Molecular Diagnostics for complement and hemostasis mediated diseases
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Targeted sequencing with the Ion Torrent System is able to identify single nucleotide variants, small insertions and small deletions. Variants in repeat sequences, large homopolymers and large insertions/deletions are not or difficult to identify.
The Sanquin NGS Panel complement/hemostasis (test code X001 to X006) includes 44 genes and regions of interest. Using this panel, the exones and intron/exon borders of the relevant genes are sequenced. For most proteins, functional/expression level testing is available (see request form no 10: immunodiagnostics and no 4: coagulation, in Dutch)
Table 1: Genes covered by the complement/hemostasis targeted NGS panel.
|
Encoded protein |
Context |
Gene |
Chr |
Chr. Positie |
OMIM |
|
alpha 2-antiplasmin |
Hemostasis/trombosis gene |
SERPINF2 |
17 |
17p13 |
613168 |
|
Factor IX |
Hemostasis/trombosis gene |
F9 |
X |
Xq27.1-27.2 |
300746 |
|
Factor V |
Hemostasis/trombosis gene |
F5 |
1 |
1q23 |
612309 |
|
Factor VII |
Hemostasis/trombosis gene |
F7 |
13 |
13q34 |
613878 |
|
Factor VIII |
Hemostasis/trombosis gene |
F8 |
X |
Xq28 |
300841 |
|
Factor XI |
Hemostasis/trombosis gene |
F11 |
4 |
4q35 |
264900 |
|
Tissue factor |
Hemostasis/trombosis gene |
F3 |
1 |
1p22-21 |
134390 |
|
Von Willebrand factor |
Hemostasis/trombosis gene |
VWF |
12 |
12p13.3 |
613160 |
|
Factor XIIIa |
Hemostasis/trombosis gene |
F13A1 |
6 |
6p25.3-24.3 |
134570 |
|
Factor XIIIb |
Hemostasis/trombosis gene |
F13B |
1 |
1q31-32.1 |
134580 |
|
gamma-glutamyl carboxylase |
Hemostasis/trombosis gene |
GGCX |
2 |
2p12 |
137167 |
|
a Disintergrin and metalloproteinase wih thrombospondin repeats |
Hemostasis/trombosis gene |
ADAMTS13 |
9 |
9q34.2 |
604134 |
|
Antithrombin |
Hemostasis/trombosis gene |
SERPINC1 |
1 |
1q23-25.1 |
107300 |
|
Plasminogen |
Hemostasis/trombosis gene |
PLG |
6 |
6q26 |
173350 |
|
Protein C |
Hemostasis/trombosis gene |
PROC |
2 |
2q13-14 |
612283 |
|
protein C receptor, endothelial |
Hemostasis/trombosis gene |
PROCR |
20 |
20q11.2 |
600646 |
|
Protein S |
Hemostasis/trombosis gene |
PROS1 |
3 |
3q11.2 |
176880 |
|
tissue factor pathway inhibitor -1 |
Hemostasis/trombosis gene |
TFPI |
2 |
2q32 |
152310 |
|
Trombomoduline |
Hemostasis/trombosis gene |
THBD |
20 |
20p11.21 |
188040 |
|
Methylmalonic aciduria and homocystinuria, cblC type |
complement genes |
MMACHC |
1 |
1p34.1 |
609831 |
|
Complement component 8 alpha chain |
complement genes |
C8A |
1 |
1p32.2 |
120950 |
|
Complement component 8 beta chain |
complement genes |
C8B |
1 |
1p32.2 |
120960 |
|
Complement component 8 gamma chain |
complement genes |
C8G |
9 |
9q34.3 |
120930 |
|
Complement factor H |
complement genes |
CFH |
1 |
1q31.3 |
134370 |
|
Complement factor H related 1 |
complement genes |
CFHR1 |
1 |
1q31.3 |
134371 |
|
Complement factor H related 2 |
complement genes |
CFHR2 |
1 |
1q31.3 |
600889 |
|
Complement factor H related 3 |
complement genes |
CFHR3 |
1 |
1q31.3 |
