strand) and the introduction of a Bst XI site.
c. Restriction analysis showing the gain of a Bst XI site that cosegregates with affected individuals heterozygous for the 1137insC (625 bp, 460 bp and 165 bp)
ATG GGG AAG GTT TGG GTG AGG GTG GTG TTG ATG TTG GGG ATT TTG GTG GTG GGG GGG GGG 1 2 0 - I -- G -- K -- V -- W -- L -- T -- V -- L -■ F -- I -- F -- R -- I -- L -- V -■ L -- G -- A -- A - 4 0 GGG GAG GAG GTG TGG GGG GAT GAG GAG TGA GAG TTG AGG TGG AAG AGG GAG GAG GGG GGG 1 8 0 -A E D S AGC V W M iro i G I D E Q S D F T G N T Q Q P G 6 0
TGG GAG AAC GTG TGG TAG GAG AGG GGG TTG GGG ATG TGG GAG ATG GGG TTG TGG GGG GTG 2 4 0 G E N V G Y D R A F P I S H I -- R -- F -- W -- A -- L - 8 0 GAG ATG ATG TTG GTG TGG AGG GGG AGG GTG ATG TAG GTG GGG GAG GTG GTG GAG ATG GTG 3 0 0 -Q -- I -- I -- F -- V -- S -- T -- P -- T -- L -- I -- Y -- L -- G -- H -- V -- L H I V 1 0 0 GGG ATG GAG GAG AAG AAG AAA GAG AGG GAG GAG GAG GAG GAG GTG AAG AGA GAG AGG GGG 3 6 0 R M E E K K K E R E E E E Q L K R E S P 1 2 0 AGG GGG AAG GAG GGA GGG GAG GAG AAT GGG TTG TGG GGG GAG GAG GGG GGG AGG GTG GGG 4 2 0 S P K E P P Q D N P S S R D D R G R V R 1 4 0 ATG GGG GGG GGG GTG GTG GGG AGG TAG GTG TTG AAG ATG ATG TTG AAG AGG GTG TTG GAG 4 8 0 M -- A -- G -- A -- L -- L -- R -- T -- Y -- V -- F -- N -- I -- I -- F -- K -- T -- L -- F -- E - 1 6 0 GTG GGG TTG ATG GGG GGG GAG TAG TTT GTG TAG GGG TTG GAG GTG AAG GGG GTG TAG GGG 5 4 0 -V -- G -- F -- I -- A -- G -- Q -- Y -- F -- L -- Y -- G -- F E L Q P L Y R 1 8 0 TGG GAG GGG TGG GGG TGG GGG AAG AGG GTG GAG TGG TTG ATG TGG AGG GGG AGG GAG AAG 6 0 0 G D R W P G P N T V D G F I S R P T E K 2 0 0 AGG ATG TTG ATG ATG TTG ATG GTG GGG GTG GGG TGG GGG TGA GTG GTG GTG AAG ATG GTG 6 6 0 T -- I -- F -- I -- I -- F -- M -- L -- A -- V -- A -- G -- A -- S -- L -- L -- L -- N -- M -- L 2 2 0 GAG ATG TAG GAG GTG GGG TGG AAG AAG GTG AAG GAG GGG GTG AGG AGG GGG GTG GGG GGG 7 2 0 E I Y H L G W K K L K Q G V T S R L G P 2 4 0 GAG GGG TGG GAG GGG GGG GTG GGG AGA GGG GAT GGG GGG GGG GTG GGG GGG AGG TGG GGG 7 8 0 D A S E A P L G T A D P P P L P P S S R 2 6 0 GGG GGG GGG GTT GGG ATG GGG TTG GGG GGG TAG TAT GGG GAG AGG GGT GGG GGG GTG GGA 8 4 0 P P A V A I G F P P Y Y A H T A A P L G 2 8 0 GAG GGG GGG GGG GTG GGG TAG GGG GGG GGG GGG GGA GGA GGG GGG GAG TTG AAA ATG GTA 9 0 0 Q A R A V G Y P G A P P P A A D F K M L 3 0 0 GCG GTG AGG GAG GGG GGG GGA AAG GGG GAG TGG GGG AAG GTG TAG AAG GGG GAG GAG GAG 9 6 0 A L T E A R G K G Q S A K L Y N G H H H 3 2 0 GTG GTG ATG AGT GAG GAG AAG TGG GGG AAG GAG GGG GGG GAG GGG GAG GGG GGG GGG GTG 1 0 2 0 L L M T E Q N W A N Q A A E R Q P P A L 3 4 0 AAG GGT TAG GGG GGA GGG TGG AGG GGT GGA GGG GGG AGG GGG GTG GGG AGG AGG TGG GGG 1 0 8 0
