MUTATION ANALYSIS OF INDONESIAN MENTALLY RETARDED INDIVIDUALS WITH MICROCEPHALY AND MACROCEPHALY - Diponegoro University | Institutional Repository (UNDIP-IR)
68
Appendix I. DNA isolation according to the Manual of Center of Biomedical
Research (Faradz, 2004)
Salt saturation method was performed. EDTA frozen blood was transferred into a
50 ml tube with the addition of NH4Cl 5-10 ml lysis buffer. The tube was
incubated for 10 30 minutes at room temperature. Afterwards the tube was
centrifuged for 5 minutes at 3000-3500 RPM, followed by removal of the
supernatant and addition of NH4Cl lysis buffer. The procedure was repeated three
times. TE lysis buffer 2 ul, Proteinase-K 10mg/ml and 100 ul 10% SDS were
added and mixed until white pellet was formed. The tube was then incubated at
50oC for 24 hours. After incubation NaCl 6M approximately one third volume of
the tube was added to the suspension and centrifuged at 4000 RPM for 10
minutes. The supernatant was moved to a new tube and absolute ethanol twice the
volume of the supernatant was added. The DNA formed white viscous thread and
could be removed by fine needle, rinsed with 70% ethanol and moved to a new
1,5 ml tube. The new tube was left open to allow evaporation of ethanol. The tube
was then filled with TE buffer for dissolution of the DNA. (Faradz, 2004)
69
Appendix II. The Nellhaus charts
70
Appendix III. List of primers
MCPH1
NM_024596.3
EXON
01
01
02
02
03
03
04
04
05
05
06
06
07
07
08_01
08_01
08_02
08_02
08_03
08_03
09
09
10
10
11
11
12
12
13
13
14
14
F/R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
SEQUENCE
tgtaaaacgacggccagtcctacagagaaatcccggaaa
caggaaacagctatgaccggtgccggtcctcaaac
tgtaaaacgacggccagtaaatctattgggcaggggat
caggaaacagctatgaccccattgcttcgtccagattc
tgtaaaacgacggccagtcagaattgctggggtagagg
caggaaacagctatgaccaaaagatgaaaatcaaactttatctgg
tgtaaaacgacggccagttgtgcagatttagtgctgtgtc
caggaaacagctatgacccatacaaatgctatcgaagaagtca
tgtaaaacgacggccagtgaaatgtatgcgaaagggct
caggaaacagctatgaccgcaaattaaacaaggtgtatgga
tgtaaaacgacggccagtcattcctgaagtatgaaggca
caggaaacagctatgaccccaccataattgaatcggct
tgtaaaacgacggccagtcaagttgactttaagatcccttct
caggaaacagctatgacccagggggaaatttagaagttctag
tgtaaaacgacggccagtgaaatgagaagaactcaagtgtggt
caggaaacagctatgacccctcggcatgatagatctcc
tgtaaaacgacggccagtatcttcaacaaaaggccacc
caggaaacagctatgacccagaagtcacgcaactcgaa
tgtaaaacgacggccagtttcctgcgttggcaaaa
caggaaacagctatgaccatggatgcactgttacatgttt
tgtaaaacgacggccagtttgcttaagttgtatttggtccat
caggaaacagctatgacctgtttcattgacccagagaag
tgtaaaacgacggccagttgagtttctatctcttggcctg
caggaaacagctatgaccgcctaaaggcacccagaatta
tgtaaaacgacggccagtaactgctttgatgggcatgt
caggaaacagctatgaccacctcagggtgacccactc
tgtaaaacgacggccagtggtttattgcctgctaaggct
caggaaacagctatgacctgggcataattgtgcttgac
tgtaaaacgacggccagttcgcctacgctatggagact
caggaaacagctatgaccacatcaacccaggtgccc
tgtaaaacgacggccagtacagctcttggctatttgttttta
caggaaacagctatgacccatttgaacatccaaagacagttt
71
CDK5RAP2
NM_018249.4
EXON
01
01
02
02
03
03
04
04
05
05
06
06
07
07
08
08
09
09
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
23
23
24_01
24_01
F/R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
SEQUENCE
tgtaaaacgacggccagtagtctgaacaagagctgggg
caggaaacagctatgaccgacaccctcaagagcaaacc
tgtaaaacgacggccagtgtgcagtccagaccgtctta
caggaaacagctatgacctcacagtccaatgtctactttcc
tgtaaaacgacggccagtgaacactgggcagtttgcat
caggaaacagctatgaccagaaacacagctttcctggg
tgtaaaacgacggccagttgttggaccctctgtcttttg
caggaaacagctatgaccaatcaaatttctgcttgaaatga
tgtaaaacgacggccagtttccccattgttgacattcat
caggaaacagctatgacctttcactgaccaacccagtg
tgtaaaacgacggccagttggggctgtgaaataaccat
caggaaacagctatgaccgaaacaaaactgtgccagcc
tgtaaaacgacggccagttgtgctggagaagaagtcataa
caggaaacagctatgacccccacgctgccagataat
tgtaaaacgacggccagttttccttcctcctccagctc
caggaaacagctatgaccttccatctcccacagaggag
tgtaaaacgacggccagttgttctggagaagctgttgg
caggaaacagctatgaccaaaacaagaggcaagaataggc
tgtaaaacgacggccagttatggtgcattttggttgga
caggaaacagctatgaccattcagggtaggacaggcca
tgtaaaacgacggccagtgaaaagcatgtgggctgtg
caggaaacagctatgaccaaagtcctcacctcaccagg
tgtaaaacgacggccagtagagcccaggtttcttggtt
caggaaacagctatgaccagccacatagccccaaaag
tgtaaaacgacggccagtagcagtaatttagcaagtcaaaca
caggaaacagctatgaccaagcccaaatgacataattaaagctct
tgtaaaacgacggccagtccttaaggagtttgggacagttt
caggaaacagctatgacccaacaagaaggcatttgagga
tgtaaaacgacggccagtggaagcagtattggaagctc
caggaaacagctatgaccaaagggtgtgtgcgtgtatg
tgtaaaacgacggccagtgacataaaaatcacaggaaggc
caggaaacagctatgaccatttaggactctcctgaagttc
tgtaaaacgacggccagtttgggaggatctgtttctga
caggaaacagctatgaccggaacccaagatacaacaaaca
tgtaaaacgacggccagtgggaagctgaagtctcttgc
caggaaacagctatgaccccaagccggttgttatatttt
