Chromosome 1 Chromosome 6 Chromosome 11 Chromosome 15 Chromosome 17 Chromosome 20 Supplementary figure 1. Regions of suggestive linkage in PVOD families 1,2 and 3. Six regions were identified with suggestive evidence of linkage (Max Lod-Score= 1.8) using non parametric linkage :1p36.3, 6q24, 11q22-23, 15q15-21, 17p13, 20q13.3.
A. PVOD1 PVOD2 1 # * 2 # * c.1554-4c>a c.354_355del 1 # * 2 # * c.2319+1g>a m1/+ m2/+ m/+ m/+ 1 # * 2 # * 3 m1 m2 # * 3 # * m (HTZ) m (HMZ) Dx 31 Dx 50 Dx 49 m/+ Dx 23 Dx 16 m1/+ 1 # * 2 # * m2/+ PVOD3 c.745c>t c.2136_2139dup m1/+ 1* 2* m2/+ PVOD4 c.1392del c.3802c>t # * 3 # * Dx 23 Dx 45 m1 m2 1* 2* Dx 20 Dx 20 m1 m2 PVOD5 c.567dup 14 5 m V * 3* Dx 27 Dx 20
B. PVOD6 PVOD7 PVOD8 c.3159g>a c.3406c>t c.1754g>a 5 3 Dx 17 Dx 26 2 m 3 4 5 6 Dx 32 m 4 2 4 3 4 5 6 Dx 26 m PVOD9 c.4065+1g>c PVOD10 c.1387c>t PVOD11 c.3448c>t c.4728+1_4728+13delinsttct? 3 4 5 m? m 3 4 m1 m2 Dx 36 Dx 18 Dx 17 Dx 37 PVOD12 PVOD13 c.1387c>t c.3244c>t c.1928t>g 3 Dx 44 7 m1 m2 4 m/+ m/+ 6 m (HTZ) m (HMZ) V 3 Dx 11 4
Supplementary Figure 2. EF2AK4 mutations identified in PVOD families. Pedigree structures and Sanger sequence chromatograms from PVOD families with EF2AK4 mutations identified by (A) whole-exome sequencing or (B) targeted secondary screening, are shown. Subjects included in the linkage analysis are indicated by a hash key, and those analyzed by exome sequencing are indicated by a star. Affected subjects are denoted by filled symbols and arrows designate probands. Subjects suspected to be affected are denoted by striped symbols. Dx indicates age at diagnosis. Genotypes of screened family members are shown under each symbol, with indicating a homozygous mutation, indicating compound heterozygous mutations. Chromatograms show mutated patient or control subject sequences. Above each chromatogram, the corresponding nucleotide change is indicated according to HGVS nomenclature.
A. Wt Wt c.1554-4c>a c.3159g>a B. c.1554-4c>a Exon 9 Exon 10..AAAGAA AGAG ATGTGTGT. c.3159g>a Exon 20 Exon 21 Exon 22 AAAGAAAGATGTGTGT Exon 20 Exon 22 Exon 20 Exon 22 r.1554-2_1554-1ins p.c519dfs*17 r.2923_3159del p.k975_k1053del Supplementary figure 3. Analysis of splicing variant c.1554-4c>a and c.3159g>a.(a) n silico analysis of the two splicing variants by splicing predictions tools Splice Site Finder, MaxEntScan, Nnsplice, GeneSplicer and Human Splicing Finder. The c.1554-4c>a mutation was predicted to create a acceptor site at position c.1554-2 by all prediction tools and the c.3159g>a mutation was predicted to abolished (Splice Site Finder, Nnsplice) or dramatically reduced the score (MaxEntScan, GeneSplicer and Human Splicing Finder) of the acceptor site of intron 21.(B) Consequences at the cdna level of the two splicing variants. The c.1554-4c>a mutation leads to the inclusion of 2 bases of intron 9 into the mature mrna. The c.3159g>a mutation leads to the skipping of exon 21 into the mature mrna.
