EDNF Center for Clinical Care & Research at GBMC

2014 PHYSICIANS CONFERENCE September 15, 2014 Ehlers-Danlos Syndrome: Molecular Analysis

Clair A. Francomano, M.D. Director, EDNF Center for Clinical Care and Research at GBMC Director, Adult Genetics Harvey Institute for Human Genetics Ehlers-Danlos Syndrome

• Spectrum of monogenic disorders • Wide range of phenotypic severity • Predominantly affecting joints, skin, blood vessels and internal organs to varying degrees • Most forms are caused by defects in one of the fibrillar or of enzymes involved in fibrillar processing • Recent research has identified defects in biosynthesis of other molecules in the extraceullar matrix and molecules involved in intracellular trafficking, secretion and assembly of ECM molecules EDS: Molecular Causes

• Most forms are caused by defects in one of the fibrillar collagens or of enzymes involved in fibrillar collagen processing • Recent research has identified defects in biosynthesis of other molecules in the extraceullar matrix and molecules involved in intracellular trafficking, secretion and assembly of ECM molecules

Fibrillar Collagens

• Major structural components of the • Include collagen types I, II, III, V, IX, and XI • Trimeric molecules (three chains) • May be made up of three identical or genetically distinct chains, called alpha chains Fibrillar Collagens

Biochemical Society Transactions (1999) , - - www.biochemsoctrans.org De Paepe and Malfait, 2012 DePaepe and Malfait, 2012 http://www.epharmapedia.com/diseases/profile/167/Ehlers-Danlos-syndrome.html?lang=en

Molecular Analysis in Classical Ehlers-Danlos Syndrome

Symoens et al, 2012 Villefranche Criteria Classical Ehlers-Danlos Syndrome

Major Criteria

Smooth, velvety, hyperextensible skin Skin fragility (splitting, slow wound healing, widened atrophic scars) Joint hypermobility – small and large joints

Villefranche Criteria Classical Ehlers-Danlos Syndrome

Minor criteria

Easy bruising Mulluscoid pseudotumors Subcutaneous spherules Inguinal or umbilical hernia Muscle hypotonia Delayed gross motor development Joint dislocations Molecular Analysis of Classical EDS

• Of 126 patients, 102 met all 3 major Villefranche criteria for Classical EDS • Of these 102, 93 (91%) were found to have a mutation • 24/126 met 2/3 of the Villefranche criteria for Classical EDS • Of these, none were found to have a Type V collagen mutation • None of the 126 patients had mutations in X Symoens et al, 2012 Deficiency

• Autosomal recessive form of EDS • Marked skin hyperextensibility, • Easy bruising • Joint laxity. • Not present: atrophic scarring or poor wound healing. • Other medical problems: – severe diverticular intestinal disease – mitral valve prolapse requiring valve replacement – obstructive airway disease. Autosomal Recessive Tenascin X Deficiency

Lindor and Bristow, 2005 Autosomal Dominant Tenascin X Deficiency

• Zweers et al (2003) examined 20 obligate heterozygotes with Tenascin-X mutations • Generalized joint hypermobility in 45% (all women) • None had skin hyperextensibility or easy bruising • Of 80 patients with hypermobile EDS, six had low levels of tenascin-X in the serum and 2 had heterozygous tenascin-X mutations Figure 1: Clinical and biochemical characteristics of the vascular Ehlers-Danlos syndrome. Typical facial features with diminished subcutaneous fat, thin nose and lips (a, b), acrogeria (c) and easy bruising (d). Biochemical analysis of the collagens reveals a diminished amoun of collagen Type III (e).

Byers PH, 2009 Vascular EDS: Molecular Analysis

• Vascular EDS is caused by mutations in COL3A1, which encodes the alpha1 chain of Type III collagen

• COL3A1 sequence analysis: 95% • Deletion/duplication analysis ~2%

• Severity of phenotype depends upon the type of mutation present

Type III Collagen is a Homotrimer

a1(III) a1(III) a1(III)

Biochemical Society Transactions (1999) , - - www.biochemsoctrans.org

Pepin M et al., 2014 Survival Curves: Vascular EDS

Pepin M et al., 2014 Vascular EDS: Survival Curves Based on Mutation Type and Altered Amino Acids

Pepin M et al., 2014 Kyphoscoliotic Type of EDS

• Neonatal kyphoscoliosis • Generalized joint laxity • Skin fragility • Severe muscle hypotonia at birth • Biochemically attributed to deficiency of • Mutations in PLOD1, which encodes lysyl hydroxylase, cause the Kyphoscoliotic form of EDS Why is Important to Hydroxylate Lysine?