tgtaaaacgacggccagtttcatacatattcatgctgcacac
caggaaacagctatgaccvtccagactcgaagactgatttg
tgtaaaacgacggccagtcatctccatcctctctccaca
caggaaacagctatgaccgcaaaatgagtttgttctccc
tgtaaaacgacggccagttaggggacaaggcctagca
caggaaacagctatgaccagtggtgagattaaaggtgatttt
tgtaaaacgacggccagttgactgtgcaaccaaaggaa
caggaaacagctatgaccgtttgtctagctcacagggaa
tgtaaaacgacggccagttgcttggctaagtttcaggtc
caggaaacagctatgaccaaaacatccaaatggtgcct
tgtaaaacgacggccagtggctgtgaggaaggagagag
caggaaacagctatgacctcagaaccattcttgggctt
72
24_02
24_02
25
25
26
26
27
27
28
28
29
29
30
30
31
31
32
32
33
33
34
34
35
35
36
36
37
37
38
38
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
tgtaaaacgacggccagttggaaggctaccagaatttca
caggaaacagctatgaccacctagtggcaatactgggg
tgtaaaacgacggccagttttccaattagtgttaaggtcacaa
caggaaacagctatgacctccaagaagtcctgggtcaa
tgtaaaacgacggccagttgagcaggaaattattagggaa
caggaaacagctatgaccgctggcaggtaaatcagacc
tgtaaaacgacggccagtgtcatagtcctggcttggga
caggaaacagctatgacctgcttactaaggttaagatggcaa
tgtaaaacgacggccagtaggatccctgcataatccat
caggaaacagctatgacccgtcacagtgaagcactcaca
tgtaaaacgacggccagtgaaagttctgtctatactgatctggag
caggaaacagctatgaccaatgcctgcattctttccat
tgtaaaacgacggccagtttttgtattcttgaagtgctgtattt
caggaaacagctatgaccgtgctgattccacgactgc
tgtaaaacgacggccagttgctctgtggggtttgaata
caggaaacagctatgaccagctgtcgggtggtcttaca
tgtaaaacgacggccagtgagttccttcgatggctgac
caggaaacagctatgacctgtcacacacagatgctcaga
tgtaaaacgacggccagtaatgtgaggggcttgactgt
caggaaacagctatgaccaaataccctcctgagttggga
tgtaaaacgacggccagtaagcgtcttgaacgcagagt
caggaaacagctatgacctfcgccatttgactcccc
tgtaaaacgacggccagtgctttcgctcagcgtgtga
caggaaacagctatgaccaaactgagacacaggcccc
tgtaaaacgacggccagttggctttgtaattcttcctgtg
caggaaacagctatgaccttacaaaccatctgggctcc
tgtaaaacgacggccagttttcacacagctctcctggat
caggaaacagctatgaccttcaagaggtcctttctggc
tgtaaaacgacggccagtcacctctgtcggggtcttac
caggaaacagctatgacctggaacaaagagacagcgtg
73
STIL
NM_003035.2
EXON
02
02
03
03
04
04
05
05
06
06
07
08
08
09
09
10
10
11
11
12_01
12_01
12_02
13
13
14
14
15
15
16
16
17_01
17_01
17_02
17_02
F/R
F
R
F
R
F
R
F
R
F
R
F
F
R
F
R
F
R
F
R
F
R
F
F
R
F
R
F
R
F
R
F
R
F
R
SEQUENCE
tgtaaaacgacggccagtttttgcctgtaattttaatatgtgg
caggaaacagctatgacccttccaacctaccttcccaa
tgtaaaacgacggccagtccaaatgtctaccaagtttgc
caggaaacagctatgaccgtaatcaactttgccttatacatcttt
tgtaaaacgacggccagtcctccgtatcccctgcct
caggaaacagctatgaccctccagcctcccaacgtg
tgtaaaacgacggccagtttgctataatgtagtcttgcttgg
caggaaacagctatgaccggttcacaataattcaaccaaat
tgtaaaacgacggccagtgagaactgaaatgctgtaaatcaga
caggaaacagctatgaccagccaccatatctctggcat
tgtaaaacgacggccagttgtgtgcaagacaacgttca
tgtaaaacgacggccagttggctgataaaagccatacg
caggaaacagctatgaccaaggttacggcattacatgaattt
tgtaaaacgacggccagtttgggagactcacatgaagtt
caggaaacagctatgacctgccaaagtgcaaaacccta
tgtaaaacgacggccagtaatcagaagtctgccactcca
caggaaacagctatgaccggaatgcatttaaaacaaagacat
tgtaaaacgacggccagtggtgcccagtgacttgattt
caggaaacagctatgaccccaatctatgtctcagtttactctagg
tgtaaaacgacggccagttgggaaatactggtctgagg
caggaaacagctatgaccacagtaagatggcagtgggg
tgtaaaacgacggccagtagtcttctcttgccccgc
tgtaaaacgacggccagtactgctgccagttttgttga
caggaaacagctatgaccctactgcaccatgcaccaat
tgtaaaacgacggccagtcattgcacccagccaatatg
caggaaacagctatgaccatctaaacattgtgccccac
tgtaaaacgacggccagtaccaggtgctgcttgagtct
caggaaacagctatgaccggtctcaatcagtttaaacctaggaa
tgtaaaacgacggccagtgggattataggtgtgaac
caggaaacagctatgaccagccaatgatgggcaaaa
tgtaaaacgacggccagttgcttgtatagaatggaacacattg
caggaaacagctatgacctcttagaaggctctttctgacc
tgtaaaacgacggccagtaagaggaacctcccgaca
caggaaacagctatgaccggctcctgttttccctaagta
74
CENPJ
NM_018451.3
EXON
03
02
03
04
04
05
05
02
06
06
07_01
07_01
07_02
07_02
07_03
07_03
07_04
07_04
08
08
09
09
10
10
11
11
16
16
17
17
12_13
12_13
14_15
14_15
F/R
F
R
R
F
R
F
R
F
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
R
F
F
R
SEQUENCE
tgtaaaacgacggccagtgaggtgtccatgaagaattgtaaa
caggaaacagctatgacccaagaagacctagaacaaatgacttta
caggaaacagctatgaccggaacgatagaaataacgtgtcaa
tgtaaaacgacggccagtgagaaaatgttgaaggaagt
caggaaacagctatgaccaaaaaacatggaaaggcttc
tgtaaaacgacggccagtgcttttcttgagctaaattg
caggaaacagctatgaccataaaggctagctattgtac
tgtaaaacgacggccagtgctttacctaatacattttgttggcta
tgtaaaacgacggccagttccatgttgtcaagggtttct
caggaaacagctatgaccttttggtatatccaagtgatctcaa
tgtaaaacgacggccagtggtcagcaattgtgctcttct
caggaaacagctatgacccagccagtatcgcaaggttt
tgtaaaacgacggccagtacagacggggaagaaatgtg
caggaaacagctatgacccttcacactcactctccttc
tgtaaaacgacggccagtcggatgtcttccacccctgt
caggaaacagctatgaccgatctcgagggctcagacac
tgtaaaacgacggccagttaaggacaccaggacccaag
caggaaacagctatgaccttgagcaataaggaaagttagga
tgtaaaacgacggccagtttttgctgcagtaatgcct
caggaaacagctatgacccagactgtttatagatggtcttaaagg
tgtaaaacgacggccagtcagcaggttctgcacatcac
caggaaacagctatgaccgatgcttacaagattgtcaccttta
