Download Familial Aortopathy — Gene Panels

Familial Aortopathy — Gene Panels
(Reference – 2013.03.002)
Notice Of Assessment
April 2014
DISCLAIMER: This document was originally drafted in French by the Institut national d'excellence en santé et en
services sociaux (INESSS), and that version can be consulted at
http://www.inesss.qc.ca/fileadmin/doc/INESSS/Analyse_biomedicale/Avril_2014/Aortopathies_familialesPanels_genes.pdf. It was translated into English by the Canadian Agency for Drugs and Technologies in Health
(CADTH) with INESSS’s permission. INESSS assumes no responsibility with regard to the quality or accuracy of the
translation.
While CADTH has taken care in the translation of the document to ensure it accurately represents the content of the
original document, CADTH does not make any guarantee to that effect. CADTH is not responsible for any errors or
omissions or injury, loss, or damage arising from or relating to the use (or misuse) of any information, statements, or
conclusions contained in or implied by the information in this document, the original document, or in any of the
source documentation.
1 GENERAL INFORMATION
1.1


Requestors
CHU Sainte-Justine (panel of 15 aortopathy genes)
CHUS-Hôpital Fleurimont (panel of 7 TAAD2 genes)
1.2


Application for Review Submitted to MSSS
CHU Sainte-Justine: September 30, 2013
CHUS-Hôpital Fleurimont: August 30, 2012
1.3
Application Received by INESSS: November 1, 2013
1.4
Notice Issued: February 28, 2014
Note:
This notice is based on the scientific and commercial information submitted by the requestor and on
a complementary review of the literature according to the data available at the time that this test
was assessed by INESSS.
2 TECHNOLOGY, COMPANY, AND LICENCE(S)
2.1
Name of the Technology
High-throughput or next-generation sequencing (NGS3).
2.2
Brief Description of the Technology, and Clinical and Technical Specifications
Next-generation sequencing (NGS) enables rapid and simultaneous sequencing of various genes,
exomes, or whole genomes, at a lower cost. Several platforms employing different types of technology
and chemistry are currently available on the market. However, regardless of the technology used, NGS
is performed in three steps: sample preparation (and gene library4 generation), sequencing, and data
analysis.
During sample preparation, genomic DNA is fragmented, generating a gene library attached to a solid
support. The requestors use a technology (Illumina MiSeq and HiSeq) and approach (gene panel) that
require the enrichment of selected regions by PCR in preparation for sequencing.
The sequencing is performed according to a basic principle similar to that in the Sanger method: the
nucleotides of DNA fragments are detected during the synthesis of a strand complementary to the
template. Nucleotides added at each cycle are identified by their specific fluorescence (type and
intensity). However, unlike conventional sequencing, NGS performs and deciphers millions of reactions
simultaneously.
Lastly, the resulting sequences can be assembled and aligned to a reference sequence. The variations
are then confirmed by sequencing, using the Sanger method—as recommended by the American
College of Medical Genetics and Genomics—and annotated.
2
Thoracic aorta aneurysms and dissections.
The term "next-generation sequencing" refers to the various recently developed sequencing platforms, which all have a very high reading
accuracy compared with standard sequencing methods (Sanger).
4
Gene library or bank.
3
1
CHU Sainte-Justine
Following a review of the tests available on the market (primarily in the Unites States) for familial or
syndrome-related aortopathies, 15 genes, for which exons and junction points will be examined, were
selected to be included in the panel: ACTA2, CBS, COL1A1, COL3A1, COL5A1, COL5A2, FBN1, FBN2,
MYH11, MYLK, SLC2A10, SMAD3, TGFB2, TGFBR1, TGFBR2 (85 kb). The template library is enriched and
prepared using the circularization of DNA fragments with oligonucleotide synthesized according to the
requestor’s specifications (custom) (HaloPlex system by Agilent) and by sequencing with the HiSeq
2500 (Illumina) system.
Figure 1: Enrichment by DNA fragment circularization (Haloplex system by Agilent)5
Digest and denature sample DNA
Hybridize probe library to DNA
targets
Purify and ligate targets
Amplify enriched fragments
by PCR
Source: Agilent Technologies. HaloPlex Custom Kits - Details & Specifications [website]. Available at:
http://www.genomics.agilent.com/article.jsp?crumbAction=push&pageId=3061.
CHUS-Hôpital Fleurimont
The enrichment of the exons and junctions of 7 genes, ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1,
and TGFBR2, is carried out through hybrid capture with oligonucleotides (custom) (SeqCap Ez system
by Roche NimbleGen). MiSeq (Illumina) is used for sequencing. The requestor states that the
oligonucleotides were chosen in consultation with experts from the company in order to maximize
enrichment. The minimum quality requirements for sequencing are Q30. It should be noted that exons
1 of the genes TGFBR1 and TGFBR2 are sequenced using the Sanger method, due to the lower
sequence coverage resulting from the high %GC in these regions.6 Moreover, Sanger sequencing will be
5
Adapted from: http://www.genomics.agilent.com/article.jsp?crumbAction=push&pageId=3061.
Personal electronic and telephone communications with M. Serge Gravel, Ph. D., who is responsible for the technical aspect of the analysis
(November 27 and 28, 2013).
6
2
used to confirm the variants observed. The genes were selected following a review of the scientific
evidence supporting their clinical value.7
Figure 6: Enrichment by hybrid capture (SeqCap EZ system by Roche NimbleGen)
Image courtesy of Roche Diagnostics Canada for Roche NimbleGen, from the document SeqCap EZ Choice Library, http://www.roche-appliedscience.com/wcsstore/RASCatalogAssetStore/Articles/06445497001_05.11.pdf.
2.3
Company or Developer: Illumina (sequencer).
2.4
Licence(s): Not applicable.
2.5
Patent, If Any: Not applicable.
2.6
Approval Status (Health Canada, FDA)
The MiSeqDx (Illumina) sequencing platform was approved by the FDA for use in clinical diagnosis.
2.7


Weighted Value
CHU Sainte-Justine: 604.48
CHUS-Hôpital Fleurimont: 782.13 (includes the cost of Sanger sequencing)
3 CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES
3.1
Targeted Patient Group
Targeted patient groups are those with:
 a dilation of the ascending aorta or of the sinuses of Valsalva;
 an aneurysm or dissection of the ascending or descending thoracic aorta;
 a positive family history;
 a possible diagnosis of Marfan syndrome, Loeys-Dietz syndrome, or another connective tissue
abnormality to be confirmed or ruled out.
Other people targeted by the test or clinical situations in which the test is indicated:
 parents, brothers, or sisters, and children of a carrier of a mutation associated with an aortopathy;
 prenatal screening when a parent is known to be a carrier of a mutation associated with
aortopathy.
