c© Indian Academy of Sciences

RESEARCH NOTE

Molecular genetic analysis of consanguineous families with primarymicrocephaly identified pathogenic variants in the ASPM gene

MUZAMMIL AHMAD KHAN1,2, CHRISTIAN WINDPASSINGER3, MUHAMMAD ZEESHAN ALI2,MUHAMMAD ZUBAIR2,4, HADIA GUL5, SAFDAR ABBAS2, SAADULLAH KHAN1,6,

MUHAMMAD BADAR2, RAMZI M. MOHAMMAD1 and ZAFAR NAWAZ1,7∗

1Translational Research Institute, Academic Health System, Hamad Medical Corporation, Doha 3050, Qatar2Gomal Centre of Biochemistry and Biotechnology, and 5Faculty of Sciences, Department of Biological Sciences,

Gomal University Dera Ismail Khan, Khyber-Pakhtoonkhwa 29050, Pakistan3Institute of Human Genetics, Medical University of Graz, Graz 8010, Austria

4Department of Cell and Developmental Biology, School of Life Sciences, University of Scienceand Technology China, Hefei 230026, China

6Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology,Kohat 26000, Khyber Pakhtunkhwa, Pakistan

7Diagnostic Genomic Division, Department of Laboratory Medicine and Pathology,Hamad Medical Corporation, Doha 3050, Qatar

AbstractAutosomal recessive primary microcephaly is a rare genetic disorder that is characterized by reduced head circumferenceand a varying degree of intellectual disability. Genetic studies on consanguineous families with primary microcephaly haveidentified 15 (MCPH) causative genes that include MCPH1, WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135,CEP152, ZNF335, PHC1, CDK6, CENPE, SASS6 MFSD2A ANKLE2 and CIT (Khan et al. 2014; Yamamoto et al. 2014;Alakbarzade et al. 2015; Morris-Rosendahl and Kaindl 2015; Basit et al. 2016). Physiologically, most of these MCPH proteinsare involved in cell cycle and its regulation. In the present clinical genetic study, we have present two consanguineous Pakistanifamilies segregating primary microcephaly and intellectual disability. These families were ascertained from the Saraiki ethnicpart of Khyber-Pakhtunkhwa province in Pakistan. Whole exome sequencing in one family revealed a novel 1-bp deletionNM_018136.4: c.10013delA (p.Asp3338Valfs*2), while the other family showed a previously reported nonsense mutationNM_018136.4: c.9730C>T (rs199422195 (p.Arg3244*)) in ASPM gene. The novel frame-shift mutation (p.Asp3338Valfs*2)in ASPM presumably truncates the protein synthesis that results in loss of armadillo-type fold domain.

[Khan M. A., Windpassinger C., Ali M. Z., Zubair M., Gul H., Abbas S., Khan S., Badar M., Mohammad R. M. and Nawaz Z. 2017 Moleculargenetic analysis of consanguineous families with primary microcephaly identified pathogenic variants in the ASPM gene. J. Genet. 96, xx–xx]

Introduction

In the broad sense, most of the identified MCPH genes haveeither structural or physiological role in cell cycle and itsregulation, neurogenesis and ciliogenesis (Barbelanne andTsang 2014). Morphologically, microcephalic patients havenormal brain architecture and the reduced brain volume which

∗For correspondence. E-mail: ZNAWAZ@hamad.qa.Muzammil Ahmad Khan and Christian Windpassinger contributed equallyto this work.

is due to small cerebral cortex (Woods et al. 2005). Thegenetic studies on cohorts and consanguineous families haveshown that ASPM (MCPH5 locus) and WDR62 (MCPH2) arethe most frequent genes reported in primary microcephaly(Mahmood et al. 2011). At cellular level, ASPM is requiredfor normal functioning of mitotic spindle in embryonic neu-roblasts. The ASPM knockout studies in mice have shownthat reduction in brain size is due to misorientation of mitoticspindle fibres, which also affects the ratio of symmetric toasymmetric cell divisions and thus, decrease the number ofneuronal cells (do Carmo Avides and Glover 1999).

Keywords. primary microcephaly; Pakistani families; exome sequencing; ASPM gene; armadillo-type fold.

Journal of Genetics, DOI 10.1007/s12041-017-0759-x

Muzammil Ahmad Khan et al.