K A Y P A A S T P A A P S P V G S S S P
Q
CAG
3 6 0
GGA GTG GGG GAG GAG GGT GAG GGG GGG GGG GGG GGG GTG GTG GTG GAT GGG AGG GGG AGG 1 1 4 0 P L A H E A E A G A A P L L L D G S G S 3 8 0
Bst X I
;
Q S G G E R P G R D P R G GAG TCT GGA GGG GAG CGC CCT GGC AGG GAC CCC CGA GGf AGT CTG GAG GGG AGC GGC CTG GGA GGG AGG GGG GAG GAG
S L E G S A L A G T P E E R G P D A P A A L A P R R CGC GGC CCA GAT GCA CCA GCC GCC CTT GCC CCT CGG AGÎ GGG GGG GAG ATG GAG GAG GGG GGG T T G GGG GTG GGA GAC
A A Q M H Q P P L P L G D Q Q G Q Q R A G Q T G G L CAG CAG GGC CAG CAG CGG GCG GGC CAG ACC GGA GGA CTT AGG AGG GGG AGG AGG GGG GGG GGG AGA GGG GAG GAG TTG
S R A S S G R A R P E D L L P P D S C V V I F F L E CTG CCT CCA GAC AGC TGT GTA GTG ATC TTT TTC TTA GAf
P P A D R T * 4 6 6
CCA CCA GCA GAT AGA ACC TGA 1 3 9 8
Figure 5.10 Human GJA3 sequence showing both mutations (N63S and 1137insC), restriction sites for Mwo I and Bst XI. Dashes indicate putative transmembrane
G G A G R D H 4 0 0 GGA GGA GCA GGC CGT GAC CAC 1 2 0 0 GAG GAG GAG GGG GTG AGG AGG 1 2 0 0 E E Q A V T T 4 0 0 P R S G Q Q G 4 2 0 CCC AGG TCG GGC CAG CAA GGC 1 2 6 0 GGA GGT GGG GGG AGG AAG GGG 1 2 6 0 P G R A S K A 4 2 0 G H L V P G C 4 4 0 GGC CAT CTA GTG CCC GGG TGC 1 3 2 0 GGG ATG TAG 1 3 0 5 A I 4 3 5 T K T K V H F 4 6 0 ACC AAA ACC AAA GTG CAC TTC 1 3 8 0
CZP3 locus and GJA3 on chromosome 13q
5.10 Summary
A whole genome search identified a novel locus for autosomal dominant pulverulent cataract in families A and E.
CZP3 (Cataract Zonular Pulverulent 3) mapped to the pericentric region of chromosome 13 to the microsatellite marker D13S175. D13S175 has been physically mapped to 13 q ll.
The Cx46 (GJA3) gene had been mapped to 1 3 q ll-q l2 (Mignon et al. 1996), providing a candidate for CZP3.
A search o f the databases showed that the coding sequence for human GJA3 was unknown.
Using primers designed from alignments for GJA3 sequences from other species, the N-terminus o f the coding region of human GJA3 was identified.
A P I clone mapping to 13q was found to contain GJA3 allowing identification of the C- terminal sequence of GJA3.
Human GJA3 coding sequence is 1305 bp in length, encoding a protein of 435 amino acids with a predicted molecular mass of 47,435 Da.
Mutation screening in family A identified an A ^ G transition at nucleotide 188, changing codon 63 from an asparagine to a serine and introducing a Mwo I restriction site.
In family E, an insertion of a cytosine at position 1137 was found to segregate with the disease. This 1137insC mutation is predicted to cause a frameshift extending the coding region by 31 amino acids and changing the last 87 amino acids o f the protein.