Primary microcephaly case from the Karachay-Cherkess Republic poses an additional support for microcephaly and Seckel syndrome spectrum disorders

Abstract

Background
Primary microcephaly represents an example of clinically and genetically heterogeneous condition. Here we describe a case of primary microcephaly from the Karachay-Cherkess Republic, which was initially diagnosed with Seckel syndrome. Case presentationClinical exome sequencing of the proband revealed a novel homozygous single nucleotide deletion in ASPM gene, c.1386delC, resulting in preterm termination codon. Population screening reveals allele frequency to be less than 0.005. Mutations in this gene were not previously associated with Seckel syndrome. Conclusions
Our case represents an additional support for the clinical continuum between Seckel Syndrome and primary microcephaly.

Full text

C A S E R E P O R T Open Access
Primary microcephaly case from the
Karachay-Cherkess Republic poses an
additional support for microcephaly and
Seckel syndrome spectrum disorders
Andrey V. Marakhonov
1,2,7*
, Fedor A. Konovalov
3
, Amin Kh. Makaov
4
, Tatyana A. Vasilyeva
1
, Vitaly V. Kadyshev
1
,
Varvara A. Galkina
1
, Elena L. Dadali
1
, Sergey I. Kutsev
1,5,6
and Rena A. Zinchenko
1,5,6
From Belyaev Conference
Novosibirsk, Russia. 07-10 August 2017
Abstract
Background: Primary microcephaly represents an example of clinically and genetically heterogeneous condition.
Here we describe a case of primary microcephaly from the Karachay-Cherkess Republic, which was initially diagnosed
with Seckel syndrome.
Case presentation: Clinical exome sequencing of the proband revealed a novel homozygous single nucleotide deletion
in ASPM gene, c.1386delC, resulting in preterm termination codon. Population screening reveals allele frequency to be
less than 0.005. Mutations in this gene were not previously associated with Seckel syndrome.
Conclusions: Our case represents an additional support for the clinical continuum between Seckel Syndrome and
primary microcephaly.
Keywords: ASPM, Clinical continuum, Clinical heterogeneity, Allelic disorders, Seckel syndrome
Background
Primary, or congenital, microcephaly (MCPH) is character-
ized by a decrease in the head circumference more than
four standard deviations (SD) below age and sex-specific
means [1]. Often, microcephaly is accompanied by a
psychomotor retardation. Primary microcephaly could be
caused by either hereditary or environmental factors,
including maternal exposure to toxoplasma or Zika virus
[2], to alcohol or excessive amounts of the phenylalanine
[3,4]. The presence of facial dysmorphism points at the
need for differentiating this condition from the Seckel syn-
drome as well as from lissencephaly and Rubenstein-Taybi
and Norman-Roberts syndromes. Hereditary primary micro-
cephaly is a genetically heterogeneous group of conditions
inherited mainly in autosomal recessive mode, though sev-
eral dominant forms have been described [5]. Although
MCHP and Seckel syndrome werepreviouslydistinguished
by height (maximum height in Seckel syndrome was
equivalent to the minimum height in MCPH), stature is no
longer a discriminating feature, leading to the conclusion
that these phenotypes constitute a spectrum rather than
distinct entities [6]. The Seckel syndrome is characterized
by more severe intellectual disability as well as more often
the presence of characteristic facial features. To date, 17
different genes associated with autosomal recessive MCPH
are identified. Nine genes are associated with Seckel syn-
drome, of them 2 (CENPJ and CEP152) could cause both
MCPH and Seckel syndrome.
In consanguineous populations, the prevalence of primary
microcephaly was estimated to be 1 in 10,000—6.8 per
10,000 [7]. Homozygous and compound heterozygous mu-
tations in ASPM gene (MCPH5; OMIM #605481) account
* Correspondence: marakhonov@generesearch.ru
1
Research Centre for Medical Genetics, Moscow, Russia
2
Moscow Institute of Physics and Technology, Dolgoprudny, Russia
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Marakhonov et al. BMC Medical Genomics 2018, 11(Suppl 1):8
DOI 10.1186/s12920-018-0326-1
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

