Kappa-Casein (kappa-CN) is the milk protein that determines the size and specific function of milk micelles, and its cleavage by chymosin is responsible for milk coagulation. We have previously detected and characterized four variants of the goat kappa-CN in Spanish, French, and Italian breeds by screening the major part of the coding region in exon 4. Here we have sequenced and analyzed the full coding region of the kappa-CN gene which includes exons 3 and 4. No additional mutations were found, with exception of a single nucleotide substitution in exon 3, which had no amino acid change. However, the analysis of the association between the different mutations resulted in two new variants designated kappa-CN F and G. The novel variants are present in the Italian breeds Teramana, Girgentana, and Sarda (variant F). A protocol for rapid simultaneous genotyping of all known kappa-CN variants using the primer extension method was described, and a total of 210 animals from nine European breeds were genotyped. Alleles A and B are the most frequent variants occurring in the majority of breeds with highest prevalence of the B variant, except for the Canaria breed where the A allele is more frequent. Sequence data suggest that the F variant is the original type of caprine kappa-CN, other alleles being derived from this type following two different trunks by successive mutations.
Polymorphism in the goat kappa casein gene was studied using the base excision sequence scanning (BESS) method and sequencing. Seven polymorphic sites, corresponding to single nucleotide transversions were detected. Three of these were silent mutations while the other four produced amino acid substitutions. The association between these polymorphic sites was investigated, which resulted in the identification of three goat kappa casein alleles, designated A, B, and C. Protocols for rapid genotyping of the C variant were developed by polymerase chain reaction–restriction fragment length polymorphism using Alw44I and BseNI restriction endonucleases. The occurrence of this allele was found to be very low in Spanish breeds but more frequent in the French Saanen goat. Further studies among different goat populations are necessary to establish the distribution of these alleles and their effects on the quality and functional properties of milk.
A new polymorphism has been identified in the goat kappa-casein gene by evaluating genomic DNA from the Montefalcone breed in Italy. The polymorphic site consists of a single nucleotide substitution A to G at position 242 of the exon 4 and produces an amino acid substitution Asp/Gly. A polymerase chain reaction-restriction fragment length polymorphism protocol for rapid genotyping of the variant has been developed, using the HaeIII enzyme. Animals from Italian, Spanish, and French breeds have been analyzed to investigate the occurrence of the allele in other populations. The allele appears to be exclusive to the Montefalcone breed.
Lafora disease (LD) can be diagnosed by skin biopsy, but this approach has both false negatives and false positives. Biopsies of other organs can also be diagnostic but are more invasive. Genetic diagnosis is also possible but can be inconclusive, for example, in patients with only one heterozygous EPM2A mutation and patients with apparently homozygous EPM2B mutations where one parent is not a carrier of the mutation. We sought to identify occult mutations and clarify the genotypes and confirm the diagnosis of LD in patients with apparent nonrecessive disease inheritance. We used single nucleotide polymorphism, quantitative PCR, and fluorescent in situ hybridization analyses. We identified large EPM2A and EPM2B deletions undetectable by PCR in the heterozygous state and describe simple methods for their routine detection. We report a coding sequence change in several patients and describe why the pathogenic role of this change remains unclear. We confirm that adult-onset LD is due to EPM2B mutations. Finally, we report major intrafamilial heterogeneity in age at onset in LD.
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