tgtaaaacgacggccagtcattgctgggtctctattcttc
caggaaacagctatgacctgctcagatattgaagctccc
tgtaaaacgacggccagtaacccacagcattcttagcac
caggaaacagctatgaccagggtagaaattaactggcgg
tgtaaaacgacggccagtggtacaaacttgctccaccc
caggaaacagctatgaccgagcacagatgaacaggtgtc
tgtaaaacgacggccagtccaagggatgtctccagaag
caggaaacagctatgacctgccacagggtaaacttttattt
caggaaacagctatgaccgcatcttgtccaaagagccc
tgtaaaacgacggccagtaaaggacagcagttcacagga
tgtaaaacgacggccagtgtaggcagttgggaggagaa
caggaaacagctatgaccttcttccatctgggtaatgtttt
75
ASPM
NM_018136.4
EXON
01
01
02
02
03_01
03_01
03_02
03_02
03_03
03_03
03_04
03_04
04
04
05
05
06
06
07
07
08
08
09
09
10
10
11-12
11-12
13
13
14
14
15-16
15-16
17
17
18_01
18_01
18_02
18_02
18_03
18_03
18_04
18_04
18_05
18_05
18_06
18_06
DIR
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
SEQUENCE
tgtaaaacgacggccagtaagcggtcagcgtaagtcc
caggaaacagctatgacctctccaatcgtcaaccttcc
tgtaaaacgacggccagtttcttataattaagcagatagggtagg
caggaaacagctatgacctggtatcccaaagactcctctg
tgtaaaacgacggccagttgttctgaaacaccaagctatattac
caggaaacagctatgacctcaaagttgaagaacagttggg
tgtaaaacgacggccagtcttcatgcatcagaaaataggg
caggaaacagctatgacctttgataatggtactttacatgtttgc
tgtaaaacgacggccagtcagcctgtgcatttggaatc
caggaaacagctatgaccgaagctgttgtcgaagaggg
tgtaaaacgacggccagtaaaggaaggccacctgtacc
caggaaacagctatgaccaaaccaaaaggaagtatcccc
tgtaaaacgacggccagtgaatatgattgtgaagaacccaaac
caggaaacagctatgaccttcttccaggctgttattcaac
tgtaaaacgacggccagtttgttcagtgttttaaagatggtattg
caggaaacagctatgaccgctaatgaacagggaattatgc
tgtaaaacgacggccagttgaaattgcattttattgctgg
caggaaacagctatgaccatatgtcaataaagccgggg
tgtaaaacgacggccagtcatgctttagctttgctgcc
caggaaacagctatgaccatggcatagtctatttacctaataagc
tgtaaaacgacggccagttcctcagtcacttccctttg
caggaaacagctatgaccaaacaggaagaatgacaataagcc
tgtaaaacgacggccagttttgtgcttgctaccctacac
caggaaacagctatgaccgcaagcaaaagtcgtaaatgg
tgtaaaacgacggccagtcagaatgatttggaggatttg
caggaaacagctatgaccaaaagtgttttccagaaaatgttagtc
tgtaaaacgacggccagtaactgttgggatttatgtggg
caggaaacagctatgaccaaatgatggttgttgttgttattc
tgtaaaacgacggccagttccatttcaggcactttattttc
caggaaacagctatgacctttgagggaaagtttgcttacac
tgtaaaacgacggccagtaacttgccattacttgccttg
caggaaacagctatgaccgaggaaaggagaaattagccg
tgtaaaacgacggccagtgatcatggttagtcccttttgg
caggaaacagctatgaccaatttatcttaataatgccatacatcc
tgtaaaacgacggccagttgtaggggtgttttatttccag
caggaaacagctatgaccaaacttcatcacattttgccttc
tgtaaaacgacggccagtggatggatttctgaattggc
caggaaacagctatgaccttctgcaaatagttggtgcg
tgtaaaacgacggccagtgcaaagagcttttagagaatgg
caggaaacagctatgaccgactgcagcacaatgacagc
tgtaaaacgacggccagttcaaatttcaggcacatgtaag
caggaaacagctatgaccaatactgccgagcctttctc
tgtaaaacgacggccagttgcacaaaagagagaagagtatatg
caggaaacagctatgaccttcaaggcagcttgatgttc
tgtaaaacgacggccagtggtacagggcgtacaagactc
caggaaacagctatgaccttcataccacgataagctgactg
tgtaaaacgacggccagtcaaaggcaacataaatgtgctatc
caggaaacagctatgaccaaattgtcaggtacttttcacgc
76
18_07
18_07
18_08
18_08
18_09
18_09
18_10
18_10
18_11
18_11
18_12
18_12
19
19
20
20
21
21
22
22
23
23
24
24
25
25
26
26
27
27
28
28
25
25
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
tgtaaaacgacggccagtgccatgtttaaaatgcatcag
caggaaacagctatgacctgtgcccgatattttctctg
tgtaaaacgacggccagtaatgatgcatatagccgcaac
caggaaacagctatgacctggtggctcgatatttcctc
tgtaaaacgacggccagtaggctgcattcaggggtatg
caggaaacagctatgaccctgcctatacattctgtaggtgc
tgtaaaacgacggccagtaagagaaaattatatcagacaatggc
caggaaacagctatgaccctgaattagtgtggcagccc
tgtaaaacgacggccagtaaaataaggaagcattatctccac
caggaaacagctatgaccgaactgaatccgtagggcag
tgtaaaacgacggccagtaagctgttcaaagtgaaggtg
caggaaacagctatgacctgacctgcttaagctcattttatac
tgtaaaacgacggccagtttttgtctgcactaaccttattttaac
caggaaacagctatgaccaaagcaagacagtacgagagatg
tgtaaaacgacggccagttgcgtgtgtaaattctgattg
caggaaacagctatgacctgtgtgaaataaatgcatacttaggtc
tgtaaaacgacggccagtgaacttctagtgggttggaaatc
caggaaacagctatgaccttcttaacactatttaacatcaagtgc
tgtaaaacgacggccagtccttctctaatagggcagtctagttg
caggaaacagctatgaccctcaagactttgctggcagg
tgtaaaacgacggccagtggggaagtaatgcctctgtg
caggaaacagctatgaccaaagagcttagcaatgaaattatgg
tgtaaaacgacggccagttttggtcgataaatgctgtcc
caggaaacagctatgaccagaaattgctccactctggg
tgtaaaacgacggccagtgaggccttaaaacacctaggttag
caggaaacagctatgacccaaacttaatttgcaggggc
tgtaaaacgacggccagtttggttgggttgtttgtaaatg
caggaaacagctatgacccaggtttgaacacacataaaacc
tgtaaaacgacggccagtgcgacagagcaagagagacc
caggaaacagctatgacctttctccactgaaaagcacatc
tgtaaaacgacggccagtatgaccaaaaggcagtggtc
caggaaacagctatgacctgccattgatatgaatttgtgag
tgtaaaacgacggccagtcctttctgccattcttgagg
caggaaacagctatgaccagagatttaagccctagtgatgag
77
WDR62
NM_001083961
EXON 1
EXON 2
EXON 3
EXON 4-5
EXON 6
EXON 7
EXON 8
EXON 9
EXON 10
EXON 11
EXON 12
EXON 13
EXON 14-15
EXON 16
EXON 17-18
EXON 19
EXON 20
EXON 21
EXON 22
