Nat. Genet. 41, 936–940 (2009). Mutations affecting the ... · Nat. Genet. 41, 936–940 (2009)....

correct ion not ice

Nat. Genet. 41, 936–940 (2009).

Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type IIKlaus Schwarz, Achille Iolascon, Fatima Verissimo, Nikolaus S Trede, Wyatt Horsley, Wen Chen, Barry H Paw, Karl-Peter Hopfner, Karlheinz Holzmann, Roberta Russo, Maria Rosaria Esposito, Daniela Spano, Luigia De Falco, Katja Heinrich, Brigitte Joggerst, Markus T Rojewski, Silverio Perrotta, Jonas Denecke, Ulrich Pannicke, Jean Delaunay, Rainer Pepperkok & Hermann HeimpelIn the version of this supplementary file originally posted online, Supplementary Table 2 was incomplete. The errors have been corrected in this file as of 28 September 2009.

nature genetics

Supplementary Information to the manuscript

Mutations in the human secretory COPII coat component

SEC23B cause congenital dyserythropoietic anemia type II

(CDA II)

By

Klaus Schwarz*, Achille Iolascon*, Fatima Verissimo, Nikolaus S. Trede, Wyatt Horsley, Wen

Chen, Barry H. Paw, Karl-Peter Hopfner, Karlheinz Holzmann, Roberta Russo, Maria Rosaria

Esposito, Daniela Spano, Luigia De Falco, Katja Heinrich, Markus T. Rojewski, Silverio Perotta,

Jonas Denecke, Ulrich Pannicke, Jean Delaunay, Rainer Pepperkok, Hermann Heimpel

*To whom correspondence should be addressed:

Email: [email protected]

Email: [email protected]

Nature Genetics: doi:10.1038/ng.405

Supplementary Note:

Results

Previously, the COPII component SEC23A has been shown to be essential for craniofacial

chondrocyte maturation. A human SEC23A Phe382Leu mutation causes the autosomal recessive

cranio-lenticulo-sutural dysplasia (CLSD) with persistent ossification defectsS1

. Primary fibroblasts of

CLSD individuals show a marked dilatation of the ER with SEC31 mislocalization and procollagen I

accumulation. Sec23a zebrafish morphants and the sec23a zebrafish mutant crusher variant

recapitulate the human phenotypeS2

. Most importantly, sec23b zebrafish morphants are similarily

affected (this study and ref. S1,S2). For this reason, we studied the phenotype and function of primary

dermal fibroblasts of SEC23B mutated individuals. Staining and FACS analysis of numerous surface

proteins, including N-glycosylated, O-glycosylated and GPI-anchored proteins are indistinguishable

when compared to wild type (Supplementary Figure 1a). Osteogenic, chondrogenic and adipogenic

differentiation of F6P1 fibroblasts and control cells is similarly effective (Supplementary Figure 1b).

No apparent difference was found between control and patient (F4P1) cells when endogenous cellular

markers for COPI, Golgi, ER exit sites, endoplasmic reticulum, early endosomes and lysosomes were

investigated (Supplementary Figure 1c) or when the transport of the CFP-tagged temperature-sensitive

cargo marker vesicular stomatits virus glycoprotein (ts-O45-G) was analyzed (Supplementary Figure

1d). In mice, dyserythropoiesis with loss of erythrocyte complex glycans has been noted when

MAN2A1 was inactivatedS3

. All other tissues were spared due to the presence of the compensating

MAN2A2 protein. Hybrid glycan dysglycosylation is a hallmark of CDAII although it may be a

secondary phenomenon since in other forms of CDA (CDAI and CDAIII) less distinct

hypoglycosylation was describedS4

. Mannosidases may represent candidates for SEC23/SEC24

selective cargo transport. When we analyzed subcellular expression of MAN2A1 versus MAN2A2 in

fibroblasts of F4P1 and a control, no difference in expression patterns was detected, (Supplementary

Figure 1e) excluding a general MAN2A-glycosidase transport disturbance. In order to explain the

mechanisms that may compensate for the loss of SEC23B function we conducted knock-down

experiments. A knock-down of the single SEC23 paralogs, either SEC23A or SEC23B does not reduce


the number of endoplasmic reticulum exit sites (ERES) in HeLa cells and does not have a significant

effect on the traffic of the cargo (CFP-tagged ts-O45-G) towards the plasma membrane. However, the

knock-down of both paralogs simultaneously decreases drastically the number of ERES and leads to a

nearly complete block of secretion from the endoplasmic reticulum (Supplementary Figure 2). This

shows that the function of SEC23A and SEC23B in the maintenance of ERES and moreover in

secretion of the cargo molecule CFP-tagged ts-O45-G is redundant. Together, these experiments

underline the erythrocyte specificity of SEC23B deficiency.

Supplementary References

S1. Boyadjiev, S.A. et al. Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation

leading to abnormal endoplasmic-reticulum-to-Golgi trafficking. Nat. Genet. 38, 1192-1197

(2006).

S2. Lang, M.R., Lapierre, L.A., Frotscher, M., Goldenring, J.R. & Knapik, E.W. Secretory COPII

coat component Sec23a is essential for craniofacial chondrocyte maturation. Nat. Genet. 38,

1198-1203 (2006).

S3. Chui, D. et al. Alpha-mannosidase-II deficiency results in dyserythropoiesis and unveils an

alternate pathway in oligosaccharide biosynthesis. Cell 90, 157-167 (1997).

S4. Zdebska, E. et al. Glycoconjugate abnormalities in patients with congenital dyserythropoietic

anaemia type I, II and III. Br. J. Haematol. 114, 907-13 (2001).


Supplementary Table 1: Clinical and Laboratory Investigations of CDAII Patients

Identifier

Gender

f(emale)

m(ale)

Ethnicity Consanguinity

Age at

Diagnosis

(Years)

Hb

g/L

Reticulocytes

%

Bilirubin

µmol/L

SDS-PAGE

N(ormal)

A(bnormal)

BM Bi-

nucleated

cells

%

F1P1 m German n.a. 35.9 106 3.1 38.5 A 16

F2P1 f Turkish Yes 38.3 114 1.4 82.0 A n.a.

F2P2 f Turkish Yes 32.0 118 1.6 47.9 A n.a.

F2P3 f Turkish Yes 14.0 112 1.2 108.0 A 11

F3P1 m Austrian n.a. 15.9 91 3.0 42.6 n.a. 10

F4P1 m Italian n.a. 16.3 94 1.4 49.0 A 11

F5P1 m German n.a. 62.5 115 2.4 104.0 n.a. 13

F6P1 f German Yes 17.5 100 3.0 28.8 A 12

F7P1 f German No 6.9 107 4.4 73.0 A 15

F7P2 f German No 10.0 121 5.2 61.0 A 16

F8P1 f Turkish No 6.8 83 2.4 103.0 A 20

F9P1 m German n.a. 0.2 63 0.3 24.0 A 15

F10P1 f Romanian No 23.3 97 2.0 43.0 A 16

F11P1 m Czech Yes 0.2 56 3.2 23.9 A 16

F12P1 m Turkish n.a. 0.2 68 10.9 9.5 A 10

F13P1 m German n.a. 12.0 103 2.0 21.7 A 10

F14P1 m Italian No 0.5 101 5.0 11.5 A 10

F15P1 f Italian No 9 88 2.8 17.8 A n.a.

F15P2 f Italian No 4 106 4.5 20.7 A n.a.

F16P1 m Pakistani Yes 9 84 1.0 32.0 A 12

F16P2 f Pakistani Yes 4 102 3.4 54.5 A n.a.