3.2
Targeted Disease(s)
Aortopathies are characterized by dilatations, aneurysms, and tortuosity of the aorta. Thoracic aortic
aneurysms and dissections (TAAD8) are responsible for 15,000 deaths annually in the United States
[Pomianowski and Elefteriades, 2013; Clouse et al., 2004]. The annual prevalence of TAAD is estimated
7
Among others: http://www.ncbi.nlm.nih.gov/books/NBK1120/ (Thoracic aortic aneurysms and aortic dissections, in GeneReviews).
Aneurysm: a permanent, localized dilatation of the aorta of at least 50% of its normal diameter, caused by a weakening of the aortic wall.
Dissection: a tear in the intimal lining of the aorta that creates a false lumen of varying length [Callewaert et al., 2009].
8
3
at 10 cases per 100,000 population [Clouse et al., 1998]. Despite progressive dilatation, thoracic aortic
aneurysms (TAAs) remain asymptomatic until dissection or rupture occurs. The annual prevalence of
TAA dissection/rupture is approximately 3 cases per 100,000 population [Clouse et al., 2004; Meszaros
et al., 2000]. The risk of mortality associated with TAAD depends primarily on how far the disorder has
progressed at the time of diagnosis. Therefore, for TAA detected prior to rupture or dissection, the risk
of operative mortality is < 15%, whereas in acute forms mortality can reach 40%, in dissection, or
exceed 50%, in rupture [Shores et al., 1994]. There are two types of TAAD: hereditary syndromic forms
(Marfan syndrome, Loeys-Dietz syndrome, Ehler-Danlos syndrome, etc.), reported in young patients;
and idiopathic and degenerative forms, which are more common in older patients. Approximately 20%
of TAADs are caused by a genetic defect commonly inherited in an autosomal dominant manner with
varying degrees of penetrance [Albornoz et al., 2006; Milewicz et al., 1998]. Although several mutations
have been identified, the cause of most nonsyndromic familial forms remains unknown [Radu et al.,
2009]. (See the appendix for a more detailed description of the diseases.)
3.3
Number of Patients Targeted
According to the requestors:
 CHU Sainte-Justine: 1 to 5 per week, or 52 to 260/year, including children, adults, and relatives.
 CHUS-Hôpital Fleurimont: 20/year (Quebec City and Sherbrooke).
3.4
Medical Specialties and Other Professions Involved
Molecular genetics, genetic counselling, cardiology, pathology.
3.5
Testing Procedure
The test is carried out using genomic DNA extracted from a blood sample (a saliva sample or buccal
swab may also be used).
4 TECHNOLOGY BACKGROUND
4.1
Nature of the Diagnostic Technology
According to the requestors, this is a new test.
4.2
Brief Description of the Current Technological Context
While NGS offers a promising alternative to Sanger sequencing, its clinical utility in the analysis of gene
panels or exomes depends on the ability to accurately enrich or amplify the regions of interest
[Wooderchak-Donahue et al., 2012]. The enrichment phase must yield high read depths,9 while keeping
errors introduced through amplification and other sample manipulations to a minimum, thus ensuring
optimal specificity and sensitivity. Several enrichment strategies, with varying advantages depending
on the intrinsic characteristics of the target sequences, are currently available. Basically, there are
three types of enrichment: the first is by PCR (in small compartments such as RainDance, Fluidigm, or
AmpliSeq), the second is by a hybridization and extension method (HaloPlex, TruSeq), and the third is
by a hybridization-only method (SureSelect, SureCap EZ). Studies comparing various enrichment
techniques showed large differences, particularly regarding the read depths generated and the number
of variants identified [Hedges et al., 2011; Kiialainen et al., 2011; Teer et al., 2010].
9
Read depth is the ratio of the length of the end-to-end alignment of all the sequence fragments read to the length of the target sequence.
For example, if 25 million bases (Mb) are sequenced for a 5 Mb genome, the resulting depth is equivalent to 5 times the genome. The greater
the depth, the greater the number of overlapping reads that can be assembled, and the greater the fraction of the genome covered. This
allows the most complete final sequence possible to be obtained, with a minimum number of “gaps” or unsequenced regions (Furelaud G. and
Esnault Y. Le séquençage des génomes – compléments [website]. Available at:
www.snv.jussieu.fr/vie/dossiers/genomes/plus/fragm_stats.htm).
4
The technology used by Illumina sequencing platforms involves the synthesis of a strand
complementary to each of the millions of template strands attached to a solid support, using primers
and (reversible) terminator nucleotides labelled with fluorochromes specific to the nucleotide type.
The sequencing reaction comprises a series of cycles that allows the successive addition of nucleotides
that are detected at each cycle.
The resulting sequences are assembled and compared with the reference sequence. The variations
identified are then analyzed individually to assess their clinical relevance, with or without a prior
automated filtering step using bioinformatic tools.
In the short term, Sanger sequencing will continue to be used in conjunction with NGS, particularly for
regions that are difficult to target (GC-rich stretches or repetitive sequences) and to confirm variations
identified by NGS [Rehm et al., 2013; Ware et Jefferies, 2012].
4.3
Brief Description of the Advantages Cited for the New Technology
Compared with Sanger sequencing, NGS performed in a clinical laboratory enables a set of genomic
regions associated with a known hereditary disease to be targeted more rapidly, as it offers greater
sequence coverage at a lower cost.
4.4
Cost of Technology and Options
TECHNOLOGY
Sequences covered by each test
Sequencing cost per test
Run time
HiSeq2000 – MiSeq (Illumina)
8.5 Gb to 600 Gb
$41/Gb to $502/Gb
39 hours to 11 days
SANGER
0.006 Gb
$5,000/Gb
1 day
Source: table adapted from Makrythanasis and Antonarakis, 2012.
5 EVIDENCE
5.1
Clinical Relevance
Despite the gradual enlargement of the aorta over time, aneurysms are usually asymptomatic, until a
dissection or rupture occurs, with devastating consequences. TAAD has been described as having the
features of several clinical conditions that are sometimes difficult to distinguish from one another. The
tests in question would allow a definitive diagnosis, stratification of the associated risks, and
adaptation of the medical approach in an effort to limit or avoid life-threatening events. If a pathogenic
mutation has been identified in the proband, other family members may be asked to undergo targeted
screening for the same mutation. Carriers of the mutation may then be closely monitored or given the
appropriate treatment, whereas those who are not carriers will be spared costly and stressful tests
[Burton et al., 2010].
Despite some overlap, certain genetic mutations are disease-specific. The genes most commonly
associated with causing FTAAD are ACTA2, TGFBR1, TGFBR 2, MYH11, MYLK, and SMAD3, whereas
those associated with Marfan syndrome (MFS), Loeys-Dietz syndrome (LDS), and Ehlers-Danlos
syndrome (EDS) are primarily FBN1, TGFBR1, TGFBR2, TGFB2, SMAD3, COL1A1, COL3A1, COL5A1, and
COL5A2.
The panel proposed by the CHUS group is composed of the genes most commonly associated with
syndromic and nonsyndromic TAAD, whereas that of the CHU Sainte-Justine group is more
comprehensive, as it contains genes for which mutations have rarely been described and genes not
associated with TAAD but that allow the differential diagnoses of the syndromes in certain cases.