• Hydroxylysine residues – Serve as attachment sites for galactose and glucosylgalactose – Act as precursors of the crosslinking process for collagen Fibrillar Collagens

Biochemical Society Transactions (1999) , - - www.biochemsoctrans.org EDS, Arthrochalasia Type

• Bilateral hip dislocations • Doughy, redundant skin • Contracture of fingers and toes • Umbilical hernia • Thoraco-lumbar kyphosis • Muscular hyptonia

Giunta C et al., 2008 Arthochalasia Type EDS

• Caused by mutations in either the alpha 1 or alpha 2 chain of type 1 collagen that interfere with the cleavage of the N-propeptide

• These are autosomal dominant mutations EDS, Dermatosporaxis Type

• Epicanthal folds • Downsloping palpebral fissures • Blue sclerae • Micrognathia • Prominent lips • Facial scarring • Easy brusing.

De Paepe and Malfait, 2012 Molecular Analysis of Dermatosporaxis Type, EDS

• ADAMTS-2 encodes the procollagen N- proteinase enzyme, which cleaves the N- propeptide of types I and II collagen • Mutations in ADAMTS-2 cause the dermatosporaxis type of EDS • These are autosomal recessive mutations is a Heterotrimer

a1(I) a1(I) a2(I)

Biochemical Society Transactions (1999) , - - www.biochemsoctrans.org EDS, Musculocontractural Type

• Malar hypoplasia • Downslanting palpebral fissures • Blue sclerae • Microcornea • Long philtrum • Thin Upper lip • Macrognathia • Pointed chin

De Paepe and Malfait, 2012 EDS, Musculocontractural Type

• Caused by mutations in CHST14 • Encodes Dermato-4-sulfotransferase-1 • Key enzyme in the synthesis of Dermatan sulfate • Disrupts the ratio of Dermatan sulfate to chondroitin sulfate – important regulator for multiple glycoprotein components of the connective tissue EDS, Cardiac Valvular Type

• Cardiac valvular insufficiency (mitral, aortic) • Joint hypermobility • Hyperextensible skin • Biochemical lack of a2(I) chains in type I collagen Type I Collagen without COL1A2 is a Homotrimer

a1(I) a1(I) a1(I)

Biochemical Society Transactions (1999) , - - www.biochemsoctrans.org Absent COL1A2

Maltfait et al., 2006 Byers and Murray, 2014 Connective Tissue Gene Tests http://www.ctgt.net/

Next Generation Sequencing Panels

Tests & Panels Genes Ehlers-Danlos syndrome NGS COL1A1 / COL1A2 / COL3A1 / COL5A1 / COL5A2 panel – Dominant Ehlers-Danlos syndrome NGS ATP7A / CHST14 / COL1A1 / COL1A2 / COL3A1 / panel - Dominant & Recessive COL5A1 / COL5A2 / FKBP14 / PLOD1 / SLC39A13 Ehlers-Danlos syndrome NGS ATP7A / CHST14 / FKBP14 / PLOD1 / SLC39A13 panel - Recessive Greenwood Genetic Center NGS Connective Tissue Panel http://www.ggc.org/diagnostic/tests-costs/test-finder/ngs-connective-tissue-panel.html

ABCC6 COL3A1 PKD2 ACTA2 COL5A1 PLOD1 ACVR1 COL5A2 PRDM5 ADAMTS2 ELN SLCSA1- ATP6V0A2 FBLN5 SLC39A13 CBS FBN1 SMAD3 CHST14 FBN2 TGFBR1 COL11A1 FKBP14 TGFBR2 COL1A1 MYLK TNXB COL1A2 NOTCH1 ZNF469 COL2A1