Identifier

Gender

f(emale)

m(ale)

Ethnicity Consanguinity

Age at

Diagnosis

(Years)

Hb

g/L

Reticulocytes

%

Bilirubin

µmol/L

SDS-PAGE

N(ormal)

A(bnormal)

BM Bi-

nucleated

cells

%

F17P1 f Italian No 4 78 2.8 26.2 A 12

F18P1 f Indian Yes 23 101 2.0 n.a. A 15

F19P1 f Algerian No 41 77 11.0 52.0 A 6

F20P1 m Italian No 0.5 60 3.1 27.5 A 9

F21P1 f French

Caucasian No 23 117 0.8 n.a. A 20

F22P1 m Italian No 11 117 3.5 43.1 A 15

F23P1 f Italian No 14 120 3.1 43.1 A 10

F24P1 f French

Polynasian No 10 96 1.6 59.0 A 20

F25P1 m Italian No 4.6 95 1.9 32.7 A 11

F26P1 m Italian No 0.1 57 3.2 53.6 A 8

F27P1 f Italian No 20 95 3.4 49.7 A 10

F28P1 f Italian No 9 86 6.6 73.2 A 12

n.a.: not available


Supplementary Table 2: Single-nucleotide polymorphism analysis of the chromosome 20 minimal

homozygous overlap region of consanguinous CDAII patients F6P1 and

F11P1

Chromosome

Physical

Position dbSNP RS ID F11P1 Call

F11P1

Confidence F6P1 Call

F6P1

Confidence Strand Cytoband Allele A Allele B

20 15967252 rs10485544 AA 0,1743749 - p12.1 C T

20 15974862 rs6135653 AA 0,2047054 - p12.1 A G

20 15975036 rs6131768 BB 0,08784609 + p12.1 C T

20 15984666 rs10485545 BB 0,08675808 + p12.1 C T

20 16124203 rs10485547 AA 0,1693824 + p12.1 C T

20 16197746 rs2282053 BB 0,07828979 + p12.1 C G

20 16199668 rs6043844 AA 0,03293836 - p12.1 C G

20 16204299 rs10485550 BB 0,04566524 + p12.1 C T

20 16204912 rs6111005 AA 0,02484916 + p12.1 A G

20 16239006 rs6111042 BB 0,004852836 + p12.1 A G

20 16272849 rs6043904 AA 0,01273769 + p12.1 C T

20 16273140 rs2144883 AA 0,01984673 + p12.1 C T

20 16293233 rs2144891 BB 0,006347965 + p12.1 A C

20 16314615 rs2876428 AA 0,1061389 - p12.1 C G

20 16351505 rs10485551 AA 0,000616553 + p12.1 C T

20 16352080 rs4814495 AA 0,01122473 - p12.1 C G

20 16454321 rs10485555 AA 0,000488 AA 0,01160656 + p12.1 C T

20 16454369 rs720592 AA 0,000488 AA 0,007857852 + p12.1 C T

20 16474628 rs10485557 AA 0,000488 AA 0,01606515 + p12.1 A G

20 16552854 rs6080334 AA 0,009766 BB 0,03634312 - p12.1 A T

20 16602155 rs2328024 BB 0,005859 AA 0,08638997 + p12.1 C T

20 16648989 rs10485561 AA 0,000488 AA 0,02228742 - p12.1 A C

20 16675859 rs6135875 BB 0,008301 BB 0,1948919 + p12.1 A C

20 16761101 rs10485563 BB 0,000488 BB 0,1100595 + p12.1 A G

20 16821265 rs2328111 BB 0,000488 BB 0,06408723 + p12.1 A C

20 16915281 rs6034709 AA 0,000488 AA 0,01232689 + p12.1 A C

20 16930918 rs6044565 AA 0,000488 BB 0,01466588 + p12.1 A G

20 16931801 rs10485564 AA 0,000488 AA 0,1121717 + p12.1 C T


20 16933601 rs724053 BB 0,000488 BB 0,02100348 - p12.1 A G

20 16952565 rs763406 BB 0,003906 BB 0,009442927 + p12.1 C G

20 16967146 rs10485565 BB 0,000488 BB 0,0105446 - p12.1 C T

20 16968974 rs10485566 BB 0,002441 AA 0,06349582 - p12.1 C T

20 17019681 rs956110 BB 0,002441 AA 0,003061618 + p12.1 C T

20 17019895 rs1885582 AA 0,001465 BB 0,02521404 - p12.1 A G

20 17020043 rs10485567 AA 0,000488 BB 0,08041599 - p12.1 A G

20 17020088 rs10485568 AA 0,000488 BB 0,005223914 + p12.1 C G

20 17024476 rs1570341 AA 0,000488 BB 0,02299678 - p12.1 C T

20 17032201 rs10485569 BB 0,000488 BB 0,1020963 + p12.1 A G

20 17043210 rs1101130 AA 0,001465 AA 0,01084096 - p12.1 A G

20 17077082 rs2010316 BB 0,000488 AA 0,01241422 + p12.1 A G

20 17131484 rs6080603 AA 0,000977 AA 0,02009541 + p12.1 G T

20 17169640 rs951843 AA 0,001465 BB 0,01410236 + p12.1 C T

20 17194614 rs1883530 BB 0,000488 AA 0,1463927 + p12.1 A G

20 17194651 rs1883531 AA 0,000488 BB 0,07286775 - p12.1 C G

20 17195793 rs6044710 BB 0,000488 BB 0,1528602 + p12.1 A C

20 17303748 rs10485570 AA 0,000488 BB 0,01373123 - p12.1 A G

20 17305573 rs4814615 BB 0,002441 BB 0,06833433 + p12.1 A G

20 17306778 rs1570008 BB 0,000488 BB 0,04969802 + p12.1 A G

20 17337877 rs2859875 AA 0,000488 AA 0,03288371 + p12.1 C T

20 17488152 rs763394 AA 0,008301 AA 0,01315379 - p12.1 C T

20 17491337 rs6080737 BB 0,001465 BB 0,08352254 + p12.1 C T

20 17536805 rs1051234 AA 0,000488 AA 0,02928853 - p12.1 A G

20 17538620 rs10485571 AA 0,000488 AA 0,02923629 + p12.1 C T

20 17739042 rs1831159 BB 0,000488 BB 0,1017207 + p12.1 A G

20 17739064 rs16823 AA 0,000488 AA 0,04091457 + p12.1 A G

20 17743053 rs10485572 BB 0,000488 BB 0,03223947 + p12.1 A G

20 17793675 rs3929674 NoCall 0,3125 BB 0,003131601 - p12.1 C T

20 17793755 rs3929675 BB 0,002441 BB 0,02475752 - p12.1 A G

20 17793921 rs2328223 AA 0,037109 AA 0,06289576 + p12.1 A C

20 17907386 rs10485576 BB 0,008301 BB 0,1058211 + p11.23 C T

20 18094703 rs1205200 AA 0,000488 AA 0,006580547 + p11.23 A G

20 18118297 rs2024673 AA 0,000488 BB 0,03175713 + p11.23 C T

20 18199839 rs2255184 AA 0,023438 AA 0,2788808 + p11.23 G T

20 18201318 rs2255412 BB 0,000488 BB 0,163718 - p11.23 A C


20 18287493 rs10485579 AA 0,000488 BB 0,03909579 + p11.23 C T

20 18309635 rs10485580 BB 0,000488 BB 0,02151024 + p11.23 A G

