Calpastatin (CAST) gene polymorphism in Kajli, Lohi and Thalli sheep breeds

Calpastatin-encoding gene (<i>CAST</i>) is located on the fifth chromosome of sheep and it plays an  important role in the development of muscles and in meat tenderness. The present study was conducted to  investigate a calpastatin (CAST) gene polymorphism in Pakistani Thalli, Lohi and Kajli sheep breed. Random blood samples were collected from 300 animals (100 samples from each Thalli, Lohi and Kajli breeds).  Genomic DNA was extracted using phenol-chloroform extraction method. A 622 bp CAST gene segment (exon 1C/1D region) was amplified by polymerase chain reaction (PCR) using ovine specific primers. Restriction  fragment length polymorphisms (RFLPs) in the amplified fragments were studied using Msp1 restriction  enzyme. Frequencies of MM, MN and NN genotypes were found to be 77, 20 and 3% in Lohi breed and 68, 26 and 6% in Kajli breed respectively. In Thalli sheep, only the MM (80%) and MN (20%) genotypes were  detected. Chi-Square test (p < 0.05) showed that all the three populations used in this study were in Hardy-Weinberg equilibrium. By comparing the results of this study with those of previous studies, it seems that the MM genotype is the dominant genotype and the M allele is the dominant allele in small ruminant breeds belonging to different geographical locations.Key words: Thalli, Lohi, CAST gene, Kajli, polymorphism, Msp1, PCR-RFLP

Pathogenic variants of AIPL1, MERTK, GUCY2D, and FOXE3 in Pakistani families with clinically heterogeneous eye diseases.

Significant number out of 2.2 billion vision impairments in the world can be attributed to genetics. The current study is aimed to decipher the genetic basis of Leber congenital Amaurosis (LCA), Anterior Segment dysgenesis (ASD), and Retinitis Pigmentosa (RP), segregating in four large consanguineous Pakistani families. The exome sequencing followed by segregation analysis via Sanger sequencing revealed the LCA phenotypes segregating in families GCUF01 and GCUF04 can be attributed to c.465G>T (p.(Gln155His)) missense and novel c.139_140delinsA p.(Pro47Trhfster38) frameshift variant of AIPL1 and GUCY2D, respectively. The c.1843A>T (p.(Lys615*) truncating allele of MERTK is homozygous in all the affected individuals, presumably suffering with RP, of the GCUF02 family. Meanwhile, co-segregation of the ASD phenotype and the c.289A>G (p.(Ile97Val)) variant of FOXE3 was found in the GCUF06 family. All the identified variants were either absent or present in very low frequencies in the control databases. Our in-silico analyses and 3D molecular modeling support the deleterious impact of these variants on the encoded proteins. Variants identified in MERTK, GUCY2D, and FOXE3 were categorized as "pathogenic" or "likely pathogenic", while the missense variant found in AIPL1 was deemed to have "uncertain significance" based upon the variant pathogenicity guidelines from the American College of Medical Genetics and Genomics (ACMG). This paper highlights the genetic diversity of vision disorders in the Pakistani population and reports the identification of four novel mutations in families who segregate clinically heterogeneous eye diseases. Our results give insight into the genotype-phenotype correlations of AIPL1, FOXE3, MERTK, and GUCY2D variants