Supplementary Table 1. Genetic, clinical, functional and hemodynamic characteristics of patients analyzed by exome sequencing at the time of PVOD diagnosis. Family number PVOD1 PVOD2 PVOD3 PVOD4 PVOD5 ndividual -1-2 -3-2 -3-2 -3-1 -2 V-2 V-3 EF2AK4 mutation status Compound HTZ HMZ Compound HTZ Compound HTZ HMZ Mutation - Nucleotide change - Protein effect c.[354_355del];[1554-4c>a] p.[c118wfs*7];[c519dfs*17] c.[2319+1g>a];[2319+1g>a] p.[?];[?] c.[745c>t];[2136_2139dup] p.[r249*];[s714hfs*21] c.[1392del];[3802c>t] p.[r465vfs*38];[q1268*] c.[567dup];[567dup] p.[l190efs*8];[l190efs*8] Consanguinity Age at PVOD diagnosis, yrs 31 50 49 23 16 23 23 20 20 20 27 Gender F F F M F F M M F F F NYHA Functional Class - 3 2 3 3 4 2 3 3 3 4 6-MWD, m - 284 506 580 525 331 478 447 500 468 0 mpap, mmhg - 58 25 47 39 65 48 39 38 30 65 RAP, mmhg - 7 2 4 4 4 9 7 6 3 13 PCWP, mmhg - 6 4 7 8 5 12 9 11 5 5 CO, L/min - 3.45 6.40 4.94 7.00 - - 3.35 6.70 5.53 3.65 C, L/min/m² - 2.11 3.49 2.72 4.40 2.02 3.33 2.06 4.58 3.18 2.25 PVR, mmhg/l/min - 15 3 8 4 13 6 9 4 6 16 SvO 2, % - 46 77 68 81 56 70 - - 72 41 DLCO, % - - 35 30 37 24 34 33 39 27 22 CT scan : - Centrilobular GGO - septals lines - enlarged mediastinal lymph nodes PAH therapy: - CCB - PDE5-i - ERA - Prostanoids - - - Outcome at 12 months at 8 months Alive at 1 month at 36 months at 21 months at 26 months at 17 months Alive at 70 months Alive at 17 months Died at 28 months at 1 month
Mutations are described according to HGVS nomenclature. CCB: calcium channel blocker, C: cardiac index, CO: cardiac output, ERA: endothelin receptor antagonist, GGO: ground glass opacities; : Lung transplantation; mpap: mean pulmonary artery pressure, NYHA: New York Heart Association, PCWP: pulmonary capillary wedge pressure, PDE5-i: phosphodiesterase type 5 inhibitor PVR: pulmonary vascular resistance, RAP: right atrial pressure, SvO 2 : mixed venous oxygen saturation, 6-MWD: 6-minute walk distance, HTZ: heterozygous, HMZ: homozygous, : lung transplant.
Supplementary Table 2. Genetic, clinical, functional and hemodynamic characteristics of index cases from PVOD families at the time of PVOD diagnosis. Family number ndividual PVOD6 PVOD7 PVOD8 PVOD9 PVOD10 PVOD11 PVOD12 PVOD13-2 -1-3 -5-1 -1-1 V-3 EF2AK4 mutation status HMZ HMZ HMZ HMZ HMZ Compound HTZ Compound HTZ HMZ Mutation -Nucleotide change - Protein effect c.[3159g>a];[3159g>a] p.[k975_k1053del];[k975_k1053del] c.[3406c>t];[3406c>t] p.[r1136*];[r1136*] c.[1754g>a];[1754g>a] p.[r585q];[r585q] c.[4065+1g>c];[4065+1g>c] p.[?];[?] c.[1387c>t];[1387c>t] p.[r463*];[r463*] c.[3448c>t(;)4728+1_4728+ 13delinsTTCT] p.[r1150*(;)?] c.[1387c>t];[3244c>t] p.[r463*];[q1082*] c.[1928t>g];[ 1928T>G] p.[l643r];[l643r] Consanguinity N/A Age at PVOD diagnosis, yrs 26 32 26 36 19 37 44 11 Gender M M M M M F M M NYHA Functional Class 2 3 2 3 3 3 - N/A 6-MWD, m 230 388 500 486 323 340-582 mpap, mmhg 44 68 41 53 70 54-22 RAP, mmhg 7 11 4 6 6 6 - - PCWP, mmhg 13 13 8 3 6 4 - - CO, L/min 4.20 3.75 6.77 2.58 3.68 3.96 - - C, L/min/m² 2.03 2.13 3.83 1.35 2.20 2.04 - - PVR, mmhg/l/min 7 15 5 19 17 13 - - SvO 2, % - 64 72 49 70 44-74 DLCO, % 32-30 22 33 22 - rmal CT scan: - Centrilobular GGO - septals lines - enlarged mediastinal lymph nodes - N/A N/A N/A PAH therapy: - CCB - PDE5-i - ERA - Prostanoids Outcome at 59 months at 23 months at 33 months Died at 27 months Died at 2 months Died at 63 months at 37 months Alive at 6months
Mutations are described according to HGVS nomenclature., CCB: calcium channel blocker, C: cardiac index, CO: cardiac output, ERA: endothelin receptor antagonist, GGO: ground glass opacities; : Lung transplantation; mpap: mean pulmonary artery pressure, NYHA: New York Heart Association, PCWP: pulmonary capillary wedge pressure, PDE5-i: phosphodiesterase type 5 inhibitor PVR: pulmonary vascular resistance, RAP: right atrial pressure, SvO 2 : mixed venous oxygen saturation, 6-MWD: 6-minute walk distance, HTZ: heterozygous, HMZ: homozygous, : lung transplant.