20 18310297 rs2328293 BB 0,000488 BB 0,04530538 + p11.23 C T

20 18358714 rs10485581 BB 0,000488 BB 0,2224395 - p11.23 A T

20 18359665 rs4813327 BB 0,000488 BB 0,03353312 - p11.23 A G

20 18380926 rs717581 AA 0,000488 AA 0,05947937 + p11.23 A G

20 18385799 rs6035046 BB 0,005859 BB 0,03418946 - p11.23 A C

20 18477233 rs3736775 AA 0,001465 AA 0,002801258 - p11.23 A T

20 18537737 rs6045501 BB 0,000488 BB 0,1245648 - p11.23 A G

20 18623206 rs10485585 AA 0,002441 AA 0,1980028 - p11.23 A G

20 18623480 rs6081303 AA 0,016113 AA 0,1057923 - p11.23 A C

20 18652367 rs6045570 BB 0,001465 BB 0,1031606 - p11.23 A G

20 18654481 rs911012 BB 0,000977 BB 0,1784837 + p11.23 A G

20 18833682 rs731667 BB 0,000488 AA 0,002737956 - p11.23 A G

20 18833772 rs731668 AA 0,000488 BB 0,01003682 + p11.23 C T

20 18835204 rs2038205 AA 0,021484 BB 0,2893227 + p11.23 A G

20 18835431 rs1997824 AA 0,001465 BB 0,06147721 + p11.23 A G

20 18898921 rs10485587 AA 0,000488 AA 0,02056367 + p11.23 A G

20 18916197 rs874468 AA 0,001465 AA 0,211034 - p11.23 G T

20 18936095 rs6514960 BB 0,002441 AA 0,02642055 - p11.23 C T

20 18941114 rs2876507 AA 0,174316 BB 0,07431348 - p11.23 A C

20 19132044 rs1074615 AA 0,000977 BB 0,03542522 + p11.23 C T

20 19144096 rs6136651 BB 0,008301 BB 0,008397616 + p11.23 A G

20 19226320 rs10485588 BB 0,000488 BB 0,1579734 - p11.23 C T

20 19288719 rs7265616 BB 0,00293 BB 0,147121 + p11.23 C T

20 19368901 rs2328384 BB 0,000488 BB 0,0193854 + p11.23 C G

20 19415882 rs10485591 BB 0,000488 BB 0,136229 + p11.23 C T

20 19429841 rs6046148 BB 0,016113 AA 0,1143576 - p11.23 A G

20 19495213 rs2328411 BB 0,000488 BB 0,004148554 + p11.23 C T

20 19495270 rs2328412 AA 0,000488 AA 0,04598513 + p11.23 A G

20 19495298 rs2328413 AA 0,011719 AA 0,0335556 + p11.23 A C

20 19550042 rs728173 BB 0,001465 AA 0,03874704 + p11.23 A G

20 19558736 rs722850 BB 0,000488 AA 0,03884655 + p11.23 A G

20 19579221 rs725862 AA 0,05421775 + p11.23 A G

20 19601543 rs720436 AA 0,001128774 + p11.23 C T

20 19646783 rs2145104 AA 0,05782693 + p11.23 A C


20 19647148 rs2180583 BB 0,04127184 + p11.23 A G

20 19722604 rs6046326 BB 0,008181145 + p11.23 C T

20 19757473 rs6035452 AA 0,1638093 - p11.23 C T

20 19757524 rs6075577 BB 0,1010811 - p11.23 C T

20 19759114 rs2876544 BB 0,1536223 - p11.23 A T

20 19772393 rs10485594 BB 0,03691566 - p11.23 A G

20 19772870 rs4814909 AA 0,03575054 - p11.23 C T

20 19777492 rs6106144 BB 0,06256936 - p11.23 C T

20 19809017 rs10485595 BB 0,008560983 + p11.23 A T

20 19906303 rs199577 AA 0,005237735 - p11.23 A G

20 19982495 rs6046575 BB 0,000560297 - p11.23 C T

20 20105767 rs4814955 AA 0,003410004 + p11.23 A C

20 20156850 rs10485596 AA 0,07027097 + p11.23 C T

20 20168869 rs6112828 AA 0,04864342 + p11.23 C T

20 20170978 rs1003356 BB 0,1012234 - p11.23 C T

20 20186324 rs6081940 BB 0,04277991 + p11.23 C T

20 20205020 rs2424316 BB 0,02681981 - p11.23 C T

20 20287310 rs721424 AA 0,01078402 + p11.23 G T

20 20291554 rs932885 AA 0,004217919 + p11.23 C T

20 20483833 rs1074440 AA 0,02388855 + p11.23 A G

20 20709015 rs6137147 BB 0,08167151 + p11.23 C T

20 20709606 rs6075709 BB 0,07612286 + p11.23 A G

20 20825930 rs6047140 AA 0,02414163 + p11.23 A G

20 20826383 rs2038383 BB 0,05582721 + p11.23 A C

20 20904222 rs1555471 BB 0,09664367 + p11.23 A G

20 20919318 rs6047186 BB 0,4475312 - p11.23 A G

20 20930386 rs755963 BB 0,09867376 + p11.23 A G

20 21018717 rs6113129 AA 0,1958241 - p11.23 C T

20 21255107 rs2296728 BB 0,03341687 - p11.22 C T

20 21273222 rs202847 BB 0,003938209 - p11.22 A C

20 21276387 rs202849 BB 0,1139255 + p11.22 C T

20 21307665 rs6132420 BB 0,1740236 - p11.22 C T

20 21307787 rs6047413 BB 0,03554758 - p11.22 C T

20 21313558 rs1012155 AA 0,3581002 - p11.22 C T

20 21514717 rs2424383 AA 0,0717807 - p11.22 C G

20 21518837 rs1014889 BB 0,0125806 + p11.22 A G


20 21521722 rs1074606 BB 0,06439041 + p11.22 C T

20 21570078 rs6047533 BB 0,009029434 + p11.22 A G

20 21803904 rs6047685 AA 0,03014364 + p11.22 A T

20 21814547 rs6075855 AA 0,008958558 - p11.22 A G

20 22014991 rs10485628 BB 0,1320559 + p11.22 A G

20 22096476 rs804562 AA 0,1155007 - p11.22 A C

20 22132614 rs804591 AA 0,08069614 - p11.22 C T

20 22193867 rs10485630 AA 0,148312 + p11.22 G T

20 22288710 rs199836 BB 0,01656749 - p11.22 C T

20 22330952 rs199773 AA 0,02852823 - p11.21 A G

20 22342312 rs1346655 AA 0,05133111 + p11.21 C T

20 22547686 rs10485632 BB 0,2040928 + p11.21 C G

20 22640624 rs10485634 AA 0,08055725 - p11.21 C T

20 22640980 rs1072708 AA 0,06525435 - p11.21 A C

20 22641136 rs2404489 BB 0,008420831 - p11.21 A C

20 22645162 rs10485635 AA 0,01345692 + p11.21 A C

20 22645757 rs10485636 BB 0,06048739 + p11.21 A G

20 22672714 rs6075942 BB 0,1825384 - p11.21 A G

20 22701695 rs2424471 BB 0,005958036 - p11.21 C T

20 22705960 rs926744 BB 0,002493925 + p11.21 C T

