ANSCI 361 1st Edition Lecture 3Outline of Last Lecture I. Genomic testinga. SNPsb. Elite breedingc. Commercial Farmsd. Imputatione. Phenotyping femalesII. SummaryOutline of Current Lecture I. Learning Goalsa. BLADb. CLADII. BLADa. Causeb. Prevalencec. Who is responsible?III. CLADIV. DUMPSa. DetectionCurrent LectureLearning goals:• Bovine Leukocyte Adhesion Deficiency (BLAD)Understand the causes of the diseaseFrom single nucleotide change to phenotypeUnderstand how simple molecular methods (PCR) can help eliminate a frequent disease• CLAD (dogs)Understand the molecular mechanisms of CLAD Conservation of genes across species leads to diagnosis and treatmentThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Bovine Leukocyte Adhesion Deficiency –BLADLAD in humans; BLAD in cattle; CLAD in dogsMost calves with BLAD die before one year of age. Recurrent bacterial infections of soft tissuesDeficiency in leukocyte surface glycoproteins known as integrinsIntegrins: family of cell surface receptors, expressed by leukocytes that play roles in host defense. Transmembrane proteins that bind ligands found in the extracellular matrix (molecular glue of life).Heterodimers: 2 interacting polypeptide chainsBLAD: White blood cells fail to migrate to the point of infection to destroy invading pathogensWhat is the cause of BLAD? CD18 Mutation. D128G mutation in cattle: Aspartic acid at amino acid 128 changed to glycine in CD18The mutation changes the leukocyte so it cannot attach to the vessel wall and reach infected tissues“The mutation occurs near the center of 26 consecutive amino acids that are identical in normalbovine, human, and murine CD18” Prevalence of BLAD in Holsteins: • USA (1993)- frequency of BLAD allele: 15% in bulls and 6% in cows• USA (1994)- 14.1% carriers• Denmark (1993)- 21.5% BLAD carriers• Japan (1995)- 2.6 to 23.5% carriers• Taiwan- 5.8% carriers• Poland (2000)- 4.8% carriersWho is responsible for spread of disease? • Bull selection: progeny testing• The sperm is collected for use in A• One bull of superior genetics can improve the performance of herds on many farms• If a sire is a heterozygous carrier for an undesirable recessive allele, that allele can be spread undetected to many progeny.Who is guilty?All of the affected calves have been traced to a common sire, Osborndale Ivanhoe, born in 1952 Sired over 79,000 daughters and over 1,200 sons (produced additional female cows)The carrier frequency of the D128G CD18 allele Active AI bulls: 15%Cows: 8%Control of BLAD in the Holstein population using restriction fragment length polymorphism (RFLP)A mutation in the recognition sequence of a restriction enzyme can lead to the gain or loss of a cutting site in a DNA sequence.RFLP Test used to eliminate BLADIn Holsteins, AI increased the frequency of the defective allele1988: 28% of young bulls were found to be BLAD carriers1993: BLAD carrier bulls for AI sire proof evaluations were eliminated using genetic testingCanine Leukocyte Adhesion Deficiency (CLAD):Pups with CLAD usually die early in life from multiple severe infections (skin and bone marrow, even when treated with massive doses of antibiotics.Leif Andersson (University of Uppsala, Sweden): CLAD mutation was easily identified because of the similarity to the huamn LAD and bovine BLAD Kijas et al. (1999): CLAD disorder is due to a mis-sense mutation in the CD18 gene (Cys36Ser) in Irish Setters.Affected animals die because of extreme susceptibility to infections, caused by an inability of white blood cells (leukocytes) to pass from the blood stream into infected tissue.Lack of a membrane glycoprotein called the leukocyte integrin beta-2 subunit or CD18.Inheritance is autosomal recessive.Deficiency of uridine monophosphate synthetase (DUMPS):• Autosomal recessive disorder that results in early embryonic mortality at day 40 of gestation• No living animals that are homozygous for the mutated allele• Heterozygous animals live normally, have 50% of normal enzyme activity, increased orotic acid in urine and milkDUMPS: Orotic acid  Uridine monophosphate synthetase (UMPS)  Uridine monophosphate monomer in RNAHeterozygous x heterozygous matings require ~3.1 services per calving, compared to ~2.0 for normal x normal matings. All carriers are descendants of Skokie Sensation Ned born in 1957.Testing for DUMPS has greatly reduced the frequency of heterozygous sires and of homozygous recessive embryos.Detection of DUMPS mutation• Measure the UMPS activity in erythrocytes• Heterozygotes show half of the normal activity• Enzyme activity may be affected by different factors• Schwenger et al. 1993: • Objective: detection of the point mutation responsible for DUMPS • Blood samples collected from 67 offspring of DUMPS carriers bulls• Enzymatic activity measures• RNA was extracted from livers of carriers and normal individuals (Why RNA?)• Reverse transcription into cDNA• Sequence of RT-PCR products• Heterozygous individuals: C/T in codon 405; arginine CGA to stop codon TGA.• Result: truncated proteinDUMPS allele = uncut, Normal allele =