ORIGINAL INVESTIGATION

Genetic mapping of an autosomal recessive postaxial polydactylytype A to chromosome 13q13.3–q21.2 and screeningof the candidate genes

Umm-e-Kalsoom • Sulman Basit •

Syed Kamran-ul-Hassan Naqvi •

Muhammad Ansar • Wasim Ahmad

Received: 5 June 2011 / Accepted: 20 August 2011 / Published online: 30 August 2011

� Springer-Verlag 2011

Abstract Postaxial Polydactyly (PAP) is characterized

by fifth digit duplication in hands and/or feet. Two types of

PAP including PAP-A, representing the development of

well-formed extra digit, and PAP-B, representing the

presence of rudimentary fifth digit, have been described.

Both isolated and syndromic forms of PAP have been

reported. Isolated forms of PAP usually segregate as an

autosomal dominant trait and to date four loci have been

identified. In the present study, we have described mapping

of the first locus of autosomal recessive PAP type A on

chromosome 13q13.3–13q21.2 in a consanguineous Paki-

stani family. Using polymorphic microsatellite markers,

the disease locus was mapped to a 17.87-cM (21.13 Mb)

region flanked by markers D13S1288 and D13S632, on

chromosome 13q13.3–13q21.2. A maximum multipoint

LOD score of 3.84 was obtained with several markers

along the disease interval. DNA sequence analysis of exons

and splice-junction sites of ten candidate genes (CHM-I,

TSC22D1, FOXO1, DIAPH3, CCDC122, CKAP2, SUGT1,

RANKL, LPAR6, C13ORF31) did not reveal potentially

causal variants.

Introduction

Polydactyly, occurrence of supernumerary digits, is the

most frequent of congenital hand and foot deformities.

Based on the location of extra digits, polydactyly can be

classified into: preaxial, involving thumb or great toe;

postaxial, affecting little finger; and central polydactyly, in

which the three central digits are affected (Swanson 1976;

Buck-Gramcko 1998). Postaxial polydactyly (PAP) is fur-

ther sub-classified into two types. In type A, a well-formed

extra digit articulates with fifth or sixth metacarpal, while

in type B a rudimentary poorly developed extra digit is

present (Temtamy and Mckusick 1978). The occurrence of

PAP in the general population varies among different racial

groups and is about ten times more frequent in Africans

than in European ancestries, with an incidence rate of

1/300 to 1/100 and 1/3,300 to 1/630 live births, respec-

tively (Frazier 1960; Temtamy 1990).

Both syndromic and non-syndromic (isolated) forms of

PAP have been reported in the literature. In syndromic forms,

PAP has been found in association with other features in

conditions such as Ellis–van Creveld (EVC, MIM 225500),

Smith–Lemli–Optiz (SLOS, MIM 270400) and McKusick–

Kaufmann syndromes (MKKS, MIM 236700), short

rib-polydactyly syndrome I (SRPS, MIM 263530), orofa-

ciodigital syndrome III (OFD III, MIM 258850), Bardet–

Biedel syndrome (BBS, MIM 209900), Meckel–Gruber

syndrome (MKS1, MIM 249000), Greig cephalopolysyn-

dactyly (GCPS, MIM 175700) and Pallister–Hall syndrome

(PHS, MIM 146510) (Schwabe and Mundlos 2004).

Isolated (non-syndromic) forms of PAP, in which pol-

ydactyly is the only clinical condition, usually segregate as

an autosomal dominant trait mostly with variable expres-

sion. To date, three non-syndromic autosomal dominant

loci for PAP have been mapped on different human chro-

mosomes. This includes PAPA1 (MIM 174200) with PAP-

A features on chromosome 7p13 and caused by mutations

in gene GLI3, (Radhakrishna et al. 1997a, b) PAPA2 (MIM

602085) on chromosome 13q21–q32 with features of

PAP-A (Akarsu et al. 1997) and PAPA3 (MIM 607324)

with features of PAP-A/B on chromosome 19p13.1–13.2

Umm-e-Kalsoom � S. Basit � S. Kamran-ul-Hassan Naqvi �M. Ansar � W. Ahmad (&)

Department of Biochemistry, Faculty of Biological Sciences,

Quaid-i-Azam University Islamabad, Islamabad, Pakistan

e-mail: wahmad@qau.edu.pk

123

Hum Genet (2012) 131:415–422

DOI 10.1007/s00439-011-1085-7

(Zhao et al. 2002). In a Dutch family, Galjaard et al. (2003)

have mapped another autosomal dominant PAP with fea-

tures of PAP-A/B and partial cutaneous syndactyly on

chromosome 7q21–q34. Several isolated autosomal reces-

sive cases of PAP have also been described in the literature

(Mohan 1969; Cantu et al. 1974; Mollica et al. 1978);

however, no gene has been mapped in any such case.

