CA S E R E P O R T Open Access
Molecular characterization of a rare
analphoid supernumerary marker
chromosome derived from 7q35 → qter: a
case report
Bárbara Marques
1
, Cristina Ferreira
1*
, Filomena Brito
1
, Sónia Pedro
1
, Cristina Alves
1
, Teresa Lourenço
2
,
Marta Amorim
2
and Hildeberto Correia
1
Abstract
Background: Analphoid supernumerary marker chromosomes (aSMC) constitute one of the smallest groups of
SMC, and are characterized by a centromeric constriction but no detectable alpha-satellite DNA. These marker
chromosomes cannot be properly identified by conventional banding techniques alone, and molecular cytogenetic
methods are necessary for a detailed characterization. Analphoid SMC derived from chromosome 7 are extremely
rare, with only five cases reported so far.
Case presentation: In this work we report an aSMC involving the terminal long arm of chromosome 7 in a
10-year-old boy with multiple dysmorphic features and severe development delay. Cytogenetic analysis revealed a
mosaic karyotype with the presence of an extra SMC, de novo, in 20% of lymphocytes and 73% of fibroblast cells.
Next, we performed FISH analysis with multiple DNA probes and cCGH analysis. This identified the origin of the
SMC as an analphoid marker resulting of invdup rearrangement of 7q35-qter region.
Affimetrix CytoScan HD array analysis redefined the aSMC as a 15.42 Mb gain at 7q35-q36.3 (minimum tetraplicated
region-chr7: 143,594,973-159,119,707; GRCh37/hg19) of maternal origin that encloses 67 OMIM genes, 16 of which
associated to disease. Uniparental disomy of chromosome 7 (UPD 7) has been excluded.
Conclusions: We report the first patient with an aSMC(7) derived from the terminal 7q region who has been
molecularly and clinically full characterized. The use of SNParray in the characterization of SMC reveals to be a powerful
tool, giving information not only about copy number variation but also about loss-of-heterozygosity and parental
origin. We conclude that an integrated genome-wide copy number variation analysis, if possible associated to FISH and
gene expression studies, could facilitate in the future the difficult task of establishing accurate
genotype-phenotype correlations and help to improve genetic counselling.
Keywords: Chromosome 7, Analphoid supernumerary marker chromosome, Neocentromere, Partial 7q tetrasomy,
7q duplication
* Correspondence: cristina.ferreira@insa.min-saude.pt
1
Unidade de Citogenética, Departamento de Genética Humana, Instituto
Nacional de Saúde Doutor Ricardo Jorge, I.P, Avenida Padre Cruz, 1649-016
Lisboa, Portugal
Full list of author information is available at the end of the article
© The Author(s). 2016 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.
Marques et al. Molecular Cytogenetics (2016) 9:87
DOI 10.1186/s13039-016-0295-z
Background
Analphoid supernumerary marker chromosomes (aSMC)
constitute one of the smallest groups of SMC, and are
characterized by a centromeric constriction but no detect-
able alpha-satellite DNA [1]. These marker chromosomes
cannot be clearly identified by conventional banding tech-
niques alone, with molecular cytogenetic methods being
necessary for their detailed characterization.