Supplementary Table 3. Genetic, clinical, functional and hemodynamic characteristics of mutation carrier patients with sporadic PVOD at the time of PVOD diagnosis. ndividual 112160 091769 05220 05498 06734 EF2AK4 mutation status HMZ HMZ Compound HTZ HMZ HMZ Mutation -Nucleotide change - Protein effect c.[560_564del];[560_564del] p.[k187rfs*9];[k187rfs*9] c.[3159g>a];[ 3159G>A] p.[k975_k1053del];[k975_k1053del] c.[2857c>t]( ;)[ 3576+1G>T] p.[q953*]( ;)[?] c.[4205dup];[4205dup] p.[s1403kfs*45];[s1403kfs*45] c.[2458c>t] ;[2458C>T] p.[r820*] ;[R820*] Age at PVOD diagnosis, yrs 32 15 20 20 28 Gender F F M M F NYHA Functional Class V V 6-MWD, m 0 0 358 507 450 mpap, mmhg 55 54 75 34 55 RAP, mmhg 5 6 9 1 7 PCWP, mmhg 15 7-3 4 CO, L/min 4.5 2.5 2.71-3.66 C, L/min/m² 2.4 1.7 1.94 2.70 2.54 PVR, mmhg/l/min 9 19 - - 14 SvO2, % 51 45 57 73 73 DLCO, % - 32 65 32 24 CT scan: - Centrilobular GGO - septals lines - enlarged mediastinal lymph nodes PAH therapy: - CCB - PDE5-i - ERA - Prostanoids Outcome at 1 month at 5 months at 2 months at 4 months at 36 months
Mutations are described according to HGVS nomenclature. CCB: calcium channel blocker, C: cardiac index, CO: cardiac output, ERA: endothelin receptor antagonist, GGO: ground glass opacities; : Lung transplantation; mpap: mean pulmonary artery pressure, NYHA: New York Heart Association, PCWP: pulmonary capillary wedge pressure, PDE5-i: phosphodiesterase type 5 inhibitor PVR: pulmonary vascular resistance, RAP: right atrial pressure, SvO 2 : mixed venous oxygen saturation, 6-MWD: 6-minute walk distance, HTZ: heterozygous, HMZ: homozygous, : lung transplant.