Materials and methods

Family recruitment and sample collection

Prior approval was obtained from ethical review board ofGomal Centre of Biochemistry and Biotechnology, GomalUniversity, D. I. Khan, Pakistan, to conduct the presentmolecular study, and the families were enrolled after takingthe informed written consent. The exome data analysis wasperformed in Hamad Medical Corporation, Doha, Qatar, withthe approval of data sharing agreement. For molecular inves-tigation, we were able to collect the peripheral blood samplesfrom individuals III-1, IV-1, IV-3 and IV-5 of family A, whileindividuals IV-3, IV-4, V-4, III-1, III-2 and V-3 were amongthe volunteers in family B. The DNA was isolated from thesesamples using standard phenol–chloroform assay.

Whole exome sequencing and prioritization of candidatepathogenic variant

Paired-end whole exome sequencing was performed onNextSeq500 plate-form (Illumina, San Diego, USA), usingNextera rapid capture exome kit for library construction, fol-lowing the manufacturer protocol. The raw data analyseswere accomplished through BaseSpace integrated applica-tions to get BAM and annotated VCF files. Subsequently,the variant filtration was carried out on genetalk softwareto identify the pathogenic variants (Kamphans and Krawitz2012). In addition to that, computational-based predictionof most probable candidate genes/variants and its associa-tion with disease phenotype was performed through exome-walker (Smedley et al. 2014), Exomiser (Robinson et al.2014) and PhenIX softwares (Zemojtel et al. 2014). Thesesoftwares perform phenotype and protein interaction-basedprioritization of most plausibly implicated genes.

Sanger sequencing and mutation analysis

For subsequent mutation detection and segregation analysis,Sanger DNA sequencing of ASPM gene was performed.Primers sequences were designed through Primer3websoftware, ver. 4.0.0 (http://bioinfo.ut.ee/primer3/). DNAsequencing was carried out by using BigDye Terminatorver. 3.1 cycle sequencing kit (Applied Biosystems, FosterCity, USA) and the products were analysed on ABI 3130xlGenetic Analyzer (ABI, USA).

Results

In this study, two consanguineous families were recruitedfrom the rural areas of Pakistan. The genealogical analysisof both families revealed their Saraiki-based ethnicity. Pedi-gree analysis demonstrated autosomal recessive mode of dis-ease segregation. The general clinical description of patientsfrom both families presented nonsyndromic congenitalprimary microcephaly. The patients presented reduced occip-itofrontal head circumference along with moderate intellec-tual disability. The patients had severe speech disability, andthus, were facing difficulty in conveying their message. Psy-chologically, the patients were having aggressive attitude dueto which they had tendency of self-beating behaviour. Thepatients never attended any training institute and were unableto perform arithmetic measures or any conceptual task, butstill they had milder category of recognition power, like rec-ognizing route to their home and family relatives. The addi-tional clinical features of all families are described in table 1.

Molecular findings

Exome sequence analysis of individual IV-3 and thesubsequent segregation study through Sanger sequencing

Table 1. Genotype associated phenotypic descriptions of consanguineous microcephalic families.

Family ID Family A Family B

c.10013delA c.9730C>TMutation (p.Asp3338Valfs*2) (p.Arg3244*) (rs199422195)

Individual ID IV-3 IV-3 IV-4 V-4Age (years) 21 19 24 6Sex Male Male Male MaleHead circumference (cm) 41 46 46 39Intellectual disability Yes Yes Yes YesSpeech disability Yes Yes Yes YesEpileptic shocks No No No NoMuscular dystrophy No No No NoSkeletal abnormality No No No NoNeurologic defect No No No NoBehavioral expression Hyperactive with Hyperactive with Hyperactive with Hyperactive with

jolly mood jolly mood jolly mood jolly moodOcular defect No No Strabismus NoDermal lesions No No No NoAny visceral organ defect No No No No

Journal of Genetics

Molecular analysis of MCPH genes

(III-1, IV-1, IV-3 and IV-5) identified a novel deletionmutation NM_018136.4:c.10013delA in the 26th exon ofASPM. The identified pathogenic variant was also pri-oritized by the candidate gene identification softwaresusing exome variant file (VCF file). Protein sequenceanalysis revealed that mutation p.Asp3338Valfs*2 ispresent in the armadillo-type fold domain. This out-of-frame deletion shifts the reading frame and truncatethe protein synthesis (p.Asp3338Valfs*2) due to prema-ture stop codon and produces 3338 amino acids-shortened

protein with deleted armadillo-type fold domain. The identifiedsequence variant is not present in 100 normal con-trols, 1000 genome browser (http://www.1000genomes.org/1000-genomes-browsers), exome variant server (http://evs.gs.washington.edu/EVS/) and exome aggregation consor-tium databases (http://exac.broadinstitute.org/). The muta-tion analysis of second family (IV-4 and V-4) revealedthe previously reported nonsense mutation c.9730C>T(rs199422195 (p.Arg3244*)) in 24th exon (NM_018136.4)of ASPM gene (figure 1).