In the present study, we report mapping of the first

autosomal recessive PAP type A locus in a consanguineous

Pakistani family on chromosome 13q13.3–q21.2. DNA

sequence analysis of ten candidate genes failed to identify

potentially causal variants.

Materials and methods

Human subjects

A consanguineous Pakistani family, segregating an autoso-

mal recessive form of postaxial polydactyly type A, was

ascertained from the Sindh Province of Pakistan. At the time

of the study, the family had four individuals affected with

PAP-A including two males (VI-2, VI-3) and two females

(VI-4, VI-5). All these four individuals underwent careful

clinical examination at the local government hospital.

The family pedigree (Fig. 1) provided convincing evi-

dence of an autosomal recessive mode of inheritance of the

phenotype, and consanguineous matings accounted for all

the affected individuals being homozygous for the mutant

allele. Approval of the study was obtained from the Quaid-

i-Azam University (QAU) Institutional Review Board

(IRB), Islamabad, Pakistan. Informed written consent was

obtained from all the family members who participated in

the study.

Extraction of genomic DNA and genotyping

Venous blood samples were collected from four affected

(VI-2, VI-3, VI-4, VI-5) and four unaffected (III-1, V-1,

VI-1, VI-6) family members in EDTA containing Vacu-

tainer sets. Genomic DNA was extracted using Sigma-

Aldrich GenElute Blood Genomic DNA Kit (St. Louis,

MO, USA). Polymerase chain reaction (PCR) was carried

out in 0.2-ml tubes (Axygen, Inc, CA, USA) in a total

volume of 25 ll, which contained 40 ng of human genomic

DNA, 2.5 ll of PCR reaction buffer, 1.5 ll MgCl2 (MBI

Fermentas, Life Sciences, York, UK), 200 lM of each

deoxynucleoside triphosphate (dNTP), 20 pmol of each

forward and reverse primer, and 1 unit of Taq DNA

polymerase (MBI Fermentas, Life Sciences, York, UK).

The thermal cycling conditions used included 95�C for

5 min, followed by 40 cycles of 95�C for 1 min, 55–58�C

for 1 min, 72�C for 1 min and a final extension at 72�C for

10 min. PCR was performed using T3 thermocycler

(Biometra GmbH, Goettingen, Germany) and the products

generated were resolved on 8% non-denaturing polyacryl-

amide gels. The average heterozygosity of each marker was

above 70%, suggesting that these markers were highly

informative for genotyping pedigree members.

Exclusion of linkage to previously reported PAP loci

The family was first tested for linkage to previously reported

autosomal dominant PAP loci. These included GLI3 gene on

chromosome 7p13 (D7S2541, D7S2469, D7S3043, D7S691,

D7S2428, D7S1488, D7S2427, D7S670, D7S1818), PAP-A2

on chromosome 13q21–q32 (D13S279, D13S1318, D13S800,

D13S166, D13S269, D13S162, D13S1306, D13S160, D13

S921, D13S170, D13S317, D13S628, D13S1283, D13S265,

D13S886, D13S1300, D13S71, D13S122, D13S1280), PAP-

A3 on chromosome 19p13.1–13.2 (D19S583, D19S1169,

D19S584, D19S1165, D19S221, D19S558, D19S840,

D19S226, D19S556, D19S929, D19S252) and PAP with par-

tial cutaneous syndactyly on chromosome 7q21–q34 (D7S820,

D7S2410, D7S2409, GATA63F08, D7S2480, D7S2509,

D7S2459, D7S692, D7S523, D7S655, D7S635, D7S2519,

D7S512, D7S500, D7S2560, D7S2513). Examination of the

haplotypes did not reveal any region of homozygosity among

affected individuals, thus excluding the family from linkage to

the four loci tested here.

Human genome scan

Human genome scan was conducted using 534 highly

informative microsatellite markers selected from linkage

mapping set (Invitrogen Co, San Diego, CA, USA). These

markers were spaced approximately at 5–6 cM apart on 22

autosomes and X and Y chromosomes. The National

Center for Biotechnology Information (NCBI) built 36.2

sequence-based physical map was used to determine the

order of the genome scan and fine mapping markers (Ma-

tise et al. 2007). Two-point linkage analysis was carried out

using the MLINK program of the FASTLINK computer

package (Cottingham et al. 1993), while multipoint linkage

analysis was performed using Allegro version-2 (Gudbj-

artsson et al. 2005). Two-point LOD scores were computed

for recombination fraction values of h = 0.0, 0.01, 0.05,

0.10, 0.20, 0.30 and 0.40. An autosomal recessive mode of

inheritance with 100% penetrance and a disease allele

frequency of 0.001 were used. Equal allele frequencies

were assumed in two-point and multipoint analysis for fine

mapping markers because it was not possible to estimate

allele frequencies from the founders, as these markers were

only genotyped in this family. Mendelian incompatibilities

and unlikely genotypes were searched by PEDCHECK

(O’Connell and Weeks 1998) and MERLIN (Abecasis

416 Hum Genet (2012) 131:415–422

123

et al. 2002), respectively. Haplotypes were constructed

using Haplopainter (Thiele and Nurnberg 2004).

