Journal of Pharmacy and Pharmacology 10 (2022) 72-78

doi: 10.17265/2328-2150/2022.03.002
D DAVID PUBLISHING

Cytogenetic Investigation in Patients with Clinical

Suspicion of Genetic Syndrome in Manaus, Brazil

Débora Pinto1,2, Denise Corrêa Benzaquem2, Rachel Cardoso Nunes2,3, Vânia Mesquita Gadelha Prazeres3 and
Cleiton Fantin2
1. Hospital Universitário Getúlio Vargas,Manaus, Amazonas 69020-170, Brasil
2. Human Genetics Laboratory, State University of Amazonas, Manaus, Amazonas 69065-001, Brazil
3. Federal University of Amazonas.Manaus, Amazonas 69067-005, Brazil

Abstract: Chromosomal alterations can cause a number of diseases; therefore, early detection of these alterations is fundamental in
the prevention and treatment of various abnormalities. In this sense, the objective of this study was to perform cytogenetic analysis of
patients with phenotypic characteristics not yet clarified in order to determine the frequency of chromosomal alterations in patients
treated in the public health system in Manaus, Brazil. A total of 98 patients with clinical suspicion of genetic syndrome were referred
to the public health system in Manaus, Amazonas state. Genetic analyses were performed from lymphocyte culture and subsequent
G-banding. Patient data were obtained through referral files and chromosomal a Analyses. Of the 98 karyotypes analyzed, 65 (66.3%)
presented normal karyotype, 18 (18.4%) presented chromosomal alterations and 15 (15.3%) were inconclusive. Of the alterations
found, 11 (61.1%) corresponded to alterations of the numerical type and 7 (38.9%) of the structural type. Of the chromosomal
alterations identified, the one with the highest frequency was aneuploidy, which was significantly represented by 9 patients with
Down’s syndrome, and different karyotypic mechanisms related to this syndrome were observed. There was also one case of deletion,
one case of pericentric inversion, and two involving the sex chromosomes. The etiological confirmation of congenital anomalies is
extremely important for the patient’s prognosis, and it is necessary to invest in cytomolecular tests, such as FISH and microarray in
order to increase the rate of diagnosis.

Key words: Karyotype, chromosomes, banding, karyotypic formula, Down’s syndrome.

