Genetic heterogeneity in Pakistani microcephaly families revisited

Ahmad I.a,b,c, Baig S.M.d, Abdulkareem A.R.b,e, Hussain M.S.a,b,c, Sur I.b, Toliat M.R.a,
Nrnberg G.a, Dalibor N.a, Moawia A.a,d, Waseem S.S.a,d, Asif M.d, Nagra H.d, Sher M.d, Ali
Khan M.M.d, Hassan I.f, ur Rehman S.d, Thiele H.a, Altmller J.a,g, Noegel A.A.b,c,h and
Nrnberg P.a,c,h
Accepted Article

a
Cologne Center for Genomics (CCG), University of Cologne, D-50931 Cologne, Germany;

b
Institute of Biochemistry I, Medical Faculty, University of Cologne, D-50931 Cologne,
Germany;

c
Center for Molecular Medicine Cologne (CMMC), University of Cologne, D-50931 Cologne,
Germany;

d
Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering
(NIBGE), Faisalabad 38000, Pakistan;

e
Genetic Engieneering and Biotechnology Institute, University of Baghdad, Baghdad, Iraq

f
Plant Biotechnology Division, National Institute for Biotechnology and Genetic Engineering
(NIBGE), Faisalabad 38000, Pakistan;

g
Institute of Human Genetics, University of Cologne, Cologne, D-50931, Germany;

h
Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases
(CECAD), University of Cologne, D-50931 Cologne, Germany

Correspondence: Peter Nrnberg, PhD

Cologne Center for Genomics (CCG),

University of Cologne;

Weyertal 115b,

50931 Cologne, Germany;

Phone: +49-221-478-96801; Fax: +49-221-478-96803

Email: nuernberg@uni-koeln.de

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1111/cge.12955

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 Conflict of interest

P.N. is a founder, CEO, and shareholder of ATLAS Biolabs GmbH. ATLAS Biolabs GmbH is
a service provider for genomic analyses.
Accepted Article
Acknowledgements
We express our gratitude to the patients and their families for their participation in this study.
We thank Elisabeth Kirst and Gerti Meyer zu Altenschildesche for their excellent technical
assistance. This work was supported by a grant from the Center for Molecular Medicine
Cologne (CMMC) to A.A.N. and P.N.
Abstract

Autosomal recessive primary microcephaly (MCPH) is a rare and heterogeneous genetic

disorder characterized by reduced head circumference, low cognitive prowess and, in
general, architecturally normal brains. As many as 14 different loci have already been
mapped. We recruited 35 MCPH families in Pakistan and could identify the genetic cause of
the disease in 31 of them. Using homozygosity mapping complemented with whole-exome,
gene panel or Sanger sequencing, we identified twelve novel mutations in three known
MCPH-associated genes nine in ASPM, two in MCPH1 and one in CDK5RAP2. The two
MCPH1 mutations were homozygous microdeletions of 164,250 bp and 577,594 bp,
respectively, for which we were able to map the exact breakpoints. We also identified four
known mutations three in ASPM and one in WDR62. The latter was initially deemed to be
a missense mutation but we demonstrate here that it affects splicing. As to ASPM, as many
as 17 out of 27 MCPH5 families that we ascertained in our sample were found to carry the
previously reported founder mutation p.Trp1326*. This study adds to the mutational spectra
of four known MCPH-associated genes and updates our knowledge about the genetic
heterogeneity of MCPH in the Pakistani population considering its ethnic diversity.

Key words: MCPH, WDR62, splicing error, CDK5RAP2, ASPM, founder mutation

Introduction

Autosomal recessive primary microcephaly (MCPH, MIM 251200) is a genetically

heterogeneous human brain disorder and a rare subgroup of congenital microcephaly (1). It
is defined as a reduced occipital frontal circumference (OFC) at birth that is less than four
standard deviations (-4SD) below the age, sex and ethnicity matched control group (2).