Screening candidate genes

University of California Santa Cruz (UCSC) Human

Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGate

way) was used to search for the genes located in the linkage

interval of PAP-A locus on chromosome 13q13.3–q21.2,

mapped in the present study. From the genes present in the

linked interval, we prioritized ten genes for sequencing. These

included CHM-I also known as LECT1 (MIM 605147);

TSC22D1 (MIM 607715); FOXO (MIM 136533); DIAPH3

(NM_001042517); CCDC122 (MIM 613408); CKAP2 (MIM

611569); SUGT1 (MIM 604098); RANKL also known as

TNFSF11 (MIM 602642); LPAR6 (MIM 609239) and

C13ORF3 (MIM 613409). These genes for mutation screen-

ing were selected on the basis of their function in bone

Fig. 1 Pedigree of a

consanguineous Pakistani

family with autosomal recessive

PAP type A. Circles and

squares represent females and

males, respectively. Clearsymbols represent unaffected

individuals while filled symbolsrepresent affected individuals.

Symbols with crossed linesrepresent deceased individuals.

Double lines are indicative of

consanguineous unions. For

genotyped individuals,

haplotypes of the closely linked

microsatellite markers on

chromosome 13q13.3–q21.2 are

shown beneath each symbol.

Genetic distances in

centimorgans (cM) are depicted

according to the Rutgers

combined-linkage physical map

(build 36.2) (Matise et al. 2007)

Hum Genet (2012) 131:415–422 417

123

formation, expression in bones or digits, structural homology

to genes involved in limb development or bone growth, and

involvement in signaling pathways or cellular processes

essential for shaping the limb (Table 1). Specific information

about each gene, provided in Table 1, was obtained from the

UCSC human genome database, OMIM and PUBMED

(http://www.ncbi.nlm.nih.gov/omim/pubmed).

Primers to amplify exons and splice sites were designed

from intronic sequences of the genes using primer3 software

(Rozen and Skaletsky 2000). DNA from two affected (VI-3,

VI-4) and one unaffected individual (III-1) of the family

were amplified by PCR under standard conditions. Primer

sequences and conditions used for amplification of the genes

are available upon request. The PCR-amplified products

were purified with commercially available kits (Marligen

Bio Sciences, Ijamsville, MD, USA) and, using DTCS Quick

Start Sequencing Kit (Beckman Coulter, Brea, CA, USA),

sequenced directly on CEQ8800 DNA sequencer (Beckman

Coulter, Brea, CA, USA). Sequence variants were identified

via BIOEDIT sequence alignment editor version 6.0.7

(http://www.mbio.ncsu.edu/BioEdit/bioedit.html).

Results

Clinical features

All four affected individuals of the family, presented here,

underwent detailed clinical investigation at the local

government hospital. By birth, all four affected individuals

had bilateral postaxial polydactyly of hands and feet;

however, extra fingers were amputated on both hands

(Fig. 2a, b). Skeletal vestige of extra fingers was not visible

on the radiograph of the hands (Fig. 2c). Radiograph of

feet of an affected individual (VI-2) showed malformed,

partially duplicated fork-shaped fifth metatarsal, separate

metatarsophalangeal joint and interphalangeal joints in the

extra toes (Fig. 2d). One of the affected individuals (VI-3)

had hallux valgus deformity of the right foot that resulted

in inward leaning of the great toe toward the second toe

(Fig. 2e).

Teeth, nails, sweating and hearing were normal in all the

affected individuals. Neurological problems and facial

dysmorphism were not observed in any of the affected

individuals. Heterozygous carrier individuals had normal

hands and feet, and were clinically indistinguishable from

genotypically normal individuals.