5
It should be noted that association studies linking various diseases with genes are not available for
several of these genes [Pomianowski and Elefteriades, 2013]. These studies are difficult to conduct, due
to the low prevalence of various diseases, their incomplete penetrance, and, at times, the frequent
occurrence of de novo mutations. Phenotype-genotype correlation studies are more common.
ACTA2
This 5 kb (3 exons) gene located on chromosome 10q23.31 was associated with FTAAD, Moyamoya
disease, and multisystemic smooth muscle dysfunction syndrome. It encodes the vascular smooth
muscle cell-specific alpha-actin. Mutations in ACTA2 were identified in 10% to 15% of patients with
nonsyndromic familial TAAD and in 2.5% of patients with sporadic TAAD, especially at younger ages
[Guo et al., 2009; Morisaki et al., 2009; Guo et al., 2007]. Despite the autosomal dominant nature of
the mutations associated with this gene, reduced penetrance has been reported (50%). However, when
all of the vascular phenotypes are combined, the penetrance increases to 80% [Pomianowski and
Elefteriades, 2013; Guo et al., 2009]. According to the Human Gene Mutation Database (HGMD10)—
Professional 2013.4 version—38 individual mutations have been identified to date. Of these, 25 are
associated with TAAD and 10 others are associated with aortopathies or other cardiovascular
phenotypes. These variations are primarily amino acid substitutions that result in nonsense or missense
mutations.
CBS
This 18 kb gene composed of 15 exons is associated with homocystinuria, a metabolic autosomal
recessive disease [Yap, 2003]. The clinical presentation of this disease is very similar to that of Marfan
syndrome (tall stature, ectopia lentis, severe myopia, and skeletal abnormalities). Unlike Marfan
syndrome, homocystinuria is associated with severely impaired cognitive development and an
increased risk of stroke at a young age. Sequencing for this gene is sometimes included in panels for
the differential diagnosis of homocystinuria and Marfan syndrome. The professional version of HGMD
reports 181 mutations, nearly all of them associated with homocystinuria (172/181). No association
between the CBS gene and TAAD has been reported.
COL1A1
This gene is composed of 51 exons and spans 17.5 kb on chromosome 17q21.33. Mutations of the
COL1A1 gene are associated with osteogenesis imperfecta (OI) type I to type IV, Ehlers-Danlos
syndrome (EDS) type I and type VIIA, Caffey disease, and idiopathic osteoporosis. Mutations linking the
gene with Ehlers-Danlos syndrome type VIIA are rare, identified in approximately twenty case reports
describing point mutations in the coding regions and junctions. Malfait et al. [2007] have suggested
that mutations involving three arginine-to-cysteine substitutions could confer a predisposition to
arterial rupture. However, this observation remains anecdotal. Public data from HGMD report
609 different mutations, 569 of which are associated with OI, 16 with EDS, 6 with reduced bone
density, 1 with Caffey disease, and the remaining 17 with incomplete clinical phenotypes.
COL3A1
This gene, composed of 5 exons that span 2.6 kb on chromosome 2q32.2, carries mutations in 98% of
EDS type IV (vascular) cases. EDS type IV accounts for 5% to 10% of EDS cases and is caused by
autosomal dominant mutations. Mutations of this gene have also been reported as the cause of EDS
type II and type III. Half of the mutations observed are de novo [Mayer et al., 2010].
10
The information drawn from HGMD provides the reader with an idea of the frequency of the lesions found in each gene. These data were
not source-verified or corroborated by other similar databases.
6
No fewer than 254 pathogenic mutations in COL3A1 have been described in the professional version of
HGMD. The mutations lead to amino acid substitutions as well to the deletion of one or more exons in
unusually high proportions. Moreover, 2% to 3% of patients are carriers of a deletion or insertion.
COL5A1 and COL5A2
Classic EDS (type I and type II) accounts for 90% of EDS cases. Approximately 50% of cases show
autosomal dominant mutations in these genes. COL5A1 mutations occur in 85% to 90% of cases,
whereas COL5A2 mutations occur in 10% to 15% of cases [Symoens et al., 2012]. Approximately 50% of
the mutations identified are de novo mutations [Malfait et al., 2010].
Genes COL5A1 and COL5A2 span 200 kb (66 exons) and 146 kb (54 exons), respectively, and are located
on chromosomes 9q34.3 and 2q32.2. The point mutations described lead to amino acid substitutions
and the premature termination of the protein. Deletions and insertions have also been reported. The
HGMD database reports 119 and 21 pathogenic mutations for COL5A1 and COL5A2, respectively.
FBN1
This gene, located on chromosome 15q21.1, spans more than 600 kb and possesses a highly
fragmented coding sequence (65 exons). The mutations are autosomal dominant and a variable
penetrance of the phenotype is observed [Akutsu et al., 2010].
The HGMD database reports 1,492 pathogenic mutations, 86% of which are associated with classic
Marfan syndrome (MFS), while the remainder are associated with incomplete clinical manifestations of
MFS. The mutations located between exons 24 and 32 of the gene, as well as the de novo mutations
identified in 25% of the patients, are associated with a more severe phenotype of the disease
(neonatal) [Morse et al., 1990]. The point mutations described lead to amino acid substitutions,
premature protein truncations, and splicing errors. Deletions and insertions have also been reported.
FBN2
FBN2 is located on chromosome 5q23.3; it spans 209 kb and contains 33 exons. All of the 47 mutations
indexed in HGMD are associated with congenital contractural arachnodactyly (CCA) or Beals syndrome,
except for one. Contrary to the conclusions of the first published studies, a dilatation of the aortic root
was reported in a few cases. CCA is a disease inherited in an autosomal dominant manner. The
reported mutations are substitution mutations that affect the coding sequences (missense/nonsense)
and splice sites.
MYH11
MYH11 is located on chromosome 16p13.11 and encodes smooth muscle myosin heavy chain 11
[Pannu et al., 2007; Zhu et al., 2006]. This gene, composed of 40 exons (134 kb), has been associated
with FTAAD and certain acute myeloid leukemias (M4). Certain autosomal dominant mutations in this
gene are involved in 2% of nonsyndromic TAAD and consist primarily of splice-site mutations and
amino acid substitutions. Kuang et al. [2011] reported a duplication of the chromosome 16p13.1
region, in which MYH11 is found, in cases of individuals with FTAAD. Deletions were also identified [Zhu
et al., 2006]. HGMD reports 29 mutations, 24 of which are associated with TAAD.
MYLK
MYLK is located on chromosome 3q21.1 and encodes myosin light chain kinase specific to vascular
smooth muscle cells. This gene comprises 30 exons that span 180 kb.