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 MCPH appears to be due to abnormal developmental processes rather than regression or
degeneration of the neuronal tissue. In general, the architecture of the brain is not grossly
affected (3), although structural defects due to impaired neuronal migration resulting from
mutations of WDR62 and CDK6 have been proposed (4). As compared to Caucasians, the
prevalence of MCPH is higher in populations with widely practiced intermarriages such as
those from South Asian and Arabic countries (1, 2).
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To date, thirteen genes have been reported to be associated with MCPH, including MCPH1
(MIM 251200), WDR62 (MIM 604317), CDK5RAP2 (MIM 604804), KNL1 (MIM 604321),
ASPM (MIM 608716), CENPJ (MIM 608393), STIL (MIM 612703), CEP135 (MIM 614673),
CEP152 (MIM 614852), ZNF335 (MIM 615095), PHC1 (MIM 615414), CDK6 (MIM 616080)
SASS6 (MIM 616402) and CIT (605629) (4-11). Among these, ASPM and WDR62 are most
commonly implicated in MCPH, with mutations of these genes causing up to 50% and 14%,
respectively, of all MCPH cases (1, 2). We omitted CENPE (MIM 117143), MFSD2A (MIM
614397) and ANKLE2 (MIM 616062), the genes reported to underlie the disease loci
MCPH13 (MIM 616051), MCPH15 (MIM 616486) and MCPH16 (MIM 616062), respectively,
from the listing above because patients with mutations in these genes display different
phenotypes, namely microcephalic primordial dwarfism (CENPE) and other syndromic forms
of microcephaly (MFSD2A and ANKLE2), in which patients show microcephaly associated
with dysmorphic facial features and hypopigmented macules (12-14). To date heterogeneity
studies were performed in Pakistani (3, 15-17), Indian (18) and Iranian populations (19). In
continuation of our previous report (16) we recruited additional 35 Pakistani families with
numerous affected individuals. Their investigation resulted in a considerable expansion of
our knowledge about the mutational spectra of MCPH-associated genes and also the
incidence of particular mutations in the different ethnicities living in Pakistan.

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 Materials and methods

Subjects

The new MCPH cohort consists of 35 consanguineous families recruited from different
geographical origins in Pakistan according to the diagnostic criteria described elsewhere (3).
Head circumference (HC) of probands was compared with a graph of age-dependent HC
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values of the normal population published by Nellhaus in 1968 (20). The phenotypic features
of the current cohort, which comprises 148 patients with a male to female ratio of 92:56, are
summarized in Table S1.2 of the Supplementary Information. Neuroimaging by MRI of two
different affected individuals showed profound reduction in the cerebral cortex (Fig. S1,
Supplementary Information). The study adhered to the Tenets of the Declaration of
Helsinki and was approved by the ethics review board at the National Institute for
Biotechnology and Genetic Engineering (NIBGE), Pakistan. Informed consent was obtained
from all participating family members.

Linkage analysis

For homozygosity mapping, we used two different array platforms with genome-wide
resolutions of 587K SNPs (Axiom Genome-Wide CEU 1 Array, Affymetrix, Santa Clara, CA)
and 700K SNPs (HumanCoreExome 12v1-1 bead chip, Illumina Inc., San Diego, CA). In
some MCPH families, we performed exclusion mapping of known loci by STR marker typing.
More detailed information on the procedures used for linkage analysis is described in the
Appendix S1 (Supplementary Information).

cDNA synthesis, RT-PCR and quantitative PCR

To extract RNA, we collected blood samples in PAXgene Blood RNA tubes (QIAGEN) from
two affected individuals carrying the mutation WDR62:c.332G>C and a control individual. We
synthesized cDNA as described (7). PCR primers were placed in exons 2 and 4 of WDR62
(Table S1.1, Supplementary Information). The amplified products were purified by PCR
clean-up gel extraction (Macherey-Nagel) and re-amplified for Sanger sequencing.

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 Mutation Analysis

All coding exons and flanking intronic sequences of candidate genes were bidirectionally
sequenced using the Sanger approach. For some families, whole-exome sequencing (WES)
was performed. Variants were excluded from further consideration as potential disease-
causing variants when known to be polymorphisms or repeatedly found in ExAC, dbSNP138
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and/or our in-house database of >1600 exomes. For the exact break point mapping of
MCPH1 microdeletions, two families were analysed with the Genome-wide Human CytoScan
HD Array from Affymetrix. For further information see Appendix S1 (Supplementary
Information).

