Mutations in axonemal dynein assembly factor DNAAF3 cause primary

Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia. Hannah M. Mitchison, Miriam Schmidts, Niki T. Loges, Judy Freshour, Athina Dritsoula, Rob A. Hirst, Christopher O’Callaghan, Hannah Blau, Maha Al Dabbagh, Heike Olbrich, Philip L. Beales, Toshiki Yagi, Huda Mussaffi, Eddie M.K. Chung, Heymut Omran, and David R. Mitchell

Supplementary Table 1. SNP haplotypes in PCD families across the DNAAF3/C19orf51 locus. Homozygous markers in affected individuals are highlighted in orange. The position of the DNAAF3 gene is indicated, with the homozygous mutations in all affected individuals and heterozygous changes in all unaffected parents and siblings shown.

Nature Genetics: doi:10.1038/ng.1106

Supplementary Table 2. Clinical features of patients with DNAAF3 mutations. ID

Nationality

CF/IE/TB excluded

Situs inversus

sinusitis

Bronchiectasis

UCL71 IV.1

UKPakistani

N/A

-

-

+

UCL71 IV.2

UKPakistani

N/A

+

-

+

UCL66/67 III.1

Saudi Arabia

+

+

+

+

UCL66/67 III.2

Saudi Arabia

N/A

+

+

N/A

UCL66/67 IV.1

Saudi Arabia

N/A

-

+

+

UCL66/67 IV.2

Saudi Arabia

N/A

-

+

-

UCL89 IV.2

Israeli Arabic

+

-

+

+ (bilateral)

UCL89 IV.3

Israeli Arabic

+

+

+

+ (bilateral)

UCL89 IV.4

Israeli Arabic

+

-

+

+ (bilateral)

UCL89 IV.5

Israeli Arabic

+

-

+

+ (bilateral)


Other documented symptoms Neonatal resp. distress, chronic cough, recurrent chest infections Chronic cough, recurrent chest infections, nasal polyps Chronic cough, recurrent chest infections Neonatal respiratory distress, chronic cough, recurrent chest infections, hearing loss, finger clubbing, seizures. Chronic cough, recurrent chest infections, otitis media, hearing impairment, bronchiolitis Chronic cough, recurrent chest infections Severe pulmonary disease, otitis media, infertility (immotile sperm) Severe pulmonary disease, otitis media Severe pulmonary disease, recurrent haemoptysis Severe pulmonary disease, otitis media

Ciliary defect

Dysmotility

N/A

+

N/A

+

N/A

+

N/A

N/A

N/A

+

N/A

+

ODA + IDA

+

ODA + IDA

+

ODA + IDA

+

ODA + IDA

+

Supplementary Table 3 Oligonucleotide sequences used for detection of human mutations, knockdown of dnaaf3 expression in zebrafish, analysis of splice blocking efficiency, and fish in situ hybridization. Primer

Forward sequence

Reverse sequence

UCL89 exon 3

5'-TGCAGGCTGAAAGTGAGTCC-3

5’-CTCAGGTCCAGGAATGAAGG-3’

UCL71 exon 5

5'-TGGGATGGAAGAGAGGGTCA-3'

5’-GTCTGAGGGAGGCTGGTGTA-3’

dnaaf3 ex3 MO

5’-GTCATGTTAGGATACGTACTGTGCA-3’

dnaaf3 ex8 MO

5’-TTCTGTGGATTGACAGCAGGAACTG-3’

dnaaf3 5'UTR MO dnaaf3 exons 2 and 5 dnaaf3 exons 7 and 10 gapdh

5'-AGTAATCAGCAAATTGTCTGCGTGT-3'

dnaaf3 in situ hybrid. probe

5’-CACACACAAAGTGAGGCTACAA-3’

5’-TCATGTTGAGATCCCAGTCG-3’

5’-ATCTGTCCTTCGGCATTG-3’

5’-GAGCAGAATCCAGCTGATGA-3’

