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Mutations in lectin complement pathway genes COLEC11 and MASP1 cause 3MC syndrome.

Rooryck C, Diaz-Font A, Osborn DP, Chabchoub E, Hernandez-Hernandez V, Shamseldin H, Kenny J, Waters A, Jenkins D, Kaissi AA, Leal GF, Dallapiccola B, Carnevale F, Bitner-Glindzicz M, Lees M, Hennekam R, Stanier P, Burns AJ, Peeters H, Alkuraya FS, Beales PL - Nat. Genet. (2011)

Bottom Line: Zebrafish morphants for either gene develop pigmentary defects and severe craniofacial abnormalities.Finally, we show that CL-K1 serves as a guidance cue for neural crest cell migration.Together, these findings demonstrate a role for complement pathway factors in fundamental developmental processes and in the etiology of 3MC syndrome.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Unit, University College London Institute of Child Health, London, UK.

ABSTRACT
3MC syndrome has been proposed as a unifying term encompassing the overlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. These rare autosomal recessive disorders exhibit a spectrum of developmental features, including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability and genital, limb and vesicorenal anomalies. Here we studied 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP1, both of which encode proteins in the lectin complement pathway (collectin kidney 1 (CL-K1) and MASP-1 and MASP-3, respectively). CL-K1 is highly expressed in embryonic murine craniofacial cartilage, heart, bronchi, kidney and vertebral bodies. Zebrafish morphants for either gene develop pigmentary defects and severe craniofacial abnormalities. Finally, we show that CL-K1 serves as a guidance cue for neural crest cell migration. Together, these findings demonstrate a role for complement pathway factors in fundamental developmental processes and in the etiology of 3MC syndrome.

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Related in: MedlinePlus

a. General morphology of colec11 zebrafish morphants at higher (4 ng) and lower (3 ng) doses of colec11 MO. Higher doses give rise to pronephric cysts, curved body axis, and cardiac oedema not present at 3 ng doses. A dose dependent loss of medial trunk pigmentation (insets – 1.5x magnification ) was observed (scale bar is 500 μm) b. Alcian blue cartilage staining in colec11 morphants (3 and 4 ng) at 5 dpf showing generalised cartilage defects (scale bar: 200 μm) c. Palate measurements in the colec11 morphants (3 ng) demonstrate shortening of length and width of the ethmoid plate, abnormal Meckel's cartilage (mc) and abnormal angulation of the ceratohyal cartilage (ch). Scale bar: 200 μm. d. Graph of ethmoid plate length and width measurements expressed as a ratio against cranial length and width, respectively, in the presence or absence of colec11 morpholino. There is clear evidence of significant morphological effects on both length and width, indicating shortening in both axes. (con length 3.164 ± 0.062 N=8, colec11 MO length 3.702 ± 0.245 N=8, con width 2.563 ± 0.0441 N=8, colec11 MO width 2.819 ± 0.168 N=8, each value indicates the mean ± SEM). e. Alcian blue cartilage staining of colec11 morphants rescued with human RNA COLEC11 (75 pg) compared to colec11 morphants (3ng), control uninjected and COLEC11 RNA injected alone embryos (scale bar: 200 μm). pq: palatoquadrate, mc: Meckel's cartilage. f. Graph showing the length ratio of both pq cartilages plus mc standardised over the cranial length. Con (0.821 ± 0.004, n=51), colec11 MO (0.896 ± 0.009, n=50), COLEC11 RNA (0.844 ± 0.007, n=21), colec11 MO plus COLEC11 RNA (0.820 ± 0.014, n=16). Values shown as mean ± SEM. Data was analysed by one way ANOVA with Tukey's Multiple Comparison Test comparing column data against control measurements. Morphants have a significantly shorter pq+mc length compared to uninjected controls (p<0.001), whilst no significant (ns) difference was observed in controls versus colec11MO plus COLEC11 RNA or versus COLEC11 RNA injected alone.Key angles and measures are indicated to illustrate cartilage defects. ep = ethmoid plate, t=trabeculae, n=notochord, bp=basal plate, pq=palatoquadrate, cb=ceratobranchials
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Figure 3: a. General morphology of colec11 zebrafish morphants at higher (4 ng) and lower (3 ng) doses of colec11 MO. Higher doses give rise to pronephric cysts, curved body axis, and cardiac oedema not present at 3 ng doses. A dose dependent loss of medial trunk pigmentation (insets – 1.5x magnification ) was observed (scale bar is 500 μm) b. Alcian blue cartilage staining in colec11 morphants (3 and 4 ng) at 5 dpf showing generalised cartilage defects (scale bar: 200 μm) c. Palate measurements in the colec11 morphants (3 ng) demonstrate shortening of length and width of the ethmoid plate, abnormal Meckel's cartilage (mc) and abnormal angulation of the ceratohyal cartilage (ch). Scale bar: 200 μm. d. Graph of ethmoid plate length and width measurements expressed as a ratio against cranial length and width, respectively, in the presence or absence of colec11 morpholino. There is clear evidence of significant morphological effects on both length and width, indicating shortening in both axes. (con length 3.164 ± 0.062 N=8, colec11 MO length 3.702 ± 0.245 N=8, con width 2.563 ± 0.0441 N=8, colec11 MO width 2.819 ± 0.168 N=8, each value indicates the mean ± SEM). e. Alcian blue cartilage staining of colec11 morphants rescued with human RNA COLEC11 (75 pg) compared to colec11 morphants (3ng), control uninjected and COLEC11 RNA injected alone embryos (scale bar: 200 μm). pq: palatoquadrate, mc: Meckel's cartilage. f. Graph showing the length ratio of both pq cartilages plus mc standardised over the cranial length. Con (0.821 ± 0.004, n=51), colec11 MO (0.896 ± 0.009, n=50), COLEC11 RNA (0.844 ± 0.007, n=21), colec11 MO plus COLEC11 RNA (0.820 ± 0.014, n=16). Values shown as mean ± SEM. Data was analysed by one way ANOVA with Tukey's Multiple Comparison Test comparing column data against control measurements. Morphants have a significantly shorter pq+mc length compared to uninjected controls (p<0.001), whilst no significant (ns) difference was observed in controls versus colec11MO plus COLEC11 RNA or versus COLEC11 RNA injected alone.Key angles and measures are indicated to illustrate cartilage defects. ep = ethmoid plate, t=trabeculae, n=notochord, bp=basal plate, pq=palatoquadrate, cb=ceratobranchials

