2. Gene mapping: [Peter Snustad, M. Simmons – Principles of Genetics]
• Mapping: physical and genetic [463-464]
• Strategies in identifying human disease genes
• Human Genome project – History and Reality
• Techniques and Technology involved in genome mapping
• Low and high resolution mapping
• Strategies and milestones in mapping and sequencing of human genome
approaches to physical and genetic mapping
• Principles & strategies for identifying unknown disease or susceptibility
genes
• Beyond genomics – the physical and genetic mapping in the post genomic
era [Restriction mapping 444, contig map – 465,
3. How to identify a
human disease gene
To achieve [B3]:
• Test for mutations in
affected people [D5]
• Known homologue in
other species [A1, A3]
• Segregation of phenotype
and chromosome
alteration [C2]
• Linkage analysis [C1, D1]
4. How to pick a possible candidate gene?
A: Model organisms
• Mapped Candidate homologue
• Cloned Candidate homologue
C: Clinical input
• Collect families for mapping
• Chromosomal translocations or
deletions
• Collect unrelated patients
B: Database searching
• Candidate chromosomal region
• Check databases for gene
• Possible candidate gene?
• Has it been fully characterized?
D: Laboratory work
• Genetic mapping: Genome search
• Clone the chromosomal breakpoints
• Identify new human genes
• Work out full sequence and
structure
• Look for mutations
E: Interpreting the
results
• Successfully located?
• Think again about mode
of inheritance,
heterogeneity
• Think again about
candidate genes
• Pathogenic mutations
found?
Figure 16.1, Strachan & Read, Human
Molecular Genetics, 4th Edition,
page 499
5. Figure 16.1, Strachan & Read, Human Molecular
Genetics, 4th Edition, page 499 [figure legend]
• How to identify a human disease gene? As this figure emphasizes, there is
no single pathway to success, but the key step is to arrive at a plausible
candidate gene (box B3).
• This can then be tested for mutations in affected people (D5).
• The candidate gene might be identified through knowledge of a homolog in
another species (A1 and A3), through a patient who has both the
phenotype of interest and a chromosome structural abnormality (C2), or
through linkage analysis (C1 and D1).
• Success depends on an interplay between clinical work, laboratory bench
work, and database analysis, with the latter becoming steadily more crucial
as genome information accumulates.
6. Gene identification
• Position Dependent Approach:
• Positional cloning; approximate chromosomal position
known; study in animal models; chromosome abnormality
at cytogenetic or SNP array level indicate position making
candidate gene identification easier
• Position- Independent Approach:
• Functional studies of cells in culture; direct identification
of altered cells in culture; direct identification of altered
proteins or changed gene expression; extrapolation from
animal models
7. GENOMICS mapping:
• Genomics is a SUBDISCIPLINE OF GENETICS DEVOTED TO THE
MAPPING, SEQUENCING, & FUNCTIONAL AND COMPARATIVE
ANALYSES OF THE GENOME
• Structural Genomics: Study of genome structure, complete
nucleotide sequence is available
• Functional Genomics: Study of genome function; analyses of
transcriptome i.e. complete set of RNA transcribed from genome &
Proteome i.e. complete set of proteins encoded by a genome
8. Mapping: Determining location of genes on
chromosomes
Various approaches for mapping
• Positional cloning: The isolation of a clone of a gene or other DNA
sequence based on its map position in the genome
• Genetic Map: A diagram of a chromosome with distances based on
recombination frequencies (centiMorgans)
• Genetic map – distance in terms of centiMorgan units
• Cytogenetic map – distance in terms of bands and sub-bands
• Physical map – distance in terms of Kb or Mb
9. Various approaches for mapping (Determining location of genes on chromosomes)
• Positional cloning: The isolation of a clone of a gene or other DNA sequence
based on its map position in the genome
• Chromosome walking: A procedure that uses overlapping clones to move
