GENETIC ANALYSIS OF CONGENITAL LIMB MALFORMATIONS

SupervisorDr. Akhtar AliCentre for Genetic DisordersBanaras Hindu University

Presented bySantosh Kumar YadavDepartment of Biotechnology VBS Purvanchal University

• 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

(a) Progress zone model

(b) Early specification model

Congenital Limb Malformations

• genetic or environmental factor during embryogenesis• Clinically variable and genetically heterogeneous

• Syndactyly, monodactyly, ectrodactyly, Brachydactyly

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-linkedincomplete penetrance, variable expressivityexcessive transmission from affected males to sons

SHFM Loci

SHFM1 7q21 --SHFM2 Xq26.3 -- SHFM3 10q24-q25 --SHFM4 3q27 TP63 geneSHFM5 2q31 --SHFM6 12q13.11 WNT10B gene

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

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

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

Genomic DNA Quantification

By agarose gel electrophoresis and Spectrophotometer (Nanodrop®)

DNA 1 2 3 4 5 6 7 8 9

Photograph showing the genomic DNA electrophoresis in 0.8% Agarose Gel

.

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

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

PCR reaction

Components Vol per reaction (l )

DNA (50 ng/l) 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

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 secExtensions 72 ºC for 30 secMelt 89 ºC for15 sec 20 Anneal 55 ºC for 15 sec Extensions 72 ºC for 30 secFinal extensions 72 ºC for 10 min 1 Hold 4 ºC hold

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 0.5µl FAM : VIC : NED

1 : 1 : 2

1µl 9µl

Mixed PCR product same panel Master mix

D10S1693, D10S192 , D10S1686, D10S597 RESULTS

D10S587 Heterozygous D10S537 Heterozygous

D10S185 D10S597

D10S192

250bp

Transmission of alleles of microsatellite markers

287153255217261291223102210

------------215245289219102----

D10S1652 D10S537 D10S1686 D10S185 D10S192 D10S597 D10S1693 D10S587 D10S1651

----161269217257289219106----

------------21725928922198 ----

III-2

----14727121725928922198----

IV-2 IV-3273273291

210258291219106216

29114725520625429121998

210

IV-1 IV-4 IV-5 IV-6 IV-7 IV-8 IV-9 IV-10291147259

217259289221102216

289161259217259291221106212

291147273

21725928922198212

289147273

21725928922198216

289159255210259285223102210

V-1 V-2 V-3

10q21.210q23.210q2410q2410q2410q25.110q24.210q25.310q26

• 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.

• 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

No gross chromosomal anomaly was detected in this patient

RESULT

G-banding Karyotype of the patient

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.

• 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, University Medical Centre, Nijmegen, The Netherlands Am. J. Hum. Genet. 69:481–492, 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 2165-2170.

• Robert Lyle, Uppala Radhakrishna, Jean-Louis Blouin, Sarantis Gaos, David 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.

• 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