A Cross Correlation Between Karyotyping and Anthropometric Measurement of Down Syndrome at Malang, Indonesia

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Please cite this article as: Christianto A, Cahyono HA, Soeatmadji DW, Fatchiyah F. A Cross Correlation Between Karyotyping and

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A Cross Correlation Between Karyotyping and Anthropometric Measurement of Down

Syndrome at Malang, Indonesia

Antonius Christianto

1

,

Haryudi Aji Cahyono

2

, Djoko Wahono Soeatmadji

3

, Fatchiyah Fatchiyah

4†

1_{Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, East Java, Indonesia}

2_{Department of Paediatrics, Syaiful Anwar Hospital, Malang, East Java, Indonesia.}

3

Department of Internal Diseases, Syaiful Anwar Hospital, Malang, East Java, Indonesia

4Head of of Research Group of Smart Molecule on Natural Genetics Resources, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, East Java, Indonesia

A R T I C L E I N F O

Article history: Received Accepted Available online

Keywords: Anthopometric Down syndrome Karyotyping Mosaic Trisomy 21

†_{Corresponding author:}

Fatchiya@ub.ac.id

A B S T R A C T

1. Introduction

Down syndrome (DS) is an abnormality that majority caused by addition of chromosome

number 21 in all individual’s cells, called trisomy

21 [Devlin and Morrison, 2004]. Chromosome number 21 contains about 47 million nucleotides and 300-400 protein coding gene [Hattori et al., 2000, Gardiner et al., 2003, Megarbane, 2009]. This syndrome often to occurred with frequency on population is 1.32:1000-2:1000 in every birth. Childs that having DS are showed abnormality in their growth and high risk to mortality in the first year of their birth. In general, it could be suggested that Childs with DS are born from maternal gestation 4-5 years older than average gestation in a population. Approximately 80%

infants with DS were born from mother with ages over 35 years, or about 1 from 400 infants neonatal from women with ages over 35 years are having DS [Fisch et al., 2003, Malini and Ramachandra, 2006, Malkova et al., 2007].

There are other types of chromosome abnormalities that could cause a DS. Lower percentages of cases, an addition of chromosomes 21 was only found on several cells, not in all

individual’s cells, called mosaicism trisomy 21.

Around 3-4% from DS cases, individual has normal numbers of chromosome, but certain chromosome was brought several materials from chromosome number 21 and resulting in an appearance similar with DS. That event is called translocation [Devlin and Morrison, 2004, Jyothy

Objective: Herein, we reported a first study of Down syndrome chromosome analysis in Malang, Indonesia and correlation between phenotype, anthropometric measurement and karyotyping data

Material and methods: Karyotyping was conducted using peripheral blood culture.

Result:

The karyotyping result showed that from nine patients with DS, we found two types of DS, trisomy 21 in seven patients and mosaicism in two patients. Interestingly, two patients with mosaicism have different percentages of cells with abnormal chromosomes (trisomy 21). In our cases, lower percentages of cells with abnormal chromosomes, the morphologies of patient were more likely normal control.

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et al., 2000, Lightner, 2006, Lovering and Percy, 2007, Ellaithi et al., 2008].

The percentage of DS prevalence is higher compare to other syndrome that cause an intellectual disability [Ellaithi et al., 2008]. In Malang area, East Java-Indonesia, currently there was no study which observes the type of chromosome abnormalities related DS in children. Although, several foundations and special schools for children with mental retardation were found in this area. Therefore, study about typical prevalence of chromosome abnormalities is needed to conduct as a basic information. Herein, we reported the first study of DS chromosomes analysis in Malang. Then

suggested correlation between qualitative

(physical characteristics) and quantitative

(anthropometric measurement) of phenotype analysis with the karyotyping result.

2. Patient and methods

2.1. Case report

Total nine patients (five males, four females)

and two normal controls (female and male) were subjected for this study. Patients and control were 8-12 years old children. Patients were suggested prior to DS based on common physical

characteristics such as slightly open mouth, large tongue, flat nasal bridge, epicanthal folds,

brushfield spots, single transverse palmar crease, low IQ, big space between the first and second toe (sandal gap), rounded face, flat facial profile, short stature, and slanted palpebral fissures [Roizen and Patterson, 2003, Bertelli et al., 2009, Steingass, 2011, Bull, 2011]. Before all patients were included in this study, they obtained and signed the inform concern.

