An FBN1 Deep Intronic Mutation in a Familial Case of Marfan Syndrome: An Explanation for Genetically Unsolved Cases?

Marfan syndrome (MFS) is caused by mutations in the FBN1 (fibrillin‐1) gene, but approximately 10% of MFS cases remain genetically unsolved. Here, we report a new FBN1 mutation in an MFS family that had remained negative after extensive molecular genomic DNA FBN1 testing, including denaturing high‐performance liquid chromatography, Sanger sequencing, and multiplex ligation‐dependent probe amplification. Linkage analysis in the family and cDNA sequencing of the proband revealed a deep intronic point mutation in intron 56 generating a new splice donor site. This mutation results in the integration of a 90‐bp pseudo‐exon between exons 56 and 57 containing a stop codon, causing nonsense‐mediated mRNA decay. Although more than 90% of FBN1 mutations can be identified with regular molecular testing at the genomic level, deep intronic mutations will be missed and require cDNA sequencing or whole‐genome sequencing.     

Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys‐Dietz syndromes

The Marfan (MFS) and Loeys‐Dietz (LDS) syndromes are caused by mutations in the fibrillin‐1 (FBN1) and Transforming Growth Factor Beta Receptor 1 and 2 (TGFBR1 and TGFBR2) genes, respectively. With the current conventional mutation screening technologies, analysis of this set of genes is time consuming and expensive. We have tailored a cost‐effective and reliable mutation discovery strategy using multiplex PCR followed by Next Generation Sequencing (NGS). In a first stage, genomic DNA from five MFS or LDS patient samples with previously identified mutations and/or polymorphisms in FBN1 and TGFBR1 and 2 were analyzed and revealed all expected variants. In a second stage, we validated the technique on 87 samples from MFS patients fulfilling the Ghent criteria. This resulted in the identification of 75 FBN1 mutations, of which 67 were unique. Subsequent Multiplex Ligation‐dependent Probe Amplification (MLPA) analysis of the remaining negative samples identified four large deletions/insertions. Finally, Sanger sequencing identified a missense mutation in FBN1 exon 1 that was not included in the NGS workflow. In total, there was an overall mutation identification rate of 92%, which is in agreement with data published previously. We conclude that multiplex PCR of all coding exons of FBN1 and TGFBR1/2 followed by NGS analysis and MLPA is a robust strategy for time‐ and cost‐effective identification of mutations. Hum Mutat 32:1–10, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Highly sensitive detection of GATA1 mutations in patients with myeloid leukemia associated with Down syndrome by combining Sanger and targeted next generation sequencing

Myeloid leukemia associated with Down syndrome (ML‐DS) is characterized by a predominance of acute megakaryoblastic leukemia, the presence of GATA1 mutations and a favorable outcome. Because DS children can also develop conventional acute myeloid leukemia with unfavorable outcome, detection of GATA1 mutations is important for diagnosis of ML‐DS. However, myelofibrosis and the significant frequency of dry taps have hampered practical screening of GATA1 mutations using bone marrow (BM) samples. In response to those problems, 82 patients were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML‐D11 study. GATA1 mutations were analyzed by Sanger sequencing (SS) using genomic DNA (gDNA) from BM and cDNA from peripheral blood (PB) followed by targeted next‐generation sequencing (NGS) using pooled diagnostic samples. BM and PB samples were obtained from 71 (87%) and 82 (100%) patients, respectively. GATA1 mutations were detected in 46 (56%) and 58 (71%) patients by SS using BM gDNA and PB cDNA, respectively. Collectively, GATA1 mutations were identified in 73/82 (89%) patients by SS. Targeted NGS detected GATA1 mutations in 74/82 (90%) patients. Finally, combining the results of SS with those of targeted NGS, GATA1 mutations were identified in 80/82 (98%) patients. These results indicate that SS using BM gDNA and PB cDNA is a rapid and useful method for screening for GATA1 mutations in ML‐DS patients. Thus, a combination of SS and targeted NGS is a sensitive and useful method to evaluate the actual incidence and clinical significance of GATA1 mutations in ML‐DS patients.     