5′-TTAAGGCAGAAGGCGGCAAA-3′

5′-AAGGAGCCAGGCAGTTGGTG-3’

5’-GCTGGTCGGACTTTTGAGG-3’

5’-TCATGTTGAGATCCCAGTCG-3’


Supplementary Figure 1. Cloning the Chlamydomonas DAB1 gene. Top, diagram showing the location of three AFLP probes on scaffold 53 of the Chlamydomonas genome v3.1 (arrows). Beneath each probe is recorded the number of recombinants seen between that probe and the pf22 locus in genomic samples from 68 random zygotic products from a cross between pf22 and AFLP strain S1D2. Middle, location on scaffold 53 of BAC clones that were tested for their ability to rescue the pf22 phenotype. Beneath each is shown the number of rescued colonies and the total number of co-transformants examined. Only BAC ptq14096 rescued the pf22 strain back to wild type motility. Bottom, a map of coding exons for the DAB1 gene in a 10 kb restriction fragment subcloned from BAC ptq14096 to form plasmid pBPF22SE.


Supplementary Figure 2. Location of the mutations in Chlamydomonas pf22 and in human DNAAF3 PCD patients. A multiple sequence alignment of PF22/DNAAF3 homologs is shown that includes seven of the species included in the dendrogram in Figure 1 B. The nonsense mutation in Chlamydomonas pf22 occurs at Trp79 (green); mutations in the human patients (red) include a miss-sense at Leu108, a nonsense at Arg136, and a frameshift at Val255. Accession numbers for all sequences used in this alignment are reported in the legend to Figure 1.


Supplementary Figure 3. Disruption of DNAH9 assembly in airway cilia of patients with DNAAF3 mutations. Nasal respiratory epithelial cells labeled for acetylated anti-alpha tubulin to label the entire ciliary axoneme (green) and DNAH9 (red) show that DNAH9 localizes only to the distal portion of the cilia in cells from an unaffected control (upper panels), highlighting the distal type 2 ODAs defined in Fliegauf et al. 200540. DNAH9 is absent from the cilia of two affected individuals from family UCL89 (IV.3, middle panels, and IV.4, bottom panels), demonstrating the disruption of distal type 2 dynein arm assembly in airway cilia of these patients. Scale bar, 10 μm.


Supplementary Figure 4. Disruption of DNAI2 assembly in airway cilia of patients with DNAAF3 mutations. Cells double-labeled for anti-alpha/beta tubulin to label the ciliary axoneme (red) and DNAI2 (green) show that both proteins co-localize along the axoneme of cilia in cells from an unaffected control (upper panels), but DNAI2 is absent from the cilia of two affected individuals from family UCL89 (IV.3, middle panels, and IV.4, bottom panels), demonstrating the disruption of outer dynein arm assembly in airway cilia of these patients. Scale bar, 10 μm.



Supplementary Figure 5. Morpholino knock-down and in situ hybridization of dnaaf3 confirm the function of dnaaf3 in cells with motile cilia. (a) Representative images of dnaaf3MOex8 embryos at 3 dpf show variable otolith numbers ranging from 13, while wildtype fish display the normal pattern of two otoliths. (b) RT-PCR of cDNA extracted from morphant fish to confirm effects of morpholinos on dnaaf3 gene expression. To assess the dnaaf3 MO ex3 morpholino, primers were designed in exon 2 and exon 5. Wildtype fish (WT) have the expected RTPCR product of ~450bp (lane 3) but morphant embryos had no product (lane 4), possibly reflecting an inability to amplify a large PCR product resulting from a large intronic insertion within the cDNA of all or part of intron 3 which is 2,282 bp in size. GAPDH RT-PCR (lane 1, 2) confirmed an equivalent amplification of this housekeeping gene in the morphants compared to controls suggesting dnaaf3 gene expression is disrupted. (c) In situ hybridization of wildtype embryos with a dnaaf3-specific antisense probe showed expression (arrows) in ciliated cells of the spinal cord floor plate (at 36 hpf) and the olfactory placodes, pronephros and lateral line organs (at 4.5 dpf). The control sense probe gave no signal.