Autosomal dominant mutations in MYLK have rarely been identified in cases of TAAD, and only in
patients with nonsyndromic FTAAD (approximately 1%) [Wang et al., 2010]. Of the 9 mutations
described to date, 5 are associated with TAAD: point mutations resulting in amino acid substitution and
a truncation of the resulting protein. The phenotype associated with the mutations in MYLK is
7
characterized by the occurrence of an aortic dissection without prior dilatation or by a slight
enlargement of the artery. A wide phenotypic variability was observed among carriers of a mutation
who were studied, from a dissection occurring at a young age to the complete absence of clinical
manifestations at an advanced age. A few cases showed gastrointestinal complications such as
diverticulitis, polyps, colorectal cancer, and inflammatory bowel disease [Wang et al., 2010].
SLC2A10
This gene containing 5 exons that span 24 kb is associated with arterial tortuosity syndrome, a rare
disease for which fewer than 80 cases have been described in literature. It is located on chromosome
20q13.1 and encodes the GLUT10 glucose transporter. To date, 23 autosomal recessive mutations of
the gene SLC2A10 have been reported. Point mutations lead to amino acid substitutions and to
deletions. Individuals affected have a propensity to aneurysm formation, vascular dissection, and
pulmonary artery stenosis.
SMAD3
A total of 12 mutations of the 17 identified in this gene containing 8 exons (25.5 kb) have been
reported in individuals with Loeys-Dietz type III syndrome (aneurysm-osteoarthritis syndrome). The
mutations described are hereditary and composed of point mutations leading to amino acid
substitutions and to deletions and insertions of 1 or 2 base pairs [Van de Laar et al., 2012].
The remaining 5 mutations in SMAD3, located on chromosome 15q22.38, are associated with familial
TAAD, or approximately 2% of cases.
TGFB2
TGFB2 is located on chromosome 1q41 and encodes the transforming growth factor beta. This gene
contains 7 exons that span 95 kb. It was recently associated with TAAD [Renard et al., 2013; Boileau et
al., 2012; Lindsay et al., 2012]. Of the 15 reported mutations, 14 were identified in individuals with
TAAD.
The described mutations consist of missense mutations and insertions/deletions (5 to 15 pb) that lead
to a change in the reading frame or the introduction of a premature stop codon.
TGFBR1 and TGFBR2
Mutations in the genes TGFBR1 and TGFBR2 have been associated with MFS, LDS, and several types of
tumours. In individuals with LDS, 95% are carriers of a mutation in TGFBR1 or TGFBR2. In the case of
Marfan syndrome, the mutations in TGFBR1 and TGFBR2 are not as common.
The genes TGFBR1 and TGFBR2 are located on chromosomes 9q22.33 and 3p24.1, respectively [Loeys
et al., 2005]. These genes are relatively small in size; TGFBR1 contains 9 exons and spans 44 kb, and
TGFBR2 contains 2 coding exons and spans 22 kb.
Many mutations were reported as being pathogenic: 46 for TGFBR1 and 108 for TGFBR2. The majority
of these lead to amino acid substitution, but protein truncations, splicing errors, and
deletions/insertions also were identified in these two genes in cases of TAAD. In cases of LDS,
approximately 75% of associated mutations were detected in TGFBR1 as opposed to 25% in TGFBR2.
Mutations were observed in 1% to 4% of FTAAD cases [Stheneur et al., 2008; Sakai et al., 2006; Singh et
al., 2006]. In inherited cases, despite the autosomal dominant feature of the mutations, penetrance
varied widely. It should be noted that 75% of individuals with LDS have de novo mutations.
8
Table 1: Diseases with aortopathies and associated genes
PATHOLOGY
PREVALENCE
Marfan syndrome
1:5,000
Loeys-Dietz syndrome (includes
aneurysmosteoarthritis syndrome)
˂ 1:1,000,000
(˂ 200,000
individuals affected
in the US)
Ehlers-Danlos syndrome
1:75,000
Arterial tortuosity syndrome
Prevalence
unknown
Congenital contractural
arachnodactyly
Prevalence
unknown
FTAAD
20% of TAAD
DIFFERENTIAL
DIAGNOSIS
Congenital contractural
arachnodactyly
Loeys-Dietz syndrome
MASS syndrome
Shprintzen-Goldberg
syndrome
Mitral valve prolapse
Ehlers-Danlos syndrome
Marfan syndrome
Ehlers-Danlos syndrome
Shprintzen-Goldberg
syndrome
Among others:
Silverman syndrome
Marfan syndrome
Loeys-Dietz syndrome
Loeys-Dietz syndrome,
Vascular Ehlers-Danlos
syndrome (type IV), and
Marfan syndrome
ASSOCIATED GENES
FBN1 (86% of cases)
TGFBR1 and TGFBR 2
TGFBR1 and TGFBR2
(95% of cases)
SMAD3
COL1A1
COL3A1
COL5A1 and COL5A2
SMAD3
SLC2A10
Marfan syndrome
FBN2
Sporadic TAAD
Syndromic TAAD
ACTA2
TGFBR1 and TGFBR2
TGFB2
MYLK
SMAD3
5.1.1 Other Tests Replaced
This test is not listed in the Index. Requests are sent outside Quebec. According to the data from MSSS,
18 tests were performed outside the province, amounting to $44,000 for the period from April 2012 to
March 2013.
The tests may be performed in several centres that offer the sequencing of panels or individual genes.
Depending on the gene, the type of procedure offered may include sequencing with a
deletions/duplication analysis or sequencing only. A summary of the tests offered by nine accredited
centres is given in the following table. Ten laboratories were assessed: Ambry Genetics; ARUP
Laboratories; Center for Human Genetics; Centogene; GeneDX; Fulgent Therapeutics; Health in Code;
Mayo Medical Laboratories; National Institute for Health Research; Prevention Genetics.
Centres offering the test
ACTA2
9/10
MYH11
CBS
5/10
MYLK
COL1A1
1/10
SLC2A10
COL3A1
8/10
SMAD3
COL5A1/2
7/10
TGFB2
Centres offering the test
9/10
6/10
7/10
8/10
5/10
FBN1
10/10
TGFBR1 and
TGFBR2
10/10
FBN2
7/10
9
5.2
Clinical Validity
Semi-automated Sanger sequencing has been used in diagnostic testing for many years and is still
considered the gold standard. However, developments in sequencing technology (NGS) now enable
several genes, exomes, or whole genomes to be simultaneously tested using a high-throughput, lowcost approach. For these reasons, NGS is in the process of being adopted in a clinical context. In
addition, the FDA approved Illumina’s MiSeqDx platform for clinical use in late 2013.
Using the NGS approach for multigenic diseases has allowed several variations described as pathogenic
to be identified and subsequently confirmed by classic sequencing [Lepri et al., 2014; Umbarger et al.,
2014; Li et al., 2013; Pomianowski and Elefteriades, 2013]. In terms of analytical capacity, NGS
surpasses Sanger sequencing, as the simultaneously sequencing of many genes would be difficult, if not
impossible, using the traditional approach in a diagnostic laboratory.