Mapping autosomal recessive PAP type

A on chromosome 13q13.3–q21

After excluding the linkage to previously reported autosomal

dominant PAP loci, the present family was subjected to

genome wide scan using highly polymorphic microsatellite

markers. The genome scan was conducted using DNA

samples from one unaffected parent (III-1) and three affected

sibs (VI-2, VI-3, VI-4) of the family. The affected individ-

uals showed homozygosity with six microsatellite markers,

Table 1 Genes present in the linkage interval of postaxial polydactyly type A mapped on chromosome 13q13.3–q21.2, and sequenced in the

present study

Genes Functions Principles of selection Homologs

CHM1 Bone remodeling through regulating osteoclast

and osteoblast numbers and functions.

(Nakamichi et al. 2003)

Function in bone formation

TSC22D1 Enhances TGF-b signaling. (Choi et al. 2005) Involvement in signaling pathways

essential for shaping the limb

FOXO1 Mesenchymal cell differentiation into osteoblasts

through regulation and interaction with Runx2

(Teixeira et al. 2010)

Function in bone formation

DIAPH3 Action filament induction. Regulation of

contractile ring during cytokinesis (Watanabe

et al. 2008; Paul and Pollard 2009)

Expression in digits in mouse homology

based

Formin (ld)

CCDC122 Not known Expression in digits in mouse homology

based (Glover and Harrison 1995)

Jun, Fos, Matrilins

SUGT1, CKAP2 Required for proper chromosome segregation

(Hong et al. 2009). Microtubule stabilization

and apoptosis (Tsuchihara et al. 2005)

Involvement in cellular processes

essential for shaping the limb

RANKL Bone remodeling through osteoclast

differentiation (Lacey et al. 1998)

Function in bone formation

LPAR6 Encodes a G protein-coupled receptor (GPCR) Homology based GPCRs, expressed by

osteoblasts and osteoclasts

C13ORF31 Not known Expression in digits in mouse

418 Hum Genet (2012) 131:415–422

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including D19S246 (19q13.3), D19S206 (19q13.41), D13

S263 (13q14.11), D13S1312 (13q14.13), D13S153

(13q14.2), D13S233 (13q21.1). Upon testing DNA from the

rest of the family members, linkage to two of these markers

(D19S246 and D19S206) was excluded, while the remaining

four markers, D13S263 (43.02 cM), D13S1312 (48.03 cM),

D13S153 (52 cM) and D13S233 (56.13 cM), showed

homozygous pattern of alleles in affected and heterozygous

in unaffected individuals of the family. The region encom-

passing these four markers was further saturated by geno-

typing 40 additional microsatellite markers, selected from

the Rutgers combined-linkage physical map (Build 36.2) of

the human genome (Matise et al. 2007). Of the 40 markers,

13 (D13S1246, D13S218, D13S1288, D13S1233,

D13S1247, D13S1276, D13S168, D13S1807, D13S119,

D13S632, D13S889, D13S1317, D13S276) were informa-

tive and considered for further analysis. Analysis of the

genome scan and fine mapping markers genotypes within

this region with PEDCHECK and MERLIN did not

elucidate any genotyping errors. The results of the two-

point and multipoint linkage analyses are presented in

Table 2. The maximum two-point LOD score of 2.52 at

0 recombination fraction (h = 0.00) was achieved for a

marker D13S119 located at the 55.92-cM region.

However, a maximum multipoint LOD score of 3.84 was

obtained with several markers (D13S1233, D13S263,

D13S1247, D13S1276, D13S1312, D13S168, D13S153,

D13S1807, D13S119, D13S233) along the disease

interval (Table 2).

Haplotypes using Haplopainter (Thiele and Nurnberg

2004) were constructed to define the centromeric and

telomeric boundaries of the mapped region. A historic

recombination event occurred between the markers

D13S1288 (39.29 cM) and D13S1233 (42.63 cM),

observed in all affected subjects (VI-2, VI-3, VI-4, VI-5)

and defined the centromeric boundary of the linkage

interval on chromosome 13q13.3. The telomeric bound-

ary of the interval on this chromosomal region was

defined by a recombination event between markers

D13S233 (56.13 cM) and D13S632 (57.16 cM) observed

in two affected individuals (VI-2, VI-3). The linkage

interval flanked by markers, D13S1288 (39.29 cM) and

D13S632 (57.16 cM), contains 21.13 Mb and is 17.87-

cM long according to Rutgers combined-linkage physical

map of the human genome (Matise et al. 2007).

Haplotype analysis revealed that interesting recombi-

nation events occurred in the maternal chromosome

meiosis in individual V-1 giving rise to generation VI.

