S. Irshad et al / Research Journal of Biology (2011), Vol. 01, Issue 01, pp.

1-6

ISSN 2049-1727

Research Paper

Linkage Study of Primary Microcephaly in Pakistani

Kindred
Saba Irshad*, Sana Anwar and Blessy Shehzad
* Institute of Biochemistry and Biotechnology University of the Punjab Lahore 54590, Pakistan
Tel.:+92-42-99230355, Fax:+92-42-9923242
E-Mail (s): saba.ibb@pu.edu.pk , sabairshad2003@yahoo.com

Abstract
Microcephaly is heterogeneous, autosomal recessive trait with reduced head circumference of at least 4 SD below age and
sex means due to reduction in neuron production. The brain of microcephalic patient is architecturally normal but severe to
mild mental retardation. It is rare disease affecting 2-2.5% of total population specifically in Asia and Arab where the
incidence of cousin marriages is relatively high. From seven known currently mapped loci ASPM is found to be the
frequent causative agent. In the current investigations exclusion mapping of a microcephalic family was done. DNA from
all blood samples was extracted using standard procedure and after gene specific PCR amplifications, 8% non-denaturing
PAGE was done. Linkage was observed at MCPH5 locus where ASPM is a candidate gene on chromosome 1q31. The
results of DNA sequencing showed G to A transition and Leucine (CTG) to Leucine (CTA) was noted. There are six triplet
codons which differ by single nucleotide encoding for Leucine. Hence, no overall change in the effect of protein expression
was observed due to the degeneracy of codons. Therefore, the sequencing of the entire ASPM gene with intervening
sequences was suggested in order to find the actual cause of microcephaly.
Keywords: Exclusion, Mapping, Microcephaly, ASPM

1. Introduction
Microcephaly (MCPH) is an autosomal recessive neurodevelopmental disorder which is described by various
terms that explain the same phenotype in literature such as
True microcephaly
microcephaly vera and
autosomal recessive primary microcephaly. The cerebral
cortex is severely affected due to decreased production of
neurons leading to overall reduction in brain size (Wood et
al, 2005).
The early clinical features which included; congenital
microcephaly at least 4 SD below age and sex means,
mental retardation but no other neurological finding such
as spasticity, seizures, or progressive cognitive decline and
normal height and weight, appearance (Jackson et al,
2002).
Genetic heterogeneity is well established in MCPH with

seven loci which have been currently mapped.

MCPH1/Microcephalin localized at 8p23.1 in Northern
Pakistani families (Jackson et al, 2002). MCPH2/WDR62
localized at 19q13.1-13.2 in Northern Pakistani families
(Nicholas et al,2010).MCPH3/CDKRAP2 localized at
9q33.2 in Northern Pakistani families (Bond et al, 2005).
MCPH4/CEP152 localized at 15q15-21 in Canadian
families (Guernsey et al, 2010). MCPH5/ASPM localized
at 1q31.1 in Turkish/Pakistani families (Bond et al, 2002).
MCPH6/CENPJ localized at 13q12.12 in Brazilian
families (Bond et al, 2005). MCPH7/STIL localized at
1p33 in Indian families (Kumar et al, 2009).
MCPH5 is localized on chromosome 1q31 and is mapped
to an 8-cM region (Pattison et al, 2000). The ASPM gene
comprises 10,434 bp long coding sequence with 28 exons
and traverse 65 kb of genomic DNA. ASPM is upregulated

Available online at www.scientific-journals.co.uk

S. Irshad et al / Research Journal of Biology (2011), Vol. 01, Issue 01, pp. 1-6

in malignant cells and is widely expressed in fetal and

adult tissues. MCPH is the consequence of impairment in
mitotic spindle regulation in cortical progenitors due to
mutations in ASPM (Gul et al, 2006). ASPM mutations
have been strongly isolated (Kumar et al, 2004).
Until now 57 ASPM mutations have been reported. Out Of
which 17 mutations are frequently occurring. One of the
mutations involved premature termination codon in the
ASPM gene. 23 nonsense mutations, 28 small deletions or
insertions mutation which lead to an alteration in the
reading frame, and five were splice site mutations, once
more lead to the use of a premature stop codon (Nicholas
et al, 2009).
The aim of the study is linkage analysis of primary
microcephaly in Pakistani population.