605336 |
|
Complement factor H related 4 |
complement genes |
CFHR4 |
1 |
1q31.3 |
605337 |
|
Complement factor H related 5 |
complement genes |
CFHR5 |
1 |
1q31.3 |
608593 |
|
Cluster of diffentiation 46 |
complement genes |
CD46 |
1 |
1q32.2 |
120920 |
|
Cluster of diffentiation 55 |
complement genes |
CD55 |
1 |
1q32.2 |
125240 |
|
Cluster of diffentiation 59 |
complement genes |
CD59 |
11 |
11p13 |
107271 |
|
Serine proteinase inhibitor G1 (C1-inhibitor) |
complement genes |
SERPING1 |
11 |
11q12.1 |
606860 |
|
Diacyl glycerol kinase epsilon |
complement genes |
DGKE |
17 |
17q2 |
601440 |
|
Complement factor D |
complement genes |
CFD |
19 |
19p13.3 |
134350 |
|
Complement component 3 |
complement genes |
C3 |
19 |
19p13.3 |
120700 |
|
Complement factor I |
complement genes |
CFI |
4 |
4q25 |
217030 |
|
Complement component 9 |
complement genes |
C9 |
5 |
5p13.1 |
120940 |
|
Complement component 7 |
complement genes |
C7 |
5 |
5p13.1 |
217070 |
|
Complement component 6 |
complement genes |
C6 |
5 |
5p13.1 |
217050 |
|
Coagulant factor 12 |
complement genes |
F12 |
5 |
5q35.3 |
610619 |
|
Complement factor B |
complement genes |
CFB |
6 |
6p21.33 |
138470 |
|
Complement component 5 |
complement genes |
C5 |
9 |
9q33.2 |
120900 |
|
Complement factor properdin |
complement genes |
CFP |
X |
xp11.23 |
300383 |
The complement/hemostasis targeted NGS panel is covering 99, 09% of the desired design (exons, flanking intronic regions, untranslated regions and promotor areas). In table 2, the missed regions and their genomic coordinates are listed. In total 9 disease causing variants listed in HGMD (version 2017-4) are not covered in the design.
Table 2: List of missed bases in the design of complement/hemostasis targeted NGS panel
|
gene |
Chr |
number missing bases |
Position |
coverage% |
missing HGMD-DM variants |
|
F7 |
13 |
6 |
113,765,157-113,765,163 |
99,81 |
|
|
vWF |
12 |
253 |
6,233,587-6,233,840 |
98,55 |
|
|
F13A1 |
6 |
105 |
6,320,819-6,320,924 |
97,46 |
|
|
PROS1 |
3 |
94 |
93,647,546-93,647,640 |
98,05 |
|
|
TFPI |
2 |
305 |
188,418,927-188,419,232 |
84,97 |
|
|
|
|
121 |
188,394,090-188,394,211 |
|
|
|
CD59 |
11 |
92 |
33,744,181-33,744,273 |
94,34 |
|
|
DGKE |
17 |
163 |
54,911,945-54,911,622 |
94,98 |
|
|
CFI |
4 |
24 |
110,679,091-110,679,115 |
99.49 |
|
|
C6 |
5 |
216 |
41,213,479-41,213,695 |
95.69 |
|
|
F12 |
5 |
9 |
176,830,994-176,831,003 |
99,44 |
1 |
|
|
|
13 |
176,831,810-176,831,823 |
|
|
|
ADAMTS13 |
9 |
67 |
136,288,136-136,288,287 |
97.11 |
8 |
|
|
|
146 |
136,291,319-136,291,465 |
|
|
|
CFP |
X |
177 |
47,489,527-47,489,704 |
96.19 |
|
The percentage of target bases that is covered 20 times (%Base20x), is at least 98.9% for the recommended Mapped Reads of 1.750.000. With this acceptance criteria, 15 amplicons failed to yield >20times coverage over the full amplicon length,listed in Table 3a). 52 published disease making mutations (DM) and 2 likely pathological mutations with”some degree of doubt” (DM?) were missed in these regions (HGMD 2018-2), listed in Table 3b.