for up to 40% of primary MCPH cases in both consanguin-
eous and non-consanguineous families [8]. ASPM (Abnor-
mal Spindle Microtubule Assembly) protein is a part of a
mother centriole complex; it regulates centriole biogenesis
during neurogenesis, apical complex, and cell fate [9].
Here we present a case of primary microcephaly with
family recurrence. This case was found in Khabezsky
district of the Karachay-Cherkess Republic, Russia, inhab-
ited by approximately 30,000 dwellers of predominantly
Circassian origin (95.2%).
The Circassians belong to the Northwest Caucasian
ethnic group [10] speaking the mutually intelligible
continuum of Circassian language with two literary
standards, Adyghe (West Circassian) and Kabardian or
Kabardino-Cherkess (East Circassian). In its narrowest
sense, the term “Circassian”is restricted to twelve
Adyghe tribes [11]. Importantly, documented calamities
of the 19th and 20th centuries, including the Caucasian
War of 1817–1864, resulted in the forcible eviction of a
large part of the Circassians into the Ottoman Empire.
Further administrative transformations carried out by
the tsarist government and then by the Soviet authorities
led to the formation of four territorially isolated groups
of the Circassian people, with separate ethnographic des-
ignations: Kabardian (Circassians of the Kabardino-
Balkar Republic), Cherkess (Circassians of the Karachay-
Cherkess Republic), Adyghe (Circassians of the Kuban
including the Republic of Adygea and Krasnodar Krai),
and Shapsug (the indigenous historical inhabitants of
Shapsugia) [12]. These four Circassian populations,
including northwestern Adyghe people, do not differ in
common mtDNA haplogroup frequencies [13]. The Y-
chromosomal markers data suggested a direct origin of
Caucasus male lineages from the Near East, followed by
high levels of isolation, differentiation and genetic drift
in situ [14].
Case presentation
Here we describe a Circassian family with three affected
siblings: a proband (eхamined at the age of 66 years old),
and his two sisters, examined at the age of 58 and 56 years
old, all were ascertained with the primary incoming diag-
nosis of Seckel syndrome. The family also included three
healthy siblings, two sisters and a brother. The patients
were examined during a field expedition to the Karachay-
Cherkess Republic with the help of local Ministry of
Health Care. Detailed clinical examination detected
following phenotypic features: mental retardation, marked
decrease in the circumference of the head (proband and
one siblings –46 cm, another sib –44 cm), pronounced
predominance of the facial part of the skull over the cere-
bral, large protruding low-set ears, narrow beveled fore-
head, low hair growth on the forehead, high roof of the
mouth, microgenia, muscular hypertonus, contractures in
the elbow joints without pathological reflexes (Fig. 1).
Epileptic seizures were not observed. All affected family
members also demonstrated short stature (142–144 cm),
kyphoscoliosis (1–2 degree), and a serious deficiency of
the cognitive component of behavior with the preserva-
tion of the response to simple commands (eating, taking
hygienic procedures). They have no reading, writing, and
arithmetic skills, and demonstrated monosyllabic speech
resembling that of a 3–4 years old children. Archival
medical records have indicated that all these children were
born with low weight (below 3000 g), while their skull
circumferences were at the lower limit of the norm until
6–7 months of life, with progressive declines in its per-
centile observed subsequently. Developmental milestones
were, at first, correspondent to the age. The delay, then
the stop in the growth of the cerebral cranium was
observed at by 5 years, with the lag at −4 SD. The height
of healthy father and brother were at 190 cm and above.
One healthy sister has height of 176 cm, while other –of
171 cm. One of the healthy sisters gave birth to healthy
children (Fig. 2).
Due to the known genetic heterogeneity of Seckel syn-
drome, DNA diagnosis in the proband was carried out by
targeted high-throughput sequencing (HTS) of clinically
relevant genes (clinical exome sequencing, CES). CES was
performed on Illumina NextSeq 500 instrument in 2 ×
151 bp paired-end mode. A total of 13.7 million reads were
obtained, corresponding to 99.9× on-target average sequen-
cing depth based on TruSight One Sequencing Panel target
region list. The raw sequencing data have been processed
with a custom pipeline based on popular open-source
bioinformatics tools BWA, Samtools, Vcftools, as well as
in-house Perl scripts, using hg19 assembly as a reference
sequence. In total 49,772 nucleotide variants were found.
Variant annotations were added by SnpEff/SnpSift software
using public databases (dbSNP, ExAC, ClinVar, dbNSFP).
After filtering the variants by functional consequence and
Fig. 1 Proband’s phenotype
Marakhonov et al. BMC Medical Genomics 2018, 11(Suppl 1):8 Page 92 of 95
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