EXON 23
DIR
sequence
F
gctgttcgctgttccagtg
R
gggccacagttctcagga
F
tgggtgttgaatgtagcagg
R
gaggctcagagcgatttacg
F
agaaccgagggagcttgttt
R
tatctccaggctgtgtaggg
F
gcagaagagggcagagtcct
R
catgtagctctccctgtgcc
F
gacgagcagggagttccag
R
ccctcaaatctcagggagaa
F
agtgttttcttcccttctcca
R
acctgtgtggtcaatgctga
F
ctctggaggcctaaggctg
R
agatggatctggaagcatgg
F
gatggatggccacaaatacc
R
tgatgcataggagggagtca
F
ttgcacgatacctgtttatcttt
R
ctctcctgcccctgcat
F
agagactggctatggaggga
R
cagggcttctgcccact
F
cctggccattctgacctatt
R
caccagacaccaactacccc
F
gaaccaaagaccccttctcc
R
cacagatactgtattcacttgtttgg
F
ttgattgatggctcttgcag
R
accagaggaacagagggagg
F
ggaggacagcaagagcca
R
gtgcactgaggaaggaccaa
F
agtcccatgtgctgtgcat
R
tgacaccaagactcccacaa
F
ctgtggtgtgggtggctt
R
ctgtcttggccaccactgt
F
acatggagcttagcacaggg
R
tatgtgccaggccctgtaaa
F
gttgcctctttggggactg
R
agggaaaagagggacatgga
F
agagtccagatgggctgtgt
R
tggagtgggagtcagcctaa
F
gttcacagcctccagcagt
R
ggcagagagcacttcttcca
78
EXON 24
EXON 25-26
EXON 27-29
EXON 30_1
EXON 30_2
EXON 31
EXON 32
F
gtacaggtgcctaggggctc
R
gcttcagtactgggtcaaagtca
F
taccatccctcctccagatg
R
ggagccaactccagggac
F
gggtttctggggagtgc
R
acgccccacagctaggc
F
gatgtccagcctagctgtgg
R
cagggtcagtctcaggttgg
F
gcttcaggccatcaccac
R
gaatgaatggcacagtccct
F
agtacagcatggagtccaccc
R
gactgagctggtctccacctt
F
ctaagccccagagttggga
R
caggaaccattcctcaaagc
79
Appendix IV. List of Single Nucleotide Polymorphisms
The list of Single Nucleotide Polimorphism (SNP) of ASPM gene among children
with microcephaly
rs Number
SNP
n
Cohort
Present
European %
Total
study %
43
48
89.6
95.8
rs1571964 c.441+14C>T
29
48
60.4
91.2
rs6677082 c.849C>T
43
48
89.6
6.8
rs4915337 c.3579T>A
48
93.8
99.1
c.7480T>C
45
rs964201
c.7566A>G
46
48
95.8
91.2
rs1412640
42
48
87.5
14.3
rs10922162 c.7605G>A
48
91.7
91.6
rs10754213 c.10331+8A>G 44
8
48
16.7
18.8
rs4915344 c.2174-20T>C
c.3138G>A
7
48
14.6
38.3
rs6676084
10
48
20.8
38.9
rs2878749 c.4449A>G
10
48
20.8
NA
rs41310925 c.5961A>G
9
48
18.8
NA
rs41308365 c.7674C>T
c.7684A>G
10
48
20.8
NA
rs41310927
9
48
18.8
NA
rs3762271 c.7939C>A
1
48
2.1
6.7
rs12138336 c.7860G>C
The list of Single Nucleotide Polimorphism (SNP) of WDR62 gene among children
with microcephaly
rs Number
SNP
n
Present study
Present European %
total
study
%
16
48
30
17.5
c.180G>A
rs61742664
7
48
14.6
20.4
c.186C>T
rs11538454
c.562-13C>T
18
48
37.5
1.7
rs78138007
20
48
41.7
5.8
c.1233+13C>T
rs76130844
c.1641G>A
11
48
22.9
21
rs2301734
20
48
41.7
5
c.3401T>G
rs61741470
c.3639C>T
20
48
41.7
5
rs45470992
42
48
87.5
60.3
c.3929A>T
rs2074435
c.4170A>C
10
48
20.8
70.5
rs1008328
c.2549T>C
31
48
64.6
70.4
rs2285745
c.700-18C>T
15
48
31.3
35
rs10423651
21
48
43.8
NA
c.1641G>A
rs2301734
c.3033G>A
5
48
10.4
NA
rs118175551
*NA = not available
80
The list of Single Nucleotide Polimorphism of MCPH1 gene among microcephaly
children
rs Number
SNP
n
Present
Present
European %
study
study %
total
c.228G>T
9
20
45
77.9
rs2305022
c.-58G>C
rs2305023
c.513T>A
rs2445313
c.940G>C
rs930557
c.1175A>G
rs2515569
c.2215-19A>G
rs2936531
c.2226C>T
rs2912010
c.2282C>T
rs1057090
c.2482C>T
rs1057091
c.513T>G
rs2442513
c.1428C>T
rs2920676
c.647T>C
rs75741316
c.1428C>T
rs2920676
c.911G>T
rs2083914
c.2215-15C>G
rs11137040
*NA = not available
9
10
12
19
18
16
16
4
5
1
5
4
1
1
20
20
20
20
20
20
20
20
20
20
20
20
20
20
45
50
60
95
90
80
80
20
25
5
25
20
5
5
22.5
0
78.3
98.6
46.6
44.7
39.2
73
38.4
2.7
NA
15.8
19
26.7
The list of Single Nucleotide Polimorphism of CDK5RAP2 gene among children
with microcephaly
rs Number SNP
n
Present
Present
European %
study
study
Total
%
c.-18T>C
4
20
20
100
rs932974
c.-2G>T
6
20
30
100
rs932975
6
20
30
3.3
rs4836822 c.865G>C
6
20
30
1.71
rs2501727 c.2274T>C
2
20
10
2
rs4837768 c.4618G>C
1
20
5
0.07
rs35909061 c.4665G>T
1
20
5
NA
rs77100552 c.5578T>C
3
20
15
3.4
rs3750494 c.480A>C
1
20
5
4.2
rs34523498 c.3065G>A
1
20
5
99
rs3780679 c.3134G>C
1
20
5
0.9
rs6478475 c.4041G>A
4
20
20
20.8
rs4837768 c.4618G>C
1
20
5
1.7
rs3739822 c.5418C>T
1
20
5
5.3
rs37504945 c.480A>C
*NA = not available
81
The list of Single Nucleotide Polimorphism of CENPJ gene among children with
microcephaly
rs Number
SNP
n
Present
Present
European
study
%
study %
Total
c.3042A>G
9
20
45
4.5
rs3742165
c.61A>G
11
20
55
1.2
rs35498994
4
20
20
5
c.3216+7A>G
rs9318917
c.4125A>G
3
20
15
100
rs9318911
c.2992-6delT
5
20
25
50
rs35599563
c.3367-12T>C
4
20
20
0
rs3742163
*NA = not available
The list of Single Nucleotide Polimorphism of STIL gene among children with
microcephaly
SNP
cDNA
n
Present
Present
European %
study %
study
Total
16
20
80
5.4
c.257C>T
rs3125630
c.1452C>G
5
20
25
8.8
rs10789505