So far 141 aSMC have been reported encompassing 20
of the 22 autossomes and both sexual chromosomes,
with 41% of these being acrocentric derivatives (from
chromosomes 15 and 13) [2]. The most common aSMC
are supernumerary inverted duplications (invdup) of the
distal arm of a chromosome. The first aSMC derived
from chromosome 7 was reported using multicolour-
FISH in a patient with learning and de velopmental delay
as well dysmorphic features [3]. Cases subsequently re-
ported include an inverted-duplication-shaped aSMC de-
rived from the long arm terminal region, and a
derivative chromosome 7 with an inte rstitial deletion of
the short and long arms segments showing a neocentro-
mere in the p14 region. None of these reports included
the patients’ clinical description [4, 5]. Recently, Kumar
et al. reported a child with dysmorphic features and de-
velopmental delay, who presented a complex chromo-
some rearrangement involving chromosome 7 and
resulting of a class II/McClintock mechanism [6]. Louvr-
ier et al. [7] reported a mosaic neocentric ring involving
the region 7q22.1q31.1, in a child with a severe global
retardation and dysmorphic features. In this case, the
aSMC charact erization, using arrayCGH, and the clin-
ical description are very detailed establishing a clear
genotype-phenotype correlation. In our study we used
different methods in order to fully characterize an aSMC
involving the terminal long arm of chromosome 7 in a
patient presenting several dysmorphic features, unstable
assisted broad-based walk and severe developmental
delay without language acquisition. To our knowledge
this is the first case reported of an aSMC(7) with an
entire molecular chara cterization and clinical description
involving the region 7q35-qter. With this stud y we hope
to contribute for a better genotype-phenotype correl-
ation and improved genetic counselling.
This case was submitted to the sSMC database (http://
ssmc-tl.com/chromosome-7.html#N), No. 07-N-q35/1-1.
Case presentation
The patient was a newborn male, the first child of a
healthy and non-consanguineous couple, born at
28 weeks of gestation with normal somatometric param-
eters (weight 1167 g corresponding to P50). In the neo-
natal period the patient showed respiratory distress
syndrome and feeding difficulties, and was diagnosed
with bronchopulmonary dysplasia probably due to
prematurity.
On physical examination, open and wide anterior fon-
tanelle, bilateral eyelid edema, low-set ears, short nose
with wide and depressed root, open tented, and fingers
and toes nail hypoplasia were described.
The patient also showed bilateral hip dysplasia, crypt-
orchidism and a bilateral inguinal hernia that has already
been corrected. Furthermore, the child was submitted, at
4 months of age to a colectomy for ne crotizing entero-
colitis, 1 month later to a tracheotomy, and at 18 months
of age to a percutaneous endoscopic gastrostomy.
The patient is currently 10 years old and has severe
developmental delay without language acquisition,
presenting with bruxism and auto-aggressive behaviour
(which started at the age of 8). The child has an
unstable, assisted broad-based walk. He still depends on
enteral feeding, has a tracheostomy tube for breathing
and suffers from recurrent respiratory infections. The
physical exam reveals an accentuation of the dysmorphic
features with frontal bossing, low-set ears, straight eye-
brows with synophrys and hypertelorism. There are also
supernumerary teeth, divergent strabismus and anterior
chamber asymmetric optic malformation (Fig. 1). Renal
and cardiac malformations were excluded by ultrasound.
Material and methods
Cytogenetics
Cytogenetic analyses were performed on conventional
giemsa-trypsin-leishman (GTL) banded metaphase chro-
mosomes obtained from phytohemagglutinin-stimulated
peripheral blood lymphocytes, from the patient and his
parents, as well as skin fibroblasts from the patient by
standard techniques. A karyotype was established
according to the International System for Human
Cytogenetic Nomenclature [8].
Fluorescent in situ hybridization (FISH)
FISH analysis was performed on chromosome spreads
prepared from lymphocyte and skin fibroblast cultures.
FISH probes included alpha-satellite probes for all chro-
mosomes (home made), ELN and D7S427 probes (Oncor,
Gaithersburg, MD), whole chromosome painting probe
(WCP) for chromosomes 2, 3, 7, 9, 10, 11,16, 17, 19 and
20 (Cambio, Cambridge, UK), subtelomeric 7q probe
(TelVysion, Abbott Molecular, Abbott Park, IL, USA) and
the bacterial artificial chromosome (BAC) clone RP11-
298A10 (kindly supplied by Wellcome Trust Sanger
Institute, Cambridge, UK). Extraction of alpha-satellite
DNA for FISH probes, labelling and FISH analysis were
performed following standard protocols. Extraction of
BAC DNA for FISH probe, labelling and FISH analysis
were performed as described earlier [9, 10].