Table 3a: Failed (low coverage) amplicons and their missing genomic locations and published mutations (HGMD, 2018-2)
Gene |
Amplicon |
%Base20x amplicon |
Locations with base coverage below 20x |
missing HGMD |
|
|
|
|
|
chromosomal coordinates |
description |
|
VWR |
ES7.VWF_60 |
30% |
chr12: 6167087 - 6167247 (160base) |
5' exon 14 (124bp) + flanking region (36bp) |
8 DM + 2 DM? |
GGCX |
ES7.GGCX_8 |
0% |
chr2: 85779508 - 85779723 (215base) |
volledig exon 10 (chr2: 85779539 - 85779690) + both flanking region |
2 DM |
F7 |
ES7.F7_9 |
24% |
chr13: 113771935 - 113772112 (177base) |
only intronic region (exon 8 + flanking regions well covered) |
none |
PROC |
ES7.PROC_5 |
25% |
chr2:128180681 - chr2:128180870 (189 base) |
3' exon 5 (chr2:128180681-128180747) + 5' exon 6 (chr2:128180850-128180871) |
34 DM |
PLG |
ES7.PLG_21 |
10% |
chr6:161173191 - 161173373 (182 base) |
3' exon 18 (chr6:161173191-161173292) + flanking region |
1 DM |
CFHR3 |
AMPL7160434353 |
40% |
chr1:196749193 - 196749383 (190 base) |
only intronic region (exon 3 + flanking regions well covered) |
none |
CFP |
ES10.CFP_4 |
13% |
chrX:47485795 - 47485956 (161 base) |
5' exon 8 (chrX:47485795-47485918) + flanking region |
5 DM |
F8 |
ES7.F8_6 |
0% |
chrX:154114288 - 154114495 (208 base) |
exon 1 (chrX:154114409-154114433(24 base) + UTR + flanking region |
none |
CFHR3 |
AMPL7160434378 |
59% |
chr1:196748218 - 196748346 (129 base) |
5' exon 2 (chr1:196748292-196748346(55 base)) + flanking region |
none |
CFHR3 |
AMPL7160434330 |
55% |
chr1:196759179 - 196759326 (149 base) |
almost complete exon 5 (chr1:196759179 - 196759326) |
none |
CFHR4 |
AMPL7160434334 |
87% |
chr1:196882107 - 196882146 (40 base) |
only intronic region 3 (exon 3 + flanking regions well covered) |
none |
CFHR2 |
AMPL7160434361 |
63% |
chr1:196920144 - 196920250 (107 base) |
3' exon 3 (chr1:196920144 - 196920158 (15 base) + flanking region |
none |
SERPING |
AMPL7156549776 |
42% |
chr11:57364916 - 57365109 (194 base) |
UTR + 5' exon 1 (chr11:57365027-57365109 (84 base)) |
2 DM |
PLG |
ES7.PLG_14 |
78% |
chr6:161152738 - 161152791 (54 base) |
5' exon 12: chr6:161152777-chr6:161152791 (14 base) + flanking region |
none |
VWF |
ES7.VWF_24 |
9% |
chr12: 6104972 - 6105179 (208 base) |
3' exon 35: chr12:6105168-6105179 (12 base) + flanking region |
none |
Table 3b: List of missing published HGMD variants in amplicons with low coverage (HGMD 2018-2)
GRCh37/hg19 |
Gene |
HGMD Ref |
HGVS |
Variant class |
Phenotype |
Reference |
chr12:6167208-10 |
VWF |
CD1714155 |
