Contributed equally
The etiology of thoracic aortic aneurysm and dissection (TAAD) is complex and heterogeneous. Emerging evidence has demonstrated that genetic causes may be a consideration in early-onset TAAD. Owing to overlapping clinical phenotypes and the genetic heterogeneity of TAAD, it is challenging for clinicians to make a molecular diagnosis of TAAD, particularly in those who present with non-specific syndromic features. In order to identify the causative mutation in two young patients with acute type B aortic dissection without syndromic features, whole exome sequencing (WES) was performed in the present study. A missense mutation (c.G6953A:p.C2318Y) and a nonsense mutation (c.C4786T:p.R1596X) were identified in the fibrillin 1 gene in patients T287 and T267, respectively. The present study emphasized the necessity of genetic testing for young patients with type B aortic dissection. WES is a timely, robust and inexpensive technique for molecular diagnosis, particularly for TAAD caused by numerous genes. Genetic diagnosis of Marfan syndrome could aid in periodic surveillance, prophylactic surgical measures, and genetic counseling.
Thoracic aortic aneurysm and dissection (TAAD) is associated with marked cardiovascular morbidity and mortality. The incidence of acute aortic dissection has been suggested to be 3–6 cases/100,000 individuals/year, with an increasing incidence in recent decades (
MFS, as an autosomal dominant disease, is the most frequent heritable connective tissue disorder, associated with mutations in the fibrillin 1 (FBN1) gene (
Whole exome sequencing (WES) is an efficient tool to identify the underlying genetic cause in TAAD (
A total of two male patients, not related by birth, were respectively admitted in Marth and May 2015 to the department of Vascular Surgery, Nanjing Drum Tower Hospital, Nanjing, China. The two individuals, designated T287 and T267, complained of acute severe chest and back pain. They were reported to be in good health prior to this acute incidence. Regular physical examination and clinical testing was performed on the two patients, including electrocardiography, computed tomography angiography (CTA) and echocardiography. Z-score was used to estimate the degree of aortic root dilatation (
Genomic DNA was extracted from peripheral whole blood samples from the subjects using the QIAamp DNA Blood Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. WES analysis was performed by Beijing Novogene Bioinformatics Technology, Co., Ltd. (Beijing, China). Target enrichment was performed to construct the exome library using the Agilent SureSelect Human All Exon V5 kit (Agilent Technologies, Inc., Santa Clara, CA, USA), according to manufacturer's protocol, and sequenced on the Illumina HiSeq 2000 platform (Illumina, Inc., San Diego, CA). The clean reads from the Illumina Genome HiSeq 2000 were aligned to the human genome reference (University of California Santa Cruz hg19;
All annotated variants were screened with databases including the SNP database (dbSNP142,
For Sanger sequencing verification of variants detected by WES, oligonucleotide primers were designed using the Primer3 program (primer3.ut.ee). The regions containing the suspected variants were amplified by standard polymerase chain reaction (PCR) analysis using GoTaq polymerase (Promega). Primers used to amplify the mutant sequence were FBN1-Ex39-F (5′-AACTTACTTCAGACGGGCAGAG-3′), FBN1-Ex39-R (5′-TAGCTCCTGGCACTCATCAATA-3′), FBN1-Ex57-F (5′-ATGTGAGAGAGGGAAGGAAGGT-3′), FBN1-Ex39-R (5′-GTCAATACGGCATCTCCAAAAT-3′). The amplification reaction mixture (50 µl) was subjected to denaturation at 98°C for 3 min followed by 35 cycles at 95°C for 30 sec, annealing temperature 60°C for 30 sec, 72°C for 30 sec and by a final extension at 72°C for 15 min. PCR products were purified and sequenced on the ABI PRISM3730 automated sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using the BigDye terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific, Inc.).