2. Materials and Methodology

2.1. Sample Collection
The data of the family of autosomal recessive primary
microcephaly was collected to generate an individuals
medical and developmental history considering the
inheritance and chance occurrence of disease in offspring.
Informed consent was obtained from all family members
who participated in the study.MCP Family having six
individuals was collected from Shirkpur, out of which two
sons were affected with primary microcephaly (as shown
in figure 1), while father was unapproachable.

(a)

(b)

Figure 1: Photographs of Microcephalic individuals of MCP

family from Shirkpur. (a) II: 2; (b) II: 4.

The clinical findings of the individuals were evaluated.

Individual II: 2 (6yrs 10months) had slopping forehead
,prominent ears, mild to moderate mental retardation and
the Head circumference was measured to be 36.5 cm.
Individual II: 4 (8 1/2 months) had slopping forehead,
small head size and prominent ears. The Head
circumference was found to be 36 cm.
2.2. DNA Extraction and Genotyping with MCPH5

ISSN 2049-1727

2.2.1. Microsatellite Markers

The patients were clinically evaluated and the information
was collected individually. The extraction of Genomic
DNA from peripheral blood was performed by using
standard
phenol-chloroform
extraction
procedure
(Sambrook et al, 2001). Microsatellite markers used for
genotyping were; D1S2757, D1S2840, D1S1660,
D1S2622, D1S373, D1S1723, D1S2655, D1S2686 and
D1S1724. The master mixture for PCR (Thermocyler I
Biorad) was prepared for five reactions. 65 l autoclaved
distilled water was added first and then 12.5 l of 10X
PCR buffer (NH4) SO4, 12.5 l of (25 mM) MgCl2 , 10.0 l
of (2.5 mM) dNTPs , 5.0 l of (50 pM) forward and 5.0 l
(50 pM) reverse primer solutions , 10.0 l of Taq DNA
polymerase were added to the master mixture. Master
mixture was mixed well and was equally distributed in
PCR tubes (24 l each). In the last 1 l of DNA template
was added and final volume was made up to 25 l in each
PCR tubes. This method of setting reactions saves times an
also minimizes the pipetting errors. Amplification was
performed with an initial denaturation for 4 min at 95C,
followed by 35 cycles of denaturation at 94C for 45 sec,
annealing at 57C for 45 sec, elongation at 72 oC for 45
sec, final extension at 72C for 10 mins and hold at 4 oC
for . The PCR products were separated on 8% nondenaturing polyacrylamide gels . The staining of gel was
done with ethidium bromide and photographed under UV
illumination Gel doc system (Syngene).
2.3. DNA sequencing
Polymerase chain reaction (PCR) amplification of exon 18
of ASPM gene was done by using gene-specific primer
D1S1660. The amplification was done following the
previously described thermocycler profile. The PCR
products were analyzed by 2% agarose gel .PCR product
were then purified using PCR Purification Kit ( Fermentas
GeneJETTM Gel Extraction Kit # K0691, # K0692).
Sequenced directly using Big Dye Terminator v3.1 cycle
sequencing kit in an ABI 3130 genetic analyzer (Applied
Biosystems, Foster City, CA, USA). Potential mutations
were confirmed by bi-directional sequencing and analyzed
by using BioEdit viewer software.

3. Results
After pedigree construction and DNA extraction,
genotyping with MCPH5 microsatellite markers was done
and linkage was observed on the basis that all the affected
individuals had the same homozygous banding pattern
whereas the normal individuals showed heterozygous
banding pattern with the MCPH5 locus. MCP family
showed complete linkage on chromosome 1q31 which
harbours ASPM gene, with the microsatellite marker
D1S1660 and D1S2840 as shown in the figure 2 and 3.