NM_000552.3: c.1534_1536delCTG |
DM |
Von Willebrand disease 3 |
Borràs (2017) Haematologica epub |
chr12:6167213 |
VWF |
CS066319 |
NM_000552.3: c.1534-3C>A |
DM |
Von Willebrand disease 1 |
Gallinaro (2006) Thromb Haemost 96: 711 |
chr12:6167196 |
VWF |
CM1718319 |
NM_000552.3: c.1548T>A; NP_000543.2: p.Y516* |
DM |
Von Willebrand disease 1 |
Lavin (2017) Blood 130: 2344 |
chr12:6167161 |
VWF |
CM941396 |
NM_000552.3: c.1583A>G; NP_000543.2: p.N528S |
DM |
Von Willebrand disease 2c |
Haberichter (2010) Blood 115: 4580 |
chr12:6167137 |
VWF |
CM147756 |
NM_000552.3: c.1607T>C; NP_000543.2: p.L536P |
DM |
Von Willebrand disease 2a |
Brehm (2014) Thromb Haemost 112: 96 |
chr12:6167098 |
VWF |
CM1714100 |
NM_000552.3: c.1646T>C; NP_000543.2: p.F549S |
DM |
Von Willebrand disease |
Borràs (2017) Haematologica epub |
chr12:6167096 |
VWF |
CM951301 |
NM_000552.3: c.1648G>A; NP_000543.2: p.G550R |
DM |
Von Willebrand disease 2c |
Brehm (2014) Thromb Haemost 112: 96 |
chr12:6167090 |
VWF |
CM1714102 |
NM_000552.3: c.1654G>A; NP_000543.2: p.A552T |
DM |
Von Willebrand disease 2n |
Borràs (2017) Haematologica epub |
chr12:6167173 |
VWF |
CM134325 |
NM_000552.3: c.1571G>A; NP_000543.2: p.C524Y |
DM? |
Von Willebrand disease 2a |
Flood (2013) Clin Chem 59, 684 |
chr12:6167119 |
VWF |
CM107439 |
NM_000552.3: c.1625C>G; NP_000543.2: p.A542G |
DM? |
Von Willebrand disease |
Veyradier (2016) Medicine (Baltimore) 95: e3038 |
chr2:85779552 |
GGCX |
CM078087 |
NM_000821.5: c.1426C>T; NP_000812.2: p.R476C |
DM |
Pseudoxanthoma elasticum |
Vanakker (2007) J Invest Dermatol 127: 581 |
chr2:85779551 |
GGCX |
CM078088 |
NM_000821.5: c.1427G>A; NP_000812.2: p.R476H |
DM |
Pseudoxanthoma elasticum |
Vanakker (2007) J Invest Dermatol 127: 581 |
chr2:128180686 |
PROC |
CD104283 |
NM_000312.3:c.339delC |
DM |
Protein C deficiency |
Pai (2010) ANN HAEMAT 89, 835 |
chr2:128180687 |
PROC |
CM950991 |
NM_000312.3:c.340G>C; NP_000303.1:p.G114R |
DM |
Protein C deficiency |
Lind (1995) TH 73, 186 |
chr2:128180687 |
PROC |
CM950992 |
NM_000312.3:c.340G>T; NP_000303.1:p.G114C |
DM |
Protein C deficiency |
Reitsma (1995) TH 73, 876 |
chr2:128180687-701 |
PROC |
CD931043 |
NM_000312.3:c.340_354delGGCATCGGCAGCTTC |
DM |
Protein C deficiency |
Poort (1993) BCF 4, 273 |
chr2:128180691-702 |
PROC |
CX005563 |
NM_000312.3:c.344_355delTCGGCAGCTTCAinsCGT |
DM |
Protein C deficiency |
Alhenc-Gelas (2000) TH 83, 86 |
chr2:128180693 |
PROC |
CM179761 |
NM_000312.3:c.346G>A; NP_000303.1:p.G116S |
DM |
Thrombosis |
Chen (2017) TH 117, 1358 |