20 22854446 rs1888610 BB 0,00844828 - p11.21 A C

20 22881104 rs955667 BB 0,1591553 + p11.21 A G

20 22925175 rs2424494 BB 0,07556926 + p11.21 A T

20 22944500 rs10485638 AA 0,04548272 + p11.21 C G

20 22944601 rs10485639 BB 0,009931466 + p11.21 A C

20 23041773 rs844850 BB 0,1257145 - p11.21 C G

20 23097739 rs10485640 AA 0,02930468 + p11.21 A G

20 23270400 rs2153987 BB 0,1392775 - p11.21 A G

20 23295553 rs999072 BB 0,015968 + p11.21 C T

20 23311989 rs1936013 BB 0,1883727 + p11.21 A G

20 23406390 rs6114139 BB 0,05805413 + p11.21 G T

20 23410022 rs6114144 AA 0,1748925 - p11.21 A T

20 23470628 rs16985462 AA 0,1021471 + p11.21 A G

20 23477910 rs1003203 BB 0,05460254 - p11.21 A G

20 23526189 rs1158167 BB 0,06142441 - p11.21 C T

20 23532116 rs726217 BB 0,01197501 - p11.21 C T


20 23573547 rs2145231 BB 0,07109149 + p11.21 A G

20 23573746 rs911122 BB 0,04043518 - p11.21 C T

20 23599589 rs10485647 BB 0,04292555 + p11.21 A G

20 23862024 rs2257471 BB 0,2826663 + p11.21 C T

20 23862371 rs1797032 AA 0,1151244 - p11.21 A T

20 23937652 rs3843776 AA 0,01788822 - p11.21 C T

20 23937782 rs3843777 BB 0,003277627 - p11.21 A G

20 23937930 rs3848799 AA 0,05134372 - p11.21 A G

20 24033110 rs761863 BB 0,1002184 - p11.21 A T

20 24048021 rs1005527 AA 0,128699 - p11.21 C T

20 24069103 rs761870 BB 0,06055103 - p11.21 A G

20 24093613 rs7268544 AA 0,01447496 + p11.21 C T

20 24093833 rs6036670 BB 0,06854122 - p11.21 A G

20 24122028 rs487665 BB 0,05946254 - p11.21 A G

20 24330313 rs2206648 AA 0,008854469 - p11.21 A T

20 24346140 rs10485832 BB 0,02621556 - p11.21 A C

20 24371416 rs722834 AA 0,000964947 + p11.21 A G

20 24373449 rs10485833 AA 0,06680652 + p11.21 C T

20 24480613 rs1326298 AA 0,0105672 - p11.21 A G

20 24480786 rs1326299 AA 0,004314346 - p11.21 A T

20 24537790 rs6049811 AA 0,02731291 + p11.21 G T

20 24726455 rs226693 BB 0,359002 + p11.21 A G

20 24996940 rs2387577 BB 0,02006178 + p11.21 C G

20 24997283 rs2207631 BB 0,02270125 + p11.21 A G

20 25180406 rs3787078 AA 0,04261045 + p11.21 A G

20 25454582 rs402120 AA 0,0867606 - p11.21 A G

20 25455495 rs10485776 AA 0,07906912 - p11.21 A G

20 26088200 rs6107147 AA 0,009222552 + p11.1 A G

20 26147643 rs703762 NoCall 0,6538487 + p11.1 A C

20 26148028 rs703761 BB 0,2423839 + p11.1 A C


Supplementary Table 3: SEC23B Mutations in CDAII Patients

Identifier

Allele

m(aternal)/

p(aternal)

Exon Mutation Predicted effect on RNA or protein Detectable protein

(Western Blot)

F1P1 n.a.

n.a.

2

14

c.40C>T

c.1588C>T

p.Arg14Trp

p.Arg530Trp

< 5% of WT expression in transient transfections

F2P1 m*

p*

4

4

c.325G>A

c.325G>A

p.Glu109Lys

p.Glu109Lys



F2P2 m*

p*

4

4

c.325G>A

c.325G>A

p.Glu109Lys

p.Glu109Lys



F2P3 m

p*

4

4

c.325G>A

c.325G>A

p.Glu109Lys

p.Glu109Lys



F3P1 n.a.

n.a.

2

7

c.40C>T

c.790C>T

p.Arg14Trp

p.Arg264X


F4P1 n.a.

n.a.

2

3 and 4

c.40C>T

c.222-817_366+4242del

p.Arg14Trp

p.Gln75GlufsX7


F5P1 n.a.

n.a.

4

4

c.325G>A

c.325G>A

p.Glu109Lys

p.Glu109Lys



F6P1 m*

p*

4

4

c.325G>A

c.325G>A

p.Glu109Lys

p.Glu109Lys



F7P1 n.a.

n.a.

6

11

c.649C>T

c.1276G>A

p.Arg217X

p.Val426Ile

F7P2 n.a.

n.a.

6

11

c.649C>T

c.1276G>A

p.Arg217X

p.Val426Ile

F8P1 n.a.

n.a.

8

8

c.938G>A

c.938G>A

p.Arg313His

p.Arg313His

F9P1 n.a.

n.a.

4

14

c.325G>A

c.1571C>T

p.Glu109Lys

p.Ala524Val


F10P1 n.a.

n.a

4

4

c.325G>A

c.325G>A

p.Glu109Lys

p.Glu109Lys



F11P1 m

p

7

7

c.716A>G

c.716A>G

p.Asp239Gly

p.Asp239Gly

WT expression level in transient transfections

WT expression level in transient transfections

F12P1 n.a.

n.a.

4

11

c.325G>A

c.1276G>A

p.Glu109Lys

p.Val426Ile


F13P1 n.a.

n.a.

2

8

c.40C>T

c.970C>T

p.Arg14Trp

p.Arg324X


F14P1 n.a.

n.a.

13

14

c.1489C>T

c.1571C>T

p.Arg497Cys

p.Ala524Val

F15P1 n.a.

n.a.

2

10

c.40C>T

c.1201C>T

p.Arg14Trp

p.Arg401X



Identifier

Allele

m(aternal)/

p(aternal)

Exon Mutation Predicted effect on RNA or protein Detectable protein (Western Blot)

F15P2 n.a.

n.a.

2

10

c.40C>T

c.1201C>T

p. Arg14Trp

p. Arg401X


F16P1 n.a.

n.a.

12

12

c.1385A>G

c.1385A>G

p. Tyr462Cys

p. Tyr462Cys

F16P2 n.a.

n.a.

12

12

c.1385A>G

c.1385A>G

p. Tyr462Cys

p. Tyr462Cys

F17P1 n.a.

n.a.

2

3

c.40C>T

c.235C>T

p. Arg14Trp

P. Arg79X


F18P1 n.a.

n.a.

12

12

c.1385A>G

c.1385A>G

p. Tyr462Cys

p. Tyr462Cys

F19P1 n.a.

n.a.

9

10

c.1063delG

c.1157A>G

p. Asp355IlefsX8

p. Gln386Arg

F20P1 n.a.

n.a.

8

8

c.953T>C

c.953T>C

p. Ile318Thr

p. Ile318Thr

F21P1 n.a.

n.a.

4

4

c.325G>A

c.325G>A

p. Glu109Lys

p. Glu109Lys



F22P1 m

p

2

3

c.40C>T

c.235C>T

p. Arg14Trp

P. Arg79X


F23P1 n.a.

n.a.

2

8

c.40C>T

c.953T>C

p. Arg14Trp

p. Ile318Thr


F24P1 n.a.

n.a.

2

14

c. 53G>A

c.1571C>T

p. Arg18His

p. Ala524Val

F25P1 m

p

2

6

c.40C>T

c.689+1G>A

p. Arg14Trp

Aberrant splicing


F26P1 n.a.

n.a.

