Results o f CNG A3 Mutation Screening

CNGA3 was screened for mutations in the panel of achromats by direct sequencing

of coding exons plus around 50bp of flanking UTR and intronic sequence amplified by

PCR. The results of this analysis are summarised in table 5.5.

Thirteen different alterations of CNGA3 were found in the panel of achromats.

Three of these nucleotide changes are single nucleotide polymorphisms (SNPs; figure 5.3).

The first, detected in individuals RM4, RM5 and RM8, is a silent T/C SNP at nucleotide

position 72 and has been reported previously (Wissinger et al., 2001). The second is a

silent nucleotide polymorphism identified as a C ->T heterozygous change at nucleotide

position 1443 in RM34 and RM35. The third, not previously reported, represents a

Metll211e polymorphism and was detected in 13 individuals including 8 affected subjects

(RM4, RM8, RM9, R M ll, RM22, RM28, RM29, RM38). Analysis of additional family

members where possible confirmed that this change did not represent a disease causing

mutation, that is, the alteration does not segregate with disease (figure 5.3h).

O f the other CNGA3 alterations found (figure 5.4), six have not previously been

reported - Arg23Term, Gln251Term, Arg276Ser, Thr3(X)Met, Ile537fs and Gly603Arg

(highlighted in blue in table 5.5). For three affected subjects (RM 15, family 7, RM18,

family 8 and RM35, family 17) for whom heterozygous mutations were found, no second

mutation of CNGA3 could be identified using this approach. The Arg278Trp (RM15 and

RM 16, family 7), Arg491Trp (RM35 and RM36, family 17), Phe602Leu (RM39, family 20

and RM43, family 24) and Arg624His (RM27, family 13) mutations identified have been

described in earlier studies (Kohl, et at., 1998; W issinger et al. 2(X)1).

In summary, the molecular genetic basis for disease in the panel of twenty-five

achromat families studied can be confirmed as due to mutations in CNGA3 in seven of

11, 12, 15, 20 and 24) the mutation identified was homozygous while compound

heterozygous mutations were found in one family (family 13). Two of the ten disease-

associated alterations found were protein truncation mutations (Arg23Term and

Gln251Term), one resulted in a frameshift mutation (Ile537fs) and the remainder were

amino acid substitution mutations (Arg278Trp, Thr300Met, Gly603Arg, Arg624His,

Arg276Ser. Arg491Trp and Phe602Leu) all of which may be described as non-conservative

(table 5.6). The mutations found are mostly located in structurally and functionally

important region of CNGA3 (figure 5.5).

Table 5.5: CNGA3 mutations identified in achromats

Previously unreported mutations are highlighted in blue. Nucleotide num bering is from the first nucleotide o f the ATG start codon

Family/Affected 7 /R M 1 5 , RM16 Nucleotide Alteration Ht. 8 3 2 C ^ T Polypeptide Alteration A rg 2 7 8 T rp 8 /R M 1 8 Ht. 8 9 9 C ^ T Thr300Met 10 / RM21 H m . 1 8 0 7 G ^ A Gly603Arg 11/R M 22 H m . 6 7 C - * T Arg 23Term 12 / RM25 H m . 7 5 1 C ^ T Gln251Term 13/R M 27 Ht. 164 7 in sC He537fs Ht. 1 8 7 1 G -* A A rg 6 2 4 H is 15/R M 29 H m . 8 6 5 C ^ T Arg276Ser 17/R M 35 H t. 1 4 7 1 C -* T A rg 4 9 1 T rp 2 0 /R M 3 9 H m . 1 8 0 6 C -> A P h e 6 0 2 L e u 24 / RM43 H m . 1 8 0 6 C -^ A P h e 6 0 2 L e u - 1 5 7-

Figure 5.3: Electropherograms showing nucleotide polymorphisms in CNGA3.

N u c e o tid e 7 2 T /C sile n t S N P ; a n o rm al se q u e n c e , b s e q u e n c e o f h e te ro z y g o te a n d c se q u e n c e o f h o m o z y g o te ; N u c le o tid e 1443 C /T sile n t S N P : d n o rm al s e q u e n c e , e se q u e n c e o f h e te ro z y g o te ; N u c le o tid e 3 3 6 C /T M e t l l 2 I l e p o ly m o rp h ism : f n o rm a l re v e rse se q u e n c e , g re v e rse s e q u e n c e o f h e te ro z y g o te , h g e n o ty p e s o f fa m ilie s w ith M e t l l 2 I l e p o ly m o rp h ism d e m o n stra tin g th a t th is a m in o a c id c h a n g e c a n n o t b e a s s o c ia te d w ith d ise a se .

C C G A G 250 A T C T C : C G A G A N C T 2 6 0 C C G A G A C C T C 60 T T C C G C A A G 2 8( T T C 2 7 0 N G C A A G

A G A G C