However, as this technology has limitations, NGS approaches are not recommended to be used
exclusively to identify and validate the presence of variants. Sanger sequencing is necessary to explore
regions in which read depth is inadequate and to validate the identified variants. However, considering
that the benefits associated with the use of NGS outweigh the associated risks, NGS technology is
recommended for clinical settings (FDA memorandum for the introduction of MiSeqDx, ACMG [Rehm
et al., 2013; Sikkema-Raddatz et al., 2013]).
The enrichment of targets to be sequenced is an important step in ensuring appropriate read depth.
Therefore, several studies have compared the various kits available on the market.
Baetens et al. [2011] tested an emulsion-based multiplex PCR method coupled with next generation
sequencing (NGS) to identify mutations associated with Marfan syndrome (FBN1, TGFBR1, and
TGFBR2). In a first phase, the technology allowed 5 known mutations in confirmed cases of MFS or
Loeys-Dietz syndrome (LDS) to be identified. In a second phase, the technology was tested on
87 patients fulfilling the Ghent criteria. In all, 75 mutations in FBN1, of which 67 were unique, were
identified. The use of multiplex ligation-dependent probe amplification (MLPA) for cases still declared
negative allowed the detection of 4 large deletions/insertions. Lastly, Sanger sequencing detected a
missense mutation in exon 1 of FBN1. Exon 1 of the gene had been deliberately omitted from the test.
The overall detection rate of the mutations was calculated at 92% [Umbarger et al., 2014].
Wooderchak-Donahue et al. [2012] compared two enrichment protocols, the first involving an
emulsion PCR (Raindance) technique and the second, an in-solution hybrid capture method
(SureSelect). An aortopathy panel of 9 genes (FBN1, FBN2, TGFBR1, TGFBR2, COL3A1, MYH11, ACTA2,
SLC2A10, and NOTCH1) totalling 194 exons (100 kb of sequence) was used. The coverage rate of target
sequences and the efficiency of variant identification were compared. The read depth generated by the
two enrichment technologies was compared with that obtained with whole-exome enrichment. Six
different samples were selected; 4 controls were positive, and, in 2 test samples, no mutations were
detected in the FBN1 and TGFBR1/2 genes (Sanger/MLPA). The two approaches showed good
sequence coverage: Hybridization: 472 X (190 to 592) and PCR: 415 X (303 to 554). Hybridization
showed better sequence coverage, but greater interlocus variation than the PCR method. Wholeexome enrichment generated a sequence coverage equivalent to 150 X. Moreover, when each exon is
considered individually, hybridization attains a coverage of 50 X for 98.5% of exons (PCR: 85.5% and
whole exome: 70%). Hybridization offers predictable coverage variation, affecting certain exons in
particular, while PCR variability is dependent on the sample and less predictable. In total, 252 variants
were identified, of which 195 are common to both methods (concordance of 77%). The validity of the
discordant variants was not examined by Sanger sequencing. However, it should be noted that a major
proportion of these were found in the introns (where coverage is low) and in the gene NOTCH1, which
has a GC-rich sequence and is highly homologous to other NOTCH genes. A new pathogenic mutation
10
(COL3A1) was identified in 1 of the 2 test samples. Considering design flexibility, performance, and
costs, the authors concluded that hybrid capture enrichment for mutations associated with
aortopathies is a method that can easily be adapted to clinical laboratories. Their work highlights the
importance of including a step to confirm the variations observed [Baetens et al., 2011].
Sakai et al. [2012] used two types of technology, targeted sequencing with a biochip (resequencing
array-ResAT) and NGS (multiplex PCR and Illumina platform) to test 8 genes associated with syndromic
forms of aortic aneurysm and dissection (AAD) in 70 patients who had surgery for thoracic AAD (35),
abdominal AAD (30), or AAD in both sites (5). None of the patients included in the study had a clinical
presentation that corresponded to one or the other syndromic form of TAAD. The genes selected were
FBN1, TGFBR2, TGFBR1, COL3A1, PLOD1, MYH11, SLC2A10, and ACTA2. The two technologies allowed
the detection of 18 new variants, of which 1 was detected solely by ResAT and 2 solely by NGS. Of the
18 new variants, 3 were identified as pathogenic, affecting 4.3% of all patients with AAD and 8.6% of
patients from the thoracic subgroup (TAAD). ResAT and NGS performed similarly in detecting most, but
not all, of the variants. ResAT technology is rapid and effective for the identification of nucleotide
substitutions but ineffective for short insertions/deletions. Moreover, it is very impractical to
frequently update the information contained in custom microarrays. NGS enabled the detection of
every type of mutation, but it requires specialized and validated digital tools [Wooderchak-Donahue et
al., 2012].
The utility and validity of each platform, for various diseases, were reported over the last few years as
part of an extensive body of literature. To summarize, compared with the Sanger method, MiSeq and
HiSeq sequencing achieve a sensitivity, specificity, and reproducibility of nearly 100% in regions with
adequate coverage [Lepri et al., 2014; Umbarger et al., 2014; Li et al., 2013; Sikkema-Raddatz et al.,
2013].
COMPONENT
Sensitivity
Specificity
Positive predictive value (PPV)
Negative predictive value (NPV)
Likelihood ratio (LR)
ROC curve
Accuracy
5.3
PRESENCE
ABSENCE
x
x
NOT APPLICABLE
x
x
x
x
x
Analytical (or Technical) Validity
Several comparative studies were carried out to assess the performance and validity of SeqCap EZ
enrichment kits by Roche NimbleGen and compare them with the Sure Select kit by Agilent and the
TruSeq and Nextera Rapid Capture Exome kits by Illumina. Publications report that the Roche
NimbleGen kit allows the capture of fewer exons; however, it ensures a greater sequence coverage
uniformity of 20X and a more efficient capture of flanking sequences [Sakai et al., 2012].
McInerney-Leo et al. [2013] compared the coverage of exons in the genes COL1A1 and COL1A2, FBN1,
TGFBR1, and TGFBR2 with various commercially available kits [McInerney-Leo et al., 2013; Asan et al.,
2011; Clark et al., 2011; Sulonen et al., 2011]. The Roche NimbleGen kit allowed 85.5%, 91%, 95%, 95%,
and 92% coverage of these genes, respectively. It should be noted that exon 1 of TGFBR1 was not
covered.
The HaloPlex kit by Agilent was put on the market in 2013 but has not been the subject of any
publication to date.
11
A review of the literature seems to indicate that each kit has its limitations. Consequently, the user
must ensure sufficient coverage of the exons to be characterized and confirm genetic variations before
they are reported (with Sanger sequencing or other methods) [McInerney-Leo et al., 2013].
Sakai et al. [2012] used two technologies, resequencing array technology (ResAT) and NGS, to analyze
8 genes associated with syndromic forms of aortic aneurysm and dissection (AAD) in 70 patients who
had surgery for thoracic AAD (35), abdominal AAD (30), or AAD in both sites (5) [Sikkema-Raddatz et al.,
2013].
ResAT/NGS Performance
The effectiveness of the base reads (base call) for the 70 samples was 95.7% (87.3% to 97.6%). NGS
allowed coverage of all the targeted sequences (100%) with a read depth of approximately 600 X for
each gene.
