Aortopathy Gene Testing by Sanger sequencing

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Wilfred Gaines
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Department of Molecular Genetics Aortopathy Gene Testing by Sanger sequencing Mutation screening for Marfan syndrome and related disorders has grown to include many genes with overlapping phenotypes.

We now offer some of these genes in a panel to be sequenced simultaneously to reduce the turnaround of screening each gene consecutively.

1 Department of Molecular Genetics Aortopathy Gene Testing by Sanger sequencing Mutation screening for Marfan syndrome and related disorders has grown to include many genes with overlapping phenotypes. We have recently added SMAD3, TGFB2, COL3A1 and SKI to our genes for Sanger sequencing. We now offer some of these genes in a panel to be sequenced simultaneously to reduce the turnaround of screening each gene consecutively. We are developing an aortopathy gene panel utilising next generation sequencing (NGS) or massively parallel sequencing (MPS). Gene FBN1 (Fibrillin-1) TGFBR1&2 (Transforming growth factor beta receptor 1 and 2) COL3A1 (Collagen type III, alpha-1) TNXB (tenascin-x) Testing by MLPA only ACTA2 (Actin alpha-2, smooth muscle) SMAD3 TGFB2 (Transforming growth factor beta 2) SMAD4 To be developed Genes tested and associated phenotypes Associated phenotype MIM number Marfan syndrome Ectopia lentis, familial MASS syndrome Acromicric dysplasia (exons 41 & 42) Geleophysic dysplasia 2 (exons 41 & 42) Stiff skin syndrome (exons 37 & 38) Weill-Marchesani syndrome 2, dominant Aortic aneurysm, ascending, and dissection Loeys-Dietz syndrome, type 1A Loeys-Dietz syndrome, type 2A Loeys-Dietz syndrome, type 1B Loeys-Dietz syndrome, type 2B Ehlers-Danlos syndrome, type III Ehlers-Danlos syndrome, type IV Ehlers-Danlos-like syndrome due to tenascin-x deficiency Aortic aneurysm, familial thoracic Moyamoya disease Loeys-Dietz syndrome, type 3 (Aneurysms- Osteoarthritis syndrome (AOS)) Loeys-Dietz syndrome, type Juvenile polyposis/hereditary Hemorrhagic Telangiectasia syndrome/aortopathy SKI Shprintzen-Goldberg syndrome Dept Molecular Genetics CHW. Version 1 Page 1 of 6

2 Pricing and turnaround s (updated May 2013) Index case (proband) Sanger sequencing panels (genes are screened and reported together) Panel Genes included Price AUD$ Marfan panel FBN1 sequencing & MLPA $ weeks Loeys-Dietz syndrome panel TGFBR1 TGFBR2 SMAD3 TGFB2 +/- COL3A1 $1200 or with COL3A1 $ weeks Fast TAT extra $ weeks Individual genes (screening is performed consecutively) Please clearly state the order of the genes to be tested if more than one gene is required is for each gene 10% discount for the 2 nd and subsequent gene tested Gene Price AUD$ FBN1 $ weeks COL3A1 $ weeks TGFBR1 & 2 $900 6 weeks ACTA2 $500 6 weeks SMAD3 $500 6 weeks TGFB2 $500 6 weeks SKI (exon 1 only) $250 4 weeks FBN1 exons 24 to 32 for neonatal Marfan syndrome $ weeks FBN1 exons 41 & 42 for Geleophysic and acromicric dysplasia $250 4 weeks FBN1 exons 37 & 38 for Stiff skin syndrome $250 4 weeks Fast TAT extra $ weeks MLPA (for the detection of deletions and insertions) Gene Price AUD$ FBN1 (2 kits) $400 6 weeks FBN1 & TGFBR2 (2 kits) Analysis of both genes will be performed together $400 6 weeks TGFBR1 & TGFBR2 (1 kit) $300 6 weeks FBN1 & TGFBR1 & TGFBR2 (3 kits) Analysis of all 3 genes will be performed together $500 6 weeks COL3A1 & TNXB (1 kit) $300 6 weeks Dept Molecular Genetics CHW. Version 1 Page 2 of 6

