Results markers analysis was done using standard PCR reaction

Results

Family Information

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In the present study three individuals are affected with primary
microcephaly belongs to district Karak, Khyber Pakhtunkhwa region of Pakistan.
This family has four generations comprising of 16 members including two
affected males and four affected females but two females and one male were
died. Now the family having five individuals having three affected individuals
(S-167-3) (S-167-4) and (S-167-5). Consanguineous marriage has been shown in
the pedigree in 3rd generation and the disease appeared in 4th generation. The
affected individuals showed primary autosomal recessive microcephaly. Five
samples of blood were collected from the family including three affected
(S-167-3) (S-167-4) and (S-167-5) and two normal (S-167-1) and (S-167-2)
individuals, and were processed for DNA extraction.

 

Genetic
Mapping

To date, 17 loci has been
identified forautosomalrecessive primary microcephaly. The family which is
selected for current study was tested by using different microsatellite markers
(D1S1660, D1S2738, D1S2840,D1S2816 and GATA135F02) mapped for ASPM gene on
chromosome 1q31. Genotyping was carried out by using these markers which having
average heterozygosity. Microsatellite markers analysis was done using standard
PCR reaction and electrophoresis on 8 % non-denaturing Polyacrylamide gel. The
markers were visualized by staining the gel with ethidium bromide and then
visual assessment of genotype was carried out. Analysis of the result shows
that the affected individuals are homozygous for their parental allele. And
also the analysis shows that the family is linked on one marker which is D1S21660
at the temperature of 550C.Therefore, it shows the family linkage
for ASPM gene, so it means that this locus is responsible for the tested
phenotype.The other marker shows no linkage on this family at every temperature
we tried.

 

 

 

 

 

 

 

 

Figure 3.1: Pedigree illustration of the
family segregating autosomal recessive Microcephaly. Affected males and females
are denoted by shaded squares and circles, respectively. Normal males and
females are denoted by empty squares and circles respectively. Double lines
between individuals signify consanguineous marriage. The black shaded circle
and square with black line denoting affected peoples of this family but they
were dead. And from which blood is collected it is shown with star.

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.2: Photographs from different
angles of the affected member (IV-9) of the family with primary microcephaly.

 

 

`

Figure3.3: Photographs from different
angles of the affected member (IV-6) of the family with primary microcephaly.

 

 

 

 

 

 

 

 

 

 

A   A  A  
N   N

 

Figure 3.4: Electrophorogram obtained
with UV Transilluminator of the extracted DNA samples

 

 

 

NNA  AA

 

Figure 3.5: The given electrophorograms
stained with ethidium bromide as a non-denaturing 8% polacrylamide gel showing
linkage allele pattern obtained with marker D1S1660 for ASPM gene on chromosome
1q31.5.

 

 

 

 

 

 

 

 

 

 

 

NNAAA

 

Figure 3.6: The given electrophorograms
stained with ethidium bromide as a non-denaturing 8% polacrylamide gel showing
linkage allele pattern obtained with marker D1S2840 for ASPM gene on chromosome
1q31.5.

 

 

 

N  N A     
AA

 

Figure 3.7: The given electrophorograms
stained with ethidium bromide as a non-denaturing 8% polacrylamide gel showing
linkage allele pattern obtained with marker D1S2816 for ASPM gene on chromosome
1q31.5.

 

 

 

 

 

 

 

 

 

 

                                               
N         N            A        AA

 

Figure 3.8: The given electrophorograms
stained with ethidium bromide as a non-denaturing 8% polacrylamide gel showing
linkage allele pattern obtained with marker D1S2738 for ASPM gene on chromosome
1q31.5.

 

 

 

                                                            
N                      A

 

Figure

3.9:Electrophorogram obtained with UV Transilluminator of the
amplified product of exon 17 by 2% Agarose gel.

 

 

 

 

 

 

 

 

 

 

 

Figure
3.10: Haplotypefigure
shows autosomal recessive MCPH inheritance. 1-1 represents homozygous (linked),
1-2 represents heterozygous (unlinked) marker of ASPM gene. Homozygous states
represents affected while heterozygous state of marker represents healthy
normal or carrier individual.

 

 

Discussion

One of the most obvious
structural characteristics of the human brain, when compared with that of other
mammals, is its large size. Cognitive abilities decrease with a significant
decrease in brain size and most striking example of it is Autosomal Recessive
Primary Microcephaly (MCPH), in which brain size reduces to one third of its
original volume with a significant cognitive decline. The MCPH proteins are
involved in different molecular pathways including DNA damage response
signaling, cell division, proper spindle orientation, microtubule dynamics,
cell cycle regulation etc. Recent functional studies confirm the presence of
MCPH proteins at centrosome for at least part of cell cycle. Therefore, one may
conclude that centrosome is the final integration point for many regulatory
pathways affecting prenatal neurogenesis in mammals.