Role of ADAMTSL4 mutations in FBN1 mutation‐negative ectopia lentis patients

Ectopia lentis (EL) is genetically heterogeneous with both autosomal‐dominant and ‐recessive forms. The dominant disorder can be caused by mutations in FBN1, at the milder end of the type‐1 fibrillinopathies spectrum. Recently in a consanguineous Jordanian family, recessive EL was mapped to locus 1q21 containing the ADAMTSL4 gene and a nonsense mutation was found in exon 11 (c.1785T>G, p.Y595X). In this study, 36 consecutive probands with EL who did not fulfill the Ghent criteria for MFS were screened for mutations in FBN1 and ADAMTSL4. Causative FBN1 mutations were identified in 23/36 (64%) of probands while homozygous or compound heterozygous ADAMTSL4 mutations were identified in 6/12 (50%) of the remaining probands. Where available, familial screening of these families confirmed the mutation co‐segregated with the EL phenotype. This study confirms that homozygous mutations in ADAMTSL4 are associated with autosomal‐recessive EL in British families. Furthermore; the first compound heterozygous mutation is described resulting in a PTC and a missense mutation in the PLAC (protease and lacunin) domain. The identification of a causative mutation in ADAMTSL4 may allow the exclusion of Marfan syndrome in these families and guide the clinical management, of particular relevance in young children affected by EL. © 2010 Wiley‐Liss, Inc.     

Quantitative mouse renal perfusion using arterial spin labeling

Information on renal perfusion is essential for the diagnosis and prognosis of kidney function. Quantification using gadolinium chelates is limited as a result of filtration through renal glomeruli and safety concerns in patients with kidney dysfunction. Arterial spin labeling MRI is a noninvasive technique for perfusion quantification that has been applied to humans and animals. However, because of the low sensitivity and vulnerability to motion and susceptibility artifacts, its application to mice has been challenging. In this article, mouse renal perfusion was studied using flow‐sensitive alternating inversion recovery at 7 T. Good perfusion image quality was obtained with spin‐echo echo‐planar imaging after controlling for respiratory, susceptibility and fat artifacts by triggering, high‐order shimming and water excitation, respectively. High perfusion was obtained in the renal cortex relative to the medulla, and signal was absent in scans carried out post mortem. Cortical perfusion increased from 397 ± 36 (mean ± standard deviation) to 476 ± 73 mL/100 g/min after switching from 100% oxygen to carbogen with 95% oxygen and 5% carbon dioxide. The perfusion in the medulla was 2.5 times lower than that in the cortex and changed from 166 ± 41 mL/100 g/min under oxygen to 203 ± 40 mL/100 g/min under carbogen. T₁ decreased in both the cortex (from 1570 ± 164 to 1377 ± 72 ms, p < 0.05) and medulla (from 1788 ± 107 to 1573 ± 144 ms, p < 0.05) under carbogen relative to 100% oxygen. The results showed the potential of the use of ASL for perfusion quantification in mice and in models of renal diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

Ten novel FBN2 mutations in congenital contractural arachnodactyly: Delineation of the molecular pathogenesis and clinical phenotype

Congenital contractural arachnodactyly (CCA) is an autosomal dominant condition that shares skeletal features with Marfan syndrome (MFS), but does not have the ocular and cardiovascular complications that characterize MFS. CCA and MFS result from mutations in highly similar genes, FBN2 and FBN1, respectively. All the identified CCA mutations in FBN2 cluster in a limited region similar to where severe MFS mutations cluster in FBN1, specifically between exons 23 and 34. We screened exons 22 through 36 of FBN2 for mutations in 13 patients with classic CCA by single stranded conformational polymorphism analysis (SSCP) and then by direct sequencing. We successfully identified 10 novel mutations in this critical region of FBN2 in these patients, indicating a mutation detection rate of 75% in this limited region. Interestingly, none of these identified FBN2 mutations alter amino acids in the calcium binding consensus sequence in the EGF‐like domains, whereas many of the FBN1 mutations alter the consensus sequence. Furthermore, analysis of the clinical data of the CCA patients with characterized FBN2 mutation indicate that CCA patients have aortic root dilatation and the vast majority lack evidence of congenital heart disease. These studies have implications for our understanding of the molecular basis of CCA, along with the diagnosis and genetic counseling of CCA patients. Hum Mutat 19:39–48, 2002. © 2001 Wiley‐Liss, Inc.     

Pathogenic FBN1 variants in familial thoracic aortic aneurysms and dissections

Marfan syndrome (MFS) due to mutations in FBN1 is a known cause of thoracic aortic aneurysms and acute aortic dissections (TAAD) associated with pleiotropic manifestations. Genetic predisposition to TAAD can also be inherited in families in the absence of syndromic features, termed familial TAAD (FTAAD), and several causative genes have been identified to date. FBN1 mutations can also be identified in FTAAD families, but the frequency of these mutations has not been established. We performed exome sequencing of 183 FTAAD families and identified pathogenic FBN1 variants in five (2.7%) of these families. We also identified eight additional FBN1 rare variants that could not be unequivocally classified as disease‐causing in six families. FBN1 sequencing should be considered in individuals with FTAAD even without significant systemic features of MFS.