Molecular genetic diagnostic techniques in choroideremia

Abstract

Purpose: To optimize and streamline molecular genetics techniques in diagnosing choroideremia (CHM).

Methods: PCR primers were designed for exons 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 15 of the CHM gene. Each PCR protocol was optimized so that all exons could be amplified with the same component ratio and PCR conditions. Sense and antisense primers were tested for their ability to be used as sequencing primers. Fibroblast cells were cultured, and an immunoblot analysis was performed to detect the presence or absence of Rab escort protein 1 (REP-1) in a suspected CHM patient sample when no mutation was detected with sequencing. Multiplex ligation-dependent probe amplification (MLPA) of the CHM gene was performed and used to detect deletions and duplications in affected males and female carriers. RNA analysis using cDNA was used to detect the presence or absence of the CHM transcript and to search for splice defects.

Results: The newly designed PCR primers allow for more efficient PCR preparation and sequencing to detect point mutations in affected males and female carriers. Immunoblot successfully detects the absence of REP-1 in a CHM patient. MLPA identifies deletions and duplications spanning multiple exons in the CHM gene. RNA analysis aids in detecting splice variants.

Conclusions: The development of new molecular biology techniques and ongoing optimization of existing methods allows for an improved integrated approach to confirm CHM diagnosis and carrier status in consideration of patient family history and available patient sample materials. CHM can be confirmed with an immunoblot assay. To detect the molecular cause of CHM, an examination of the genomic DNA or the mRNA must be performed. Presymptomatic carriers with no identifiable fundus signs can be identified only through molecular analysis of genomic DNA or through quantitative assays.

Figure 1

Lysates from a male healthy control subject (C) and from a male choroideremia patient (P) were analyzed with immunoblot as described in the Materials and Methods section, and probed with 2F1 antibody (upper panel) and anti-beta-actin (bottom panel). The figure demonstrates the absence of REP-1 in a CHM patient.

Figure 2

QiaXcel capillary electrophoresis (QIAxcel DNA Screening Kit, Qiagen) demonstrates the PCR products obtained using all the CHM genomic primers under the same conditions, increasing the efficiency of this analytic tool. The sample analysed is from a choroideremia patient with no detectable mutation in the exon sequences.

Figure 3

The multiplex ligation-dependent probe amplification peak ratios of choroideremia exons 2, 3 and 4 are significantly lower than those of the other exons for this patient, indicating a deletion on exons 2 through 4 in the CHM gene.

Figure 4

The RT-PCR result with primers specific to the coding region of the CHM mRNA showing a 2.2 kb amplicon. Size marker (“M”): GeneRuler 1kb+ DNA Ladder (Thermo Fisher Scientific, Waltham, MA), water control (“N”), RNA used from healthy male control subject (“C”). This demonstrates the ability to detect the presence of CHM RNA when it is expected to be present by creating and amplifying cDNA.