Analytical Sensitivity
To validate the sensitivity of the microarray, amplicons containing the following known mutations were
tested: 16 substitutions, 3 short deletions (1 bp to 2 bp), and 1 insertion (1 pb). ResAT detected 87.5%
of the substitutions in automatic mode (14/16) and 93.8% (15/16) in manual and automatic modes.
None of the short insertions/deletions (indels) were detected in either manual or automatic mode.
ResAT allowed the identification of 70 different substitutions, or between 0 and 3 per patient. Of these,
51 had already been indexed (SNP131 database or local data). The 19 new mutations were verified
using Sanger sequencing. One mutation (PLOD1) was homozygous, while the others were
heterozygous.
NextGENe software detected an average of 876 variants (581 to 1,209), with a mutation score of 10 or
more for all of the 70 samples. MAQ (quality mapping) and SeattleSeq (annotation) detected an
average of 271 variants (111 to 384). The semi-automatic exclusion of the variants found outside the
regions of interest, and of those already known, allowed the numbers of these variants to be reduced
to between 0 and 6 per sample. Moreover, 20 new variants were detected with MAQ and NextGENe.
Teer et al. (2010) compared three genomic enrichment methods conducted prior to NGS on the
Illumina platform, by evaluating genotype sensitivity and the accuracy of variant detection. The
methods were: molecular inversion probe (MIP), which is derived from hybridization and PCR
extension; solution hybrid selection (SHS); and microarray-based genomic selection (MGS). A group of
exons consisting of highly conserved non-contiguous sequences from 528 genes (2.612 Mb) were
targeted [Sakai et al., 2012].
Performance Comparison
Probe design for each method provided sequence information for 95.9% (2.506 Mb), 92.6% (2.419 Mb),
and 93.8% (2.450 Mb) of the bases in the regions of interest, for the MIP method, solution hybrid
selection, and microarray-based genomic selection, respectively.
Fraction of Sequences Aligned to the Regions of Interest
The fraction reflects the method’s capacity to enrich the appropriate target sequences. The test
focused on 6 different samples (2 per method). MIP generated approximately 30 million fragment
reads (1,100 Mb in total) and hybridization (SHS and MGS) between 10 million and 14 million fragment
reads (approximately 400 Mb to 500 Mb in total). Among these sequence reads, 57% (600 Mb), 55% to
59% (≈ 290 Mb), and 52% to 55% (193 Mb to 270 Mb) concerned the MIP, SHS, and MGS methods,
respectively.
12
Depth and Uniformity of Coverage
The MIP, SHS, and MGS enrichment methods attained a 10X minimum coverage depth for 79%, 87%,
and 93% and 20X for 74%, 83%, and 89% of the regions of interest, respectively.
Sensitivity
The overall test sensitivity was calculated at 78% for MIP, 86% for SHS, and 92% for MGS. A genotype
was assigned by the 3 methods for 70% of the bases found in the regions of interest, whereas one
genotype was assigned by only one method for 25% of the bases. Five percent of the bases were not
assigned a genotype by any method.
Genotype Concordance
There is no gold standard for each of the bases to be tested. Two data sources were used to verify the
accuracy of the assigned genotype (Infinium 1M SNP and 30X coverage shotgun sequencing). Each of
the methods achieved a concordance rate of 99.84% to 99.99% between the assigned genotype and
the reference genotype.
COMPONENT
PRESENCE
Repeatability
Reproducibility
Analytical sensitivity
Analytical specificity
Matrix effect
Concordance
Correlation between test and comparator
Other, depending on type of test
5.4
ABSENCE
NOT APPLICABLE
x
x
x
x
x
x
x
Recommendations from Other Organizations
The American Heart Association published guidelines in 2010 for patients with thoracic aortic diseases
that provide recommendations for diagnosis and management [Teer et al., 2010].
If a mutation in a gene associated with TAAD (FBN1, TGFBR1, TGFBR2, COL3A1, ACTA2, or MYH11) is
identified in a patient, first-degree relatives should be referred for genetic counselling and undergo
testing.
6 ANTICIPATED OUTCOMES OF INTRODUCING THE TEST
6.1
Impact on Material and Human Resources
A considerable amount of bioinformatic and human resources are needed to interpret the identified
variants. Databases for known mutations, software to predict variations in protein function, and
scientific literature must be consulted for variant characterization.
The complexity of the technology and interpretation of the results require specialized personnel with
the appropriate initial and continuing vocational training.
6.2
Economic Consequences of Introducing Test Into Quebec’s Health Care and Social
Services System
The identification of individual mutations for each gene in the panel is more costly using the Sanger
method than using large-scale sequencing. However, as current technologies have limitations,
conventional technologies for mutational analysis (Sanger and MLPA) cannot be discarded altogether.
13
6.3
Main Organizational, Ethical, and Other (Social, Legal, Political) Issues
The dividing line between the usefulness of NGS for clinical or research purposes is difficult to
establish.
NGS identifies mutations of variable, probable, or uncertain pathogenicity that can influence clinical
and personal decision-making [Hiratzka et al., 2010].
NGS provides opportunities for incidental findings of pathogenic mutations outside the initial clinical
context [Evans and Rothschild, 2012; Makrythanasis and Antonarakis, 2012].
7 IN BRIEF
7.1
Clinical Relevance
TAADs are common medical conditions with a high risk of mortality and morbidity. Approximately 20%
of cases are inherited and caused by a single-gene alteration. As the mutations are dominant and
transmissible, relatives should undergo preventive screening and carriers should be monitored closely.
NGS is an emerging technology in clinical environments that provides an opportunity to assess the
status of several genes at the same time with high sensitivity. NGS is faster and less expensive than
Sanger sequencing. The clinical priority given to the mutations so identified remains a key issue.
7.2
Clinical Validity
A review of the literature revealed few validation studies that used high-throughput sequencing
approaches such as those that have been proposed. However, the correlation between TAAD and the
genes proposed by the CHUS was validated individually for all the genes with the Sanger method. The
CHU Sainte-Justine proposes the use of a larger panel for a more comprehensive approach to diseases
of the aorta (TAADs and other connective tissue syndromes).
7.3
Analytical Validity
Few studies addressing the use of the proposed enrichment strategies were identified. Nevertheless,
NGS is a powerful technology that has shown high test sensitivity. The variants found must be
confirmed by Sanger sequencing.
7.4
Recommendations from Other Organizations
Current recommendations mainly address the importance of identifying pathogenic variants, regardless
of the technology used. The American College of Medical Genetics and Genomics recommends that the
identified variants and the regions inadequately covered by NGS be examined using a different
approach (usually semi-automated Sanger sequencing).