1. Introduction processes, and can occur in autosomal or sex

chromosomes, thus leading to a modification in gene
Chromosomal alterations represent one of the largest
expression, and the production of a phenotypically
categories of genetic diseases and are responsible for
abnormal individual [6].
most cases of intellectual disability, congenital
Detection of chromosomal alterations can be carried
malformations, facial dysmorphisms and repeated
out using cytogenetic methods that allow the
miscarriages [1, 2]. These represent more than 100
identification of various chromosomal dysfunctions
syndromes that have already described and, statistically,
and provide the diagnosis of abnormal phenotypes. An
chromosomal alterations affect approximately 7.5% of
important method, widely used by laboratories, is the
pregnancies (most of which are miscarried in the first
classical karyotype, which involves the culture of cells
trimester).Chromosomal alterations occur 0.6% of the
in metaphase and GTG banding. By means of a
time in live births (many are related to certain genetic
pattern of chromosomal bands, it is possible to assess
diseases or congenital malformations) [3-5]. These
the integrity of the chromosome and identify possible
alterations stem from any change in the structure or
abnormalities [7, 8].
number of chromosomes during mitotic or meiotic
In Brazil, there are few studies on the presence of
Corresponding author: Cleiton Fantin, PhD, research fields:
genetic abnormalities in the population and there are
genetics and cytogenetics. no official databases that record this distribution [9].
 Cytogenetic Investigation in Patients with Clinical Suspicion of Genetic Syndrome in Manaus, Brazil
The lack of official databases and the lack of genetic
services and professionals have largely led to the lack
of care for patients with chromosomal diseases in the
country [10]. In the state of Amazonas, the situation is
no different. The capital, Manaus, is the only city that
has a medical genetics service, which is offered by the
public health system, and receives patients from all
over the state. However, in the state of Amazonas, it
was implemented through a research project, namely
the cytogenetic service at the Cytogenetics Laboratory
of the Amazonas State University, for patients with
suspected genetic syndrome, and aims to contribute to
diagnosis, collection of cytogenetic information and
health care of these patients. In this sense, the objective
of this study was to perform cytogenetic analysis in
patients with phenotypic characteristics not yet defined
and determine the frequency of chromosomal
alterations in these patients attended by the public
health network in Manaus.
2. Material and Methods
A retrospective descriptive study was carried out
based on the results of peripheral blood karyotype
tests performed at the Cytogenetics Laboratory of the
Amazonas State University over almost 3 years, from
its emergence in November 2018 to October 2021.
The study was approved by the Ethics Committee of
the Amazonas State University (UEA), under approval
No. 2.949.936. Each participant or their legal guardian
was informed about the nature of the study and all
signed an informed consent form (ICF).
The cytogenetic examination performed by the
laboratory was done via the culture of peripheral blood
lymphocytes, following the methodology described by
Moorhead et al. [11], with some modifications. The
technique of G-banding was according to Seabright
[12], with some modifications. The resolution obtained
by G-banding was 400-500 bands per genome. For
cytogenetic analyses, 50 metaphases per patient were
observed and analyzed using an optical microscope
(Nikon eclipse E200). At least 10 metaphases were
73
photographed and karyotyped using the GeneALL
program. The karyotype images generated were saved
in the program and one of them was attached to the
exam report. The results of the G-banding were
interpreted according to the norms present in the
International System for Human Cytogenetic
Nomenclature [13] in order to issue the reports of the
patients under study.
Statistical analyses were performed based on data
obtained from patient records. They were compiled
and analyzed in EXCEL® software. The relative
frequency of the following parameters were obtained:
chromosomal changes found, normal karyotypes and
karyotypes that could not be identified; types of
changes (structural and numerical); and chromosomes
involved (autosomal or sexual). The relative
frequency of karyotypic mechanisms involved in
Down's syndrome was also obtained.
3. Results
In the Cytogenetics Laboratory of the Amazonas
State University, between November 2018 and October
2021, 98 karyotypes of patients with phenotypic
characteristics were analyzed. Of the 98 patients
analyzed, 65 (66.3%) presented a normal karyotype,
18 (18.4%) presented chromosomal alterations and 15
(15.3%) had undefined data (Figure 1).
The chromosomal alterations found involving the
autosomal and sex chromosomes were classified as
either numerical or structural. Of these, 11 (61.1%)
were numerical and 7 (38.9%) were structural (Figure 2).
Fig. 1 Distribution of normal, altered and undefined
karyotype results.
74 Cytogenetic Investigation in Patients with Clinical Suspicion of Genetic Syndrome in Manaus, Brazil

occurred in the sex chromosomes (Figure 3).

Fig. 2 Distribution of chromosomal alterations classified
as either numerical or structural.
Of the chromosomal alterations identified, the one
with the highest frequency was aneuploidy, which was
significantly represented by nine patients (50%) with
Down’s syndrome. In addition, different karyotypic
mechanisms related to Down’s syndrome were
observed (Figure 4).
Table 1 summarizes all the chromosomal alterations
found.

Fig. 3 Distribution of chromosomal alterations for the

chromosomes involved.

Regarding the chromosomes involved in the

chromosomal alterations found, 16 (88.9%) occurred
in the autosomal chromosomes and 2 (11.1%) Fig. 4 Analysis of the frequency of the karyotypic.