Results

In an effort to find novel genetic causes of primary microcephaly, 17 consanguineous families

segregating this disorder were genotyped using STR markers and/or SNP arrays.
Homozygosity mapping resulted in the mapping to already published linkage intervals of 13
of these whereas 4 families could not be linked to any of the 14 known MCPH loci. Among
the families showing linkage to known loci, 2 were linked to MCPH1, 1 to MCPH2, 1 to
MCPH3 and as many as 9 to the MCPH5 locus. Further, ASPM was sequenced in 18
additional families from Northern Pakistan based on the fact that mutations of ASPM are the
most common cause of congenital microcephaly among families from this region (3).

MCPH1 mutations

Genome-wide analysis in families MCP118 and MCP125 revealed linkage to the MCPH1
locus (Appendix S2, Fig. S2A-1a,b, S2A-2a,b, Supplementary Information). Interestingly,
PCR failed to amplify exons 1-2 of MCPH1 in patient samples from MCP118 and exons 1-11
in patient samples from MCP125, suggesting partial homozygous deletions in both families.
We also performed WES for one patient from each family and excluded pathogenic variants
in other known MCPH genes, but rather corroborated the homozygous deletions in MCPH1
by read depth evaluation (Fig. S2A-1d, S2A-2e, Supplementary Information). To map the
breakpoints, we then performed high-resolution molecular karyotyping using the CytoScan
HD array from Affymetrix. An abrupt drop of the hybridization signal over a 164 kb DNA
segment in MCP118 and 577 kb segment for MCP125 revealed the true extent of the
deletions (Fig. S2A-1c and S2A-2d, Supplementary Information). To fine-map the exact
positions of the breakpoints, several PCR primer pairs were designed (Table S1.1,

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 Supplementary Information). Sequencing of breakpoint spanning amplification products
revealed that the microdeletion in family MCP118 comprises the first 2 exons and extends
~160 kb upstream of MCPH1 whereas in MCP125, the microdeletion removed the first 11
coding exons and ~493 kb upstream of MCPH1 (Fig. 1a, b). Sanger sequencing of the
hemizygous DNA samples of the parents from both families did not reveal any point mutation
in the deleted exons. To complete the report, the overlapping deletions observed in the two
Accepted Article
MCP families were absent from the structural variant data of the large cohort of healthy
individuals deposited in the database of genome variants (DGV).

WDR62 mutation

Only one family (MCP129) mapped to the MCPH2 locus. Sequence analysis of WDR62
revealed a known missense mutation (c.332G>C, p.Arg111Thr) that we already reported
earlier in another family (MCP14) (16) yet the consequences of this apparent missense
mutation might have been misinterpreted. Here, we investigated the effect of the nucleotide
change on WDR62 transcript splicing since the nucleotide affected by this mutation
represents the last base of exon 3 of WDR62. Indeed, RT-PCR products obtained from
cDNA of two affected individuals of the MCP14 family amplified with primers upstream (exon
2) and downstream (exon 4) of the mutation in exon 3 revealed aberrant product sizes of 147
bp and 297 bp, whereas the control showed the expected product size of 210 bp (Fig. 2a).
Sanger sequencing of the small and large abnormally sized products revealed skipping of the
complete exon 3 and retention of 87 bp of intron 3, respectively, whereas only normal
splicing was observed in the control sample (Fig. 2b,c). There was no residual wild-type
WDR62 transcript visible in any of the affected individuals of the MCP14 family tested here.
Skipping of exon 3 results in the deletion of 63 bp at mRNA level and consequently a loss of
21 amino acids (p.Cys91_Arg111del) in the WDR62 protein. The analysis of the larger
transcript revealed that the c.332G>C mutation abolishes the original splice donor site and
favours a cryptic one in the flanking intron 3 at position +88 resulting in the retention of 87 bp
of intron 3 sequence in the transcript. This changes the reading frame and introduces an
early termination codon (p.Arg111Thrfs*2).

