INTRODUCTION Limb development is a complex process by which limb is formed from embryonic cell, grow and digits are formed. Limb formation begins in the limb field, as a limb "bud." Fibroblast growth factor (FGF) induces formation of an organizer, called the apical ectodermal ridge, which guide further development and controls cell death. The vertebrate skeleton is generated from three different lineages: the somites which give rise to the axial skeleton, the lateral plate mesoderm which generates the limb skeleton, and the cranial neural crest which gives rise to the pharyngeal arches as well as the craniofacial bones and cartilage . In the majority of the skeleton, osteogenesis is achieved through endochondral ossification, where a cartilage intermediate is formed from mesodermal tissue, then replaced by a calcified matrix to form bone. Apoptosis is a programmed cell death which is necessary to eliminate webbing between digits. Fig 1 shows ectrodactyly and central reduction of digit in hand from Pascal H.G. Duijf 2003.

1. GENETIC ANALYSIS OF CONGENITAL LIMB MALFORMATIONS
Supervisor
Dr. Akhtar Ali
Centre for Genetic Disorders
Banaras Hindu University
Presented by
Santosh Kumar Yadav
Department of Biotechnology
VBS Purvanchal University

2. • Embryonic limb develop, as a limb "bud." Fibroblast growth factor
(FGF) induces formation of an organizer, called the apical
ectodermal ridge (AER) , which guide further development and
controls cell death
• Apoptosis is necessary to eliminate webbing between digits
• Somites give to the axial skeleton, the lateral plate mesoderm
generates the limb skeleton
Normal Limb Development

3. (a) Progress zone model
(b) Early specification model

5. Congenital Limb Malformations
• genetic or environmental factor during embryogenesis
• Clinically variable and genetically heterogeneous
• Syndactyly, monodactyly, ectrodactyly, Brachydactyly

6. Split-Hand/Foot Malformation (SHFM)
• Characterized by deep median cleft due to absent of central
ray and fusion of remaining digits
• Crab claw like hand (Ectrodactyly)
• 1 in every 8,500 to 25,000 births
accounting for 8-17% of all limb reduction defects
• Clinically variable and genetically heterogeneous
• syndromic (e.g. EEC) and non-syndromic forms(SHFLD)
• autosomal dominant, autosomal recessive, X-linked
incomplete penetrance, variable expressivity

7. SHFM Loci
SHFM1 7q21 --
SHFM2 Xq26.3 --
SHFM3 10q24-q25 --
SHFM4 3q27 TP63 gene
SHFM5 2q31 --
SHFM6 12q13.11 WNT10B gene

8. ObjectiveObjective
Genetic Analysis of Congenital Limb Malformations
Determination of Linkage of
10q25 locus with SHFM using
microsatellite markers in a
multiplex family
Cytogenetic analysis of congenital
limb malformations

9. Family based linkage study of congenital limb
malformation
 DNA isolation from members of a multiplex family
 PCR of microsatellite markers and capillary
electrophoresis
 Analysis of inheritance of alleles
Determination of Linkage of 10q25 locus with SHFM using microsatellite
markers in a multiplex family

10. II-1 I-2I-1
II-1 II-2
III-3 III-4III-2III-1
IV-2IV-1
V-1 V-2 V-3
IV-4IV-3
V-5V-4
IV-5 IV-6 IV-7
V-9V-8V-6 V-7 V-10 V-11
VI-1 VI-2
IV-8 IV-9
V-14 V-15V-12 V-13
IV-10
Pedigree of the Family
Pedigree show autosomal dominant inheritance

11. Genomic DNA Quantification
By agarose gel electrophoresis and Spectrophotometer (Nanodrop®)
λ DNA 1 2 3 4 5 6 7 8 9
Photograph showing thegenomic DNA electrophoresisin 0.8% AgaroseGel
.