sequentially down a chromosome from one site to another site
• Chromosome jumping: A procedure that uses large DNA fragments to move
discontinuously along a chromosome from one site to another site
• RFLP (Restriction Fragment Length Polymorphism): Restriction enzymes has
specific cleavage site in terms of nucleotide sequence, which can be changed
by mutation. Enzyme will not recognize that site leading to fragments of size
that are different from the wild type. This is used to construct genetic map for
use in positional cloning [Figure:16.3]
10. Gene identification by positional cloning:
DMD example; 1987
• 1983: locus was mapped to Xp21
11. Positional cloning:
Team work of
Clinical, laboratory, bioinformatics work, animal model
1. Define the candidate region
2. Obtain clones of all the DNA of the region
3. Identify all the genes in the region
4. Prioritize them for mutation screening
5. Test candidate genes for mutation in affected people
12. Recombination & Genetic mapping
• Prophase-I of Meiotic division: Recombination takes place among any of 2 pairs
i.e. 4 chromatids following synapse producing 2 recombinant and 2 non-
recombinant gametes
• Two loci on same chromosome [SYNTENIC] will separate due to recombination if
they are placed more distant
• Two loci present very close together on a chromosome transmit as a block
through a pedigree [HAPLOTYPE], thus are used to track through pedigrees and
through populations
• RECOMBINATION FRACTION is Genetic Distance between two loci
• % frequency of recombination can be expressed as cM [1% 1cM centi Morgan]
13. Crossing over and Recombination
• The bivalents stay together due to physical entanglement between two
of the four chromatids that is crossing-over seen in late prophase
cytological preparations as Chiasma (Pl. Chiasmata) that takes place
early to mid prophase of meiosis
• Chiasmata enables Recombination to take place between homologues
that is expressed in progeny (next generation)
• No. of crossovers can be counted by; counting chiasma on cytological
preparations and by counting recombinant chromosomes on genetic
analysis
14. • If 0.02 is Recombination Fraction of two loci, what will be the frequency
of recombination between them and what will be the genetic distance?
• 2% of meiotic prophases will show recombination and Genetic Distance
between them will be 2CM
• What is LOD score?
• Logarithm of Odds Score: The ratio of probability of loci being linked or not
linked in terms of likelihood gives odds of linkage; and logarithm of odds is
LOD score [symbolized as Z].
• What is Linkage Disequilibrium?
• Sharing of haplotypes or marker alleles at a higher or lower frequency than
would be expected for individual alleles. These are ‘Allelic Association’ or
LD, It’s a statistics of population not individuals.
• How HAPMAP project addresses Linkage Disequilibrium?
• International Haplotype Mapping in terms of high density maps for SNPs in
order to aid the identification of common disease genes [Nature,
2005,437:1299-1320 / 2007,449:851-861].
15. Linkage: Genes on the same chromosomes
inherit together
• How to identify? Separation of linked genes and formation of new
gene combinations as a result of physical event of crossing over
• Relation between recombination and linkage: Tightly linked genes
seldom recombine; loosely linked genes often recombine
• How to assess if recombination has occurred?: Number of progeny not
resembling characters selected in parents are recombinants
• What is the maximum possible frequency of recombination? If two
genes are independently assorted, they are present on separate
chromosomes, and have 50% recombination frequency thus,
maximum frequency is 50%; less then that indicate the genes are
present on the same chromosome
16. • What is Likelihood Ratio?
Measurement of likelihood of a person having a susceptibility factor
for developing a disease as compared to the general population i.e.
with or without the factor. OR, as compared to a subject known to have
no factor. Data of epidemiology of disease is required here.
• What is ODDS Ratio?