c.2954A>G
3
20
15
3.4
rs13376679
*NA = not available
82
Appendix V. Ethical Clearance
Appendix I. DNA isolation according to the Manual of Center of Biomedical
Research (Faradz, 2004)
Salt saturation method was performed. EDTA frozen blood was transferred into a
50 ml tube with the addition of NH4Cl 5-10 ml lysis buffer. The tube was
incubated for 10 30 minutes at room temperature. Afterwards the tube was
centrifuged for 5 minutes at 3000-3500 RPM, followed by removal of the
supernatant and addition of NH4Cl lysis buffer. The procedure was repeated three
times. TE lysis buffer 2 ul, Proteinase-K 10mg/ml and 100 ul 10% SDS were
added and mixed until white pellet was formed. The tube was then incubated at
50oC for 24 hours. After incubation NaCl 6M approximately one third volume of
the tube was added to the suspension and centrifuged at 4000 RPM for 10
minutes. The supernatant was moved to a new tube and absolute ethanol twice the
volume of the supernatant was added. The DNA formed white viscous thread and
could be removed by fine needle, rinsed with 70% ethanol and moved to a new
1,5 ml tube. The new tube was left open to allow evaporation of ethanol. The tube
was then filled with TE buffer for dissolution of the DNA. (Faradz, 2004)
69
Appendix II. The Nellhaus charts
70
Appendix III. List of primers
MCPH1
NM_024596.3
EXON
01
01
02
02
03
03
04
04
05
05
06
06
07
07
08_01
08_01
08_02
08_02
08_03
08_03
09
09
10
10
11
11
12
12
13
13
14
14
F/R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
SEQUENCE
tgtaaaacgacggccagtcctacagagaaatcccggaaa
caggaaacagctatgaccggtgccggtcctcaaac
tgtaaaacgacggccagtaaatctattgggcaggggat
caggaaacagctatgaccccattgcttcgtccagattc
tgtaaaacgacggccagtcagaattgctggggtagagg
caggaaacagctatgaccaaaagatgaaaatcaaactttatctgg
tgtaaaacgacggccagttgtgcagatttagtgctgtgtc
caggaaacagctatgacccatacaaatgctatcgaagaagtca
tgtaaaacgacggccagtgaaatgtatgcgaaagggct
caggaaacagctatgaccgcaaattaaacaaggtgtatgga
tgtaaaacgacggccagtcattcctgaagtatgaaggca
caggaaacagctatgaccccaccataattgaatcggct
tgtaaaacgacggccagtcaagttgactttaagatcccttct
caggaaacagctatgacccagggggaaatttagaagttctag
tgtaaaacgacggccagtgaaatgagaagaactcaagtgtggt
caggaaacagctatgacccctcggcatgatagatctcc
tgtaaaacgacggccagtatcttcaacaaaaggccacc
caggaaacagctatgacccagaagtcacgcaactcgaa
tgtaaaacgacggccagtttcctgcgttggcaaaa
caggaaacagctatgaccatggatgcactgttacatgttt
tgtaaaacgacggccagtttgcttaagttgtatttggtccat
caggaaacagctatgacctgtttcattgacccagagaag
tgtaaaacgacggccagttgagtttctatctcttggcctg
caggaaacagctatgaccgcctaaaggcacccagaatta
tgtaaaacgacggccagtaactgctttgatgggcatgt
caggaaacagctatgaccacctcagggtgacccactc
tgtaaaacgacggccagtggtttattgcctgctaaggct
caggaaacagctatgacctgggcataattgtgcttgac
tgtaaaacgacggccagttcgcctacgctatggagact
caggaaacagctatgaccacatcaacccaggtgccc
tgtaaaacgacggccagtacagctcttggctatttgttttta
caggaaacagctatgacccatttgaacatccaaagacagttt
71
CDK5RAP2
NM_018249.4
EXON
01
01
02
02
03
03
04
04
05
05
06
06
07
07
08
08
09
09
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
23
23
24_01
24_01
F/R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
SEQUENCE
tgtaaaacgacggccagtagtctgaacaagagctgggg
caggaaacagctatgaccgacaccctcaagagcaaacc
tgtaaaacgacggccagtgtgcagtccagaccgtctta
caggaaacagctatgacctcacagtccaatgtctactttcc
tgtaaaacgacggccagtgaacactgggcagtttgcat
caggaaacagctatgaccagaaacacagctttcctggg
tgtaaaacgacggccagttgttggaccctctgtcttttg
caggaaacagctatgaccaatcaaatttctgcttgaaatga
tgtaaaacgacggccagtttccccattgttgacattcat
caggaaacagctatgacctttcactgaccaacccagtg
tgtaaaacgacggccagttggggctgtgaaataaccat
caggaaacagctatgaccgaaacaaaactgtgccagcc
tgtaaaacgacggccagttgtgctggagaagaagtcataa
caggaaacagctatgacccccacgctgccagataat
tgtaaaacgacggccagttttccttcctcctccagctc
caggaaacagctatgaccttccatctcccacagaggag
tgtaaaacgacggccagttgttctggagaagctgttgg
caggaaacagctatgaccaaaacaagaggcaagaataggc
tgtaaaacgacggccagttatggtgcattttggttgga
caggaaacagctatgaccattcagggtaggacaggcca
tgtaaaacgacggccagtgaaaagcatgtgggctgtg
caggaaacagctatgaccaaagtcctcacctcaccagg
tgtaaaacgacggccagtagagcccaggtttcttggtt
caggaaacagctatgaccagccacatagccccaaaag
tgtaaaacgacggccagtagcagtaatttagcaagtcaaaca
caggaaacagctatgaccaagcccaaatgacataattaaagctct
tgtaaaacgacggccagtccttaaggagtttgggacagttt
caggaaacagctatgacccaacaagaaggcatttgagga
tgtaaaacgacggccagtggaagcagtattggaagctc
caggaaacagctatgaccaaagggtgtgtgcgtgtatg
tgtaaaacgacggccagtgacataaaaatcacaggaaggc
caggaaacagctatgaccatttaggactctcctgaagttc
tgtaaaacgacggccagtttgggaggatctgtttctga
caggaaacagctatgaccggaacccaagatacaacaaaca
tgtaaaacgacggccagtgggaagctgaagtctcttgc
caggaaacagctatgaccccaagccggttgttatatttt
tgtaaaacgacggccagtttcatacatattcatgctgcacac
caggaaacagctatgaccvtccagactcgaagactgatttg