CDK5RAP2 mutation

Family MCP121 was mapped to the MCPH3 locus. WES revealed a novel nonsense
mutation in CDK5RAP2 at nucleotide position c.1279 in exon 12 (c.1279C>T,

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 ENST00000349780) (Fig. 2d). This mutation is predicted to result in a premature termination
codon (p.Arg427*). The variant was validated by Sanger sequencing and showed perfect
segregation with the phenotype when all available individuals of the family were tested
(Table 1).
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ASPM mutations

Targeted Sanger sequencing of ASPM was employed for 27 families including 9 previously
linked to MCPH5 and 18 families with no prior linkage mapping data. In total, nine novel and
three known mutations were found in these families. Interestingly, 17 families carried the
already reported founder mutation p.Trp1326* (Table 1, Table S1.4, Supplementary
Information). Moreover, two families harboured compound heterozygous mutations, which
are all novel (Fig. 2e). Each of these mutations segregated faithfully within the corresponding
family. All ASPM mutations are summarized in Table 1.

Discussion

Investigation of thirty-five consanguineous families with MCPH from Pakistan using array
mapping, Sanger sequencing of candidate genes and next generation sequencing either as
gene panel or whole-exome sequencing revealed the underlying mutations in thirty-one of
them (Table 1). As many as 27 families (77.1%) could be linked to the MCPH5 locus, two
families (5.7%) to the MCPH1 locus, one family (2.8%) to the MCPH2 locus and another one
(2.8%) to the MCPH3 locus. For four families (11.4%) we could exclude that they are linked
to any of the known MCPH loci but still not succeeded to identify the causal gene variants in
their genomes.

The two overlapping microdeletions identified in MCPH1 both strongly suggest that the
truncated gene may be no longer functional. Mutations in MCPH1 have been reported to
result in premature chromosomal condensation (PCC) at G2 and delayed decondensation at
G1 (21-25). In both of the current families, patients had normal height and weight and no
phenotypic resemblance to PCC syndrome was observed. To date a total of 18 MCPH1
mutations have been reported and only two of them in the Pakistani population (16, 26, 27).
This work adds two further MCPH1 mutations from the Pakistani population, thereby
increasing the total number of MCPH1 mutations to 20. Moreover, it is the first report
describing the precise breakpoints of microdeletions >22 kb associated with MCPH genes.

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 The mutation WDR62:c.332G>C has been reported previously in another Pakistani family
with primary microcephaly (16). Originally, we considered it an ordinary missense mutation
changing Arg111 into threonine. Here we tested its influence on splicing and noticed that the
splice donor of intron 3 is compromised by the G-to-C transversion at the last position of
exon 3. As a result about three quarters of the transcripts showed retention of part of intron
3, whereas one quarter showed skipping of exon 3. The predicted consequences at the
Accepted Article
protein level are a frame shift with premature termination of translation and a deletion of 21
amino acids, respectively. Mutation p.Arg111Thrfs*2 would be likely to impair all functions of
WDR62 due to the loss of major functional domains, whereas mutation p.Cys91_Arg111del
would result in the deletion of three amino acids of the first WD repeat of the WDR62 protein
only. Previously a missense mutation in KNL1 was also reported to impair splicing (28), but
this is the first study to show the functional consequences of a missense mutation affecting
the last base of an exon in an MCPH family.

Interestingly, in the MCP129 family, we observed a second disease phenotype that seemed
to segregate independently of microcephaly. Only one out of four microcephalic family
members, but also one sibling with normal HC, showed myotonic dystrophy and polydactyly.
The latter individual was heterozygous for the WDR62 mutation. Hence we speculate that the
phenotype of myotonic dystrophy and polydactyly may be caused by a second mutation in a
different gene.

Further, p.Arg427* was identified as a novel nonsense mutation in CDK5RAP2. This

mutation is predicated to result in a truncated gene product of 427 amino acids as compared
to 1893 amino acids of the wild-type product. If produced and stable in the patients, it would
lack most of the 215 kDa protein except for the TuRC-binding domain and a part of the N-
terminal SMC-domain. Mutations in CDK5RAP2 are rare and only 8 different mutations have
been described so far (29, 30). With this report we increase the number to nine.