14
8 INESSS NOTICE IN BRIEF
Familial Aortopathy – Gene Panels
Status of the Diagnostic Technology


Established

Experimental (for research purposes only)

Replacement for technology:
Innovative
, which becomes obsolete
INESSS Recommendation

Include test in the Index


Do not include test in the Index
Reassess test
Given the innovative nature of this sequencing technology for gene panels, more extensive
research of the clinical relevance of each gene included in the panel should be carried out
before it is added to the Index. The requestors will be asked to provide additional
information.
Additional Recommendation

Draw connection with listing of drugs, if companion test

Produce an optimal use manual

Identify indicators, when monitoring is required
15
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20
APPENDIX
Summary of the clinical presentation of the syndromes associated with TAAD
Marfan Syndrome (MFS)
MFS is the most common form of syndromic TAAD; its prevalence is estimated at 1 case per
5,000 idividuals [Makrythanasis and Antonarakis, 2012]. The clinical diagnosis is based on the Ghent
nosology criteria [Pearson et al., 2008]. MFS is characterized by cardiovascular, ocular, and skeletal
involvement. The ocular abnormalies are ectopia lentis (displacement of the crystalline lens) and high
myopia. Skeletal defects affect the thorax and extremities of the limbs (thin and fragile) and include
scoliosis.
The most commonly observed cardiac abnormalities are mitral valve prolapse and dilatation of the
pulmonary artery. Dilatations occur predominantly in the aortic root and generally develop at a young
age (75% of cases before the age of 19 years) [Loeys et al., 2010]. The dilatation is cause by a decrease
in aortic compliance and an increase in the stiffness of the aortic wall [Van Karnebeek et al., 2001].
Moreover, more than 20% of the individuals develop an aneurysm or abdominal aortic dissection.
Complete aortic imaging is indicated for adult patients. Treatment usually involves surgery, but may
also include the use of beta blockers and antihypertensives, although the efficacy of these therapies in
these cases is still being studied [Nollen et al., 2004].
Loeys-Dietz Syndrome (SLD)
The prevalence of this syndrome is reported by Orphanet as being lower than 1/1,000,000, and the
Office of Rare Diseases Research of the National Institutes of Health (NIH) estimates that fewer than
200,000 people are affected in the Unites States. The main feature of patients with LDS is the presence
of TAAD in the aortic root. Half of the patients have aneurysms or dissections in other arteries (cerebral
or abdominal). Most cases involve tortuosity of the arteries in the neck and head. Other classical
clinical features of LDS are skeletal (pectus deformity, scoliosis, joint laxity, arachnodactyly, club foot),
craniofacial (hypertelorism, bifid uvula, cleft palate, craniosynostosis), and cutaneous (translucent skin,
predisposition to bruising, dystrophic scarring) abnormalities [Chiu et al., 2013; Gersony et al., 2007].
Despite the phenotypic similarities between LDS and MFS, there are some clear distinctions, such as
generalized arterial tortuosity, cleft palate, and craniosynostosis. Moreover, in patients with LDS,
arterial dissection occurs earlier and prior to a less marked dilatation (maximum of 40 mm) compared
with patients with MFS. More complications during childbirth have also been observed [Loeys et al.,
2005]. Standard preventive treatment includes a beta blocker and restriction of intense physical
exercise [Loeys et al., 2006]. Studies are underway to test the effect of antihypertensives.
Ehlers-Danlos Syndrome (EDS)
EDS is a heterogeneous disorder comprising various subtypes of connective tissue diseases. Its
prevalence is between 1/20,000 and 1/100,000, depending on the subtype. To date, at least 14 genes
have been associated with this syndrome. It has been estimated that approximately 50% of the
mutations identified are germline mutations, as opposed to 50% de novo [Arslan-Kirchner et al., 2011].
Classic EDS (type I and type II), which accounts for 90% of cases, is characterized by skin
hyperextensibility, the presence of typical scars, and extreme joint laxity. The classic form of EDS is
caused by an abnormality of type 5 collagen, involving mutations in the genes COL5A1 and COL5A2,
and is inherited primarily in an autosomal dominant manner. EDS type IV, or vascular type, accounts for
5% to 10% of EDS cases [Malfait et al., 2010]. EDS type IV is characterized by arterial wall fragility,
which predisposes affected individuals to a high risk of rupture without necessarily presenting a prior
dilatation. This fragility also affects the gastrointestinal and uterine tissues. Other clinical features, such
as translucent skin, a predisposition to bruising, and typical craniofacial characteristics are associated
21
with EDS. The median age for the first major arterial or gastrointestinal complication is 23 years. Of the
individuals suspected of being affected with EDS type IV, due to an associated clinical manifestation,
25% with confirmed cases were at least 20 years old and 80% were at least 40 years old at the time of
the first clinical manifestation [Germain, 2007; Beighton et al., 1998]. Surgical procedures are
complicated due to the vascular system’s excessive and generalized fragility. The American Heart
Association nevertheless recommends preventive surgery for patients with a dilatation.
Aneurysm-Osteoarthritis Syndrome (AOS)
Recently described as a syndromic form of TAAD, AOS is characterized by the presence of aneurysms
and dissections of the aortic root, combined with arterial tortuosity, skeletal or skin abnormalities, and
early-onset osteoarthritis [Oderich et al., 2005]. It is now included in LDS type III. According to
Orphanet, its prevalence is less than 1/1,000,000. As with MFS, TAAD of the aortic root is the most
common clinical manifestation. Aneurysms affecting other arteries are often identified. Dissections or
ruptures generally occur at a smaller arterial diameter than with MFS or nonsyndromic familial TAAD.
Preventive surgery is recommended for these individuals [Van de Laar et al., 2012; 2011].
Arterial Tortuosity Syndrome (ATS)
ATS is mainly characterized by the elongation, deformation, stenosis, and enlargement of the arteries.
Other clinical manifestions, such as skin fragility and hyperextensibility, arachnodactyly, thoracic
abnormalities, and joint laxity, are typical of those described in other connective tissue syndromes [Van
der Linde et al., 2012].
Congenital Contractural Arachnodactyly (CCA)
CCA or Beals syndrome is a connective tissue disorder characterized by abnormal and prolonged
muscle contractions, arachnodactyly, severe scoliosis, abnormal ear cartilage, and poor muscle
development [Callewaert et al., 2008; Wessels et al., 2004]. Its prevalence is unknown. Although CCA
may be mistaken for MFS (neonatal), the presence of multiple joint contractures and the morphology
of the ear helix are distinguishing features. Contrary to the findings of the first published studies, aortic
root dilatations have been reported in a few cases. CCA is an autosomal dominantly inherited disease
caused by a mutation in the gene FBN2 found on chromosome 5q23. Prenatal ultrasound can identify
joint contractures in suspected cases. Treatment for children with CCA varies according to the
symptoms observed. Early surgical treatment of scoliosis prevents complications in adulthood. Cardiac
and ophthalmologic evaluations are recommended.