Available online at www.scientific-journals.co.uk

S. Irshad et al / Research Journal of Biology (2011), Vol. 01, Issue 01, pp. 1-6

Haplotype was constructed and the region was defined

with the microsatellite marker D1S2757 and D1S1678 as
shown in the figure 4. DNA sequence analysis revealed a
M N NN A A

ISSN 2049-1727

G to A transition [(CTG) to (CTA)] in affected individual

(II: 4) as shown in the figure 5 and 6 for exon 18 of ASPM
gene.
M NNN A A

1Kb DNA ladder

1Kb DNA ladder

Figure 2. Electropherogram of ethidium bromide stained

8% non-denaturing PAGE gel of MCP for primer
D1S1660 (212.44 cM) from Left to right, on chromosome
1q31 showing homozygosity among all affected (A) and
normal (N) individuals.

Figure 3: Electropherogram of ethidium bromide

stained 8% non-denaturing PAGE gel of MCP for
primer D1S2840 (212.04 cM) from Left to right, on
chromosome 1q31 showing homozygosity among all
affected (A) and normal (N) individuals.

Figure 4. Pedigree of family MCP showing primary microcephaly associated haplotype. The genetic map distance is according to
Marshfield genetic map in centimorgans (cM) is shown next to the marker name. Where circles shows females and squares
for males, filled for circles and squares are for affected

Available online at www.scientific-journals.co.uk

S. Irshad et al / Research Journal of Biology (2011), Vol. 01, Issue 01, pp. 1-6

ISSN 2049-1727

(Carrier, Heterozygous,
G + A)

Figure 5. Chromatogram showing the sequence of exon 18 of phenotypically normal/carrier (II: 2) individual.
Arrows indicate the site of mutation.

(Affected, Homozygous,
G > A)

Figure 6. Chromatogram showing the sequence of exon 18 of affected (II: 4) individual. Arrows indicate the site of
mutation.

4. Discussion
The viability of using a simple and defined PCR followed
by 8% native PAGE and sequencing is supportive in
screening possible mutations in ASPM and all other MCPH
genes. Family MCP was selected for screening for all
possible known loci MCPH1-MCPH7. The selection of the
locus was clearly on the basis of chance for the disease to
occur. So first of all MCPH5 was selected as it is the most
frequent causative agent. More than 50% of the MCPH
families were mapped to MCPH5 locus in Pakistan (Saleha
et al, 2011). In Pakistan the incidence of MCPH is ~ 1 in
10,000 individuals (Woods et al, 2005). In our study,
MCPH5 markers were selected which include D1S2757
(209.15 cM) to D1S1678 (218.05 cM) in centromeric to
telomeric direction. Screening was preceded with the

marker D1S2840, D1S1660, D1S2622, D1S373, D1S1723,

D1S2686, D1S2655, D1S1724 and D1S1678 which
indicated the homozygous banding pattern of the affected
individuals where as phenotypically normal or carrier
individuals showed heterozygous banding pattern thus,
linkage was observed in the region starting from 209.15cM
to 218.05 cM. Some microsatellite markers which had
been tested were non informative i.e., could not
differentiate between affected and normal individual and
same banding pattern was observed. Allelic frequency of
all five individuals was observed in MCP family by
difference in heterozygous and homozygous banding
pattern and hence showed complete linkage on
chromosome 1q31.
In present study, G to A transition [(CTG) to (CTA)] was

Available online at www.scientific-journals.co.uk

S. Irshad et al / Research Journal of Biology (2011), Vol. 01, Issue 01, pp. 1-6

noted in affected individual (II: 4) as shown in the figure 5

and 6. The observed two triplet codons were from six
triplet codons code for leucine and represented the
degeneracy of codons. Hence, no change in the overall
protein structure was observed. The observed G to A
transition was not present in all previously reported
mutations of ASPM gene (Nicholas et al, 2009).
Thus, the sequencing of entire ASPM gene including
intervening sequences is suggested to determine the real
cause of MCPH phenotype. Further investigations are
recommended for more molecular elucidations to prevent
the risk of disease. Hence, mutation screening will help in
genetic counselling and prenatal diagnosis for
microcephaly in Pakistani population, which may in turn
help reduce the incidence of microcephaly in
consanguineous population.