Sequence analysis of ASPM in 27 families revealed 12 different mutations including 3

previously published ones and 9 novel mutations. The three mutations p.Trp1326*,
p.Lys1862Glu and p.Tyr3164* (Table S1.3, Supplementary Information) were reported
previously (18, 19, 31). To date, mutation p.Trp1326* has been described in 32 families (16)
(Fig. S1-4, Supplementary Information) and proposed as a founder mutation in Northern
Pakistani MCPH families (15, 16, 31-33). Most of these families were reported to have
Pashtun ethnicity while some reports did not provide ethnicity information (34). Pashtuns
reside mainly in Southern and Eastern Afghanistan and in North-West Pakistan (34, 35). In
this study, we found the same mutation in a further 17 families of Pashtun ethnicity (Fig. S1-

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 4, Supplementary Information) including two from Afghanistan (Table S1.4) who are
currently living as refugees in Khyber Pakhtunkhwa, Pakistan. This increases the total
number of p.Trp1326* mutation reports from 32 to 49. The 9 novel mutations identified
include p.Lys412Thrfs*5, p.Gly807Glufs*7, p.Trp1404*, p.Q1987*, p.Leu2285Argfs*6,
p.Gln2377*, p.Arg2700*, p.Lys2837Metfs*34 and p.Arg3491Leufs*15. Four of them were
identified in two families with compound heterozygous patients. All novel ASPM mutations
Accepted Article
are nonsense or frameshift mutations that most probably induce nonsense-mediated mRNA
decay and thus result in a complete loss of ASPM function irrespective of the position of the
mutation. The current study increases the total number of different ASPM mutations from 133
to 142 (1, 36). The 142 mutations comprise 59 nonsense, 4 missense and 8 splice site
mutations. As many as 68 mutations are small deletions or insertions leading to a frameshift.
In addition, two microdeletions and one complex rearrangement have been found (36).

In summary, this study reports 12 novel MCPH-causing mutations and substantially adds to
the mutational spectra of known MCPH-associated genes. We confirm that ASPM is the
most frequently mutated gene in MCPH. In particular, the observed high frequency of the
ASPM mutation p.Trp1326* underscores its prominent role as a founder mutation in the
Pashtun population living in Pakistan and Afghanistan. On the basis of these findings, we
recommend to test for mutation ASPM:p.Trp1326* as a first tier when performing molecular
genetic diagnostics in MCPH families with Pashtun ethnicity.

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Figures legends

Fig. 1. Breakpoint analysis of partial MCPH1 microdeletions. Electropherograms of DNA

sequences of affected individuals of the families MCP118 (a) and MCP125 (b) are shown
along with the corresponding wild-type sequence. The homozygous deletions comprise the
first 2 or the first 11 exons of the MCPH1 gene, respectively, and a substantial part of its 5

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 upstream sequence. The exact positions of the breakpoints are marked with arrows.
Sequence coordinates are given according to the human genome assembly GRCh38.
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Fig. 2. Positions and consequences of mutations identified in various MCPH-associated

genes. (a) Effect of the mutation WDR62:c.332G>C on the transcript. RT-PCR products were
obtained with primers specific for the critical region of WDR62 from cDNA of two affected
individuals of the MCP14 family and separated by electrophoresis on a 2% agarose gel along
with a control. (b) Graphical scheme of a partial region of WDR62 to demonstrate the
aberrant splice phenomena as revealed by RT-PCR. Forward and reverse primer positions
are indicated by arrows. The larger PCR product is caused by partial retention of intron 3-4
(hatched box). (c) Sanger traces of wild-type and mutant cDNA to verify the presumed
skipping of exon 3 and retention of part of intron 3-4. (d) Schematic representation of the
exon/intron structure of CDK5RAP2. Exons (boxes) are drawn to scale whereas introns
(connecting lines) are arbitrarily sized. The nonsense mutation p.Arg427* (c.1279C>T) is
located in exon 12. (e) Schematic representation of ASPM. Homozygous mutations are
shown above the scheme while compound heterozygous mutations are displayed below.