tgtaaaacgacggccagtcatctccatcctctctccaca
caggaaacagctatgaccgcaaaatgagtttgttctccc
tgtaaaacgacggccagttaggggacaaggcctagca
caggaaacagctatgaccagtggtgagattaaaggtgatttt
tgtaaaacgacggccagttgactgtgcaaccaaaggaa
caggaaacagctatgaccgtttgtctagctcacagggaa
tgtaaaacgacggccagttgcttggctaagtttcaggtc
caggaaacagctatgaccaaaacatccaaatggtgcct
tgtaaaacgacggccagtggctgtgaggaaggagagag
caggaaacagctatgacctcagaaccattcttgggctt
72
24_02
24_02
25
25
26
26
27
27
28
28
29
29
30
30
31
31
32
32
33
33
34
34
35
35
36
36
37
37
38
38
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
tgtaaaacgacggccagttggaaggctaccagaatttca
caggaaacagctatgaccacctagtggcaatactgggg
tgtaaaacgacggccagttttccaattagtgttaaggtcacaa
caggaaacagctatgacctccaagaagtcctgggtcaa
tgtaaaacgacggccagttgagcaggaaattattagggaa
caggaaacagctatgaccgctggcaggtaaatcagacc
tgtaaaacgacggccagtgtcatagtcctggcttggga
caggaaacagctatgacctgcttactaaggttaagatggcaa
tgtaaaacgacggccagtaggatccctgcataatccat
caggaaacagctatgacccgtcacagtgaagcactcaca
tgtaaaacgacggccagtgaaagttctgtctatactgatctggag
caggaaacagctatgaccaatgcctgcattctttccat
tgtaaaacgacggccagtttttgtattcttgaagtgctgtattt
caggaaacagctatgaccgtgctgattccacgactgc
tgtaaaacgacggccagttgctctgtggggtttgaata
caggaaacagctatgaccagctgtcgggtggtcttaca
tgtaaaacgacggccagtgagttccttcgatggctgac
caggaaacagctatgacctgtcacacacagatgctcaga
tgtaaaacgacggccagtaatgtgaggggcttgactgt
caggaaacagctatgaccaaataccctcctgagttggga
tgtaaaacgacggccagtaagcgtcttgaacgcagagt
caggaaacagctatgacctfcgccatttgactcccc
tgtaaaacgacggccagtgctttcgctcagcgtgtga
caggaaacagctatgaccaaactgagacacaggcccc
tgtaaaacgacggccagttggctttgtaattcttcctgtg
caggaaacagctatgaccttacaaaccatctgggctcc
tgtaaaacgacggccagttttcacacagctctcctggat
caggaaacagctatgaccttcaagaggtcctttctggc
tgtaaaacgacggccagtcacctctgtcggggtcttac
caggaaacagctatgacctggaacaaagagacagcgtg
73
STIL
NM_003035.2
EXON
02
02
03
03
04
04
05
05
06
06
07
08
08
09
09
10
10
11
11
12_01
12_01
12_02
13
13
14
14
15
15
16
16
17_01
17_01
17_02
17_02
F/R
F
R
F
R
F
R
F
R
F
R
F
F
R
F
R
F
R
F
R
F
R
F
F
R
F
R
F
R
F
R
F
R
F
R
SEQUENCE
tgtaaaacgacggccagtttttgcctgtaattttaatatgtgg
caggaaacagctatgacccttccaacctaccttcccaa
tgtaaaacgacggccagtccaaatgtctaccaagtttgc
caggaaacagctatgaccgtaatcaactttgccttatacatcttt
tgtaaaacgacggccagtcctccgtatcccctgcct
caggaaacagctatgaccctccagcctcccaacgtg
tgtaaaacgacggccagtttgctataatgtagtcttgcttgg
caggaaacagctatgaccggttcacaataattcaaccaaat
tgtaaaacgacggccagtgagaactgaaatgctgtaaatcaga
caggaaacagctatgaccagccaccatatctctggcat
tgtaaaacgacggccagttgtgtgcaagacaacgttca
tgtaaaacgacggccagttggctgataaaagccatacg
caggaaacagctatgaccaaggttacggcattacatgaattt
tgtaaaacgacggccagtttgggagactcacatgaagtt
caggaaacagctatgacctgccaaagtgcaaaacccta
tgtaaaacgacggccagtaatcagaagtctgccactcca
caggaaacagctatgaccggaatgcatttaaaacaaagacat
tgtaaaacgacggccagtggtgcccagtgacttgattt
caggaaacagctatgaccccaatctatgtctcagtttactctagg
tgtaaaacgacggccagttgggaaatactggtctgagg
caggaaacagctatgaccacagtaagatggcagtgggg
tgtaaaacgacggccagtagtcttctcttgccccgc
tgtaaaacgacggccagtactgctgccagttttgttga
caggaaacagctatgaccctactgcaccatgcaccaat
tgtaaaacgacggccagtcattgcacccagccaatatg
caggaaacagctatgaccatctaaacattgtgccccac
tgtaaaacgacggccagtaccaggtgctgcttgagtct
caggaaacagctatgaccggtctcaatcagtttaaacctaggaa
tgtaaaacgacggccagtgggattataggtgtgaac
caggaaacagctatgaccagccaatgatgggcaaaa
tgtaaaacgacggccagttgcttgtatagaatggaacacattg
caggaaacagctatgacctcttagaaggctctttctgacc
tgtaaaacgacggccagtaagaggaacctcccgaca
caggaaacagctatgaccggctcctgttttccctaagta
74
CENPJ
NM_018451.3
EXON
03
02
03
04
04
05
05
02
06
06
07_01
07_01
07_02
07_02
07_03
07_03
07_04
07_04
08
08
09
09
10
10
11
11
16
16
17
17
12_13
12_13
14_15
14_15
F/R
F
R
R
F
R
F
R
F
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
R
F
F
R
SEQUENCE
tgtaaaacgacggccagtgaggtgtccatgaagaattgtaaa
caggaaacagctatgacccaagaagacctagaacaaatgacttta
caggaaacagctatgaccggaacgatagaaataacgtgtcaa
tgtaaaacgacggccagtgagaaaatgttgaaggaagt
caggaaacagctatgaccaaaaaacatggaaaggcttc
tgtaaaacgacggccagtgcttttcttgagctaaattg
caggaaacagctatgaccataaaggctagctattgtac
tgtaaaacgacggccagtgctttacctaatacattttgttggcta
tgtaaaacgacggccagttccatgttgtcaagggtttct
caggaaacagctatgaccttttggtatatccaagtgatctcaa
tgtaaaacgacggccagtggtcagcaattgtgctcttct
caggaaacagctatgacccagccagtatcgcaaggttt
tgtaaaacgacggccagtacagacggggaagaaatgtg
caggaaacagctatgacccttcacactcactctccttc
tgtaaaacgacggccagtcggatgtcttccacccctgt
caggaaacagctatgaccgatctcgagggctcagacac
tgtaaaacgacggccagttaaggacaccaggacccaag
caggaaacagctatgaccttgagcaataaggaaagttagga
tgtaaaacgacggccagtttttgctgcagtaatgcct
caggaaacagctatgacccagactgtttatagatggtcttaaagg
tgtaaaacgacggccagtcagcaggttctgcacatcac
caggaaacagctatgaccgatgcttacaagattgtcaccttta
tgtaaaacgacggccagtcattgctgggtctctattcttc
caggaaacagctatgacctgctcagatattgaagctccc