Table A1: Syndromic aortopathies
GENE
FBN1
Fibrillin 1
65 exons
PHENOTYPE
GENOTYPE
DIAGNOSIS
(DIFFERENTIAL)
Marfan
TAAD aortic root
Mitral valve
prolapse
1,492 mutations
(1,278 fulfill
Ghent criteria)
AD
Variable
penetrance
75% with
affected parent
Ghent Nosology
Tall stature
Arachnodactyly
Funnel chest
Displacement of
crystalline lens
Scoliosis
Joint
hypermobility
Arachnodactyly
Funnel chest
Scoliosis
Joint
Prevalence 1:5,000
TGFBR1
TGFBR2
Transforming
growth factor-
Loeys-Dietz
TAAD aortic root
and other
Tortuosity
46 and
108 mutations
(75% de novo)
AD
PROGNOSIS
FOLLOWUP/TREATMENT
TAAD < 19 years
(75%)
Rupture <
40 years
50% mortality
Imaging/surgery/
beta blocker/
antihypertensives
Median survival
age 26 years
Restricted
physical activity
*
NGS
CHUS
CHU SJ
CHUS
CHU SJ
22
GENE
PHENOTYPE
GENOTYPE
DIAGNOSIS
(DIFFERENTIAL)
beta
receptor
9 and 7 exons
PDA/ASD
Variable
penetrance
hypermobility
Cleft palate
Craniosynostosis
Bruising
Dystrophic scars
COL3A1
Collagen 3α1
51 exons
SMAD3
SMAD family
member
39 exons
Prevalence
unknown
Ehlers-Danlos
(vascular type)
5% to 10% of EDS
cases
Aneurysm (rare)
Dissection (high)
Tortuosity (none)
Prevalence
1:75,000
Aneurysm
Osteoarthritis
TAAD aortic root
and other
Tortuosity
PDA/ASD/PS
Prevalence
unknown
SLC2A10
Glucose
transporter
5 exons
TGFB2
Transforming
growth factorbeta
8 exons
COL1A1
Collagen I
51 exons
Arterial tortuosity
TAAD aortic root
and other (rare)
Tortuosity (high)
PS
Prevalence
unknown
“LDS like”
TAAD aortic root
(high)
Dissection (rare)
Tortuosity
(medium)
Prevalence
unknown
Osteogenesis
imperfecta
Frequent aortic
regurgitation
TAAD aortic root
and other (rare)
Tortuosity (none)
Prevalence
1:15,000
5% of non-LDS
FTAAD
254 mutations
(50% de novo)
AD
17 mutations
1% to 2% of
previously
reported
nonsyndromic
TAAD
AD
High penetrance
Very high intraand interfamilial
variability
23 mutations
AR
Translucent skin
Bruising
Characteristic
craniofacial
features
Gastrointestinal
perforations
Skeletal and
cutaneous
abnormalities
similar to MFS and
LDS
Osteoarthritis
Clinical
presentation
overlaps that of
LDS and EDS
PROGNOSIS
FOLLOWUP/TREATMENT
beta blocker
antihypertensives
Median survival
age 48 years
25% of cases with
TAAD rupture
before 20 years
Imaging/tissue
biopsy/complex
surgery
Median survival
age 54 years
Severe and
generalized
vascular disease
High mortality
rate
Consider
preventive
surgery
High mortality
rate at young age
due to TAAD and
risk of stroke
*
NGS
CHU SJ
CHUS
CHU SJ
CHU SJ
Imaging
15 mutations
AD
609 mutations
AD
Variable
penetrance
CHU SJ
Clinical
presentation
overlaps that of
MFS and LDS
Short stature
Blue sclerae
Osteopenia
Susceptibility to
fractures
Clinical
presentation
overlaps that of
EDS
Aneurysm
detected around
35 years
Variable survival
rate
Risk of rupture in
young adults
Cardiovascular
monitoring
Complex surgery
CHU SJ
23
GENE
PHENOTYPE
GENOTYPE
DIAGNOSIS
(DIFFERENTIAL)
COL5A1
COL5A2
Collagen 566
and 38 exons
Ehlers-Danlos
(classic)
90% of EDS cases
Prevalence
1:30,000
119 and
21 mutations
AD
75% of cases
show mutations
in COL5A1 and
25% in COL5A2
47 mutations
Differential
diagnosis of classic
type and vascular
type
FBN2
Fibrillin
233 exons
Congenital
contractural
arachnodactyly
TAAD aortic root
(rare)
Mitral valve
prolapse, PDA,
BAV, ASD
Prevalence
unknown
AD
Villefranche
classification
Differential
diagnosis with
neonatal MFS
Multiple
contractures
Hypotonia
Severe scoliosis
Stahl’s ear
PROGNOSIS
FOLLOWUP/TREATMENT
6% of cases with
mild aortic
dilatation
Treatment of
symptoms
(antalgics,
orthoses)
Cardiovascular
monitoring
Scoliosis surgery
Symptomatic
treatments
*
NGS
CHU SJ
CHU SJ
Table A2: Nonsyndromic aortopathies
GENE
PHENOTYPE
(FREQUENCY)
GENOTYPE
DIAGNOSIS
(DIFFERENTIAL)
ACTA2
alpha-actin
9 exons
FTAAD type 4
TAAD aortic root
(high)
Tortuosity (none)
PDA, BAV
38 mutations
AD
Penetrance 50%
15% of FTAAD
2.5% sporadic
TAAD
Cutaneous
manifestations
(livedo reticularis)
Ocular
abnormalities (iris
flocculi)
Systemic smooth
muscle dysfunction
MYH11
Myosin
42 exons
FTAAD PDA
TAA ascending
aorta (high)
Dissection (low)
Tortuosity (none)
PDA (high)
29 mutations
AD
Highly variable
penetrance
At least 1 parent
with PDA
2% of FTAAD
9 mutations
AD
Highly variable
penetrance
1% of FTAAD
Highly variable
clinical expression
MYLK
Myosin
kinase
33 exons
FTAAD Type 7
TAA ascending
aorta (rare)
Dissection (high)
Tortuosity (none)
Gastrointestinal
problems
PROGNOSIS
FOLLOWUP/TREATMENT
Median survival
age 67 years
High risk of stroke
and coronary
disease
Consider early
surgery
High risk of
vascular occlusion
and stroke
Sudden death
before 40 years
reported
No predissection
dilatation
Emergency
procedure
following first case
only
NGS
CHUS
CHU SJ
CHUS
CHU SJ
CHUS
CHU SJ
24
Table A3: Other
GENE
CBS
Cystathionine
beta-synthase
17 exons
PHENOTYPE
(FREQUENCY)
GENOTYPE
DIAGNOSIS
(DIFFERENTIAL)
Homocystinuria
Metabolic
disorder in
newborns
Mitral valve
prolapse
Prevalence
1:344,000
181 mutations
Variable
penetrance
AR
Exclusion of MFS
19% of cases have
Marfanoid
features
PROGNOSIS
FOLLOWUP/TREATMENT
Stroke < 16 years
(25%)
Stroke < 30 years
(50%)
Treatment to
ensure normal
development:
folate, vitamin
B12, and
pyridoxine
NGS
CHU SJ
25