Table 1 Chromosomal alterations observed in the study population. Mild ID = Mild Intellectual Disability; ASD/ID =
Autism Spectrum Disorders/Intellectual Disability; NPMD = Neuropsychomotor development.
Clinical Indication Alteration Karyotype N
Aneuploidy – Free trisomy 21 47, XY + 21 6
mos 47, XX + 21 [64]/46, XX [36]
Aneuploidy - Mosaic type mos 47, XX, + 21 [67]/46, XX [33] 3
mos 47, XY + 21 [64]/46, XY [36]
Down’s syndrome
46, XX, rob (14,21)(q10, q10) +21
Aneuploidy with Robertsonian 46, XY, rob (14,21)(q10,q10) +21
4
translocation structural change 46, XY, +21, rob (21,21)(q10,q10)
46, XY, rob (14, 21)(q10, q10) + 21
Mild ID and short stature Pericentric inversion 46, XX, inv(7)(p14;q11.2) 1
ASD/ID + microcephaly Additional heterochromatin 46, XY, 16qh+ 1
Turner’s syndrome Aneuploidy 45, X 1
Delayed NPMD, reflux, hypotonia, ocular
Deletion 46, XX del (16) (q?) 1
hypertelorism and macrostomy
Hypotonia, weak cry, dolichocephaly, congenital mos 47, XXY[10]/48,
Aneuploidy 1
heart disease. Probable Edward’s syndrome XXXY[20]/XXXXY[70]
Mechanisms of Down’s syndrome.
 Cytogenetic Investigation in Patients with Clinical Suspicion of Genetic Syndrome in Manaus, Brazil
4. Discussion
In this study, 65 (66.3%) patients had a normal
karyotype, but were referred for examination because
they had some clinical suspicion of abnormality, such
as congenital malformations, autism, intellectual
disability and developmental delay. The G-banding
karyotype examination is able to detect chromosomal
alterations greater than 5 megabases [14]. Therefore, a
more detailed investigation is necessary for these
individuals and this involves complementary
examinations using molecular cytogenetic techniques
for diagnostic confirmation. This result corroborates
that found by Moreira et al [3], who determined the
frequency of chromosomal abnormalities of individuals
examined at a public community genetics service in
the state of Bahia, Brazil, which demonstrated that
67% (546/813) presented a normal karyotype.
On the other hand, 18 (18.4%) patients presented
chromosomal alterations in their karyotype, and this
value is within the average found in other studies
carried out in genetic services in Brazil, such as in the
states of Paraná with 15.8% [6] and in Bahia with
23.35% [15].
Numerical chromosomal alterations had higher
representativeness than structural chromosomal
alterations. The predominance of numerical alterations
in relation to structural alterations, found in this study,
reinforces the fact that numerical aneuploidies are the
most frequent chromosomal disorder in the human
species; a fact widely reported in the literature [2, 6,
16]. Furthermore, alterations involving autosomal
chromosomes were also more frequent than sexual
ones, as predicted. These results are consistent
with the results found by Balkan et al [16] and
Miziara et al [17].
In this study, Down’s syndrome was the most
frequent alteration in autosomal chromosomes, and
the same was observed by Fantin et al. [5, 18] and
Pande et al. [19]. This was mostly represented by free
trisomy 21 (46.2%); though in smaller proportions,
75
Robertsonian translocation also occurred (30.8%);
while the presence of cell mosaicism was 23.1%. Free
trisomy 21 was also found more frequently in a study
conducted by Carneiro et al. [15] and Fantin [18].
For the cases involving the sex chromosomes, two
variations of the aneuploidy type were observed, and
sometimes involved the addition of chromosomes, and
sometimes their loss. The latter is the complete
monosomy of the X chromosome, characterizing
Turner’s syndrome, and was found in a single patient,
thus representing 5.6% of cases of total chromosomal
alterations. It is believed that about 50% of cases of
Turner’s syndrome result from monosomy of the X
chromosome in all cells (45, X), while mosaicism
occurs in 30% of cases and are present in other forms
such as an isochromosome, deletion and ring X
chromosome [20]. The other variation found in this
study was the mosaic type mos 47, XXY[10]/48,
XXXY[20]/49 XXXXY[70] with the presence of three
different chromosomal lineages. This karyotype
corresponds to Klinefelter syndrome. Karyotypes with