Fig. 2 Clinical features of

autosomal recessive PAP-A

phenotypes. After amputating

the extra digits, the present

status of hands of an affected

individual (VI-2) of the family

(a), bilateral PAP type A of feet

(b), radiograph of hands

depicting no skeletal vestige (c),

and radiograph of feet showing

extra digits, articulating with

partially duplicated fork-shaped

fifth metatarsals (d). An affected

individual (VI-3) showing

bilateral PAP type A in feet with

hallux valgus deformity of the

right foot (e)

Hum Genet (2012) 131:415–422 419

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Individuals VI-2 and VI-3 showed double maternal

recombination events that occurred between markers

D13S1288 and D13S1233, and D13S233 and D13S632.

Similarly, in individuals VI-4 and VI-5, maternal recom-

binations occurred between markers D13S1288 and

D13S1233. Although the probability of simultaneously

observing three independent events is low as genetic

interference restricts the multiple crossover events, this is

not impossible. Genotype data of several markers along the

disease interval including those mapped outside the

homozygous by decent region failed to detect any unlikely

genotypes.

Mutation analysis of the candidate genes

The candidate linkage interval of 21.13 Mb on chromo-

some 13q13.3–q21.2 contains many genes. To identify the

gene responsible for autosomal recessive PAP-A, coding

exons and splice-junction sites of the ten selected genes

(CHM-I, TSC22D1, FOXO1, DIAPH3, CCDC122, CKAP2,

SUGT1, RANKL, LPAR6, C13ORF31) were screened by

sequencing in two affected (VI-3, VI-4) and one unaffected

(III-1) individuals of the family. Sequence analysis with

standard sequence of the exons and splice junctions of the

ten genes (http://www.ensembl.org/Homo_sapiens) failed

to discover any potential sequence variant, which could be

responsible for the disease phenotype.

Discussion

In the present study, we have presented a consanguineous

Pakistani family segregating autosomal recessive postaxial

polydactyly type A. All four affected individuals of the

family presented sixth well-formed, functional digits in

both hands and feet. The extra digits on the hands were

amputated before the family was located and studied. In

addition to the presence of a sixth digit in hands and feet,

partially duplicated fork-shaped fifth metatarsals of feet

were observed in the affected individual (VI-2). Almost

32 years ago, Mollica et al. (1978) reported similar features

in a Sicilian family with autosomal recessive PAP-A.

However, the presence of hallux valgus deformity,

observed in an affected individual (VI-3) of our family,

was not reported in the Sicilian family. In families with

autosomal dominant forms of postaxial polydactyly, most

of the affected individuals exhibit features representing

both type A and type B PAP (Zhao et al. 2002; Galjaard

Table 2 Multipoint and two-point LOD score results between an autosomal recessive PAP type A locus and genotyped markers in the candidate

region on chromosome 13q13.3–q21.2

Markers Distance (position) mpt Two-point LOD values at recombination fraction theta

Genetic (cM) Physical (Mb) 0.00 0.01 0.05 0.10 0.20 0.30 0.40

D13S1246 25.41 30,003,436 -16.12 -0.73 -0.43 -0.06 0.04 0.04 0.003 -0.003

D13S218 38.60 37,930,230 -17.24 -0.73 -0.43 -0.06 0.04 0.04 0.003 -0.003

D13S1288 39.29 38,421,269 –Inf –Inf -5.5 -2.83 -1.77 0.80 -0.32 -0.08

D13S1233 42.63 39,975,096 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S263 43.02 40,978,919 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S1247 43.84 41,859,585 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S1276 43.84 42,313,784 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S1312 48.03 44,830,573 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S168 51.37 46,709,326 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S153 52.00 47,788,734 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S1807 55.50 54,004,097 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S119 55.92 57,446,735 3.84 2.52 2.46 2.22 1.92 1.33 0.75 0.24

D13S233 56.13 58,345,917 3.84 2.35 2.29 2.07 1.79 1.24 0.70 0.21

D13S632 57.16 59,555,958 –Inf –Inf -3.08 -1.62 -0.99 -0.42 -0.16 -0.04

D13S889 58.51 62,461,388 –Inf –Inf -3.08 -1.62 -0.99 -0.42 -0.16 -0.04

D13S1317 60.43 65,963,262 –Inf –Inf -3.08 -1.62 -0.99 -0.42 -0.16 -0.04

D13S276 61.36 67,907,806 –Inf –Inf -3.08 -1.62 -0.99 -0.42 -0.16 -0.04

Average sex distance in cM and physical position according to the Rutgers combined-linkage physical human genome map Build 36.2 (Matise

et al. 2007)

Markers D13S1288 and D13S632 in boldface flank the disease-associated region

mpt multipoint

420 Hum Genet (2012) 131:415–422

123

et al. 2003). In a Dutch family mapped on chromosome

7q21–q34, affected individuals showed additional features

of partial cutaneous syndactyly (Galjaard et al. 2003).