population frequencies, no suitable candidates were found
in a proband among the genes known to date that are
responsible for Seckel syndrome. After ranking the variants
by their functional consequences and population frequen-
cies, only one suitable candidate gene, ASPM, was identified
in a proband as previously not described homozygous vari-
ant hg19::chr1:197111995TG>T. This variant leads to mu-
tation NM_018136.4(ASPM_v001):c.1386delC in the exon
3oftheASPM gene, leading to the formation of the prema-
ture stop codon p.Tyr462*. Sanger sequencing confirmed
that two affected sisters bear the same mutation in the
homozygous state while healthy siblings were heterozygous
for the mutation (Fig. 3).
Importantly, homozygous and compound heterozygous
loss-of-function mutations in the ASPM gene were previ-
ously described in patients with autosomal recessive pri-
mary MCPH type 5 (OMIM #608716). ASPM:p.Tyr462*
mutation has not been previously found in the publicly
available control cohorts (genome Aggregation Database)
as well as in 202 population-matched control chromo-
somes (screened by PCR-RFLP). Therefore, we conclude
that, according to the ACMG criteria, on the strength of
Fig. 2 Pedigree of the family
Fig. 3 Results of Sanger sequencing
Marakhonov et al. BMC Medical Genomics 2018, 11(Suppl 1):8 Page 93 of 95
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