chr2:128180695-96 |
PROC |
CI962338 |
NM_000312.3:c.340_348dupGGCATCGGC |
DM |
Protein C deficiency |
Ireland (1996) TH 76, 867 |
chr2:128180696-99 |
PROC |
CD123690 |
NM_000312.3:c.349_352delAGCT |
DM |
Protein C deficiency |
Tang (2012) PLOS ONE 7, e35773 |
chr2:128180699 |
PROC |
CM910307 |
NM_000312.3:c.352T>C; NP_000303.1:p.F118L |
DM |
Protein C deficiency |
Reitsma (1991) BLOOD 78, 890 |
chr2:128180702-19 |
PROC |
CD910546 |
NM_000312.3: c.355_372delAGCTGCGACTGCCGCAGC |
DM |
Protein C deficiency |
Tsuda (1991) Thromb Haemost 65 647A |
chr2:128180705 |
PROC |
CM950993 |
NM_000312.3:c.358T>G; NP_000303.1:p.C120G |
DM |
Protein C deficiency |
Reitsma (1995) TH 73, 876 |
chr2:128180707 |
PROC |
CM071944 |
NM_000312.3:c.360C>A; NP_000303.1:p.C120* |
DM |
Protein C deficiency |
Rovida (2007) HUM MUT 28, 345 |
chr2:128180707-24 |
PROC |
CD910547 |
NM_000312.3:c.360_377delCGACTGCCGCAGCGGCTG |
DM |
Protein C deficiency |
Tsuda (1991) TH 65, 647A |
chr2:128180719 |
PROC |
CM014374 |
NM_000312.3:c.372C>G; NP_000303.1:p.S124R |
DM |
Protein C deficiency |
Taliani (2001) GENET TEST 5, 39 |
chr2:128180720 |
PROC |
CM961152 |
NM_000312.3:c.373G>C; NP_000303.1:p.G125R |
DM |
Protein C deficiency |
Miyata (1996) TH 76, 302 |
chr2:128180720 |
PROC |
CM004058 |
NM_000312.3:c.373G>T; NP_000303.1:p.G125C |
DM |
Protein C deficiency |
Singh (2000) HAEMATOL 85, 891 |
chr2:128180730 |
PROC |
CM1618220 |
NM_000312.3:c.383G>A; NP_000303.1:p.G128D |
DM |
Protein C deficiency |
Cheng (2016) BCF 27, 838 |
chr2:128180733 |
PROC |
CM950994 |
NM_000312.3:c.386G>A; NP_000303.1:p.R129H |
DM |
Protein C deficiency |
Reitsma (1995) TH 73, 876 |
chr2:128180735-44 |
PROC |
CD157088 |
NM_000312.3:c.388_397delTTCTGCCAGC |
DM |
Protein C deficiency |
Boey (2015) BJH epub |
chr2:128180736 |
PROC |
CM128563 |
NM_000312.3:c.389T>G; NP_000303.1:p.F130C |
DM |
Protein C deficiency |
Caspers (2012) TH 108, 247 |
chr2:128180739 |
PROC |
CM005555 |
NM_000312.3:c.392G>C; NP_000303.1:p.C131S |
DM |
Protein C deficiency, type I |
Alhenc-Gelas (2000) TH 83, 86 |
chr2:128180745 |
PROC |
CM005564 |
NM_000312.3:c.398G>C; NP_000303.1:p.R133P |
DM |
Protein C deficiency, type I |
Alhenc-Gelas (2000) TH 83, 86 |
chr2:128180746 |
PROC |
CM005565 |
NM_000312.3:c.399C>T; NP_000303.1:p.R133R |
DM |
Protein C deficiency, type I |
Alhenc-Gelas (2000) TH 83, 86 |
chr2:128180747 |
PROC |
CM950995 |
NM_000312.3:c.400G>T; NP_000303.1:p.E134* |
DM |
Protein C deficiency |
Reitsma (1995) TH 73, 876 |
chr2:128180748 |
PROC |
CS004399 |