3 lineages have already been described by Asirvatham
et al [21] in a study based on 44 cases of this
syndrome. Klinefelter syndrome has an incidence of 1
in 85,000 individuals [22-24]. The most frequent
karyotype is 47, XXY, which occurs in 90% of cases,
and mosaic 46, XY/47, XXY is the second most
frequent [25-27]. Karyotypes 48 XXYY; 48 XXXY;
49, XXXXY are considered the rarest variants for
Klinefelter syndrome and, therefore, it may be
suggested that the study patient has Klinefelter
syndrome due to presenting the variants and rare
phenotypic characteristics.
A balanced structural alteration was observed in a
patient with mild intellectual disability and short
stature who presented a pericentric inversion on
chromosome 7(46, XX, inv(7) (p14; q11.2). To date,
no case reports presenting this same reversal have
been found in the literature. Shah et al. [28] reported
that inversion involving the centromere of
chromosome 7 is a rare event. However, pericentric
76 Cytogenetic Investigation in Patients with Clinical Suspicion of Genetic Syndrome in Manaus, Brazil
inversions on chromosome 7 in other chromosomal
regions are to found in the literature. In
Williams-Beuren syndrome, a deletion occurs in the
7q11.23 region, resulting in loss of 20 to 28 genes
[29, 30]. This region coincides with one of the
inversion cutoff regions found in our analyses. Other
studies have shown that inversion in chromosome 7
may be involved with different phenotypes such as
neoplasms, infertility and autism spectrum disorder
[31, 28].
Regarding the unbalanced structural alteration, one
of the probands presented karyotype 46, XX del (16)
(q?). According to Monaghan et al. [32], the loss of
chromosomal segments in the long arm of
chromosome 16 are considered critical regions for
16q deletion syndrome. In addition, the phenotypic
characteristics presented by the patient were delayed
neuropsychomotor development (NPMD), reflux,
ocular hypotonia and macrostomy, and these
phenotypes are some of the phenotypes that are
identified in individuals with a karyotype that
evidences the 16q deletion, as cited by other studies
[32-34]. Other examinations should still be
performed with this patient since the region of the
16q deletion found was not defined; therefore,
molecular tests will be necessary.
Failures in obtaining peripheral blood cultures
with metaphase chromosomes were recorded in
15.3% of the analyzed samples, and these were
identified in this work as undefined cases. This type
of failure occurs due to several factors, including
inadequate collection of peripheral blood samples,
absence of viable cells in the collected material, use
of certain medications by the patient, and even
accidents during the processing of biological material
in the laboratory. The high failure in obtaining good
cultures for metaphase chromosome analysis
observed in this study is probably related to the high
level of controlled drugs used by the study
population, such as carbamazepine, allopurinol,
ethanol, levamisole, penicillamine, streptokinase,
clozapine. Another factor related to this issue is the
debilitated state of the patient’s health.
This epidemiological study included, to date, the
largest sample ever described in relation to other
similar works carried out in the state of Amazonas,
and the results reproduce, in part, data on the
occurrence of chromosomal alterations in the region.
The cytogenetic investigation concluded the clinical
diagnosis via the G-banding of the karyotype in
18.4% of patients, and shows that the karyotyping
was useful in the diagnosis of these patients;
however, the use of cytomolecular tests can increase
the diagnostic capacity for patients using the public
health system. It is essential that health professionals,
especially doctors, have access to these results, since
a correct and early diagnosis allows for appropriate
interventions and treatment aimed at improving the
quality of life of these individuals.
Acknowledgement
We would like to thank the Fundação de Amparo de
Pesquisa do Estado do Amazonas – FAPEAM and the
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - CAPES.
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