Genetic mapping established linkage of the family, pre-

sented here, on chromosome 13q13.3–q21.2. Significant

evidence of linkage to this chromosomal region was found

with a maximum multipoint LOD score of 3.84 with several

markers along the disease interval. Haplotype analysis

located the region of homozygosity in 17.8 cM, which cor-

responds to 21.13 Mb flanked by markers D13S1288

(39.29 cM, 38.42 Mb) and D13S632 (57.16 cM, 59.55 Mb).

Akarsu et al. (1997), in a large family with Turkish

origin, mapped an isolated form of autosomal dominant

PAP-A2 locus in a 28-cM region flanked by centromeric

marker D13S800 (67.39 cM, 72.77 Mb) and telomeric

marker D13S154 (90.12 cM, 95.06 Mb) on chromosome

13q21–q32. Several studies described syndromic forms of

polydactyly involving variable 13q segmental duplications,

which include chromosomal segments 13q14, 13q21,

13q22, 13q31 and 13q32 (Muneer et al. 1981; Nikolis et al.

1991; Prieto et al.1980; Maltby 1984; Galan et al. 1989;

Sakata et al. 2008; Pilgaard et al. 1983; Tharapel et al.

1986). Although these chromosomal regions were not

precisely mapped, still it is likely that some of these

regions coincide with the region mapped in the present and

the study reported by Akarsu et al. (1997).

Recently, Van der Zwaag et al. (2010) described a patient,

born to non-consanguineous parents, with multiple clinical

features including postaxial polydactyly type A of hands,

preauricular tag, anterior placed anus, a broad nasal bridge,

telecanthus and frontal bossing. These authors using FISH

and array-CGH, revealed an interstitial duplication of chro-

mosome 13q31.3–q32.1 in the patient. The duplicated region

spanned 5.59 Mb (89.67–95.25 Mb), which the authors

suggested contains a gene causing PAP-A2. The linkage

interval of 21.13 Mb (38.42–59.55 Mb) for autosomal

recessive PAP type A locus mapped in our family clearly

indicates that this does not overlap with the linkage interval

for PAP-A2 locus defined earlier (Akarsu et al. 1997; Van der

Zwaag et al. 2010). The linkage interval of PAP type A locus

in our family is located 30.12-Mb proximal to PAP-A2 locus,

thus suggesting that two different genes are responsible for

autosomal dominant and autosomal recessive PAP type A

disorders mapped on chromosome 13.

The candidate region of autosomal recessive PAP type A

on chromosome 13q13.3–q21.2, mapped here, contains

many genes and hypothetical proteins. Among these, protein

coding sequences and exon–intron borders of ten genes

(CHM-I, TSC22D1, FOXO1, DIAPH3, CCDC122, CKAP2,

SUGT1, RANKL, LPAR6, C13ORF31) were sequenced.

Three genes including CHM-I, FOXO1 and RANKL, due to

their direct involvement in bone formation, were screened

first for the potential sequence variants. In the second phase,

three other genes, TSC22D1, SUGT1 and CKAP2, involved

in signaling pathways and different cellular processes used

by the signaling centers to shape the limb, were screened for

the disease-causing mutation. After failing to identify

sequence variants in the above-mentioned six genes, three

other genes including, DIAPH3, CCDC122 and C13ORF31,

were selected for screening. The genes DIAPH3 and

CCDC122 shared homology to other genes involved in limb

and bone development (Zeller et al. 1999; Frank et al. 2002).

The gene LPAR6 encodes a G protein-coupled receptor

(GPCR) P2Y5. The P2Y5 protein contains seven predicted

hydrophobic transmembrane regions, a structural feature of

GPCRs (Webb et al. 1996). Several GPCRs are expressed

both by osteoblasts and osteoclasts. These GPCRs, when

activated by nucleotides, released from cells in response to

mechanical stimulation, could play an important role in

modulating the bone cell function (Orriss et al. 2010).

No functional sequence variants were discovered in the

ten candidate genes sequenced in the present study. There-

fore, their involvement in causing autosomal recessive PAP-

A at the present locus is not supported. However, we cannot

exclude the possibility of the presence of functional

sequence aberrations in the regulatory regions of the genes

that were not sequenced here. Further fine mapping and

sequencing work are required to identify the gene causing

autosomal recessive PAP-A at the present locus.

Acknowledgments We wish to thank the family members for their

invaluable participation and cooperation. This work was funded by

the Higher Education Commission (HEC), Islamabad, Pakistan.

Umm-e-Kalsoom was supported by an indigenous PhD fellowship

from HEC, Islamabad, Pakistan.

Conflict of interest We declare that we have no conflict of interest.