3 Cascade testing Once a family-specific mutation has been identified, cascade testing can be used for at risk family members. If the mutation was not identified in our laboratory a specimen from the proband will be required to test alongside the family members. Cascade testing (known mutation) Single patient (sequencing) If the index case was not tested in our laboratory it is advisable to test the index case alongside the other family members. Please provide a sample from the index case with the patient to be tested. An additional $50 will be charged. Multiple patients (sequencing) More than one patient tested at the same for the same familial mutation Predictive testing 2nd sample (sequencing) 2nd sample received after 1st sample has been tested Price AUD$ $250 4 weeks $200 4 weeks $100 4 weeks MLPA - FBN1 $400 6 weeks MLPA TGFBR1 or TGFBR2 $300 6 weeks Prenatal testing of familial mutation $600 <2 weeks Summary of phenotypes for recently added genes ACTA2: Mutations in ACTA2 associated with TAAD were first reported by Guo et al (2007) in 14 TAAD families. Other features found in some individuals with mutations included livedo reticularis, iris flocculi and patent ductus arteriosus. In addition Guo et al (2009) looked at the phenotype of 127 individuals with an ACTA2 mutation from 20 families. The primary vascular disease in these patients was TAAD. Other features included premature onset coronary artery disease and ischemic strokes. In another cohort of 237 patients with nonfamilial TAAD six mutations were identified. Other features found in this group include ischemic strokes, Moyamoya disease, coronary artery disease, bicuspid aortic valve. Guo, D.-C et al Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections. Nature Genet. 39: , Guo, D.-C. et al Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease. Am. J. Hum. Genet. 84: , SMAD3 : van de Laar et al (2011) found 2 mutations in 99 patients with thoracic aortic aneurysms and dissections (TAAD) and Marfan-like features but without FBN1, TGFBR1 or TGFBR2 mutations. Regalado et al (2011) screened 181 patients with familial TAAD and found mutations in four families. The phenotype for patients with mutations varied but included TAAD alone, TAAD with intracranial aneurysms, abdominal aortic aneurysms, osteroarthritis, arterial tortuosity and cutaneous and skeletal features of MFS, LDS and AOS. van de laar (2012) screened SMAD3 for mutations in 393 patients with aneurysms without mutations in FBN1, TGFBR1 and TGFBR2 and identified mutations in five families. They studied the phenotypic spectrum of patients with SMAD3 mutations from these families and suggest that joint abnormalities such as osteoarthritis, osteochondritis dissecans and meniscal anomalies maybe useful discriminating features from other forms of TAAD. van de Laar IM, et al Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis. Nat Genet Feb;43(2): van de Laar IM, et al Phenotypic spectrum of the SMAD3-related aneurysms osteoarthritis syndrome. J Med Genet Jan;49(1): Regalado ES, Guo DC, et al Exome sequencing identifies SMAD3 mutations as a cause of familial thoracic aortic aneurysm and dissection with intracranial and other arterial aneurysms. Circ Res.2011 Sep 2;109(6): TGFB2: Boileau et al (2012) identified TGFB2 mutations in two unrelated families with autosomal dominant TAAD using genomewide linkage analysis and whole exome sequencing. Two more mutations were identified in Dept Molecular Genetics CHW. Version 1 Page 3 of 6

4 a further 276 probands with TAAD. Genome SNP arrays for 898 individuals with thoracic aortic disease did not detect any deletions or duplications of TGFB2. Aortic dilatation at the level of the sinuses of Valsalva was present in the majority of the 19 family members with TGFB2 mutations. Other features included skeletal features, joint laxity, striae and inguinal hernias similar to MFS. Ectopia lentis was absent in these and they did not have a particular facial appearance or the craniofacial or skin findings of LDS. Joint disease described in AOS was also not seen. Lindsay et al (2012) performed SNP array analysis on 8 families with autosomal dominant aortic aneurysms and MFS-like and LDS-like features including pectus deformity, arachnodactyly, scoliosis, skin striae, hypertelorism, bifid uvula, bicuspid aortic valve, arterial tortuosity, club feet and thin skin with easy bruising. Ectopia lentis was not present. Two heterozygous microdeletions including TGFBR2 were detected in two individuals who also had developmental delay. A further 86 individual with aneurysms and no mutation in FBN1, TGFBR1 and TGFBR2 were screened and six TGFB2 mutations were detected. Renard et al (2012) initially screened TGFB2 in 40 patients with isolated aortic root dilatation and did not find any mutations. They then screened a further 146 patients with a wider TAAD phenotype including cerebrovascular disease, arterial tortuosity, marfanoid skeletal features and mitral valve prolapse and detected mutations in six patients. Clinical features in the mutation positive patients included thoracic aortic aneurysm, type A aortic dissection and mitral valve prolapse along with some features of MFS, LDS or AOS. Mutations were found in individuals with and without a positive family history of TAAD. Lindsay, M.E. et al. Loss- of function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm. Nat Genet. 44(8): (2012). Boileau, C. et.al. TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome. Nat.Genet. 44 (8): (2012). Renard, M. et.al. Thoracic aortic-aneurysm and dissection in association with significant mitral valve disease caused by mutations in TGFB2. Int J Cardiol. (2012). SMAD4: Andrabi et al (2011) report a family with a SMAD4 mutation in individuals with juvenile polyposis in addition to mitral valve prolapse, mitral valve regurgitation, and aortic dilatation. SMAD4 mutations have previously been described in patients with juvenile polyposis and/or hereditary hemorrhagic telangiectasia syndrome (see OMIM ). Andrabi, S et al SMAD4 mutation segregating in a family with juvenile polyposis, aortopathy, and mitral valve dysfunction. Am. J. Med. Genet. 155A: , Genes and loci known to be associated with TAAD: TGFBR1: TGFBR2: MYH11 ACTA2: FBN1: MYLK: SMAD3: AAT1 (FAA1) locus: AAT2 (TAAD1) locus: 1% of familial TAAD 4% of familial TAAD ~1% of familial TAAD (encoding smooth muscle-specific myosin heavy chain) ~10%-14% of familial TAAD The frequency of mutations in this gene in familial TAAD is unknown ~ 1% of familial TAAD (encoding the myosin light chain kinase (MLCK) protein) ~ 2% of familial TAAD involved gene unknown involved gene unknown Milewicz DM, Regalado E. Thoracic Aortic Aneurysms and Aortic Dissections Feb 13 [Updated 2012 Jan 12]. In: Pagon RA, Bird TD, Dolan CR, et al., editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle; Available from: Next Generation Sequencing panel We are currently validating an aortopathy NGS panel for diagnostic testing. The genes included are ACTA2, COL3A1, FBLN4, FBN1, FBN2, MYH11, NOTCH1, SLC2A10, SMAD3, SMAD4, TGFB2, TGFBR1, TGFBR2, SKI. TNXB and FLN will be added in the next design. We are aiming to have this panel ready for diagnostic testing by the end of Specimen 5 to 10mls EDTA whole blood or extracted DNA from any source with at least 2 unique identifiers. Dept Molecular Genetics CHW. Version 1 Page 4 of 6