Primary autosomal recessive microcephaly is a
clinical diagnosis of exclusion in an individual with a head circumference ?4 SDs below the expected age-and-sex mean. There is associated moderate
mental retardation, and neuroimaging shows a small but structurally normal cerebral
cortex. The inheritance pattern in the majority of cases is considered to be
autosomal recessive.

MCPH is a
neurogenic mitotic disorder, though affected patients demonstrate normal
neuronal migration, neuronal apoptosis and neural function. Seventeen MCPH loci
(MCPH1-MCPH17) have been mapped to date from various populations around the
world and contain the following genes: Microcephalin, WDR62, KNL1, CDK5RAP2,
CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1, CDK6, SASS6, MFSD2A,
ANKLE2, and CIT. It is predicted that MCPH gene mutations may lead to the
disease phenotype due to a disturbed mitotic spindle orientation, premature
chromosomal condensation, signalling response as a result of damaged DNA,
microtubule dynamics, transcriptional control or a few other hidden centrosomal
mechanisms that can regulate the number of neurons produced by neuronal
precursor cells.

The first locus is MCPH1 caused by
mutations in microcephaline gene on chromosome 8q23 33,35,36,18,19,21-23. Second
locus is MCPH2 caused by mutations in WDR62 on chromosome 19q13.12 5,37-39. Third locus is MCPH3 caused by mutations in CDK5RAP2 gene
on chromosome 9q33.2 49-51,78,80. Fourth locus is MCPH4 caused by mutations
in CEP152 gene on chromosome 15q15-q21 67,39. The fifth locus is MCPH5 caused
by mutations in ASPM gene on chromosome 1q31.3 39, 52, 53, 56. MCPH5 is the
most common locus throughout the world discovered so far. Sixth locus is MCPH6
caused by mutations in CENPJ on chromosome 13q12.12 62-64, 39.The seventh one
is MCPH7 caused by mutations in STIL/SIL gene on chromosome 1p33 70- 73. The
eight number locus is MCPH8 which is caused by mutation in CEP135 gene on
chromosome 4q12 74-76. The ninth number locus is MCPH9 which is caused by
mutation in CEP152 gene on chromosome 15q21.181-84. The tenth number locus is
MCPH10 which is caused by mutation in ZNF335 gene on chromosome 20q13.12
85,86. The eleventh number locus is MCPH11 which is caused by mutation in
PHC1 gene on chromosome 12p13.311p21.287. The twelth number locus is MCPH12
which is caused by mutation in CDK6 gene on chromosome 7q21.2 83,89,90. The
thirteen number locus is MCPH13 which is caused by mutation in CENPE gene on
chromosome 4q24 94. The fourteen number locus is MCPH14 which is caused by
mutation in SASS6 gene on chromosome 96,97. The fifteen number locus is
MCPH15 which is caused by mutation in MFSD2A gene on chromosome 1p34.2 96,97.
The sixteen number locus is MCPH16 which is caused by mutation in ANKLE2 gene
on chromosome 12q24.33 96,97. The last and final one locus identified so far
is MCPH17 caused by mutations in CIT gene on chromosome 12q24.23 92,93.

In the present study we have a
consanguineous Pakistani family of five members segregating primary autosomal
recessive microcephaly. The three affected individuals in the family having
microcephaly with slight mental retardation and their behavior was not normal.
We performed genetic mapping to find out genetic linkage for ASPM gene of the
given family. Genotyping was performed by using five polymorphic markers
(D1S1660, D1S2738, D1S2840, D1S2816 and GATA135F02) for MCPH5 (ASPM gene) on
chromosome one for five samples of the given family.

Resultantly the family showslinkage
for the affected individuals in the given region D1S1660 (marker). Therefore it
shows the family linkage for ASPM gene, so it means that this locus is
responsible for the tested phenotype.

 

Conclusions

Cognitive
capabilities are reduced with a substantial decrease in brain size in MCPH
patients, whose brain sizes decrease to one third compared with its original
size with a substantial cognitive decline. There are seventeen known genes that
cause this neurodevelopmental disorder, and recent evidence suggests that MCPH
is possibly a primary disorder of neurogenic mitosis. With the progress of MCPH
gene identification, prenatal diagnosis (detects a disorder recurrence), postnatal
diagnosis (differentiates the disorder from various differential diagnosis) and
carrier testing (for consanguineous families in which the disease is known to
occur) are increasingly available for patients. The successful creation of
mammalian models for MCPH has enabled researchers to further add to the
knowledge regarding the aetiology and pathophysiology of MCPH. Better
genotyping, neuro-physiological and neuroimaging testing, together with the
creation of genetically homogeneous groups of patients, would benefit the
identification of exact genotype phenotype correlations.