Marques et al. Molecular Cytogenetics (2016) 9:87 Page 2 of 7
Chromosomal comparative genomic hybridization (cCGH)
Metaphase spreads were prepared from phytohemagglu-
tinin-stimulated lymphocytes from healthy individuals, ac-
cording to standard procedures. Genomic DNA was
extracted from the patient’s peripheral blood sample using
the Wizard® genomic DNA purification Kit from Promega
(Promega Corporation, WI, USA). cCGH analysis was per-
formed according to standard procedures. The data was
analyzed using ISIS-CGH software (MetaSystems, Altleis-
sheim, Germany) with average ratio profiles of 1,25 e 0,75
and standard deviation limits.
SNParray
Array analysis wa s performed in genomic DNA
extracted from peripheral blood of the patient and his
parents using Affymetrix CytoScan HD® array (Affyme-
trix, California, USA) according to the manufacturer’s
recommendations (Affymetrix manual protocol Affymetrix®
Cytogenetics Copy Number Assay P / N 703038 Rev. 3).
CytoScan HD array contains 740,304 polymorphic
(SNP, single nucleotide polymorphism) and 1,953,249
non-polymorphic (copy number probes) markers with an
average intragenic marker spacing of 880 bps and
intergenic marker spacing of 1737 bps. The raw data were
processed using Genotyping Console v4.0 and Chromo-
some Analysis Suite 3.0.0.42 with NetAffx na33.1 (UCSC
GRCh37/hg19) and the output data were interpreted with
the UCSC Genome Browser (https://genome.ucsc.edu/;
GRCh37/hg19 assembly), DECIPHER (https://decipher.san-
ger.ac.uk/) and ClinGen (http://www.clinicalgenome.org).
The functions of the genes, which were located within the
region of the genomic imbalance, were retrieved from
the GeneCards (http://www.genecards.org) and OMIM
(http://www.ncbi.nlm.nih.go v/omim) databases. The trio
analysis to exclude uniparental disomy of chromosome 7
(UPD 7) was done using the CytoScanHD_Array
Fig. 1 Clinical facial features of the patient at different ages. a At 5th months: facial edema, short nose with wide and depressed root; b At
4-year-old: turricephaly, curly hair, straight eyebrows with synophrys, opened tented mouth; c and d At 10-year-old: accentuation of the
dysmorphic features with hypertelorism, underdeveloped crus helix, full lips and dental crowding
Marques et al. Molecular Cytogenetics (2016) 9:87 Page 3 of 7
Mendelian Error Check tool and the parental origin of the
aSMC was determined using MyPODFinder v.1.0.
Results
Cytogenetic analysis revealed a mosaic karyotype with
the presence of a SMC, de novo, in 20% of lympho cytes
and 73% of skin fibroblast cells (Fig. 2a).
FISH analysis with alpha-satellite probes for all chro-
mosomes indicated that the SMC was an analphoid
marker (Fig. 2b), while the presence of euchromatic
material was revealed with whole chromosome painting
probe for chromo some 7 (Fig. 2c). cCGH analysis indi-
cated that the euchromatic material had origin in the
7q35-qter region (data not shown). Hybridization with
RP11-298A10 and subtelomeric 7q probes, allowed
establishing that the aSMC results of an invdup
rearrangement of 7q35-qter region (Fig. 2d).
Affimetrix CytoScan HD array analysis redefines the
aSMC to a region of about 15.42 Mb enclosing 67
OMIM genes, 16 of which are associated to disease
(Fig. 3). Trio analysis of the patient and his parents
excluded the presence of UPD 7 and indicated a mater-
nal origin of the aSMC.
Based on these results, the karyotype was established
as:
mos 47,XY,+mar dn[73]/46,XY[27].ish invdup(7)(q-
ter → q35::q35 → neo → qter)(wcp7+,D7Z1-,RP11-298A
10++,TelVysion7q++).arr[GRCh37] 7q35q36.3(14321895
4x2,143594973_159119707x4).
