Techniques to study complex genetic diseases. Dr. Nallur B. Ramachandra Principle Investigator Professor and Coordinator for M.Sc., Genetics Genomics Laboratory, DOS in Zoology University of Mysore Manasagangotri , Mysore – 570 006 email@example.com / firstname.lastname@example.org.
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Dr. Nallur B. Ramachandra
Professor and Coordinator for M.Sc., Genetics
Genomics Laboratory, DOS in Zoology
University of Mysore
Manasagangotri, Mysore – 570 006
A catalog of human genes and genetic disorders
On average two random people differ at 3 mbp ~1/1000.
A few genes can have a big impact on appearance.
Approach – 3 Four Levels of Biomarkers
Congenital heart defects
Neural tube defects
The hemoglobin disorders
March of demise birth defect foundation, 2006
1) Chromosomal syndromes –DS,TS,KS
2) Congenital Heart diseases
Reamon-Buettner et al., 2007 BMC Med Genet.; 8: 38,
Smitha et al.,2006, Indian Journal of Human Genetics, 12,1
A four chambered heart is necessary for the separation of oxygenated and deoxygenated blood.
Garg, 2006,Cell Mol Life Sci, 63:1141-1148
Overall Prevalence = 1-2% live births
Smitha et al., Ind J Hum Genet, 2005
1 2 3 4 7 9
1 2 3 4 5 6 7
1 2 3 4 5 6 7 8
1 3 4 5 6
1 2 3 4
1 2 3
Pedigree of consanguineous marriages between parents of CHD patients:
(A) Uncle-niece marriage and(B) First-cousin marriage
Smitha et al., Annals Hum Bio, 2006
G-banded chromosomes of congenital heart disease patient with isochromosome 18p [47,XX,+i(18p)]: Metaphase showing the i(18p) (arrow).
Smitha et al., J App Genet, 2006
FISH plate showing chromosome 18p subtelomeric region on metaphase chromosome of the isochromosome 18p child. FISH probe identifies chromosome 18p (spectrum green),centromeric 18 (spectrum aqua),chromosome 11p (spectrum green) and chromosome 11q (spectrum orange). The labels 18p and arrow denote the 18p of the normal chromosome 18 (white) and isochromosome 18p (yellow) respectively.
FISH image of the proband [47,XY,+21,t(9;13)(q34.1;q34)]:(A) Trisomy 21 (Spectrum red) and (B) Subtelomeric deletion on chromosome 9q and the translocation of the deleted region on subtelomeric region of chromosome 13q on metaphase chromosomes of the proband. (a) Normal chromosome 9q region (Spectrum red); (b) Chromosome 9 with subtelomeric deletion of 9q; (c) Chromosome 13 with subtelomeric region of chromosome 9 (Spectrum red).
G-banded chromosomes of the congenital heart disease patient with complex variant translocation among chromosomes 5, 9 and 13 [t(5;9;13)(q14;p24;q34.1)]: (A) Metaphase showing (i) del9q11 (arrow) and (ii) t(5;9;13)(q14;p24;q34.1), and (B) Karyotype of the above metaphase.
FISH image showing the complex variant translocation between chromosomes 5q, 9p and 13q on metaphase chromosomes of the proband [46, XY, t(5;9;13)(q14;p24;q34.1)]:(a) Normal chromosome 5; (b) Abnormal chromosome 5 with part of 5q deleted; (c) Normal chromosome 9; (d) Abnormal chromosome 9 with the deleted part of chromosome 5q;(e) Chromosome 13 with the deleted subtelomeric region of chromosome 9p.
G-banded chromosomes of the congenital heart disease patient with inversion in chromosome 9[inv(9)(p11-q13)]: Metaphase showing inv(9)(p11- q13) (arrow).