References

Abecasis GR, Cherny SS, Cookson WO, Cardon LR (2002) Merlin

rapid analysis of dense genetic maps using sparse gene flow

trees. Nat Genet 30:97–101

Akarsu AN, Ozbas F, Kostakoglu N (1997) Mapping of the second

locus of postaxial polydactyly type A (PAP-A2) to chromosome

13q21–q32. Am J Hum Genet 6:A265

Buck-Gramcko D (1998) Angeborene Fehlbildungen der Hand. In:

Nigst H, Buck-Gramcko D, Milesi H (eds) Handchirurgie.

Thieme, Stuttgart, pp 12.1–12.115

Cantu JM, del Castillo V, Cortes R, Urrusti J (1974) Autosomal

recessive postaxial polydactyly: report of a family. Birth Defects

Orig Artic Ser 10:19–22

Choi SJ, Moon JH, Ahn YW, Ahn JH, Kim DU, Han TH (2005) Tsc-

22 enhances TGF-beta signaling by associating with Smad4 and

induces erythroid cell differentiation. Mol Cell Biochem

271:23–28

Cottingham RW Jr, Jonasson K, Frigge ML, Kong A (1993) Faster

sequential genetic linkage computations. Am J Hum Genet

53:26–252

Hum Genet (2012) 131:415–422 421

123

Frank S, Schulthess T, Landwehr R, Lustig A, Mini T, Jeno P, Engel

J, Kammerer RA (2002) Characterization of the matrilin coiled-

coil domains reveals seven novel isoforms. J Biol Chem

277:19071–19079

Frazier TM (1960) A note on race-specific congenital malformation

rates. Am J Obstet Gynecol 80:184–185

Galan F, Garcia R, Aguilar MS, Moya M (1989) Partial trisomy

13q22-qter. A new case. Ann Genet 32:114–116

Galjaard RJ, Smits AP, Tuerlings JH, Bais AG, Bertoli Avella AM,

Breedveld G, de Graaff E, Oostra BA, Heutink P (2003) A new

locus for postaxial polydactyly type A/B on chromosome 7q21–

q34. Eur J Hum Genet 11:409–415

Glover JN, Harrison SC (1995) Crystal structure of the heterodimeric

bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature

373:257–261

Gudbjartsson DF, Thorvaldsson T, Kong A, Gunnarsson G, Ingolfs-

dottir A (2005) Allegro version 2. Nat Genet 37:1015–1016

Hong KU, Kim E, Bae CD, Park J (2009) TMAP/CKAP2 is essential

for proper chromosome segregation. Cell Cycle 8:314–324

Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T,

Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J,

Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S,

Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ

(1998) Osteoprotegerin ligand is a cytokine that regulates

osteoclast differentiation and activation. Cell 93:165–176

Maltby EL (1984) Familial pericentric inversion (13) detected by

antenatal diagnosis. J Med Genet 21:149–151

Matise TC, Chen F, Chen W, De La Vega FM, Hansen M, He C,

Hyland FC, Kennedy GC, Kong X, Murray SS, Ziegle JS,

Stewart WC, Buyske S (2007) A second-generation combined

linkage physical map of the human genome. Genome Res

17:1783–1786

Mohan J (1969) Postaxial polydactyly in three Indian families. J Med

Genet 6:196–200

Mollica F, Volti SL, Sorge G (1978) Autosomal recessive postaxial

polydactyly type A in a Sicilian family. J Med Genet 15:212–216

Muneer RS, Donaldson DL, Rennert OM (1981) Complex balanced

translocation of chromosomes 2, 3, and 13. Hum Genet

59:182–184

Nakamichi Y, Shukunami C, Yamada T, Aihara K, Kawano H, Sato

T, Nishizaki Y, Yamamoto Y, Shindo M, Yoshimura K,

Nakamura T, Takahashi N, Kawaguchi H, Hiraki Y, Kato S

(2003) Chondromodulin-I is a bone remodeling factor. Mol Cell

Biol 23:636–644

Nikolis J, Ivanovic K, Diklic V (1991) Partial trisomy 13q resulting

from a paternal reciprocal Yq;13q translocation. J Med Genet

28:425–426

O’Connell JR, Weeks DE (1998) PedCheck: a program for identi-

fication of genotype incompatibilities in linkage analysis. Am J

Hum Genet 63:259–266

Orriss IR, Burnstock G, Arnett TR (2010) Purinergic signalling and

bone remodeling. Curr Opin Pharmacol 10:322–330

Paul AS, Pollard TD (2009) Review of the mechanism of processive