Exon Forward Reverse 1 5 -CCCATAGCCCGTCCCCAG-3 5 -CCAATCTGGAAGTGTCCGGG-3 2 5 -AATGATTGGCTCTACAGCTTTG-3 5 -CGAATTTCAATCTGTTTCTATGTTG-3 3 5 -GTTTAGCAAATATTAATGCAAACAGG-3 5 -TTTGAGGTGTAATGGGGAAGTT-3 4 5 -GGGCCTTCCCATTATTTGAT-3 5 -ACAGTAGTTTGCAATCCTCCA-3 5 5 -TCCATAATTTATATGTTGTATGGGTTT-3 5 -CAGATGTCATCAGTGGAAATAAAAA-3 6 5 -TTATGTACCATGATTTTCTTCCATGT-3 5 -TTCTACATCTGTCATTCTCCCAGA-3 7 5 -CAGTAATAAACGGGAGAAAATGG-3 5 -GGGAGCCAGAATGACTACCA-3 8 5 -TGTATTACCCCCTCCCTTCC-3 5 -CTCAGTCAGCCACCTTGGAT-3 9a 5 -CACTTTTGAAATGAAACCCAAG-3 5 -CGAGGCGCTTAGAAATGCT-3 9b 5 -TCAGGCCTTGATTATCTGCAC-3 5 -AGATGTCTGGCACACTCCAG-3 10 5 -AGCACTGATTGTTTGTGATGCT-3 5 -TTTAATGTCAAACAGGTTACCAAA-3 11 5 -AGCTCTTCTTCCAGGGCTTC-3 5 -CCCCACTCCCAAAATAAAAG-3 12 5 -CTGACCTTCCCCTGGCTGT-3 5 -ATCAGAGGCAGCTGGTCC-3 13 5 -TAATGATAATAGGGATTTCTGTTCCTT-3 5 -ACATCAGACGTCAATTATATCACGA-3 14 5 -TGCTTAATTTTGGCCTCCAT-3 5 -GGGTGTTGCTGTGTCACCTA-3 15 5 -CCTTCACTTAAAACTGTTGTGTAATCA-3 5 -TTACATTTGATATTTTTATGCAGTCA-3 16 5 -TGGCCTTTTAAATTAGGAAGTAGG-3 5 -TCCGCTAACACTTCCAGGAT-3 17 5 -TTCATTTAGAAACAGACAAGCCATT-3 5 -GGTTTCAGGTTAAGAAATTCTGG-3 18 5 -TTTTTAGTTTGTGTCTTCTGTAAGTGC-3 5 -TTGACTATTTTAGCCGGGAC-3 19 5 -TTCCCTGGTAAATTATACTTTCATATC-3 5 -TCCTGCTTTGAATTCTGATCTC-3 20 5 -ATGCTTTCACTGTGGCAGTC-3 5 -TGGCAGAAGATTACTAATTGAAAAA-3 21 5 -CGAATTTCAATCTGTTTCTATGTTG-3 5 -GGCTTGATCCTTTTGTGGAC-3 22 5 -GCCTACTGCAAGCCACAGAT-3 5 -TGATATGCCCGAACACTAACAG-3 23 5 -GCAATTACTTAACTGTCAGGTTTGTG-3 5 -GAGATGCCATCCCTGTGACT-3-24 5 -TTTCAATAAAGCCGCTATTAACAAAAG-3 5 -ATAAAATATTCTTTCCTGGTTGACAAA-3
25 5 -AAGCAATGACCTTAGAAATCTGG-3 5 -CTGTGGTTTTCCCCACCAC-3 26 5 -CCAGATTTGGTCATGTACCAGTAA-3 5 -TCAACATCAAGGTATTATCTTTCAACA-3 27 5 -TTGTTCAGTGCCAGATTTCG-3 5 -GGCTGCTACCCTACTGCAAC-3 28 5 -GGAGTCTTCCCCTGCTGTG-3 5 -AACACATTTCCTCCCTGTGC-3 29 5 -TTCCACATTCTAAATTTCAAACC-3 5 -GAAGAAATAAAATTAAGTCAGAGCAAA-3 30 5 -TTCTTTCCTATTTCATAACCATAACTG-3 5 -ATGACTGCAAAAGGCAAATCA-3 31 5 -CCCTCCTGATGCTGGTTTC-3 5 -GCCATGAAAGCCATAGCAA-3 32 5 -TTGGAAACAGTATTTTCATTCTCC-3 5 -CAGGTGCTCCACAGGTAACA-3 33 5 -TGTGGTGCTCTGAGCTAGTCTT-3 5 -GCACCTGGCTCAAATTGACT-3 34 5 -TTCACTGTAAGTTATGTGTTGCTTG-3 5 -GCTACAGTAATAAGTGCTTAGAGCAAA-3 35 5 -GGGACCAGATAAGGCCATAAA-3 5 -TTCCTTTCCTGTTGTTGTTTTTC-3 36 5 -GGGATAGGAAATAAGATGGCAAG-3 5 -TGCATCCAAATTCTGCTGAC-3 37 5 -TTCATACTGCTGCATTTCACG-3 5 -TGCCGTCTTCAGTATTTTGG-3 38 5 -GAATTTCATTAGTGACCCAGAAAAA-3 5 -AGGCGTCAAACCTCAACAAG-3 39 5 -GCTCTGTTCTTCACTCATTAAACTG-3 5 -AATGTACAACATTCCATTATTCCA-3 Supplementary Table 4. Oligonucleotides used for PCR amplification of the entire coding sequence and intronic junctions of the EF2AK4 gene