Smitha et al., Int J Hum Genet, 2007
M-FISH image of congenital heart disease patient with complex variant translocation chromosomes 5, 9, 11 and13 [t(5;9;11;13)(q14;p24;p15;q33)]
Smitha et al., Int J Hum Genet, 2010
Covers a spectrum of sizes 21bp to > 2000kb
microRNA – 11bps - 21bps
tRNA, snRNA genes – smallest 65-75bps
The smallest protein coding gene- 406 bp –histone H4
The longest human gene so far discovereddystrophin gene- 2400 kb with 79 introns
DNA LIBRARY PROBE
PURIFICATION OF POSTIVE CLONE
DNA SEQUENCING & ANALYSIS
CONFIRMATION OF GENE
USE AS UNIQUE MOLECULE / DRUG
…invented by Kary Mullis while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino,
"It was quiet and something just went, Click!"
Kary B. Mullis
Nobel Laureate, 1993
Bruneau, Nature, 2008
transcription factor mutations
Mass in Daltons
Congenital Heart Disease(CHD)
3-D models of mutated proteins derived from 9 nsSNPs of zinc finger region of GATA4. Each model was superimposed by control (left side of each image) and mutated protein (right side of each image). Models were done using solution structure of the n-terminal zinc finger of murine GATA-1 (PDB Id: 1gnfA) and also for mutant. Ball and stick representation in both the models showed the side chain structural differences between control and mutated proteins. (a) E216D: In control- Delta Carbon (CD), Epsilon Oxygen (OE1, OE2) and in mutant OD1, OD2. (b) G214S: In control-no changes and in mutant Gamma Carbon (CG), Gamma Oxygen (OG). (c) M223T: In control- CB, CG, Delta Sulphur (SD), CE and in mutant CG2, OG1. (d) R229S: In control- CG, CD, Epsilon Nitrogen (NE), CE2, Zeta Carbon (CZ). NH1, NH2 and in mutant OG. (e) G234S: In control- no changes and in mutant CB, OG. (f) N239D: In control-ND2, OD1 and in mutant OD1, OD2. (g) N239S: In control- CG, ND2, OD1 and in mutant OG. (h) Y244C: In control- CG, CD1, CD2, CE1, CZ, OH and in mutant Gamma Sulphur (SG). (i) N248S: In control- CG, ND2, OD1and in mutant OG.
Dinesh et al., New York Science J, 2010
Genomic Size: 3,125 bps; Exon : 2
Transcript length: 1,585 bps
Translation length: 324 residues
Heterozygous - c. 896C>A (A240A)
Dinesh et al., Genet Test Mol Bio, 2010
Remon-Buettener et al., BMC Med Genet, 2007
Analyse Gene (mRNA) or protein expression through large scale non-radioactive northern and southern hybridization analysis.
Essentially fluorescent based (Cy3, Cy5, Alexa) high throughput Northern blot analysis.
Allows expression analysis of about 30,000 genes in one experiment.
Short (20-25bp) and Long (30-70bp) Oligo arrays.
cDNA arrays printed on Glass (500-1000bp).
Genomic or EST Arrays generated by DNA amplification method.
Of Cy3 and Cy5
Hybridize on micro array
Hybridizing arrays are of two varieties;
1. Wells 2.Beads
cM (recombinant distance)
Genomic Screen[1100 markers 5 cM]
LOD = 2.1
LOD = 3.5
Multipoint LOD = 3.5
cM (recombinant distance)
Long generation time
Experimental mating is not possible
Research Fellows:Dr. Smitha Ramegowda
Ms. Kusuma L.
Mr. Dinesh S. M.
IISc, Bangalore:Dr. Arun Kumar
Pediatricians, Mysore:Dr. B. Krishnamurthy
Dr. M. R. Savitha
Dr. D. Narayanappa
Dr. S. N. Prashanth
Dr. Prasad Karat
Dr. H. Abbas
Dr. P.A. Mahesh
NIRRH, Mumbai:Dr. Zarine Patel
Mr. Harsh Gawde
CSIR, New Delhi
Patients, families participated in this study
Doctors and PG students for their kind support
Chairman, DOS in Zoology, Univ. of Mysore
Prof. H. A. Ranganath for his encouragement