Measurement of likelihood in case control studies is done using ODDS
RATIO based on the results of cases as compared to controls that are
truly representative of the population, and also free from overlapping
confounding factors
17. Calculation of ODDS Ratio
Cases Controls
A: Risk Genotype
present
a c
B: Risk Genotype
absent
b d
The ODDs of being affected for A are a/c
The ODDs of being affected for B are b/d
The ODDS RATIO is (a/c) / (b/d) = ad / bc
18. Calculation of ODDS Ratio
Cases Controls
A: Risk Genotype
present
a
800
c
700
a/c
1.143
B: Risk Genotype
absent
b
200
d
300
b/d
0.67
The ODDs of being affected for A are a/c 1.143
The ODDs of being affected for B are b/d 0.67
The ODDS RATIO is (a/c) / (b/d) = ad / bc 1.71
19. Gene identification: History in nut-shell
• Positional cloning: DMD (1987) & Cystic Fibrosis (1989)
• Bronchio-oto-renal syndrome (1997)
• Multiple Sulfatase deficiency (2003) Lactase persistence (2002-2007)
• CHARGE syndrome (2004)
• Breast Cancer
• Crohn disease
20. Duchenne muscular dystrophy
• Locus mapped to Xp21 by two different groups:
1. DNA sample from a boy with DMD and cytogenetic deletion Xp21
served to compare by subtraction cloning and ZOO blotting, deleted
region could be pin-pointed, and conserved sequence among
various species could be identified-a 14kb transcript
2. DNA sample from a female with X autosome translocation that
disrupted the disease gene; a case with Xp/21p translocation
served to identify 2kb cDNA containing exons 1-16 of dystrophin
gene [figures 16.18 and 16.9]
21. Cystic Fibrosis: Gene finding by positional
cloning without any chromosome anomaly
• Cloning from DNA library
• Human-Hamster hybrid cells containing only human chromosome 7 –
when used for cloning by a group,
• Without any clue for position of the candidate gene, and restriction of
~45kb size with cosmids, chromosome jumping approach was used to
get clones that contain the gene of interest; comparison with other
species was carried out and conserved sequences were used to
screen cDNA library from affected muscle genes, with CpG island as a
marker for the 5’ end of gene; a 6.5kb fragment was found to contain
CFTR gene
23. Bronchio-oto-renal syndrome
• Linkage, chromosome aberration, large-scale sequencing, and homology
with a drosophila gene – combined approaches led to identification of a
gene on 8q13
• 7cM candidate region was refined to 470-650kb by further mapping and
delineation of a chromosome deletion in a patient – by screening genomic
library with P1 and PAC clones; markers close to candidate regions were
used; gaps were filled by chromosome walking
• The candidate genomic seq. was searched for ORF and amino acid
sequence; compared with drosophila eya [eye absent] gene; both proteins
are transcription factors; similar biochemical functions but loss of function
phenotype is different for human and drosophila
• Autosomal Dominant syndrome
24. Multiple sulfatase deficiency
• Autosomal recessive, inability to convert cysteine into formylglycine at the
active site of the sulfatase enzymes by sulfatase-modifying factor 1
underlies the inborn errors of metabolism involving sulfatase
• Cysteine converting activity was assayed and used for purifying the protein;
bovine testis was used as a rich source of activity, pure protein sequence
was searched in genetic databases for sequence that could encode this
peptide; a single human cDNA was found. This belonged to human 3p26,
gene SUMF1; same was sequenced in a series of patients with MSD and
mutations were shown
• Different approach; microcell mediated chromosome transfer was used in
human-mouse somatic cell hybrids; each containing single human
chromosome with dominant selectable marker. Microcells were fused with
patient cells, recovery was seen in #3 monochromosomal hybrids
• To narrow down the region on #3, microcells were irradiated before, using
microsatellite markers the donor specific alleles were identified in
recovered cells, 2.6Mb candidate region defined, out of 7 genes contained,
1 always showed mutations in patients [16.21 & 22]
25. Intestinal lactase – persistence of
• Lactase in intestine becomes inactive at later life, hence adults face
lactose intolerance; mainly linked with being pastoralist
• LCT gene on2q21; no change in seq. but; one SNP –13190 C/T in
intron 13 of neighbouring gene MCM6; affected its transcription in
Northern Europeans but not in Africans – where there was another
type of SNP; in Eurasians, the length was 1.4Mb in T/T homozygotes
which was 1900 bp in C/C homozygotes suggesting recent selection
Figure:16.22; 23
26. GWAS: Genome wide association studies
• High penetrance Mendelian disease genes: Low allele frequency, can
be mapped by parametric linkage analysis to candidate regions
• Intermediate penetrance variants conferring susceptibility to complex
disease: Common in population but with weak effects, can be
mapped by non-parametric linkage analysis but with low statistical
power and hence with limited success
• Low penetrance variants with association rather than causative effect
are identified by GWAS.