5 Billing Please indicate who should be invoiced for the testing. The organisation or the individual must have agreed to pay for the testing. Testing will not commence without billing consent. Prenatal testing Prenatal diagnosis will be offered only to families in whom an existing gene mutation has already been characterised. Where a mutation has been identified by an external source, the laboratory would always seek to confirm the mutation prior to undertaking prenatal diagnosis. All prenatal testing MUST be booked with the laboratory as early in the pregnancy as possible and prior to dispatch. Presymptomatic testing in children and cascade testing Once a family-specific mutation has been identified, cascade testing can be used for at risk family members. If the mutation was not identified in our laboratory a specimen from the proband will be required to test alongside the family members. It is recommended that two independent samples, collected at different s, be sent for cascade testing. Presymptomatic testing should be classified and follow the guidelines set out in the NPAAC document Classification of Human Genetic Testing Edition. If samples are received and tested at the same there will be no additional charge. If the second sample is received and tested after the first sample there will be an additional cost of $100. Counselling and Consent Genetic counselling should be offered and written consent obtained prior to testing, consistent with the NSW Dept of Health document DNA Diagnostic Testing for Genetic Disorders, and the Consent for DNA Diagnostic Testing/ Storage form. Consent should be retained with the patient s medical record. The lab does NOT require a copy. Clinical and Counselling Services Within Australia details of the clinical and counselling services available in your area can be obtained from your State Health service or via links from the NSW Genetic Education Program ph [02] ; fax [02] ; Transport Please transport the specimens at room temperature. Referring laboratories will be responsible for arranging and paying for the transportation of specimens to the laboratory. For countries outside of Australia, blood and DNA samples without a known infectious risk, do not need quarantine inspection if external packaging is clearly labeled. i.e. "Contents: Product of human origin, non-hazardous, non-infectious, for diagnostic in-vitro testing only" and "Exempt human specimen. Diagnostic specimens packed in compliance with IATA packaging instructions 650". If package is not correctly labeled and incurs a quarantine charge, this fee will be passed onto the referring laboratory. AQIS website: e&whi chquery=go+to+full+text&intsearch=1&logsessionid= Address Postal: Department of Molecular Genetics, Western Sydney Genetics Program, The Children s Hospital at Westmead, Locked Bag 4001, Westmead NSW 2145 AUSTRALIA Courier: Department of Molecular Genetics, Western Sydney Genetics Program, The Children s Hospital at Westmead, Loading dock 5, Redbank Rd, Northmead NSW 2152 AUSTRALIA Dept Molecular Genetics CHW. Version 1 Page 5 of 6

6 Contacts For laboratory enquiries: A/Prof Bruce Bennetts - Head, Department of Molecular Genetics Ph [61-2] Fax[61-2] bruce.bennetts@health.nsw.gov.au For clinical enquiries: Dr Lesley Adès - Head, Marfan Research Group and Clinical Geneticist Ph [61-2] Fax[61-2] lesley.ades@health.nsw.gov.au Dept Molecular Genetics CHW. Version 1 Page 6 of 6

Congenital Aortopathies Marfans, Loeys-Dietz, ACTA 2, etc. DATE: October 9 th, 2017 PRESENTED BY: Cristina Fuss, MD

Congenital Aortopathies Marfans, Loeys-Dietz, ACTA 2, etc. DATE: October 9 th, 2017 PRESENTED BY: Cristina Fuss, MD 24 yof present with SoB 9/4/2017 2 24yo F Presenting to local ED with SoB No other pertinent