Discussion
When an SMC is discovered in a current cytogenetic
analysis, its identification can be a challenging ta sk if
using FISH only. The best approach to study an SMC
with euchromatic material is to use DNA microarray to
determine its origin, involved region and size, followed
by FISH for its characterisation. In our case, the SMC
identification and characterization was made by FISH
and cCGH. Afterwards, SNParray was done to mapping
at submicroscopic level the genomic imbalance, to ex-
clude the presence of UPD and to determine the paren-
tal origin. This allowed the characterization of an aSMC,
Fig. 2 Cytogenetic and FISH investigation a GTL-banded chromosomes showing the aSMC (arrow); FISH with b α-satellite probe for chromosome
7 showing absence of signal on the aSMC; c WCP(7) showing the presence of chromosome 7 euchromatin in the aSMC; d BAC RP11-298A10
(red) e subtelomeric 7q (green) probes showing that the aSMC is a invdup involving the terminal region of chromosome 7 (7q35-qter)
Marques et al. Molecular Cytogenetics (2016) 9:87 Page 4 of 7
which consists of an invdup(7) of maternal origin, result-
ing in tetrasomy of 7q35qter region, with a size of
15.42 Mb and no morbid gene disrupted at the break-
point. It contains 67 OMIM genes, 16 of which associ-
ated to disease and therefore predicted to cause
phenotypic effects.
The correlation of syndromic manifestations with spe-
cific chromosome segments duplications is not simple,
since a pure 7q partial trisomy is rare. In 2008, Scelsa
et al. reviewed 18 cases with apparently pure dup(7) and
compared them with a patient who had trisomy of 7q32-
7qter region [11]. In this study the authors classified the
patients into four groups according to the size of the re-
arrangement. The last group, were we can include our
patient, presents distal duplications and the most fre-
quent phenotypic features are: mac rocephaly, frontal
bossing, small nose, low-set ears and usually severe
developmental delay. However, from the seven cases
reported in this group, only a tandem duplication of
7q36-qter could be considered as pure dup(7) since it
does not involve another chromosome [12]. In the other
six cases the duplicated material is present as part of a
derivative chromosome that arises from a parent’s
balanced translocation, hence the contribution of an
eventual positional effect or a gene regulatory disruption
to the phenotype cannot be excluded [13–15]. In
addition, these cases were not run through genome-wide
studies so the presence of a single genomic imbalance
region could not be confirmed. DECIPHER database lists
109 patients with a 7q gain that overlaps with our pa-
tient, but only 49 have a single region of imbalance [16].
There is only one patient with a small triplicate region
of 354.50 Kb [DECIPHER ID 251853: arr[GRCh37]
7q36.3(157930785_158285281)×3] which was inherited
from a normal parent, that comprises PTPRN2
(OMIM*601698), and who presented with broad thumb,
delayed speech and language development, hypertelor-
ism and intellectual disability. Furthermore, seven of
these patients have copy number gains of more than
1 Mb, being the biggest a de novo gain of 11.50 Mb [DE-
CIPHER ID 268243: arr[GRCh37] 7q33q36.1(1379145
89_149415401)×3] that encompasses several genes,
including CNTNAP2 (OMIM*604569), whilst presenting
with developmental delay and stra bismus. Intelle ctual
disability, delayed speech and language development are
the most common phenotypic features associated with
terminal 7q gains, however global development delay
and spe cific learning disability can also be found.
The tetraplicated region of our patient encloses 16
morbid genes. The analysis of these genes permitted to
Fig. 3 SNParray profile for chromosome 7. a Copy number probe intensities (log2 ratio) are represented in the upper track, allele peak tracks in
the middle and smooth signal below indicating a mosaic gain. b Chromosome 7 ideogram showing the tetraplicated region (blue box); c OMIM
genes associated to disorder present in the aSMC: In blue = more potentially implicated in phenotype; In orange = apparently not related
to phenotype
Marques et al. Molecular Cytogenetics (2016) 9:87 Page 5 of 7