carotid artery and truncus brachiocephalicus at age 49. After 1 month of conservative treatment she underwent a Petticoat procedure at the aortic level (thoracic endovascular aortic repair [TEVAR] + bare aorta stent) and received an iliac stent for a compressed true lumen with abdominal and right leg ischemia. In a second stage, chimney TEVAR for the saccular arch aneurysm was performed. Her medical history mentioned significant hypertension, which was treated by metoprolol 10 years prior to dissection. Besides cardiovascular findings, unilateral iris flocculi, hyperopia, retrognathia and osteoarthrosis (onset age 44) were observed. A computed tomography (CT)-scan of the aorta at age 50 showed an aortic root diameter of 33 mm. The medical history of her father (III:1) involved a cerebrovascular accident (age 74), arterial hypertension (age 66) and arthrosis. Magnetic resonance (MR)-angiography showed an aortic size of 39 mm with a proximal descending aortic diameter of 25 mm. In family B, the proband (III:1) presented with an acute type B dissection at age 47, starting from the arteria lusoria to the aortic bifurcation (descending aortic diameter 42-44 mm). She was diagnosed with hypertension at the time of dissection and conservative treatment was maintained. A recent CT-scan revealed dilatation of the proximal descending aorta (46 mm). Non-cardiovascular features included myopia (1.75 dioptres), striae, delayed wound healing with mild atrophic scars, pes planus and hypercholesterolemia. She underwent hemithyroidectomy for a non-malignant nodule (38 mm). The paternal half-brother (III:2) underwent mitral valvuloplasty surgery for severe mitral insufficiency with significant P2-prolapse (age 41) and bypass surgery of the left anterior descending coronary artery. Aortic evaluation at age 48 by echocardiography and MR-angiography revealed no aortic aneurysm. He is known with hypertension which is treated by angiotensin-converting-enzym (ACE)-inhibition. Initial presentation of aortic disease was a type B dissection at relatively young ages in patients with a clear history of hypertension. Importantly, we also observed 2 mutation carriers, who are currently without aortic aneurysms (A-III:1, B-III:2). Additional risk factors (eg, hypertension) thus might be required to provoke dissections. Interestingly, 1 of the 2 previously published MYLK patients with type B dissection presented with hypertension too. As previously described, vascular disease beyond the aorta was observed in our mutation carriers. Furthermore, various systemic clinical features present, suggesting that the phenotypic spectrum of MYLK mutations might encompass non-syndromic and syndromic TAAD. Our data support that these mutations associate to aortic disease with little to no aortic enlargement prior to dissection. Importantly, we believe that the cardiovascular management strategy in MYLK mutation carriers should focus on strict treatment of cardiovascular risk factors like hypertension, as no other predictive markers for aortic dissection are available. Funding information Fund for Scientific Research Flanders (FWO), Grant/Award number: G.0221.12; Foundation Leducq, Grant/Award number: 12CVD03; European Research Council, Grant/Award number: ERC-StG-2012-30972; Agency for Innovation by Science and Technology: IWT.141429. Received: 13 December 2016 Revised: 15 February 2017 Accepted: 16 February 2017

Novel pathogenic SMAD2 variants in five families with arterial aneurysm and dissection: further delineation of the phenotype

Background Missense variants in SMAD2, encoding a key transcriptional regulator of transforming growth factor beta signalling, were recently reported to cause arterial aneurysmal disease. Objectives The aims of the study were to identify the genetic disease cause in families with aortic/arterial aneurysmal disease and to further define SMAD2 genotype–phenotype correlations. Methods and results Using gene panel sequencing, we identified a SMAD2 nonsense variant and four SMAD2 missense variants, all affecting highly conserved amino acids in the MH2 domain. The premature stop codon (c.612dup; p.(Asn205*)) was identified in a marfanoid patient with aortic root dilatation and in his affected father. A p.(Asn318Lys) missense variant was found in a Marfan syndrome (MFS)-like case who presented with aortic root aneurysm and in her affected daughter with marfanoid features and mild aortic dilatation. In a man clinically diagnosed with Loeys-Dietz syndrome (LDS) that presents with aortic root dilatation and marked tortuosity of the neck vessels, another missense variant, p.(Ser397Tyr), was identified. This variant was also found in his affected daughter with hypertelorism and arterial tortuosity, as well as his affected mother. The third missense variant, p.(Asn361Thr), was discovered in a man presenting with coronary artery dissection. Variant genotyping in three unaffected family members confirmed its absence. The last missense variant, p.(Ser467Leu), was identified in a man with significant cardiovascular and connective tissue involvement. Conclusion Taken together, our data suggest that heterozygous loss-of-function SMAD2 variants can cause a wide spectrum of autosomal dominant aortic and arterial aneurysmal disease, combined with connective tissue findings reminiscent of MFS and LDS.

Bi-allelic inactivating variants in the COCH gene cause autosomal recessive prelingual hearing impairment

Pathogenic variant in COCH are a known cause of DFNA9 autosomal dominant progressive hearing loss and vestibular dysfunction with adult onset. Hitherto, only dominant nonsynonymous variants and in-frame deletions with a presumed dominant negative or gain-of-function effect have been described. Here, we describe two brothers with congenital prelingual deafness and a homozygous nonsense c.292C>T(p.Arg98*) COCH variant, suggesting a loss-of-function effect. Vestibular dysfunction starting in the first decade was observed in the older patient. The heterozygous parents and sibling have normal hearing and vestibular function, except for the mother, who shows vestibular hyporeflexia and abnormal smooth pursuit tests, most likely due to concomitant disease. This is the first report of autosomal recessive inheritance of cochlea-vestibular dysfunction caused by a pathogenic variant in the COCH gene. An earlier onset of hearing impairment and vestibular dysfunction compared to the dominant hearing loss causing COCH variants is observed.