The two patients complained of acute episodes of chest and back pain. T267 is a 35-year-old man, 182 cm in height and 60 kg in weight. He was diagnosed with descending aortic dissection by CTA. Echocardiography of T267 demonstrated that the diameter of the aortic sinus was 4.3 cm, and identified hypertrophy of the left ventricle. T287 is a 32-year-old male, 178 cm in height and 95 kg in weight. A CTA scan revealed descending aortic dissection and ascending aortic dilatation. Echocardiography of T287 demonstrated that the diameter of the aortic sinus was 4.5 cm, in addition to identifying mild aortic valve regurgitation, and mild bicuspid and tricuspid valve regurgitation. Additionally, the grandfather of T287 died suddenly of an unknown cause at age of 40. Clinical manifestations of other systems, including the skeletal and ocular systems, were absent. The clinical data of the two patients are presented in
The present study generated a total of 58,189,201 and 50,222,226 raw reads of 180–280 bp paired-end read sequences from the patients T267 and T287, respectively. The raw depth of T267 and T287 was 346.49× and 299.06×, respectively; and the Phred-like Q20 (that is, a 99% accuracy of the base call) was 95.02 and 94.26%. The average depth of targeted sequences was 199.80× and 171.32× for T267 and T287, respectively. The sequence depth and proportion covered bases of every sequence and chromosome of T267 and T287 are presented in
In order to identify pathogenic mutations in the two patients, candidate genes were filtered with reported disease-causing genes for TAAD (data for the two patients are presented in
The two variants were confirmed by Sanger sequencing (
MFS is a common multisystem connective tissue disorder, inherited in an autosomal dominant manner, caused by mutations in the FBN1 gene on chromosome 15q21.1 (
Considering the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular genetic testing is often required for the timely and accurate diagnosis of affected individuals. Traditional Sanger sequencing is costly and laborious due to the size of the FBN1 gene. The present study identified one missense mutation (c.G6953A:p.C2318Y in exon 57) and one nonsense mutation (c.C4786T:p.R1596X in exon 39) in the FBN1 gene by WES in two early-onset affected individuals with type B aortic dissection. The two patients lacked a family history of TAAD or MFS. For T287, the diameter of the aortic sinus was measured and the Z-score was calculated to be 4.12, using the Z-score calculator at
Clinical manifestations vary between TAAD caused by MFS and non-syndromic TAAD. In previous decades, without the use of surgical aortic root replacement, the incidence of type A aortic dissection was increased compared with type B aortic dissection in patients with MFS (
In the present study, the two patients were admitted to the Department of Vascular Surgery with acute type B aortic dissection in their thirties. It was inferred that the symptoms were likely to be caused by a genetic defect. Subsequent molecular analysis confirmed this hypothesis. It is widely accepted that endovascular stent grafting may not be suitable for the treatment of type B dissection, particularly in young patients with MFS, due to weakening of the aortic walls and the lack of long-term durability of stent grafting (
In conclusion, the present study identified one missense mutation and one nonsense mutation via WES in two patients with early-onset type B aortic dissection without typical syndromic features, who were subsequently diagnosed with MFS. the present study emphasized the necessity of genetic testing for young patients with type B aortic dissection. WES is a timely, robust and inexpensive technique for genetic sequencing, particularly for TAAD which is caused by numerous genes. Genetic diagnosis of MFS may facilitate periodic surveillance, prophylactic surgical measures and genetic counseling.
The present study was supported by the Natural Science Foundation of Jiangsu Province, China (grant no. BK20140103) and the Natural Science Foundation of China (grant no. 81270396).
Sequence depth and proportion covered bases. (A) Proportion of different sequence depths. (B) Cumulative proportion of different sequence depths. (C) Mean sequence depth and proportion covered bases of every chromosome.
Sequencing analysis of FBN1 gene. (A) Sequence of heterozygous FBN1 c.G6953A (p.C2318Y) mutation of T287 and (B) the corresponding sequence of unaffected individuals. (C) Sequence of heterozygous FBN1 c.C4768T (p.R1596X) mutation of T267 and (D) the corresponding sequence of unaffected individuals. FBN1, fibrillin 1.
Clinical characteristics of the two individuals affected with acute type B aortic dissection.
Cardiovascular | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|
ID | Age, years | Sex | Height, cm | Weight, kg | Diameter of aortic sinus, cm |
Z-Score |
Aorta | Other | Skeletal | Ocular |
T267 | 35 | M | 182 | 60 | 4.3 | 3.86 | TAD | Hypertension; left ventricular hypertrophy | None | None |
T287 | 32 | M | 178 | 95 | 4.5 | 4.12 | TAD | Hypertension; dilatation of ascending aorta; mild valve regurgitation | None | None |
Diameter of the aortic sinus was measured by echocardiography.
Z-score was calculated via a calculator at
Number of variants present in the patients at different stages of the filtering process.
Number | ||
---|---|---|
Filtering criteria | T267 | T287 |
Total number of variants | 221,478 | 198,478 |
Variants not in dbSNP, MAF<1% | 22,723 | 19,655 |
Filtering using 1000 Genomes and ESP | 22,529 | 17,494 |
Variants in exonic or splicing | 1,082 | 1,021 |
Non-synonymous and frameshift variants | 950 | 917 |
Functional analysis by SIFT and Polyphen-2 | 776 | 753 |
Genes associated with TAAD | 1 | 1 |
MAF, minor allele frequency; ESP, exome sequencing project; SIFT, sorts intolerant from tolerant; Polyphen-2, polymorphism phenotyping version 2; TAAD, thoracic aortic aneurysm and dissection; SNP, single nucleotide polymorphism.