13

13

c.1489C>T

c.1489C>T

p. Arg497Cys

p. Arg497Cys

F27P1 m

p

4

4

c.325G>A

c.325G>A

p. Glu109Lys

p. Glu109Lys



F28P1 n.a.

n.a

4

4

c.325G>A

c.325G>A

p. Glu109Lys

p. Glu109Lys



When possible, analysed maternal (m) or paternal (p) alleles are indicated or deduced (*). c. denotes nucleotide sequence in SEC23B

cDNA (NM_006363). The adenosine of the initiation codon is nucleotide +1. p. denotes amino acid numbering of the SEC23B

protein. The effect of the mutations on RNA or protein is predicted. When tested, Protein levels as assessed by Western Blots after

transient overexpression in 293T cells are indicated. n.a.: not available.


Supplementary Table 4: Phylogenetic Conservation of SEC23B

Missense Mutated Residues

R14W

R18H H.sapiens SEC23B MATYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPLACLLTPLKERPD 50

P.troglodytes SEC23B MATYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPLACLLTPLKERPD 50

C.lupus fam. SEC23B MATYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPLACLLTPLKERPD 50

B.taurus SEC23B MATYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPLACLLTPLKERPD 50

M.musculus SEC23B MATYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPLACLLTPLKERPD 50

R.norvegicus SEC23B MATYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPLACLLTPLKERPD 50

G.gallus SEC23B MATYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPLACLLTPLRERPD 50

X.laevis SEC23B MATYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPLGCLFTPLKERPD 50

D.rerio SEC23B MATYQEFIQQNEDRDGIRCSWNLWPSSRLEATRLVVPVSCLYTPLKERPD 50

H.sapiens SEC23A MTTYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPVAALFTPLKERPD 50

