The containment and eradication of genetic disorders represent primary objectives in modern dog breeding. Despite its global popularity, the Chihuahua breed is predisposed to several hereditary conditions due to a constricted gene pool
5-7. In this study, we developed cost-effective PCR-RFLP primer-enzyme sets for the detection of eight Mendelian disorders that affect Chihuahuas and are often challenging to diagnose phenotypically. These sets are designed for easy implementation in veterinary clinics and diagnostic laboratories. However, the primer and enzyme combinations in this study were selected based on strict thermodynamic criteria and restriction mapping to ensure high specificity, minimizing the risk of off-target amplification or incomplete digestion in clinical applications.
While DNA sequencing technologies offer high sensitivity, they can be cost-prohibitive and time-consuming for routine screening, particularly in developing countries or resource-limited settings36,37. In these contexts, PCR-RFLP remains a reliable, rapid, and economical method for genotyping23,29. As highlighted in recent studies, PCR-based enzymatic digestion methods continue to offer flexible solutions requiring minimal instrumentation36,37. Such flexibility is crucial when screening for disorders with diverse inheritance patterns. Certain genetic disorders may cause embryonic lethality in homozygous recessive genotypes or hemizygous males (potentially associated with NSDHL). Other conditions may exhibit a dominant inheritance (COL5A2, GFAP). Regardless of the mode of inheritance, the PCR-RFLP method enables the detection of the defective allele. Moreover, this method remains feasible for analyzing samples collected from post-mortem specimens or embryos.
The hereditary skin disorders addressed in this study often share overlapping phenotypic presentations. Notably, ichthyosis has recently been shown to arise from distinct mutations in the ALOXE3 and SDR9C7 genes9,10. The primer sets designed here, compatible with PpuMI and AvaI/BsoBI digestion, allow for the molecular differentiation of these variants. Similarly, the differential diagnosis of KRT10-associated hyperkeratosis and NSDHL-associated verrucous epidermal nevi is critical for determining appropriate treatment protocols13,14. However, it must be noted that PCR-RFLP may not be applicable for all mutation types, as observed with the NSDHL missense mutation, where the single-nucleotide change did not alter a restriction site.
Beyond dermatological conditions, the early diagnosis of life-threatening systemic and neurological disorders is vital to prevent the breeding of carriers. Spinal dysraphism caused by NKX2-8 variation leads to severe neurological defects in offspring20. In the BtgZI restriction method, the resulting short 57 bp fragment in carriers (wt/mut) may be indistinguishable from primer dimers. However, the clear distinction between the 507 bp mutant and 449 bp wild-type bands is sufficient to identify heterozygotes. To achieve optimal resolution and differentiate these bands clearly, the use of higher concentration agarose gels (2% to 4%) is advisable rather than the simulated 1.4% gel (Figure 1I). Furthermore, the ability to detect rare but severe conditions such as COL5A2-associated Ehlers-Danlos syndrome and GFAP-associated Alexander disease provides breeders with a comprehensive health screening tool. Additionally, screening for CYB5R3-associated methaemoglobinaemia, a metabolic defect, provides clinicians with crucial data for pre-anesthetic risk assessment16. However, the enzymatic digestion of the mutant CYB5R3 allele generates relatively small fragments (92 bp and 78 bp). In the absence of wet-lab validation, there is a potential risk that primer dimers could mimic these low-molecular-weight bands, leading to a misinterpretation of wild-type samples as heterozygotes. Therefore, optimization of PCR conditions to minimize dimer formation and the use of high-percentage agarose gels are recommended for accurate interpretation of this primer-enzyme combination.
While PCR-RFLP sets for progressive rod-cone degeneration detection have been previously described22,26-28, including the use of ApaLI in Labrador Retrievers38, the inclusion of BsrGI in this study provides laboratories with greater flexibility regarding enzyme availability. Notably, the single primer pair designed herein permits PRCD detection using either ApaLI or BsrGI. Moreover, these new sets can serve as a confirmatory mechanism to validate results from existing methods, thereby enhancing diagnostic reliability.
Predicted electrophoresis profiles confirmed that all designed primer-enzyme pairs clearly distinguish between homozygous wild-type, heterozygous, and homozygous mutant genotypes. Notwithstanding these design considerations, experimental validation of the proposed sets is recommended prior to clinical implementation.
In conclusion, this protocol set offers a cost-effective, rapid, and robust method for screening Mendelian genetic disorders in the Chihuahua breed. The integration of this method into routine veterinary practice will significantly contribute to early diagnosis and the breeding of healthy generations by identifying carriers.
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