Acknowledgments
This study is funded by the University of the Punjab,
Pakistan. We wish to thank Dr. Muhammad Yaqoob, Head
of the Genetics department at the Childrens Hospital,
Lahore for his essential assistance in blood sample
collection and reviewing the patients clinical history and
pedigree for this study.

References
Bond, J., Roberts, E., Mochida, G.H., Hampshire, D.J.,
Scott, S., Askham, J.M., Springell, K., Mahadevan, M.,
Crow, Y.J., Markham, A.F., and Walsh, C.A. (2002)
ASPM as a major determinant of cerebral cortical size.
Nature Genetics, 32, pp. 316-320.
Bond, J., Roberts, E., Springell, K., Lizarraga, S., Scott, S.,
Higgins, J., Hampshire, D.J., Morrison, E.E., Leal, G.F.,
Silva,E.O., Costa, S.M.R., Baralle, D., Raponi, M.,
Karbani, G., Rashid, Y., Jafri, H., Bennett, C., Corry, P.,
Walsh, C.A., and Woods, C.G. (2005) A centrosomal
mechanism involving CDK5RAP2 and CENJP controls
brain size. Nature Genetics, 37, pp. 353-355.
Castiel, A., Danieli, M.M., David, A., Moshkovitz, S.,
Aplan, P.D., Kirsch, I.R., Brandeis, M., Krmer, A., and
Izraeli, S. (2011) The Stil protein regulates centrosome
integrity and mitosis through suppression of Chfr. Journal
of Cell Science, 124, pp. 532-539.
Guernsey, D.L., Jiang, H., Hussin, Julie., Arnold, M.,
Bouyakdan, K., Perry, S., Sturk, T.B., Beis, J., Dumas, N.,
Evans, S.C., Ferguson, M., Matsuoka, M., Macgillivray,
C., Nightingale, M., Patry, L., Rideout, A.L., Thomas, A.,
Orr, A., Hoffmann, I., Michaud, J.L., Awadalla, P., Meek,
D.C., Ludman, M., and Samuels, M. E. (2010) Mutations