H.sapiens SEC23B LPPVQYEPVLCSRPTCKAVLNPLCQVDYRAKLWACNFCFQRNQFPPAYGG 100

P.troglodytes SEC23B LPPVQYEPVLCSRPTCKAVLNPLCQVDYRAKLWACNFCFQRNQFPPAYGG 100

C.lupus fam. SEC23B LPPVQYEPVLCSRPTCKAILNPLCQVDYRAKLWACNFCFQRNQFPPAYTG 100

B.taurus SEC23B LPPVQYEPVLCSRPTCKAILNPLCQVDYRAKLWACNFCFQRNQFPPAYAG 100

M.musculus SEC23B LPPVQYEPVLCSRPTCKAILNPLCQVDYRAKLWACNFCFQRNQFPPAYAG 100

R.norvegicus SEC23B LPPVQYEPVLCSRPTCKAILNPLCQVDYRAKLWACNFCFQRNQFPPAYAG 100

G.gallus SEC23B LPPVQYEPVLCSRPTCKAVLNPLCQVDYRAKLWACNFCFQRNQFPPAYAG 100

X.laevis SEC23B LPPVQYEPVLCTRPTCKAVLNPLCQVDYRAKLWACNFCFQRNQFPPTYAG 100

D.rerio SEC23B LPPVQYEPVPCTRANCKAVLNPLCQVDFRAKIWACNFCFQRNSFPPSYAG 100

H.sapiens SEC23A LPPIQYEPVLCSRTTCRAVLNPLCQVDYRAKLWACNFCYQRNQFPPSYAG 100

E109K H.sapiens SEC23B ISEVNQPAELMPQFSTIEYVIQRG--AQSPLIFLYVVDTCLEEDDLQALK 148

P.troglodytes SEC23B ISEVNQPAELMPQFSTIEYVIQRG--AQSPLIFLYVIDTCLEEDDLQALK 148

C.lupus fam. SEC23B ISEVNQPAELMPQFSTIEYVLQVTSLSCSPLIFLYVVDTCLEEDDLQALK 150

B.taurus SEC23B ISEVNQPAELMPQFSTIEYVIQRG--APSPLIFLYVVDTCLEDDDLQALK 148

M.musculus SEC23B ISEVNQPAELMPQFSTIEYMIQRG--ARSPLIFLYVVDTCLEEDDLQALK 148

R.norvegicus SEC23B ISEVNQPAELMPQFSTIEYMIQRG--ARSPLIFLYVVDTCLEEDDLQALK 148

G.gallus SEC23B ISEVNQPAELMPQFSTIEYIVQRG--PQTPLIFLYVVDTCLEEEDLQALK 148

X.laevis SEC23B ISEVNQPAELMPQFSTIEYIVQRG--PQTPLVFLYVVDTCLEEEDLQALK 148

D.rerio SEC23B ISEVNQPAELMPQFSTIEYIVQRG--PPTPLIFLYLVDTCLEEEDLQALK 148

H.sapiens SEC23A ISELNQPAELLPQFSSIEYVVLRG--PQMPLIFLYVVDTCMEDEDLQALK 148

H.sapiens SEC23B ESLQMSLSLLPPDALVGLITFGRMVQVHELSCEGISKSYVFRGTKDLTAK 198

P.troglodytes SEC23B ESLQMSLSLLPPDALVGLITFGRMVQVHELSCEGISKSYVFRGTKDLTAK 198

C.lupus fam. SEC23B ESLQMSLSLLPPDALVGLITFGRMVQVHELSCEGISKSYVFRGTKDLTAK 200

B.taurus SEC23B ESLQMSLSLLPPDALVGLITFGRMVQVHELSCEGISKSYVFRGTKDLTAK 198

M.musculus SEC23B ESLQMSLSLLPPDALVGLITFGRMVQVHELSCEGISKSYVFRGTKDLTAK 198

R.norvegicus SEC23B ESLQMSLSLLPPDALVGLITFGRMVQVHELSCEGISKSYVFRGTKDLTAK 198

G.gallus SEC23B ESLQMSLSLLPADALVGLITFGRMIQVHELSCEGISKSYVFRGTKDLTAK 198

X.laevis SEC23B ESLQMSLSLLPPDALVGLITFGRMVQVHELSCDGISKSYVFRGTKDLTAK 198

D.rerio SEC23B ESLQMSLSLLPPNALVGLITFGRMIQVHELSCEGIAKSYVFRGTKELAPK 198

H.sapiens SEC23A ESMQMSLSLLPPTALVGLITFGRMVQVHELGCEGISKSYVFRGTKDLSAK 198


D239G H.sapiens SEC23B QIQDMLGLTKPAMPMQQARPAQPQEHPFASSRFLQPVHKIDMNLTDLLGE 248

P.troglodytes SEC23B QIQDMLGLTKPAMPMQQARPAQPQEHPFVSSRFLQPVHKIDMNLTDLLGE 248

C.lupus fam. SEC23B QIQDMLGLTKPAVPMQQARPGPPQEHPFVSSRFLQPVHKIDMNLTDLLGE 250

B.taurus SEC23B QIQDMLGLTKPAMPVQPMRPAQPQERPSVSSRFLQPIHKIDMNLTDLLGE 248

M.musculus SEC23B QIQEMLGLTKSAMPVQQARPAQPQEQPFVSSRFLQPIHKIDMNLTDLLGE 248

R.norvegicus SEC23B QIQEMLGLTKSAMPVQQVRPGQPQEQSAVSSRFLQPVHKIDMNLTDLLGE 248

G.gallus SEC23B QIQDMLGLSRPAVPIQQGRPLQAPEQPVISSRFLQPVHKIDMNLTDLLGE 248

X.laevis SEC23B QIQDMLGLSKAAAPAQQGRPLQPQEQPFLSSRFLQPVHKIDMNLTDLLGE 248

D.rerio SEC23B QIQEMLGLMKPATSGQQGKPLAPHD-AAASCRFLQPVQMVDMNLTDLLGE 247

H.sapiens SEC23A QLQEMLGLSK--VPLTQATRGPQVQQPPPSNRFLQPVQKIDMNLTDLLGE 246

H.sapiens SEC23B LQRDPWPVTQGKRPLRSTGVALSIAVGLLEGTFPNTGARIMLFTGGPPTQ 298

P.troglodytes SEC23B LQRDPWPVTQGKRPLRSTGVALSIAVGLLEGTFPNTGARIMLFTGGPPTQ 298

C.lupus fam. SEC23B LQRDPWPVTQGKRPLRSTGVALSIAVGLLEGTFPNTGARIMLFTGGPPTQ 300

B.taurus SEC23B LQRDPWPVPQGKRPLRSTGVALSIAVGLLEGTFPNTGARIMLFTGGPPTQ 298

M.musculus SEC23B LQRDPWPVTQGKRPLRSTGVALSIAVGLLEGTFPNTGARIMLFTGGPPTQ 298

R.norvegicus SEC23B LQRDPWPVTQGKRPLRSTGVALSIAVGLLEGTFPNTGARIMLFTGGPPTQ 298

G.gallus SEC23B LQRDPWPVTQGKRPLRSTGVALSIAVGLLEGTFPNTGARIMLFTGGPPTQ 298

X.laevis SEC23B LQRDPWPVPQGKRPLRSTGVALSIAVGLLEGTLPNTGARIMLFTGGPPTQ 298

D.rerio SEC23B LQRDPWPVPQGKRPLRSTGIALSIAVGLLEGTFPNTGARVMLFTGGPPTQ 297

H.sapiens SEC23A LQRDPWPVPQGKRPLRSSGVALSIAVGLLECTFPNTGARIMMFIGGPATQ 296

R313H

I318T H.sapiens SEC23B GPGMVVGDELKIPIRSWHDIEKDNARFMKKATKHYEMLANRTAANGHCID 348

P.troglodytes SEC23B GPGMVVGDELKIPIRSWHDIEKDNARFMKKATKHYEMLANRTAANGHCID 348

C.lupus fam. SEC23B GPGMVVGDELKVPIRSWHDIEKDNARFMKKATKHYEMLANRTATNGHCID 350

B.taurus SEC23B GPGMVVGDELKVPIRSWHDIEKDNARFMKKATKHYEMLANRTAANGHCID 348

M.musculus SEC23B GPGMVVGDELKTPIRSWHDIEKDNARFMKKATKHYEMLANRTATNGHCID 348

R.norvegicus SEC23B GPGMVVGDELKTPIRSWHDIEKDNARFMKKATKHYEMLANRTATNGHCID 348

G.gallus SEC23B GPGMVVGDELKTPIRSWHDIEKDNARFMKKATKHYETLANRTATNGHCID 348

X.laevis SEC23B GPGMVVGDELKTPIRSWHDIEKDNARFMKKATKHYEALANRTAANGHCID 348

D.rerio SEC23B GPGMVVGDELKTPIRSWHDIQKDNARHLKKATKYYEALANRAAVNGHSVD 347

H.sapiens SEC23A GPGMVVGDELKTPIRSWHDIDKDNAKYVKKGTKHFEALANRAATTGHVID 346

Q386R H.sapiens SEC23B IYACALDQTGLLEMKCCANLTGGYMVMGDSFNTSLFKQTFQRIFTKDFNG 398

P.troglodytes SEC23B IYACALDQTGLLEMKCCANLTGGYMVMGDSFNTSLFKQTFQRIFTKDFNG 398

C.lupus fam. SEC23B IYACALDQTGLLEMKCCANLTGGHMVMGDSFNTSLFKQTFQRIFSKDFNG 400

B.taurus SEC23B IYACALDQTGLLEMKCCPNLTGGYMVMGDSFNTSLFKQTFQRIFSKDFNG 398

M.musculus SEC23B IYACALDQTGLLEMKCCPNLTGGHMVMGDSFNTSLFKQTFQRIFSKDFNG 398

R.norvegicus SEC23B IYACALDQTGLLEMKCCPNLTGGLMVMGDSFNTSLFKQTFQRIFSKDFNG 398

G.gallus SEC23B IYACALDQTGLLEMKCCANLTGGHMVMGDSFNTSLFKQTFQRVFNKGLNG 398

X.laevis SEC23B IYACALDQTGLLEMKCCSNLTGGEIVIGDSFNTSLFKQTFQRVFTKDPNG 398

D.rerio SEC23B IYACALDQTGLLEMKCLSNLTGGHIVMGDSFNTSLFKQTFQRVFNKDYNG 397

H.sapiens SEC23A IYACALDQTGLLEMKCCPNLTGGYMVMGDSFNTSLFKQTFQRVFTKDMHG 396

V426I H.sapiens SEC23B DFRMAFGATLDVKTSRELKIAGAIGPCVSLNVKGPCVSENELGVGGTSQW 448

P.troglodytes SEC23B DFRMAFGATLDVKTSRELKIAGAIGPCVSLNVKGPCVSENELGVGGTSQW 448

C.lupus fam. SEC23B DFRMAFGATLEVKTSRELKVAGAIGPCVSLNVKGPCVSENELGVGGTNQW 450

B.taurus SEC23B NFRMAFGATLEVKTSRELKVAGAIGPCVSLNVKGPCVSENELGVGGTSQW 448