27. GWAS:
• Based on HAPMAP information, SNP tags for genetic location are
selected to identify each haplotype
• Disease cases and controls are genotyped with microarrays
• Statistically significant higher frequency of SNPs in disease cases
compared with controls are recorded, and analysis is repeated to
confirm
• In another population of Case / Control genotyping is done to see if
these SNPs show association here also
• Candidate SNPs are then studied for association
28. • Reticulocytes have high Hb levels, hence making C-DNA for alpha &
beta chains is easier
• How to map genes that are expressed at very low level
29.
30. Positional cloning: 16.8
Steps involved in the positional cloning of genes:
Human: Genetic mapping must be done by pedigree analysis, and
candidate genes must be screened by sequencing wild-type and mutant
alleles.
In other species, the genes of interest is mapped by appropriate genetic
crosses, and the candidate genes are screened by transforming the wild-
type alleles into mutant organisms and determining whether or not they
restore the wild type phenotype
31. Positional cloning: Steps involved
Human: Genetic mapping
must be done by pedigree
analysis, and candidate genes
must be screened by
sequencing wild-type and
mutant alleles
In other species, the genes of
interest is mapped by
appropriate genetic crosses,
and the candidate genes are
screened by transforming the
wild-type alleles into mutant
organisms and determining
whether or not they restore
the wild type phenotype
32. Understanding centiMorgan: 1 cM is one in hundred chromatids recovered
from meiosis will have undergone a crossing-over event in this interval
Mendel’s three postulates relate to single factors, 4th postulate was based on; ‘What if
there are more than one factors in a case, will the presence of one particularly affect
the presence of other?: During gamete formation, segregating pairs of unit factors assort
independently of each other hence, all possible combination of gametes can form with
equal frequency: Law of Independent assortment
• Deviation from the expected frequency indicates ‘Linkage’ between genes; if linkage is
tight, the frequency of recombination is almost zero & if linkage is absent, the frequency
of recombination is 50% i.e. two nearby genes do not recombine, whereas genes far
apart recombine more frequently, the average frequency of crossovers between two loci
is distance on genetic map
Meiosis stages; 4NN:
• Prophase-1:
• Leptonema
• Zygonema
• Pachynema
• Diplonema
chiasma forms
• Diakinesis
• Metaphase-1
• Anaphase-1
• Telophase-1
• Cytokinesis
• Prophase-2
• Metaphase-2
• Anaphase-2
• Telophase-2
33. Human Genome sequence: Variations among individuals
• Three categories of variants in terms of DNA sequence;
• No effect on phenotype
• Effect on phenotype contributing to individuality
• Pathogenic
• Types of variations between human genomes
• SNP
• Interspersed repeats: short and long
• Tandem repeats: Satellite, Minisatellite, Microsatellites
•
34. SNP: polymorphic and pathogenic
• Missense mutation
• Nonsense mutation
• Splice variants
• Frameshifts
• Silent changes that can be pathogenic
35. Short Tandem Repeats:
• Dynamic mutations
• Expanding nucleotide repeats;
• Very large, Moderate [or CAG within coding sequences];
• Dynamic expansions
36. Micro deletions & Micro duplications:
Dosage effect in cases involving
“Smaller than cytogenetics limits; Larger than single gene”
• Single gene syndromes
• Contiguous gene syndromes
• Segmental aneuploidy syndrome
38. Mechanism of pathogenesis due to variation
• Loss of function mutation
• Gain of function mutation
• Dominant negative mutation
39. Genotype – Phenotype correlations: A
challenge for researchers
• Success stories mainly in cancer and haemoglobinopathies
• Poor in mitochondrial mutations
• Importance of family studies
40. 92% genome
$3Billion
Government funded
Human Genome Project:
1990-2001,
15th Feb 2001:
Nature:409:860-921.