ISSN 2049-1727

in Centrosomal Protein CEP152 in Primary Microcephaly

Families Linked to MCPH4. The American Journal of
Human Genetics, 87, pp. 40-51.
Gul, A., Hassan, M.J., Mahmood, S., Chen, W., Rhmani,
S., Naseer, M.I., Dellefave, L., Muhammad, N., Rafique,
M.A., Ansar, M., Chishti, M.S., Ali, G., Siddique, T., and
Ahmad, W. (2006) Genetic studies of autosomal recessive
primary microcephaly in 33 Pakistani families: novel
sequence variants in ASPM gene. Neurogenetics, 7, pp.
105-110.
Jackson, A.P., Eastwood, H., Bell, S.M., Adu J., Toomes,
C., Carr, I.M., Roberts, E., Hampshire, D.J., Crow, Y.J.,
Mighell, A.J., Karbani, G., Jafri, H., Rashid Y., Mueller,
R.F., Markham, A.F., and Woods, C. G. (2002)
Identification of Microcephalin, a Protein Implicated in
determining the size of the Human Brain. The American
Journal of Human Genetics, 71, pp. 136-42.
Kaindl, A.M., Passemard, S., Kumar, P., Kraemer,
N., Issa, L., Zwirner, A., Gerard, B., Verloes, A., Mani, S.,
and Gressens, P. (2010) Many roads lead to primary
autosomal recessive microcephaly. Progress in
Neurobiology, 90, pp. 363-383.
Kumar, A., Blanton, S.H., Babu, M., Markandaya, M., and
Girimaji, S.C. (2004) Genetic analysis of primary
microcephaly in Indian families: novel ASPM mutations.
Clinical Genetics, 66, pp. 341-348.
Kumar, A., Girimaji, S.C., Duvvari, M.R., and Blanton,
S.H. (2009) Mutations in STIL, Encoding a Pericentriolar
and Centrosomal Protein, Cause Primary Microcephaly.
American Journal of Medical Genetic, 84, pp. 286-290.
Leal, G.F., Roberts, E., Silva, E.O., Costa, S.M.R.,
Hampshire, D.J., and Woods, C.G. (2003). A novel locus
for autosomal recessive primary microcephaly (MCPH6)
maps to 13q12.2. Journal of Medical Genetics, 40, pp.
540-542.
Mohamed, F., Baig, S. M., Hansen L., Hussain M.S.,
Inayat I.A., Aslam M., Qureshi, J.A., Toilat, M., Kirst, E.,
Wajid, M., Nurnberg, P., Eiberg, H., Tommerup, N., and
Kjaer, K.W. (2009) Compound heterozygous ASPM
mutations in Pakistani MCPH Families. American
Journal of Medical Genetics, 149A, pp. 926-930.
Moynihan, L., Jackson, A.P., Roberts, E., Karbani, G.,
Lewis, I., Corry, P., Turner, G., Mueller, R.F., Lench,
N.J., and Woods, C.G. (2000) A Third Novel Locus for
Primary Autosomal Recessive Microcephaly maps to
chromosome 9q34. The American Journal of Human
Genetics, 66, pp. 724-727.
Nicholas, A.K., Khurshid, M ., Desir, J., Carvalho, O.P .,

Available online at www.scientific-journals.co.uk

S. Irshad et al / Research Journal of Biology (2011), Vol. 01, Issue 01, pp. 1-6

ISSN 2049-1727

Cox, J.J, Thornton, G., Kausar, R., Ansar, M., Ahmad

W., Verloes, A., Passemard S., Misson, J.P., Lindsay, S.,
Gergely, F., Dobyns, W.B. , Roberts, E., Abramowicz, M.,
and Woods, C.G. (2010) WDR62 is associated with the
spindle pole and is mutated in human microcephaly. Nature
Genetics, 42, pp. 1010-1014.
Pattison, L., Crow, Y. J., Jayne, D.V., Jackson, A.P., Jafri,
H. Rashid, Y. Roberts, E., and Woods, C.G. (2000) A Fifth
Locus for Primary Autosomal Recessive Microcephaly
Maps to Chromosome 1q31. The American Journal of
Human Genetics, 67 (6), pp. 1578-1580.
Sambrook, J., and Russell, D.W. (2001) Commonly used
techniques in molecular cloning: a laboratory Manual.
2nd ed., pp. 54. New York, Cold Spring Harbor
Laboratory Press.
Saleha, S., Ajmal, M., Jamil, M., Nasir, M., and Hameed,
A. (2011) A nonsense (c.3978G>A) abnormal spindle-like,
microcephaly associated (ASPM) gene mutation is a major
cause of primary microcephaly in Pashtoon ethnic group of
Pakistan. African Journal of Biotechnology, 10, pp.
6396-6400.
Woods, C.G. (2004) Human microcephaly. Current
Opinion in Neurobiology, 14, pp. 112-117.
Woods, C.G., Bond, J., and Enard, W. (2005) Autosomal
Recessive Primary Microcephaly (MCPH): A Review of
Clinical, Molecular, and Evolutionary Findings. The
American Journal of Human Genetics, 76, pp. 717-728.

Available online at www.scientific-journals.co.uk