Table 1.
General description of patients and molecular tools included in this study
|
Families in study |
APEX microarray |
Indirect studies |
SSCP & |
HRM |
Sanger sequencing |
Total families characterized |
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CRB1 2 alleles | Second allele by whole CRB1 analysis * | Total characterized | Haplotypes | IBD mapping | CRB1 2 alleles | CRB1 2 alleles | CRB1 1 allele | Second allele by Sanger sequencing * | 2 alleles | CRB1 mutations | Mutations in another gene # | |
|
114 LCA |
8 /114 |
4 / 8 a |
12 (10%) |
0 / 20 |
1 b/43 |
1 / 3 |
1 / 32 |
1 / 32 |
0 / 1 |
1/51 |
16 (14%) |
32 (28%) |
|
290 EORP |
6 /290 |
15 /25 a |
21 (7%) |
0 / 60 |
0 / 23 |
1 / 20 |
0 / 193 |
1 / 193 |
1 / 1 c |
4 d/ 209 |
27 (9%) |
59 (20%) |
| 404 Total | 14 / 404 | 19 /33 | 33 (8%) | 0 / 80 | 1 / 66 | 2 / 23 | 1 / 225 | 2 / 225 | 1 / 2 | 5/ 260 | 43 (11%) | 91 (22%) |
Overview of the sequential steps performed during the mutational CRB1 analysis in a cohort of LCA and EORP Spanish patients was reflected. The number of cases characterized and studied is outlined for each molecular tool. APEX: Arrayed primer extension; IBD: Inherited-by-descent; SSCP: single-strand conformation polymorphism analysis; HRM: High resolution melting analysis. * A direct mutational scanning of CRB1 was performed using dHPLC, HRM or Sanger sequencing in patients with a first allele identified by APEX microarray. & SSCP findings were reported by Bernal S. et als, 2003. # Mutations in another gene were found by APEX microarray, whole-genome homozygosity maping, whole exome sequencing or targeted NGS (data not shown).
a A second CRB1 allele was not found in 14 patients: 2 carried a known frameshift mutation and 12 carried a uncertain or very unlikely missense variant.
b A 5 Mb-homozygous region involving CRB1 was identified in an endogamic family and a homozygous known mutation was further found by Sanger sequencing. This mutation represents a false negative of the previously LCA chip analysis.
c A second allele (c.1702C>T) was further found by Sanger sequencing, representing a false negative of the HRM analysis.
d Two heterozygous transitions (c.2291 G>A and c.4168C>T) were found in one patient that previously showed normal melting curves in the HRM analysis thus, representing false negatives.