cumulative evidence, this mutation should be regarded as
pathogenic [15]. As the mutation causes the formation of
the premature stop codon –p.Tyr462* –the mRNA
should be a target for nonsense-mediated mRNA decay
(NMD) leading to the null-allele [16].
As this mutation occurred in the homozygous state in
the proband, estimations of run-of-homozygosity (ROH)
region length were performed around the mutation
according to the states of alternate alleles of frequent
SNPs covered by clinical exome sequencing data. We
found that in this Circassian family, ROH region spreads
at least from rs79351096 to rs4950927, with the minimal
length 6.2 Mb. In fact, the length of ROH region could
be even greater as clinical exome data used for its esti-
mation cover only coding sequences of genes related to
hereditary diseases.
Discussion
Here we present a description of a Circassian family with
three out of six siblings displaying primary microcephaly,
short stature, mental retardation, and bird-like face.
Clinical exome sequencing revealed a novel homozygous
single nucleotide deletion c.1386delC in ASPM gene,
which leads to preterm stop-codon and truncating of
protein. According to The American College of Medical
Genetics and Genomics (ACMG) criteria, this single
nucleotide variant is classified as pathogenic with a
strong evidence (PM2, PVS1, PS3, PP1-S) [15]. The same
ethnic background of the parents of the index patient
could explain the homozygous state of the identified
mutation. However, population screening for the muta-
tion in 202 normal chromosomes reveals no carriership,
indicating that the frequency of this mutation is less
than 0.005. Analysis of the genetic structure of the
Circassian population shows that in the rural district of the
family’s residence the level of random Wright inbreeding
(F
ST
) was at 0.00890, while the value of local inbreeding
estimated through the isolation model by the Malecot’sdis-
tance was at 0.00933, i. е.almost1%[17,18]. In addition, it
is known that the marriages with a positive ethnic assorta-
tiveness are preferred in this population. Although the pedi-
gree does not show the consanguinity, taking into account
the genetic structure of the population, we should assume
thepresenceofconsanguinity[19]. Analysis of runs-of-
homozygosity on CES data also supports the idea of the
inbred origin of the proband. The length of ROH region
encompassed the revealed homozygous frame-shifting dele-
tion appears to be at least 6.2 Mb, which is much greater
than an average for outbred populations [20], thus, pointing
to the possible endogamous ancestry of the family.
To date, more than 400 different nucleotide variants in
ASPM gene are registered in ClinVar [21], and only 155 of
them reported to be pathogenic or likely pathogenic. A
majority of them being loss-of-function and should lead to
NMD. All reported mutations of ASPM are associated
with autosomal recessive primary MCPH type 5. To date,
17 genes are described to be associated with primary auto-
somal recessive MCPH. The vast majority of them partici-
pate in mitotic spindle assembly (ASPM,WDR62,
CDK5RAP2,KNL1,CENPJ,STIL,CEP135,CEP152,
CENPE,SASS6,CIT,andANKLE2), while others are asso-
ciated with chromosome condensation and maintenance
(MCPH1,ZNF335,PHC1), cell cycle control (CDK6), and
blood-brain barrier maintenance (MFSD2A). Mutations in
two of them, CENPJ and CEP152, could also cause an
allelic condition known as autosomal recessive Seckel syn-
drome [22,23], which is characterized by proportionate
growth and mental retardation, microcephaly, and charac-
teristic bird-like face. Other forms of Seckel syndrome are
caused by mutations in genes associated with cell growth
(TRAIP), genomic integrity and repair (ATR ,NSMCE2,
DNA2,andRBBP8), centrosome function (NIN,CEP63)
[6]. Clinical diagnosis of these conditions is also compli-
cated by the need to differentiate them from primordial
dwarfism which sometimes could lead to similar pheno-
types [24], but may be distinguished from Seckel syn-
drome by radiological assessment. Meier-Gorlin syndrome
could also manifest with microcephaly and intrauterine
and postnatal growth retardation [25]. This clinical
spectrum of overlapping phenotypes makes differential
diagnosis challenging.
Conclusions
The proband presented here was initially diagnosed with
Seckel syndrome because of primary microcephaly, severe
mental delay, and characteristic facial features. This pheno-
type is not common in described primary microcephaly
cases as intellectual disability is usually more severe in
Seckel syndrome as well as characteristic facial features,
which could correspond to the relative sparing of the mid-
facial structures compared to the rest of the head. High-
throughput sequencing of clinically relevant genes in pro-
band identified no candidate nucleotide variants in any
genes associated with Seckel syndrome to date. The only
mutation identified in this family was a frame-shifting
single nucleotide deletion affecting ASPM gene. To our
knowledge, no ASPM mutations have been associated with
Seckel-like phenotypes to date. Therefore, our observation
broadens the phenotypic heterogeneity of MCPH and
supports the view on MCPH and Seckel syndrome as a
clinical continuum.
Funding
Publication of this article was funded by the Russian Scientific Foundation
[grant number 17-15-01051].
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Marakhonov et al. BMC Medical Genomics 2018, 11(Suppl 1):8 Page 94 of 95
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About this supplement
This article has been published as part of BMC Medical Genomics Volume 11
Supplement 1, 2018: Selected articles from Belyaev Conference 2017: medical
genomics. The full contents of the supplement are available online at
https://bmcmedgenomics.biomedcentral.com/articles/supplements/volume-
11-supplement-1.
Authors’contributions
AVM performed molecular genetic experiments, analyzed and interpreted
the patient data, wrote the manuscript. FAK analyzed and interpreted the
patient’s HTS data. AKM collected samples. TAV contributed to the analysis
of patient data, prepared the manuscript. VVK, VAG, ELD performed a clinical
examination of the patient. SIK and RAZ designed the study and helped
supervise the project. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The clinical and molecular genetic study was performed in accordance with
the Declaration of Helsinki and approved by the Institutional Review Board
of the Federal State Budgetary Institution “Research Center for Medical
Genetics,”Moscow, Russia, with written informed consent obtained from
each participant and/or their legal representative, as appropriate.
Consent for publication
Consent for publication was obtained from the legal guardian of the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Research Centre for Medical Genetics, Moscow, Russia.
2
Moscow Institute of
Physics and Technology, Dolgoprudny, Russia.
3
Genomed Ltd, Moscow,
Russia.
4
Khabez central district hospital, Khabez, Russia.
5
Pirogov Russian
National Research Medical University, Moscow, Russia.
6
Moscow State
University of Medicine and Dentistry, Moscow, Russia.
7
Laboratory of Genetic
Epidemiology, Research Centre for Medical Genetics, Moskvorechie St., 1,
Moscow, Russian Federation115478.
Published: 13 February 2018
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