Mentions: As nothing is known of the specific role of COLEC11 during embryonic development and in the absence of an available mouse model, we demonstrated expression in zebrafish embryos in the pronephric duct, lateral hindbrain and liver (Fig 2d). Next we sought to determine the effects of loss of function of this protein during zebrafish embryogenesis. Two antisense morpholinos were designed; one directed against the initiation site (colec11-ATG-MO) and another against the exon 2-intron 2 splice site (colec11-SPL-MO)(Suppl Fig 7). Injection of both MOs from 1 to 8 ng doses at the one-cell stage gave rise to morphological abnormalities which could be reversed upon co-injection with full-length COLEC11 mRNA (Fig 3e,f). This effect was dose-dependent including, for higher doses (4 ng), heart edema, pronephric cyst formation, curved body axis, disorganised pigment distribution and high mortality (Fig 3a). To ensure that these phenotypes were not the result of morpholino off-target effects (e.g up regulation of p53), we co-injected p53 morpholino with colec11 MO (4 ng) but could not rescue the phenotypes (Suppl Fig 8 and not shown).


Mutations in lectin complement pathway genes COLEC11 and MASP1 cause 3MC syndrome.

Rooryck C, Diaz-Font A, Osborn DP, Chabchoub E, Hernandez-Hernandez V, Shamseldin H, Kenny J, Waters A, Jenkins D, Kaissi AA, Leal GF, Dallapiccola B, Carnevale F, Bitner-Glindzicz M, Lees M, Hennekam R, Stanier P, Burns AJ, Peeters H, Alkuraya FS, Beales PL - Nat. Genet. (2011)

a. General morphology of colec11 zebrafish morphants at higher (4 ng) and lower (3 ng) doses of colec11 MO. Higher doses give rise to pronephric cysts, curved body axis, and cardiac oedema not present at 3 ng doses. A dose dependent loss of medial trunk pigmentation (insets – 1.5x magnification ) was observed (scale bar is 500 μm) b. Alcian blue cartilage staining in colec11 morphants (3 and 4 ng) at 5 dpf showing generalised cartilage defects (scale bar: 200 μm) c. Palate measurements in the colec11 morphants (3 ng) demonstrate shortening of length and width of the ethmoid plate, abnormal Meckel's cartilage (mc) and abnormal angulation of the ceratohyal cartilage (ch). Scale bar: 200 μm. d. Graph of ethmoid plate length and width measurements expressed as a ratio against cranial length and width, respectively, in the presence or absence of colec11 morpholino. There is clear evidence of significant morphological effects on both length and width, indicating shortening in both axes. (con length 3.164 ± 0.062 N=8, colec11 MO length 3.702 ± 0.245 N=8, con width 2.563 ± 0.0441 N=8, colec11 MO width 2.819 ± 0.168 N=8, each value indicates the mean ± SEM). e. Alcian blue cartilage staining of colec11 morphants rescued with human RNA COLEC11 (75 pg) compared to colec11 morphants (3ng), control uninjected and COLEC11 RNA injected alone embryos (scale bar: 200 μm). pq: palatoquadrate, mc: Meckel's cartilage. f. Graph showing the length ratio of both pq cartilages plus mc standardised over the cranial length. Con (0.821 ± 0.004, n=51), colec11 MO (0.896 ± 0.009, n=50), COLEC11 RNA (0.844 ± 0.007, n=21), colec11 MO plus COLEC11 RNA (0.820 ± 0.014, n=16). Values shown as mean ± SEM. Data was analysed by one way ANOVA with Tukey's Multiple Comparison Test comparing column data against control measurements. Morphants have a significantly shorter pq+mc length compared to uninjected controls (p<0.001), whilst no significant (ns) difference was observed in controls versus colec11MO plus COLEC11 RNA or versus COLEC11 RNA injected alone.Key angles and measures are indicated to illustrate cartilage defects. ep = ethmoid plate, t=trabeculae, n=notochord, bp=basal plate, pq=palatoquadrate, cb=ceratobranchials
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Related In: Results  -  Collection