tgtaaaacgacggccagtaacccacagcattcttagcac
caggaaacagctatgaccagggtagaaattaactggcgg
tgtaaaacgacggccagtggtacaaacttgctccaccc
caggaaacagctatgaccgagcacagatgaacaggtgtc
tgtaaaacgacggccagtccaagggatgtctccagaag
caggaaacagctatgacctgccacagggtaaacttttattt
caggaaacagctatgaccgcatcttgtccaaagagccc
tgtaaaacgacggccagtaaaggacagcagttcacagga
tgtaaaacgacggccagtgtaggcagttgggaggagaa
caggaaacagctatgaccttcttccatctgggtaatgtttt
75
ASPM
NM_018136.4
EXON
01
01
02
02
03_01
03_01
03_02
03_02
03_03
03_03
03_04
03_04
04
04
05
05
06
06
07
07
08
08
09
09
10
10
11-12
11-12
13
13
14
14
15-16
15-16
17
17
18_01
18_01
18_02
18_02
18_03
18_03
18_04
18_04
18_05
18_05
18_06
18_06
DIR
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
SEQUENCE
tgtaaaacgacggccagtaagcggtcagcgtaagtcc
caggaaacagctatgacctctccaatcgtcaaccttcc
tgtaaaacgacggccagtttcttataattaagcagatagggtagg
caggaaacagctatgacctggtatcccaaagactcctctg
tgtaaaacgacggccagttgttctgaaacaccaagctatattac
caggaaacagctatgacctcaaagttgaagaacagttggg
tgtaaaacgacggccagtcttcatgcatcagaaaataggg
caggaaacagctatgacctttgataatggtactttacatgtttgc
tgtaaaacgacggccagtcagcctgtgcatttggaatc
caggaaacagctatgaccgaagctgttgtcgaagaggg
tgtaaaacgacggccagtaaaggaaggccacctgtacc
caggaaacagctatgaccaaaccaaaaggaagtatcccc
tgtaaaacgacggccagtgaatatgattgtgaagaacccaaac
caggaaacagctatgaccttcttccaggctgttattcaac
tgtaaaacgacggccagtttgttcagtgttttaaagatggtattg
caggaaacagctatgaccgctaatgaacagggaattatgc
tgtaaaacgacggccagttgaaattgcattttattgctgg
caggaaacagctatgaccatatgtcaataaagccgggg
tgtaaaacgacggccagtcatgctttagctttgctgcc
caggaaacagctatgaccatggcatagtctatttacctaataagc
tgtaaaacgacggccagttcctcagtcacttccctttg
caggaaacagctatgaccaaacaggaagaatgacaataagcc
tgtaaaacgacggccagttttgtgcttgctaccctacac
caggaaacagctatgaccgcaagcaaaagtcgtaaatgg
tgtaaaacgacggccagtcagaatgatttggaggatttg
caggaaacagctatgaccaaaagtgttttccagaaaatgttagtc
tgtaaaacgacggccagtaactgttgggatttatgtggg
caggaaacagctatgaccaaatgatggttgttgttgttattc
tgtaaaacgacggccagttccatttcaggcactttattttc
caggaaacagctatgacctttgagggaaagtttgcttacac
tgtaaaacgacggccagtaacttgccattacttgccttg
caggaaacagctatgaccgaggaaaggagaaattagccg
tgtaaaacgacggccagtgatcatggttagtcccttttgg
caggaaacagctatgaccaatttatcttaataatgccatacatcc
tgtaaaacgacggccagttgtaggggtgttttatttccag
caggaaacagctatgaccaaacttcatcacattttgccttc
tgtaaaacgacggccagtggatggatttctgaattggc
caggaaacagctatgaccttctgcaaatagttggtgcg
tgtaaaacgacggccagtgcaaagagcttttagagaatgg
caggaaacagctatgaccgactgcagcacaatgacagc
tgtaaaacgacggccagttcaaatttcaggcacatgtaag
caggaaacagctatgaccaatactgccgagcctttctc
tgtaaaacgacggccagttgcacaaaagagagaagagtatatg
caggaaacagctatgaccttcaaggcagcttgatgttc
tgtaaaacgacggccagtggtacagggcgtacaagactc
caggaaacagctatgaccttcataccacgataagctgactg
tgtaaaacgacggccagtcaaaggcaacataaatgtgctatc
caggaaacagctatgaccaaattgtcaggtacttttcacgc
76
18_07
18_07
18_08
18_08
18_09
18_09
18_10
18_10
18_11
18_11
18_12
18_12
19
19
20
20
21
21
22
22
23
23
24
24
25
25
26
26
27
27
28
28
25
25
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
tgtaaaacgacggccagtgccatgtttaaaatgcatcag
caggaaacagctatgacctgtgcccgatattttctctg
tgtaaaacgacggccagtaatgatgcatatagccgcaac
caggaaacagctatgacctggtggctcgatatttcctc
tgtaaaacgacggccagtaggctgcattcaggggtatg
caggaaacagctatgaccctgcctatacattctgtaggtgc
tgtaaaacgacggccagtaagagaaaattatatcagacaatggc
caggaaacagctatgaccctgaattagtgtggcagccc
tgtaaaacgacggccagtaaaataaggaagcattatctccac
caggaaacagctatgaccgaactgaatccgtagggcag
tgtaaaacgacggccagtaagctgttcaaagtgaaggtg
caggaaacagctatgacctgacctgcttaagctcattttatac
tgtaaaacgacggccagtttttgtctgcactaaccttattttaac
caggaaacagctatgaccaaagcaagacagtacgagagatg
tgtaaaacgacggccagttgcgtgtgtaaattctgattg
caggaaacagctatgacctgtgtgaaataaatgcatacttaggtc
tgtaaaacgacggccagtgaacttctagtgggttggaaatc
caggaaacagctatgaccttcttaacactatttaacatcaagtgc
tgtaaaacgacggccagtccttctctaatagggcagtctagttg
caggaaacagctatgaccctcaagactttgctggcagg
tgtaaaacgacggccagtggggaagtaatgcctctgtg
caggaaacagctatgaccaaagagcttagcaatgaaattatgg
tgtaaaacgacggccagttttggtcgataaatgctgtcc
caggaaacagctatgaccagaaattgctccactctggg
tgtaaaacgacggccagtgaggccttaaaacacctaggttag
caggaaacagctatgacccaaacttaatttgcaggggc
tgtaaaacgacggccagtttggttgggttgtttgtaaatg
caggaaacagctatgacccaggtttgaacacacataaaacc
tgtaaaacgacggccagtgcgacagagcaagagagacc
caggaaacagctatgacctttctccactgaaaagcacatc
tgtaaaacgacggccagtatgaccaaaaggcagtggtc
caggaaacagctatgacctgccattgatatgaatttgtgag
tgtaaaacgacggccagtcctttctgccattcttgagg
caggaaacagctatgaccagagatttaagccctagtgatgag
77
WDR62
NM_001083961
EXON 1
EXON 2
EXON 3
EXON 4-5
EXON 6
EXON 7
EXON 8
EXON 9
EXON 10
EXON 11
EXON 12
EXON 13
EXON 14-15
EXON 16
EXON 17-18
EXON 19
EXON 20
EXON 21
EXON 22
EXON 23
DIR
sequence
F
gctgttcgctgttccagtg
R
gggccacagttctcagga
F
tgggtgttgaatgtagcagg