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Accepted Article

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 Table 1. Mutations identified in MCPH-associated genes in 31 Pakistani families
No. Family ID Gene Mutation (nt)* Mutation (aa) Region Ethnicity status

1 MCP96 ASPM c.9492T>G p.Tyr3164* Chichawatni- Punjabi known

Punjab
2 MCP101 ASPM c.3978G>A p.Trp1326* Buner-KPK Pashtun known
3 MCP103 ASPM c.5584A>G p.Lys1862Glu Malakand-KPK Pashtun known
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4 MCP106 ASPM c.3978G>A p.Trp1326* Khushab- Pashtun known
Punjab
5 MCP117 ASPM c.2420delG p.Gly807Glufs*7 Chitral-KPK Pashtun novel
6 MCP118 MCPH1 c.1-160,656_ p.(Met1_Asn95del) Faiasalabad- Punjabi novel
114+892del164250 Punjab
7 MCP120 ASPM c.8098C>T p.Arg2700* Faiasalabad- Punjabi novel
Punjab
8 MCP121 CDK5RAP2 c.1279C>T p.Arg427* Tando Sindhi novel
Muhammad
Khan-Sindh
9 MCP123 ASPM c.6851_6854delTCTC p.Leu2285Argfs*6 Hyderabad- Sindhi novel
c.7129C>T p.Gln2377* Sindh novel
10 MCP125 MCPH1 c.1-493520_ p.(Met1_Ala761del) Tando Baloch novel
2137-9110del577594 Allahyar-Sindh
11 MCP129 WDR62 c.332G>C p.Arg111Thr Jamshoro- Baloch known
Sindh
12 MCP130 ASPM c.4212G>A p. Trp1404* Jamshoro- Baloch novel
Sindh
13 MCP131 ASPM c.1235_1239delAAGTA p.Lys412Thrfs*5 Shangla-KPK Pashtun novel
14 MCP135 ASPM c.3978G>A p.Trp1326* KohatKPK Pashtun known
15 MCP136 ASPM c.3491_3494delGTAC p.Arg3491Leufs*15 KohatKPK Pashtun novel
16 MCP137 ASPM c.3978G>A p.Trp1326* KohatKPK Pashtun known
17 MCP138 ASPM c.3978G>A p.Trp1326* KohatKPK Pashtun known
18 MCP139 ASPM c.3978G>A p.Trp1326* KarakKPK Pashtun known
19 MCP140 ASPM c.3978G>A p.Trp1326* KarakKPK Pashtun known
20 MCP141 ASPM c.3978G>A p.Trp1326* KarakKPK Pashtun known
21 MCP143 ASPM c.3978G>A p.Trp1326* KarakKPK Pashtun known
22 MCP165 ASPM c.8508_8509delGA p.Lys2837Metfs*34 Mianwali- Sariaki novel
c.5959C>T p.Gln1987* Punjab novel
23 MCP171 ASPM c.5959C>T p.Gln1987* Okara-Punjab Punjabi novel
24 MCP174 ASPM c.3978G>A p.Trp1326* Bannu-KPK Pashtun known
25 MCP175 ASPM c.3978G>A p.Trp1326* Bannu-KPK Pashtun known
26 MCP176 ASPM c.3978G>A p.Trp1326* Karak- KPK Pashtun known
27 MCP177 ASPM c.3978G>A p.Trp1326* Karak-KPK Pashtun known
28 MCP178 ASPM c.3978G>A p.Trp1326* Nowshera-KPK Pashtun known
29 MCP187 ASPM c.3978G>A p.Trp1326* Bannu-KPK Pashtun known
30 MCP190 ASPM c.3978G>A p.Trp1326* Mardan-KPK Pashtun known
31 MCP191 ASPM c.3978G>A p.Trp1326* Malakand-KPK Pashtun known

*nt indicates nucleotide; aa indicates amino acid; KPK is used as an acronym for Khyber Pakhtunkhwa, province of Pakistan.

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