a

b Heavy Chain Abundance

0.8

WT

WT

WT

pf22

pf22

0.9

oda7

0.4

pf13

0.5

oda7

0.6

0.3

0.7 0.6 0.5

pf22oda7

Relative Abundance

0.8

0.7

pf13

0.4 0.3

oda7

0.1

oda7

0.2

0.2

0.1 0

0 alpha

beta

alpha

gamma

c

d

e

HC alpha trypsin sensitivity

HC beta trypsin sensitivity 120%

100%

oda9

80%

pf22

60%

pf13

40%

oda7

20% 0%

% full length remaining


120%

HC gamma trypsin sensitivity 120%

100%


Relative Abundance

1

pf22

0.9

1.1

pf22

1

pf13

WT

1.1

HC abundance

oda9

80%

pf22

60%

pf13

40%

oda7

20%

100%

1

2

3

4

5

6

pf22

60%

pf13 40%

oda7

20% 0%

0% 0

oda9

80%

0

1

Trypsin trt

2

3

4

5

0

6

1

trypsin trt

f

2

3

4

5

6

trypsin trt

g HC alpha

HC beta

2

1.6

1.8

1.4

ODA16

1.4

PF22

1.2

PF13 ODA7

1

Expon. (ODA7)

0.8

Expon. (PF22)

0.6

Expon. (PF13) Expon. (ODA16)

0.4

1.2 Fraction remaining

Fraction remaining

1.6

oda16 1

pf22 pf13

0.8

Expon. (pf13) Expon. (pf22)

0.6

Expon. (oda16) 0.4 0.2

0.2 0

0

0

10

20 Time (hrs)

30

0

10

20

30

Time (hr)

Supplementary Figure 6. Densitometric quantification of dynein heavy chain abundance from western blot films. (a) Heavy chain abundance in cytoplasmic extracts of the indicated strains (from the blots in Fig. 6 a). (b) Relative abundance of HCα in extracts of wild type, oda7, pf22, and a pf22oda7 double mutant (from the blots in Fig. 6b). Abundance in the double mutant is intermediate between that in each single mutant. (ce) Decrease in full length heavy chain abundance following trypsin treatment (from the blots in Fig. 6 ce). Arbitrary numbers 1-5 on the X axis correspond to trypsin concentrations of 0, 1, 3, 10 and 30 μg/ml. (f, g) Changes in heavy chain abundance during cycloheximide treatment (from the blots in Fig. 6f). Exponential fits to the data are also shown. All data normalized to the density of each heavy chain in wild type cytoplasm (a, b), at zero μg/ml trypsin (c-e) or at time zero (f-g). Nature Genetics: doi:10.1038/ng.1106

Supplementary Movie 1. Wild type Chlamydomonas cells swimming under darkfield illumination. Most cells swim progressively. Occasional stationary cells have adhered to the glass surface through flagellar contact. Images were captured and displayed at 30 fps. Supplementary Movie 2. Mutant Chlamydomonas strain pf22 cells fail to swim. Most cells are nonadherent but remain stationary due to lack of flagellar motility. Images were captured and displayed at 30 fps. Supplementary Movie 3. Wild type swimming of the Chlamydomonas pf22 strain expressing a Myctagged PF22 protein. Most cells have full length flagella and swim progressively with a swimming pattern and velocity similar to wild type. Occasional stationary cells are adhering to the glass surface by their flagella. Images were captured and displayed at 30 fps. Supplementary Movie 4. Olfactory placode cilia in dnaaf3MOex8 zebrafish embryo. Supplementary Movie 5. Olfactory placode cilia in wildtype zebrafish embryo. Supplementary Movie 6. Spinal cord canal cilia in dnaaf3MOex8 zebrafish embryo. Supplementary Movie 7. Spinal cord canal cilia in wildtype zebrafish embryo.