M.musculus SEC23B DFRMAFGATLDVKTSRELKIAGAIGPCVSLNVKGPCVSENELGVGGTSQW 448

R.norvegicus SEC23B DFRMAFGATLEVKTSRELKIAGAIGPCVSLNVKGPCVSENELGVGGTSQW 448

G.gallus SEC23B EFRMAFGANLEVKTSRELKIAGAIGPCVSLNVKGPCVSENELGIGGTSQW 448

X.laevis SEC23B SFKMAFGANLDVKTSRELKVSGTIGPCVSLNVNGPCVSENELGVGGTSQW 448

D.rerio SEC23B EFRMAFGATLEVKTSRELKVSGAIGPCVSLNTKGPCVSDSEMGVGGTCQW 447

H.sapiens SEC23A QFKMGFGGTLEIKTSREIKISGAIGPCVSLNSKGPCVSENEIGTGGTCQW 446


Y462C R497C H.sapiens SEC23B KICGLDPTSTLGIYFEVVNQHNTPIPQGGRGAIQFVTHYQHSSTQRRIRV 498

P.troglodytes SEC23B KICGLDPTSTLGIYFEVVNQHNTPIPQGGRGAIQFVTHYQHSSTQRRIRV 498

C.lupus fam. SEC23B KICGLDPTSTLGIYFEVVNQHNAPVPQGGRGAIQFVTQYQHSSTQRRIRV 500

B.taurus SEC23B KICGLDPTTTLGIYFEVVNQHNAPIPQGGRGAIQFVTHYQHSSTQRRIRV 498

M.musculus SEC23B KICGLDPSSTLGIYFEVVNQHNAPVPQGGRGAIQFVTQYQHSSTQKRIRV 498

R.norvegicus SEC23B KICGLDPSSTLGIYFEVVNQHNAPIPQGGRGAIQFVTQYQHSSTQKRIRV 498

G.gallus SEC23B KICGLDPCTTLAIYFEVVNQHNAPIPQGGRGAVQFVTQYQHSSTQKRIRV 498

X.laevis SEC23B KICALDPTTTLGIYFEVVNQHNAPIPQGGRGAVQFVTQYQHSTTQKRIRV 498

D.rerio SEC23B KICSLSPATTLAMYFEVVNQHNAPVPQGGRGVVQFVTQYQHSNTQRRIRV 497

H.sapiens SEC23A KICGLSPTTTLAIYFEVVNQHNAPIPQGGRGAIQFVTQYQHSSGQRRIRV 496

A524V

R530W H.sapiens SEC23B TTIARNWADVQSQLRHIEAAFDQEAAAVLMARLGVFRAESEEGPDVLRWL 548

P.troglodytes SEC23B TTIARNWADVQSQLRHIEAAFDQEAAAVLMARLGVFRAESEEGPDVLRWL 548

C.lupus fam. SEC23B TTIARNWADAQSQLRHIEAAFDQEAAAVLMARLGVFRAESEEGPDVLRWL 550

B.taurus SEC23B TTVARNWADVQSQLKHIEAAFDQEAAAVLMARLGVFRAETEEGPDVLRWL 548

M.musculus SEC23B TTIARNWADAQSQLRHIEAAFDQEAAAVLMARLGVFRAESEEGPDVLRWL 548

R.norvegicus SEC23B TTIARNWADAQSQLRHIEAAFDQEAAAVLMARLGVFRAESEEGPDVLRWL 548

G.gallus SEC23B TTIARNWADAQSQLQHIEAAFDQEAAAVLMARLGVYRAESEEGPDVLRWL 548

X.laevis SEC23B TTVARNWADAQSQIQHIEAAFDQEAAAVLMARLGVYRAETEEGPDVLRWL 548

D.rerio SEC23B TTIARNWADAQSQIQHIESSFDQEASAVLMARLGVFRAESEEGPDVLRWL 547

H.sapiens SEC23A TTIARNWADAQTQIQNIAASFDQEAAAILMARLAIYRAETEEGPDVLRWL 546

H.sapiens SEC23B DRQLIRLCQKFGQYNKEDPTSFRLSDSFSLYPQFMFHLRRSPFLQVFNNS 598

P.troglodytes SEC23B DRQLIRLCQKFGQYNKEDPTSFRLSDSFSLYPQFMFHLRRSPFLQVFNNS 598

C.lupus fam. SEC23B DRQLIRLCQKFGQYNKEDPTSFRLSDSFSLYPQFMFHLRRSPFLQVFNNS 600

B.taurus SEC23B DRQLIRLCQKFGQYNKEDPMSFRLSDSFSLYPQFMFHLRRSPFLQVFNNS 598

M.musculus SEC23B DRQLIRLCQKFGQYNKEDPTSFRLSDSFSLYPQFMFHLRRSPFLQVFNNS 598

R.norvegicus SEC23B DRQLIRLCQKFGQYNKEDPTSFRLSDSFSLYPQFMFHLRRSPFLQVFNNS 598

G.gallus SEC23B DRQLIRLCQKFGQYNKDDPNSFRLSESFSLYPQFMFHLRRSPFLQVFNNS 598

X.laevis SEC23B DRQLIRLCQKFGQYNKDDPSSFRLSDSFSLYPQFMFHLRRSPFLQVFNNS 598

D.rerio SEC23B DRQLIRLCQKFGQFNKDDPNSFRLSESLSLYPQFMFHLRRSPFLQVFNNS 597

H.sapiens SEC23A DRQLIRLCQKFGEYHKDDPSSFRFSETFSLYPQFMFHLRRSSFLQVFNNS 596

H.sapiens SEC23B PDESSYYRHHFARQDLTQSLIMIQPILYSYSFHGPPEPVLLDSSSILADR 648

P.troglodytes SEC23B PDESSYYRHHFARQDLTQSLIMIQPILYSYSFHGPPEPVLLDSSSILADR 648

C.lupus fam. SEC23B PDESSYYRHHFARQDLTQSLIMIQPILYSYSFHGPPEPVLLDSSSILADR 650

B.taurus SEC23B PDESSYYRHHFARQDLTQSLIMIQPILYSYSFHGPPEPVLLDSSSILADR 648

M.musculus SEC23B PDESSYYRHHFARQDLTQSLIMIQPILYSYSFHGPPEPVLLDSSSILADR 648

R.norvegicus SEC23B PDESSYYRHHFARQDLTQSLIMIQPILYSYSFHGPPEPVLLDSSSILADR 648

G.gallus SEC23B PDESSYYRHHFARQDLTQSLIMIQPILYAYSFHGPPEPVLLDSSSILPDR 648

X.laevis SEC23B PDESSYYRHHFARQDLTQSLIMIQPILYSYSFYGPPEPVLLDSSSILADR 648

D.rerio SEC23B PDESSYYRHHFVRQDLTQSLIMIQPILYSYSFHGPPEPVLLDSSSILPDR 647

H.sapiens SEC23A PDESSYYRHHFMRQDLTQSLIMIQPILYAYSFSGPPEPVLLDSSSILADR 646

H.sapiens SEC23B ILLMDTFFQIVIYLGETIAQWRKAGYQDMPEYENFKHLLQAPLDDAQEIL 698

P.troglodytes SEC23B ILLMDTFFQIVIYLGETIAQWRKAGYQDMPEYENFKHLLQAPLDDAQEIL 698

C.lupus fam. SEC23B ILLMDTFFQIVIYLGETIAQWRKAGYQDMPEYENFKHLLQAPLDDAQEIL 700

B.taurus SEC23B ILLMDTFFQIVIYLGETIAQWRKAGYQDMPEYENFKHLLQAPLDDAQEIL 698

M.musculus SEC23B ILLMDTFFQIVIYLGETIAQWRKAGYQDMPEYENFKHLLQAPLDDAQEIL 698

R.norvegicus SEC23B ILLMDTFFQIVIYLGETIAQWRKAGYQDMPEYENFKHLLQAPLDDAQEIL 698

G.gallus SEC23B ILLMDTFFQIVIYLGETIAQWQKAGYQDMPEYENFKHLLQAPLDDAQEIL 698

X.laevis SEC23B ILLMDTFFQIVIYLGETIAQWRNAGYHDMPEYENFKQLLQAPLDDAQEIL 698

D.rerio SEC23B ILLMDTFFQLVIYHGETIAQWRKAGYQEMAEYENFKQLLQAPLDDAQEIL 697

H.sapiens SEC23A ILLMDTFFQILIYHGETIAQWRKSGYQDMPEYENFRHLLQAPVDDAQEIL 696


H.sapiens SEC23B QARFPMPRYINTEHGGSQARFLLSKVNPSQTHNNLYAWGQETGAPILTDD 748

P.troglodytes SEC23B QARFPMKGYINTEHGGSQARFLLSKVNPSQTHNNLYAWGQETGAPILTDD 748

C.lupus fam. SEC23B QARFPMPRYINTEHGGSQARFLLSKVNPSQTHNNLYAWGQETGAPILTDD 750

B.taurus SEC23B QARFPMPRYIHTEHGGSQARFLLSKVNPSQTHNNLYAWGQETGAPILTDD 748

M.musculus SEC23B QARFPMPRYINTEHGGSQARFLLSKVNPSQTHNNLYAWGQETGAPILTDD 748

R.norvegicus SEC23B QARFPMPRYINTEHGGSQARFLLSKVNPSQTHNNLYAWGQETGAPILTDD 748

G.gallus SEC23B QTRFPMPRYVHTEHGGSQARFLLSKVNPSQTHNNLYAWGQESGAPILTDD 748

X.laevis SEC23B QSRFPMPRYINTEHGGSQARFLLSKVNPSQTHNNLYNWGQESGAPILTDD 748

D.rerio SEC23B QTRFPMPRYIDTEHGGSQARFLLSKVNPSQTHNNLYAWGQESGAPILTDD 747

H.sapiens SEC23A HSRFPMPRYIDTEHGGSQARFLLSKVNPSQTHNNMYAWGQESGAPILTDD 746

H.sapiens SEC23B VSLQVFMDHLKKLAVSSAC 767

P.troglodytes SEC23B VSLQVFMDHLKKLAVSSAC 767

C.lupus fam. SEC23B VSLQVFMDHLKKLAVSSAC 769

B.taurus SEC23B VSLQVFMDHLKKLAVSSAC 767

M.musculus SEC23B VSLQVFMDHLKKLAVSSAS 767

R.norvegicus SEC23B VSLQVFMDHLKKLAVSSAS 767

G.gallus SEC23B VSLQVFMDHLKKLAVSSAA 767

X.laevis SEC23B VSLQVFMDHLKKLAVSTAS 767

D.rerio SEC23B VSLQVFMDHLKKLAVSSSA 766

H.sapiens SEC23A VSLQVFMDHLKKLAVSSAA 765


Supplementary Table 5: List of shRNAs, Morpholinos,

Oligonucleotides and siRNAs

Transfection Experiments:

Sequenc of non-silencing shRNAmir:

TGCTGTTGACAGTGAGCGATCTCGCTTGGGCGAGAGTAAGTAGTGAAGCCACAGATGTACTT

ACTCTCGCCCAAGCGAGAGTGCCTACTGCCTCGGA.

Sequence of silencing SEC23B shRNAmir:

TGCTGTTGACAGTGAGCGAAGCTTGATGCCCACTTTGAAATAGTGAAGCCACAGATGTATTT

CAAAGTGGGCATCAAGCTCTGCCTACTGCCTCGGA

Zebrafish Morpholino (MO) Sequences:

sec23b-translation blocker (MO A): 5’-CCATGACAGCGCGAACAACTTTTTA-3’,

sec23b-splice blocker (MO S): 5-ACAGGGTGAGTGTAAAATTACCTGC-3’,

pu.1-translation blocker (MO A): 5’-GATATACTGATACTCCATTGGTGGT-3’.

cDNA Primers for RT-PCR of Zebrafish RNA:

sec23b exon 4F: 5’–TCATACGCAGGCATTTCAGAAGTGA-3’

sec23b exon 7R: 5’– TTCATATCCACCATTTGCACAGGCT-3’

ef1α F: 5’-CCAACTTCAACGCTCAGGTCAT-3’

ef1α R: 5’-AGTAGAGTGCCCAGGTTTAGAGA-3’

Reagents for Cell Biology Studies

Control siScramble duplex: Sense 5´>3´: UUCUCCGAACGUGUCACGUtt.


CD63CD59CD55CD45CD44

CD34CD29CD10CD9CD3

contr

ol

patientF

6P

1contr

ol

patientF

6P

1

Supplementary Figure 1

a


FLAERMDRMHC IIMHC ICD271


contr

ol

patientF

6P

1contr

ol

patientF

6P

1


contr

ol

patientF

6P

1


adipogenicchondrogenicosteogenic

contr

ol

patientF

6P

1

unin

duced

unin

duced

induced

induced


b



c



d



e


Supplementary Figure 1. Analysis of SEC23B defective human

dermal fibroblasts. (a) Control and patient (F6P1) fibroblasts were

FACS-analysed for the indicated surface antigens. (b) Patient fibroblasts

(F6P1) retain the ability to differentiate into osteoblasts, chondroblasts

and adipocytes. (c) The endogenous cellular compartments COPI (A, B),

Golgi (CD), ER exit sites (E, F), endoplasmic reticulum (G, H), early

endosomes (I, J) and lysosomes (K, L) were investigated in control (A,C,

E, G, I, K) and a patient (B, D, F,H, J, L) cell line expressing SEC23B

wild type or SEC23B mutations (F4P1). Antibodies were directed against

ß´COP, GM130, SEC31, calnexin, EEA1, and LAMP-1. No apparent

difference was found with any of the markers. (d) Wild type fibroblasts

(A-C) and F4P1 mutant fibroblasts (D-F) were infected with adenovirus

encoding the temperature-sensitive cargo marker CFP-tagged ts-O45-G

and incubated at 39.5°C for 16h for accumulation of the protein in the ER;

after 1h release at the permissive temperature (32°C) the amount of cargo

at the membrane and the total amount of cargo was analyzed. Panels A

and D illustrate the total amount of cargo and panels B and E illustrate the

amount of cargo at the plasma membrane. Panels C and F show the

merged channels (false coloring). No difference on cargo transport was

detected between both cell lines. (e) GFP-tagged mannosidase 2A1 (G-L)

and 2A2 (A-F) were microinjected into control (A-C, G-I) and patient

F4P1(D-F, J-L) fibroblasts and expressed for 4h after which protein

synthesis was stopped by adding cycloheximide. The GFP-mannosidases

were chased for 1.5h or 4h, fixed and cells were stained with the Golgi

marker GM130. In both the control (A-C) and the mutant cell line (D-F)

cells expressing mannosidase 2A2 (A,D) were found co-localizing with

the Golgi Marker GM130 (B, E). In contrast mannosidase 2A1 (G, J) was

never seen co-localizing with the Golgi marker GM130 (H, K) in neither

of the cell lines (arrowheads). Enlarged regions shown in the bottom left

corner highlight the co-localization with the endogenous GM130.

.



siScramble

siSEC23A-1 +

siSEC23A-2

siSEC23B-1

siSEC23B-2

siSEC23B-2

siSEC23A-2 +

siSEC23B-1

siSEC23A-1

siRNA used


Supplementary Figure 2 . Analysis of SEC23 knock-down

experiments. HeLa Kyoto cells transfected with scramble (A, B),

SEC23A (C-F) or SEC23B (G-J) siRNA duplexes or simultaneously with

Sec23A and SEC23B (K-N) siRNA duplexes were infected with

adenovirus encoding CFP-tagged ts-O45-G and incubated at 39.5°C for

16h to accumulate the cargo marker in the ER. Thereafter, the temperature

was shifted to 32°C for 1h and the cells were fixed. A, C, E, G, I, K and

M show representative images of the COPII marker SEC31A . In A’, C’,

E’, G’, I’, K’ and M’ a zoom of the correspondent images is shown. Cells

transfected with either SEC23A (C, E) or SEC23B (G, I) siRNA duplexes

did not show a drastic reduction of ERES number when compared with

the control (A). However, when both SEC23A and SEC23B siRNA

duplexes were transfected simultaneously (K, M) the number of ERES

reduced drastically. B, D, F, H, J, L and N show representative images of

CFP-tagged ts-O45-G after 1h of release at the permissive temperature .

In B’, D’, F’, H’, J’, L’ and N’ a zoom of the correspondent images is

shown. In accordance to what was observed with ERES, cells transfected

with only SEC23A (D, F) or SEC23B (G, I) siRNA duplexes exhibited a

similar cargo transport to the plasma membrane when compared to the

control (B). In cells transfected with both SEC23A and SEC23B siRNA

duplexes simultaneously (L, N) the transport of the cargo marker CFP-

tagged ts-O45-G was severely affected being in the majority of the cells

not exported from the endoplasmic reticulum. Arrows depict CFP-tagged

ts-O45-G at the plasma membrane. Arrowheads point to the nuclear

envelope that can be visualized after 1 h at the permissive temperature

indicating that CFP-tagged ts-O45-G is retained at the ER. The bar

represents 10µm.


Nat. Genet. 41, 936–940 (2009). Mutations affecting the ... · Nat. Genet. 41, 936–940 (2009)....

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Transcript of Nat. Genet. 41, 936–940 (2009). Mutations affecting the ... · Nat. Genet. 41, 936–940 (2009)....