NM_000312.3:c.400+1G>C |
DM |
Protein C deficiency |
Miyata (1998) THROMB RES 92, 181 |
chr2:128180749 |
PROC |
CS128579 |
NM_000312.3:c.400+2T>C |
DM |
Protein C deficiency |
Caspers (2012) TH 108, 247 |
chr2:128180752 |
PROC |
CS910456 |
NM_000312.3:c.400+5G>A |
DM |
Protein C deficiency |
Reitsma (1991) BLOOD 78, 890 |
chr2:128180752 |
PROC |
CS910457 |
NM_000312.3:c.400+5G>C |
DM |
Protein C deficiency |
Reitsma (1991) BLOOD 78, 890 |
chr2:128180752 |
PROC |
CS910458 |
NM_000312.3:c.400+5G>T |
DM |
Protein C deficiency |
Reitsma (1991) BLOOD 78, 890 |
chr2:128180832-47 |
PROC |
CD001911 |
NM_000312.3:c.401-18_401-3delGCCCTCCCCTGCCCGC |
DM |
Protein C deficiency |
Millar (2000) HUM GENET 106, 646 |
chr2:128180848 |
PROC |
CS128580 |
NM_000312.3:c.401-2A>C |
DM |
Protein C deficiency |
Caspers (2012) TH 108, 247 |
chr2:128180848 |
PROC |
CS930861 |
NM_000312.3:c.401-2A>G |
DM |
Protein C deficiency |
Soria (1993) HUM GENET 92, 506 |
chr2:128180867 |
PROC |
CM071947 |
NM_000312.3:c.418T>C; NP_000303.1:p.C140R |
DM |
Protein C deficiency |
Rovida (2007) HUM MUT 28, 345 |
chr2:128180871 |
PROC |
CM961153 |
NM_000312.3:c.422C>A; NP_000303.1:p.S141* |
DM |
Protein C deficiency |
Ireland (1996) TH 76, 867 |
chr6:161173272 |
PLG |
CM981587 |
NM_000301.3: c.2251G>A; NP_000292.1: p.G751R |
DM |
Plasminogen deficiency |
Higuchi (1998) Br J Haematol 103, 867 |
chrX:47485831 |
CFP |
CM972880 |
NM_002621.2: c.1028A>G; NP_002612.1: p.Q343R |
DM |
Properdin deficiency |
Truedsson (1997) Immunopharmacology 38: 203 |
chrX:47485823 |
CFP |
CM994439 |
NM_002621.2: c.1036C>T; NP_002612.1: p.R346C |
DM |
Properdin deficiency |
Fijen (1999) Mol Immunol 36: 863 PubMed: 10698340 |
chrX:47485898 |
CFP |
CM972879 |
NM_002621.2: c.961T>G; NP_002612.1: p.W321G |
DM |
Properdin deficiency |
Truedsson (1997) Immunopharmacology 38: 203 |
chrX:47485897 |
CFP |
CM001765 |
NM_002621.2: c.962G>C; NP_002612.1: p.W321S |
DM |
Properdin deficiency |
van den Bogaard (2000) Eur J Hum Genet 8: 513 |
chrX:47485897 |
CFP |
CM171423 |
NM_002621.2: c.962G>A; NP_002612.1: p.W321* |
DM |
Immunodeficiency, primary |
Stray-Pedersen (2017) J Allergy Clin Immunol 139: 232 |
chr11:57365057 |
SERPING1 |
HR080001 |
NM_000062.2: c.-161A>G (-687 relative to initiation codon) |
DM |
Angioneurotic oedema |
Uyguner (2008) Hum Genet 124 309 |
chr11:57365055 |
SERPING1 |
CR961722 |
NM_000062.2: c.-163C>T (-689 relative to initiation codon) |
DM |
Angioneurotic oedema |
Verpy (1996) Am J Hum Genet 59: 308 |
Background information
Service pages NGS