95% genome
$300million
Private company funded
Human Genome Project:
1998-2001,
16th Feb 2001:
Science:291:1304-1351.
Craig Venter
Celera Genomics
Francis Collins
HGP consortium
41. Complementation analysis
• To check if same phenotype due to mutation has resulted
due to mutations in two different genes or in same alleles
• Cross between two mutant strains F1 phenotype
• If: Wild type phenotype: Two mutations are in separate
genes, each heterozygous i.e. one normal copy of each gene
is encoded to ‘complement’ the mutant phenotype
• If: Mutant phenotype: Two mutations on same gene,
homozygous for two mutants, no normal copy is encoded
42. Two mutants with
similar
phenotypes:
mutated gene will
be same or
different?? How
to study this?
Cross the two & analyze
the F1..
•All normal: Genes are
different, progeny all
heterozygous, mutation
complemented
•All mutant: Genes are
same, progeny
homozygous for two
alleles
45. Thomas Morgan,1910 & Calvin Bridges
• First experimental evidence of Boveri & Sutton’s theory that genes are
transmitted on specific chromosomes & also the link with sex was
observed
• Peas plants: Origin of trait was Pollen or Ova – did not matter,
but not the same for Drosophila, ratio changes depending on
mutant is male or female
• Cross A:
Red Female/White MaleRed male & Red female progeny
Red female + Red female + red male + white male
• Cross B:
White Female/Red MaleRed female & white male progeny
white female + red female + white male + red male
46. What is…..
1. Hemizygous?
2. PAR?
3. X-linkage?
1. Paired genes are
either homo or
heterozygous,
unpaired genes are
hemizygous
2. X & Y synapse &
segregate at meiosis,
pairing at a region on
each end of
chromosome i.e.
pseudo autosomal region
3. Unique inheritance
pattern for the genes
on X-chromosome
47. What is
Crisscross pattern
of inheritance
& why is it unique
to X-linked
conditions?
Traits controlled by
recessive X-linked genes
pass from homozygous
mothers to all sons, lead
to this pattern when
pedigree is plotted
49. Certain
X-linked
traits
seen only
in males…
Why???
• Only source of lethal allele is
heterozygous female carriers
• Passed to half of sons who will be
hemizygous and hence affected but
are..
• Disabled or die before reproductive
age e.g., Duchenne form of Muscular
Dystrophy;
Passed to females but they are
heterozygous hence carriers
50. Which
human
genetic
conditio
ns are
X-
linked?
• Hemophilia A & B
• Muscular dystrophy
• Glucose 6 – Phosphate dehydrogenase
• Hypoxanthine guanine phosphoribosyl
transferase (Lesch-Nyhan syndrome)
• Fabry’s disease
• Hunter syndrome
• Ichthyosis
• Color blindness – protan or deutan
type i.e. insensitivity to Red or Green
light respectively
52. Physical mapping: Based on distances in base pairs, kilo base
pairs, or mega base pairs separating markers
• Isolation and preparation of restriction maps of large numbers of
genomic clones, overlapping clones can be identified and used to
construct physical maps of chromosomes and entire genomes
53. Explain with reasons giving details of genetic
conditions
• Drinking water kept in copper utensil overnight is considered very
good for health. But in certain cases this can be wrong and even
harmful.
• Milk is good for health, but there are exceptions.
[16th Feb. 2015] to submit on 18th
![Gene mapping: [Peter Snustad, M. Simmons – Principles of Genetics]
• Mapping: physical and genetic [463-464]
• Strategies in identifying human disease genes
• Human Genome project – History and Reality
• Techniques and Technology involved in genome mapping
• Low and high resolution mapping
• Strategies and milestones in mapping and sequencing of human genome
approaches to physical and genetic mapping
• Principles & strategies for identifying unknown disease or susceptibility
genes
• Beyond genomics – the physical and genetic mapping in the post genomic
era [Restriction mapping 444, contig map – 465,](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)