12. Microsatellite markerMicrosatellite marker
• short tandem repeat sequence of nucleotide such as ‘CACACACA’ and
occur in non-coding DNA
• polymorphic and variable in population
• applications in genetics such as mapping genes in inherited disease
• detect microsatellite is to design PCR primer that is unique to one locus
in genome and base pair on either side of repeated portion
• Two PCR primer (F and R) are designed to microsatellite region

13. Microsatellite marker used
DNA Marker Dye Colour
D10S192 NED YELLOW
D10S 185 FAM BLUE
D10S 1686 VIC GREEN
D10S 587 NED YELLOW
D10S 537 NED YELLOW
D10S 1693 VIC GREEN
D10S 597 VIC GREEN
D10S 1651 NED YELLOW
D10S 1652 NED YELLOW

15. PCR reaction
Components Vol per reaction
(µl )
DNA (50 ng/µl) 0.6
True allel PCR mix (ABI, USA )
contains dNTPs, Polymerase,
buffer, H2O
4.5
Sterile deionised water 1.9
Primer (F+R) 0.5
Total 7.5

16. PCR condition
Cycle condition No. of
cycles
Initial denaturation 95ºC for 12 min 1
Denaturation 94 ºC for 15 sec 10
Anneal 55 ºC for 15 sec
Extensions 72 ºC for 30 sec
Melt 89 ºC for15 sec 20
Anneal 55 ºC for 15 sec
Extensions 72 ºC for 30 sec
Final extensions 72 ºC for 10 min 1
Hold 4 ºC hold

17. Capillary Electrophoresis
• HiDi formamide and GS500LIZ DNA size standard (ABI, USA)
• Take 1µl PCR product and 9µl Master mix in 96well sequencing plate
• Then mixed spun, denatured at 95ºC for 5 minutes, snap-chilled and
put in the 3130Genetic Analyzer (ABI, USA) for capillary
electrophoresis.
• After electrophoresis the data was analysed with Gene Mapper
software (ABI, USA).
HiDi + LIZ
9.0µl 0.5µlFAM : VIC : NED
1 : 1 : 2
1µl 9µl
Mixed PCR product same panel
Master mix

18. D10S1693, D10S192 , D10S1686, D10S597
RESULTS

19. D10S587 Heterozygous D10S537 Heterozygous

20. D10S185 D10S597

21. D10S192
250bp

22. Transmission of alleles of microsatellite markers
287
153
255
217
261
291
223
102
210
----
----
----215
245
289
219
102
----
D10S1652
D10S537
D10S1686
D10S185
D10S192
D10S597
D10S1693
D10S587
D10S1651
----
161
269
217
257
289
219
106
----
----
----
----217
259
289
221
98
----
III-2
----147
271
217
259
289
221
98
----
IV-2 IV-3
273
273
291
210
258
291
219
106
216
291
147
255
206
254
291
219
98
210
IV-1 IV-4 IV-5 IV-6 IV-7 IV-8 IV-9 IV-10
291
147
259
217
259
289
221
102
216
289
161
259
217
259
291
221
106
212
291
147
273
217
259
289
221
98
212
289
147
273
217
259
289
221
98
216
289
159
255
210
259
285
223
102
210
V-1 V-2 V-3
10q21.2
10q23.2
10q24
10q24
10q24
10q25.1
10q24.2
10q25.3
10q26

23. • A haplotype alleles 217-259-289-221 of markers D10S185,
D10S192, D10S597 and D10S1693 is co-transmitted with
the disorder suggesting its linkage with the disorder.
• A recombination happened between the markers D10S1693
and D10S587 in individual IV-8.
• Therefore mutation/other genetic error causing split-
hand/foot malformation in this family is likely to be present
somewhere between D10S185 and D10S1693 markers.

24. • Heparinized blood was received from a patient with digital anomaly
and other congenital malformations referred from the hospital for
chromosome analysis .
• The blood was cultured for 72 hours using RPMI1640 medium and
10% FBS. Phytohaemagglutinin (PHA-m) was added to induce cell
division.
• Colchicine was added 2 hour before harvesting of the culture to
arrest cells in metaphase stage.
• This culture was harvested and chromosome slides were prepared .
• The slides were incubated for overnight followed by G-banding .
• G-banded metaphase plates were observed under the microscope
and karyotyping was performed using iKaryos® software (Carl
Zeiss, Germany)
Cytogenetic analysis of congenital limb malformations