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Figure 1. (a) A four-generational consanguineous Pakistani family inheriting autosomal recessive primary microcephaly. The sequencechromatograms depict the homozygous deletion of adenine nucleotide in patient IV-2 (c.10013delA), heterozygous status in carrier IV-3and reference allele in IV-5. (b) The graphical presentation of family tree with reported ASPM mutation (c.9730C>A).

Journal of Genetics

Muzammil Ahmad Khan et al.

Discussion

Abnormal spindle-like microcephaly associated protein(ASPM) is encoded by ASPM gene that spans 62.567 Mblong region on chromosome 1q31.3 (December 2013(GRCh38/hg38)). Its longest transcript (NM_018136) consistsof 28 exons and encodes for a protein with 3477 amino acids.ASPM is reported to be crucial for the normal functioningof mitotic spindle fibres during embryonic neuroblasts for-mation. The ASPM protein has several functional domains:the calponin homology domains, P-loop containing nucle-oside triphosphatase hydrolase domains (P-loop NTPase),armadillo-type fold domain and IQ motifs (interpro acces-sion ID: Q8IZT6). Among these functional domains, thecalponin homology domain is associated with regulation ofcontractility and organization of actin cytoskeleton in smoothmuscles (Bramham et al. 2002), the P-loop NTPase domainsperform hydrolysis of β–γ phosphate bond in nucleosidetriphosphate (NTP) (Leipe et al. 2004), the IQ motif actsas an EF-hand-binding site and sometime as a phosphory-lation site of protein kinase C (Baudier et al. 1991) andthe armadillo-type fold domain is suggestively involved inassociating cadherin to the cytoskeleton (Huber et al. 1997).

Literature survey has shown that mutations in ARMdomain have previously been reported to be implicated inMCPH (Gul et al. 2007; Nicholas et al. 2009). ARM domainis a 42 amino acid-conserved entity which is present in var-ious proteins, e.g. β-catenin, α-importin and plakoglobin,which together make a distinct class of protein familycalled ARM-repeat family proteins. Tandem repetition ofARM repeats makes a superhelical fold which provide aversatile interacting platform to the protein and therefore,ARM domain containing proteins shown to have severalindependent molecular and biological functions in the cell(Coates 2003). String matching analysis using protein–protein interaction (STRING, protein interaction database;http://string-db.org/) did not show any significant change inthe interaction of ASPM with its known interactors, whichprobably indicates the involvement of some other proteininteractors involved in the neurogenesis pathway with whomASPM interacts through its ARM domain.

In conclusion, we performed mutation analysis of ASPMin two consanguineous Pakistani families presenting primarymicrocephaly. One family revealed a novel single-base dele-tion of adenine nucleotide (c.10013delA) in 26th exon ofASPM, while the other family exhibited a reported muta-tion (c.9730C>A) in its 24th exon. The novel frameshiftmutation presumably leads to a truncated protein with thedeletion of an armadillo-type fold domain. The study addeda new pathogenic variant in the mutational spectrum ofarmadillo-type fold domain of ASPM. This will help in elu-cidating the novel protein interactors involved in neuroge-nesis pathway. Moreover, screening of additional Saraikiethnic families may contribute in devising a molecular diag-nostic test that might be helpful in genetic counselling ofnonconsanguineous families.

Acknowledgements

We extend special thanks to all family members for their volunteerparticipation in this study. The current study is partially supportedby startup research grant from Higher Education Commission ofPakistan (M-IPFP/HRD/HEC/2011/346) for molecular screening ofneurological disorders. MAK appreciates the valuable contributionof his beloved nephew, Abdul Ahad Khan Gandapur in recruitingthe family.

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Received 7 June 2016, in final revised form 1 August 2016; accepted 7 September 2016Unedited version published online: 12 September 2016

Final version published online: 9 May 2017

Corresponding editor: S. GANESH

Journal of Genetics