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Figure 3: a. General morphology of colec11 zebrafish morphants at higher (4 ng) and lower (3 ng) doses of colec11 MO. Higher doses give rise to pronephric cysts, curved body axis, and cardiac oedema not present at 3 ng doses. A dose dependent loss of medial trunk pigmentation (insets – 1.5x magnification ) was observed (scale bar is 500 μm) b. Alcian blue cartilage staining in colec11 morphants (3 and 4 ng) at 5 dpf showing generalised cartilage defects (scale bar: 200 μm) c. Palate measurements in the colec11 morphants (3 ng) demonstrate shortening of length and width of the ethmoid plate, abnormal Meckel's cartilage (mc) and abnormal angulation of the ceratohyal cartilage (ch). Scale bar: 200 μm. d. Graph of ethmoid plate length and width measurements expressed as a ratio against cranial length and width, respectively, in the presence or absence of colec11 morpholino. There is clear evidence of significant morphological effects on both length and width, indicating shortening in both axes. (con length 3.164 ± 0.062 N=8, colec11 MO length 3.702 ± 0.245 N=8, con width 2.563 ± 0.0441 N=8, colec11 MO width 2.819 ± 0.168 N=8, each value indicates the mean ± SEM). e. Alcian blue cartilage staining of colec11 morphants rescued with human RNA COLEC11 (75 pg) compared to colec11 morphants (3ng), control uninjected and COLEC11 RNA injected alone embryos (scale bar: 200 μm). pq: palatoquadrate, mc: Meckel's cartilage. f. Graph showing the length ratio of both pq cartilages plus mc standardised over the cranial length. Con (0.821 ± 0.004, n=51), colec11 MO (0.896 ± 0.009, n=50), COLEC11 RNA (0.844 ± 0.007, n=21), colec11 MO plus COLEC11 RNA (0.820 ± 0.014, n=16). Values shown as mean ± SEM. Data was analysed by one way ANOVA with Tukey's Multiple Comparison Test comparing column data against control measurements. Morphants have a significantly shorter pq+mc length compared to uninjected controls (p<0.001), whilst no significant (ns) difference was observed in controls versus colec11MO plus COLEC11 RNA or versus COLEC11 RNA injected alone.Key angles and measures are indicated to illustrate cartilage defects. ep = ethmoid plate, t=trabeculae, n=notochord, bp=basal plate, pq=palatoquadrate, cb=ceratobranchials
Mentions: As nothing is known of the specific role of COLEC11 during embryonic development and in the absence of an available mouse model, we demonstrated expression in zebrafish embryos in the pronephric duct, lateral hindbrain and liver (Fig 2d). Next we sought to determine the effects of loss of function of this protein during zebrafish embryogenesis. Two antisense morpholinos were designed; one directed against the initiation site (colec11-ATG-MO) and another against the exon 2-intron 2 splice site (colec11-SPL-MO)(Suppl Fig 7). Injection of both MOs from 1 to 8 ng doses at the one-cell stage gave rise to morphological abnormalities which could be reversed upon co-injection with full-length COLEC11 mRNA (Fig 3e,f). This effect was dose-dependent including, for higher doses (4 ng), heart edema, pronephric cyst formation, curved body axis, disorganised pigment distribution and high mortality (Fig 3a). To ensure that these phenotypes were not the result of morpholino off-target effects (e.g up regulation of p53), we co-injected p53 morpholino with colec11 MO (4 ng) but could not rescue the phenotypes (Suppl Fig 8 and not shown).

Bottom Line: Zebrafish morphants for either gene develop pigmentary defects and severe craniofacial abnormalities.Finally, we show that CL-K1 serves as a guidance cue for neural crest cell migration.Together, these findings demonstrate a role for complement pathway factors in fundamental developmental processes and in the etiology of 3MC syndrome.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Unit, University College London Institute of Child Health, London, UK.

ABSTRACT
3MC syndrome has been proposed as a unifying term encompassing the overlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. These rare autosomal recessive disorders exhibit a spectrum of developmental features, including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability and genital, limb and vesicorenal anomalies. Here we studied 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP1, both of which encode proteins in the lectin complement pathway (collectin kidney 1 (CL-K1) and MASP-1 and MASP-3, respectively). CL-K1 is highly expressed in embryonic murine craniofacial cartilage, heart, bronchi, kidney and vertebral bodies. Zebrafish morphants for either gene develop pigmentary defects and severe craniofacial abnormalities. Finally, we show that CL-K1 serves as a guidance cue for neural crest cell migration. Together, these findings demonstrate a role for complement pathway factors in fundamental developmental processes and in the etiology of 3MC syndrome.

Show MeSH
Related in: MedlinePlus