actin filament elongation by formins. Cell Motil Cytoskelet

66:606–617

Pilgaard B, Jorgensen E, Knudsen VS, Mortensen E, Mikkelsen M

(1983) Familial inversion translocation (8;13) with partial trisomy

13 in several family members. Eur J Pediatr 140:105–108

Prieto F, Badia L, Asensi F, Roques V (1980) Two reciprocal

translocations (9p?;13q-) and t(13q-;21q?): a study of the

families. Hum Genet 54:7–11

Radhakrishna U, Blouin J-L, Mehenni H, Patel UC, Patel MN, Solanki

JV, Antonarakis SE (1997a) Mapping one form of autosomal

dominant postaxial polydactyly type A to chromosome 7p15–

q11.23 by linkage analysis. Am J Hum Genet 60:597–604

Radhakrishna U, Wild A, Grzeschik KH, Antonarakis SE (1997b)

Mutation in GLI3 in postaxial polydactyly type A. Nat Genet

17:269–271

Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users

and for biologist programmers. Methods Mol Biol 132:365–386

Sakata R, Usui T, Mimaki M, Araie M (2008) Developmental

glaucoma with chromosomal abnormalities of 9p deletion and

13q duplication. Arch Ophthalmol 126:431–432

Schwabe GC, Mundlos S (2004) Genetics of congenital hand

anomalies. Handchir Mikrochir Plast Chir 36:85–97

Swanson AB (1976) A classification for congenital limb malforma-

tions. J Hand Surg [Am] 1:8–22

Teixeira CC, Liu Y, Thant LM, Pang J, Palmer G, Alikhani M (2010)

FOXO1, a novel regulator of osteoblast differentiation and

skeletogenesis. J Biol Chem 285:31055–31065

Temtamy S, Mckusick V (1978) Polydactyly as an isolated malfor-

mation. In: Temtamy S, McKusick V (eds) The genetics of hand

malformations. Birth defects, vol 14. Alan R Liss Inc, New

York, pp 364–392

Temtamy SA (1990) Polydactyly, postaxial. In: Buyse ML (ed) Birth

defects encyclopedia. Blackwell Scientific, Cambridge, pp 1397–

1398

Tharapel SA, Lewandowski RC, Tharapel AT, Wilroy RS Jr (1986)

Phenotype karyotype correlation in patients trisomic for various

segments of chromosome 13. J Med Genet 23:310–315

Thiele H, Nurnberg P (2004) HaploPainter: a tool for drawing pedigrees

with complex haplotypes. Bioinformatics 21:1730–1732

Tsuchihara K, Lapin V, Bakal C, Okada H, Brown L, Hirota-

Tsuchihara M, Zaugg K, Ho A, Itie-Youten A, Harris-Brandts M,

Rottapel R, Richardson CD, Benchimol S, Mak TW (2005)

Ckap2 regulates aneuploidy, cell cycling and cell death in a p53-

dependent manner. Cancer Res 65:6685–6691

Van der Zwaag PA, Dijkhuizen T, Gerssen-Schoorl KB, Colijn AW,

Broens PM, Flapper BC, van Ravenswaaij-Arts CM (2010) An

interstitial duplication of chromosome 13q31.3q32.1 further

delineates the critical region for postaxial polydactyly type A2.

Eur J Med Genet 53:45–49

Watanabe S, Ando Y, Yasuda S, Hosoya H, Watanabe N, Ishizaki T,

Narumiya S (2008) mDia2 induces the actin scaffold for the

contractile ring and stabilizes its position during cytokinesis in

NIH 3T3 cells. Mol Biol Cell 19:2328–2338

Webb TE, Kaplan MG, Barnard EA (1996) Identification of 6H1 as aP2Y purinoceptor: P2Y5. Biochem Biophys Res Commun

219:105–110

Zeller R, Haramis AG, Zuniga A, McGuigan C, Dono R, Davidson G,

Chabanis S, Gibson T (1999) Formin defines a large family of

morphoregulatory genes and functions in establishment of the

polarising region. Cell Tissue Res 296:85–93

Zhao H, Tian Y, Breedveld G, Huang S, Zou Y, Y J, Chai J, Li H, Li

M, Oostra BA, Lo WHY, Heutink P (2002) Postaxial polydac-

tyly type A/B (PAP-A/B) is linked to chromosome 19p13.1–13.2

in a Chinese kindred. Eur J Hum Genet 10:162–166

Web resource

GenBank Accession Numbers: http://www.ensembl.org/Homo-sapiens

Online Mendalian Inheritance in Man (OMIM): http://www.ncbi.

nlm.nih.gov/omim/

PUBMED: http://www.ncbi.nlm.nih.gov/pubmed/

USCS Genome Bioinformatics website: http://genome.ucsc.edu/cgi-

bin/hgGateway

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