R
gaggctcagagcgatttacg
F
agaaccgagggagcttgttt
R
tatctccaggctgtgtaggg
F
gcagaagagggcagagtcct
R
catgtagctctccctgtgcc
F
gacgagcagggagttccag
R
ccctcaaatctcagggagaa
F
agtgttttcttcccttctcca
R
acctgtgtggtcaatgctga
F
ctctggaggcctaaggctg
R
agatggatctggaagcatgg
F
gatggatggccacaaatacc
R
tgatgcataggagggagtca
F
ttgcacgatacctgtttatcttt
R
ctctcctgcccctgcat
F
agagactggctatggaggga
R
cagggcttctgcccact
F
cctggccattctgacctatt
R
caccagacaccaactacccc
F
gaaccaaagaccccttctcc
R
cacagatactgtattcacttgtttgg
F
ttgattgatggctcttgcag
R
accagaggaacagagggagg
F
ggaggacagcaagagcca
R
gtgcactgaggaaggaccaa
F
agtcccatgtgctgtgcat
R
tgacaccaagactcccacaa
F
ctgtggtgtgggtggctt
R
ctgtcttggccaccactgt
F
acatggagcttagcacaggg
R
tatgtgccaggccctgtaaa
F
gttgcctctttggggactg
R
agggaaaagagggacatgga
F
agagtccagatgggctgtgt
R
tggagtgggagtcagcctaa
F
gttcacagcctccagcagt
R
ggcagagagcacttcttcca
78
EXON 24
EXON 25-26
EXON 27-29
EXON 30_1
EXON 30_2
EXON 31
EXON 32
F
gtacaggtgcctaggggctc
R
gcttcagtactgggtcaaagtca
F
taccatccctcctccagatg
R
ggagccaactccagggac
F
gggtttctggggagtgc
R
acgccccacagctaggc
F
gatgtccagcctagctgtgg
R
cagggtcagtctcaggttgg
F
gcttcaggccatcaccac
R
gaatgaatggcacagtccct
F
agtacagcatggagtccaccc
R
gactgagctggtctccacctt
F
ctaagccccagagttggga
R
caggaaccattcctcaaagc
79
Appendix IV. List of Single Nucleotide Polymorphisms
The list of Single Nucleotide Polimorphism (SNP) of ASPM gene among children
with microcephaly
rs Number
SNP
n
Cohort
Present
European %
Total
study %
43
48
89.6
95.8
rs1571964 c.441+14C>T
29
48
60.4
91.2
rs6677082 c.849C>T
43
48
89.6
6.8
rs4915337 c.3579T>A
48
93.8
99.1
c.7480T>C
45
rs964201
c.7566A>G
46
48
95.8
91.2
rs1412640
42
48
87.5
14.3
rs10922162 c.7605G>A
48
91.7
91.6
rs10754213 c.10331+8A>G 44
8
48
16.7
18.8
rs4915344 c.2174-20T>C
c.3138G>A
7
48
14.6
38.3
rs6676084
10
48
20.8
38.9
rs2878749 c.4449A>G
10
48
20.8
NA
rs41310925 c.5961A>G
9
48
18.8
NA
rs41308365 c.7674C>T
c.7684A>G
10
48
20.8
NA
rs41310927
9
48
18.8
NA
rs3762271 c.7939C>A
1
48
2.1
6.7
rs12138336 c.7860G>C
The list of Single Nucleotide Polimorphism (SNP) of WDR62 gene among children
with microcephaly
rs Number
SNP
n
Present study
Present European %
total
study
%
16
48
30
17.5
c.180G>A
rs61742664
7
48
14.6
20.4
c.186C>T
rs11538454
c.562-13C>T
18
48
37.5
1.7
rs78138007
20
48
41.7
5.8
c.1233+13C>T
rs76130844
c.1641G>A
11
48
22.9
21
rs2301734
20
48
41.7
5
c.3401T>G
rs61741470
c.3639C>T
20
48
41.7
5
rs45470992
42
48
87.5
60.3
c.3929A>T
rs2074435
c.4170A>C
10
48
20.8
70.5
rs1008328
c.2549T>C
31
48
64.6
70.4
rs2285745
c.700-18C>T
15
48
31.3
35
rs10423651
21
48
43.8
NA
c.1641G>A
rs2301734
c.3033G>A
5
48
10.4
NA
rs118175551
*NA = not available
80
The list of Single Nucleotide Polimorphism of MCPH1 gene among microcephaly
children
rs Number
SNP
n
Present
Present
European %
study
study %
total
c.228G>T
9
20
45
77.9
rs2305022
c.-58G>C
rs2305023
c.513T>A
rs2445313
c.940G>C
rs930557
c.1175A>G
rs2515569
c.2215-19A>G
rs2936531
c.2226C>T
rs2912010
c.2282C>T
rs1057090
c.2482C>T
rs1057091
c.513T>G
rs2442513
c.1428C>T
rs2920676
c.647T>C
rs75741316
c.1428C>T
rs2920676
c.911G>T
rs2083914
c.2215-15C>G
rs11137040
*NA = not available
9
10
12
19
18
16
16
4
5
1
5
4
1
1
20
20
20
20
20
20
20
20
20
20
20
20
20
20
45
50
60
95
90
80
80
20
25
5
25
20
5
5
22.5
0
78.3
98.6
46.6
44.7
39.2
73
38.4
2.7
NA
15.8
19
26.7
The list of Single Nucleotide Polimorphism of CDK5RAP2 gene among children
with microcephaly
rs Number SNP
n
Present
Present
European %
study
study
Total
%
c.-18T>C
4
20
20
100
rs932974
c.-2G>T
6
20
30
100
rs932975
6
20
30
3.3
rs4836822 c.865G>C
6
20
30
1.71
rs2501727 c.2274T>C
2
20
10
2
rs4837768 c.4618G>C
1
20
5
0.07
rs35909061 c.4665G>T
1
20
5
NA
rs77100552 c.5578T>C
3
20
15
3.4
rs3750494 c.480A>C
1
20
5
4.2
rs34523498 c.3065G>A
1
20
5
99
rs3780679 c.3134G>C
1
20
5
0.9
rs6478475 c.4041G>A
4
20
20
20.8
rs4837768 c.4618G>C
1
20
5
1.7
rs3739822 c.5418C>T
1
20
5
5.3
rs37504945 c.480A>C
*NA = not available
81
The list of Single Nucleotide Polimorphism of CENPJ gene among children with
microcephaly
rs Number
SNP
n
Present
Present
European
study
%
study %
Total
c.3042A>G
9
20
45
4.5
rs3742165
c.61A>G
11
20
55
1.2
rs35498994
4
20
20
5
c.3216+7A>G
rs9318917
c.4125A>G
3
20
15
100
rs9318911
c.2992-6delT
5
20
25
50
rs35599563
c.3367-12T>C
4
20
20
0
rs3742163
*NA = not available
The list of Single Nucleotide Polimorphism of STIL gene among children with
microcephaly
SNP
cDNA
n
Present
Present
European %
study %
study
Total
16
20
80
5.4
c.257C>T
rs3125630
c.1452C>G
5
20
25
8.8
rs10789505
c.2954A>G
3
20
15
3.4
rs13376679
*NA = not available
82
Appendix V. Ethical Clearance