25. No gross chromosomal anomaly was detected in this patient
RESULT

26. G-banding Karyotype of the patient

27. ReferencesReferences
• Alison M. Elliott, Martin H. Reed, and Jane A. Evans, (2007).
Triphalangeal Thumb in Association with Split Hand/Foot
Malformation : A Phenotypic Marker for SHFM3 Birth Defects
Research (Part A) 79:58–61
• Alison M. Elliott and Jane A. Evans Genotype–Phenotype
Correlations in Mapped Split Hand Foot Malformation (SHFM)
Patients Department of Biochemistry and Medical Genetics,
University of Manitoba, Winnipeg, Manitoba, Canada American
Journal of Medical Genetics Part A 140A:1419–1427 (2006).
• Christian Babbs Raoul Heller David B. Everman Mark Crocker
Stephen R. F. Twigg Charles E. Schwartz Henk Giele Andrew O.
M. Wilkie A new locus for split hand/foot malformation with long
bone deWciency (SHFLD) at 2q14.2 Wed from a chromosome
translocation Hum Genet (2007) 122:191–199.

28. • David B. Everman, Chad T. Morgan, Robert Lyle, Mary E. Laughridge, Michael J.
Bamshad, Frequency of Genomic Rearrangements Involving the SHFM3 Locus at
Chromosome 10q24 in Syndromic and Non-Syndromic Split-Hand/Foot
Malformation1Center for Molecular Studies, J.C. Self Research Institute of Human
Genetics, Greenwood Genetic Center, Greenwood, South Carolina American Journal
of Medical Genetics Part A 140A:1375–1383 (2006).
• Hans van Bokhoven ,Ben C.J.Hamel.Mike Bamshad,Eugenio Sangiorgi,fiorella
Gurrieri,P63 Gene Mutation in EEC Syndrome, Department of Human Genetics,
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492, 2001).
• Mark E.Nunes ,Gregory Schutt,Raj p.Kapur ,Frederick Luthardt ,Mary Kkolich ,Peter
Byers and Jams P.Evans (1995 ) A second autosomal split hand /split foot locus
maps to chromosome 10q24-q25 Human Molecular Genetics, 1995 Vol.4 , No. 11
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B.Everman, Corinne Gehrig, Nath ,Fiorella Gurrieri ,Giovanni Neri V.Solanki,
Charles E.Schwartz,and Stylianos E. Antonarakis (2005 ) Split-Hand /Split Foot
Malformation 3 (SHFM3) at 10q24,Development of Rapid Diagnostic Methods and
Gene Expression From the Region American General of Medical Genetics part A
140A:1384-1395.

29. • Rydvan S. Ozen, Bora E. Baysal, Bernie Devlin , Joan E. Farr ,Michael D.Ehrlich,
and Charles W. Richard, III (1999) Gorry, and Garth Fine Mapping of the Split-
hand/Split-Foot locus (SHFM3) at 10q 24: Evidence for Anticipation and Segregation
Distortion Am.j. Human. Genetic.64:1646-1654
• Mohammed Naveed, Swapan K. Nath, Mathew Gaines, Mahmoud T. Al-Ali,
Genomewide Linkage Scan for Split–Hand/Foot Malformation with Long-Bone
Deficiency in a Large Arab Family Identifies Two Novel Susceptibility Loci on
Chromosomes 1q42.2-q43 and 6q14.1 American Journal of Medical Genetics Part A
140A:1375–1383 (2006).
• Tony Roscioli, Peter J. Taylor, Andrew Bohlken, Jennifer A. Donald, John Masel,
Ian A. Glass, and Michael F. Buckley, The 10q24-linked Split Hand/Split Foot
Syndrome (SHFM3): Narrowing of the Critical Region and Confirmation of the Clinical
Phenotype American Journal of Medical Genetics 124A:136–141 (2004).
• Raas-Rothschild, S Manouvrier, M Gonzales, J P Farriaux, S Lyonner, A Munich
Refined mapping of a gene for split hand-split foot malformation on chromosome
10q25 France medical genetic 1996;33;996-1001