2.2 Anthropometric measurements

Anthropometric measurements were conducted as source of quantitative data for physical

characteristics. Several characteristics were included in anthropometric measurements in our study such as body weight (BW), height (H), hands span (HS), head circumference (HC), distance of nasal cavity (DN), eyes distance (DE), upper segment (US) and lower segment (LS).

2.3. Karyotyping analysis

Peripheral blood culture was used for

karyotyping analysis. Karyotyping was performed based on previous study [Ellaithi et al., 2008, Paramayuda et al., 2012] with some modification. Culture of blood cells was conduct using PB Karyomax (Invitrogen, Life Technology) media.

Culture was incubated in 370C for 72 hours.

Culture of blood cells was treated with colcemid (Invitrogen, Life Technology) and hypotonic solutions. Chromosomes preparat was made using treated solution that dropped above the slide glass. Giemsa (Sigma Aldrich) staining was performed after preparat treated with Tripsin-EDTA (Invitrogen, Life Technology) solution. Observation was conducted using DIC

microscope BX-50 (Olympus) and continued with analysis using software CytoVision 4.5.1.

3. Results and discussion

Physical analysis was conducted on nine patients that suggested for having DS (figure 1.). The percentages of similarity with physical characteristics of DS in six and two patients were 75% and 83.33% respectively. Only one patient had the lowest percentage of similarity, only 8.33%. Basically, all patients except one patient have almost same percentage of similarity. Physical characteristics were not found in all patients patients such as brushfield spots in eyes, a big space between the first and second toe (sandal gap), and short stature. Sandal gap was found only in two patients (P3 and P4). One patient (P1) with the lowest similarity of physical characteristics was only characterized by slightly

open mouth. Prevalence of physicals

characteristics from DS patients was varying within several studies [Bertelli et al., 2009]. It is reported from several papers related DS physical characteristics have shown a large difference of

prevalence [Devlin and Morrison, 2004,

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Furthermore, in our study, quantitative data of physical was resulted from anthropometric

measurements (Table 1). Anthropometric

measurements were conducted in all patients. The result showed that compared to control, there were only slightly different in all anthropometric measurements.

Short stature characteristic was closely related with height that measured in anthropometric measurement. Based on CDC [CDC, 2001], height of all patients were in range of normal children height, therefore all patient could not categorized as a individual with short body. DS was characterized with slowly growth. The decreasing was observed especially in pubertal period. Thus, if we compared between adult DS patients and normal adult, there will be a significantly different in height [Myrelid et al., 2002]. Previous study related with growth curve of Indonesian children, minimum height averages for 6-12 years old children were 96-128 cm for girls and 96-125 cm for boys, whereas maximum height averages were 124-154 cm for girls and 118-159 cm for boys [Batubara et al., 2006].

The difference between control and patients on anthropometric measurements was found in ratio between upper and lower segment. Interestingly, one patient that only had the lowest percentages of similarity in physical characteristics, the ratio was similar with normal control. The average ratio of control and eight patients (except one patient shorter than the lower segment.

Although one patient had the lowest percentage of similarity with DS physical

characteristics and the anthropometric

measurement also give the same result as control, but all patients were continued prior to karyotyping analysis (Figure 2). Karyotyping

result from normal control (Figure 2A) showed a total 23 pairs chromosome (46, XX/46, XY) without any addition in chromosome numbers or abnormalities in chromosome shaped. In contrast, two different results were shown. Seven patients were shown a karyotyping result with an addition of a chromosome in number 21, called trisomy 21 (Figure 2B). And two other patients were shown a mosaicism of trisomy 21, a condition in which from different cell of an individual resulting a different karyotyping result, trisomy 21 and normal. [Lovering and Percy, 2007]. Both conditions were happen because of non disjunction event, but on different stages. Trisomy 21 was resulted from non disjunction at meiosis I. However, mosaicism of trisomy 21 was appeared if non disjunction event happen at meiosis II [Ellaithi et al., 2008, Morris et al., 2002].

An interesting finding was observed in our study. Two patients (labelled with P1 and L4) with mosaicism of trisomy 21 showed different

Comparison between karyotyping and

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2009, Shin et al., 2010, Hulten et al., 2013]. Although in several studies reported that phenotype of DS patients with mosaicisms was depend rate of tissue-specifics trisomy 21 [ Papavassiliou et al., 2009, Hulten et al., 2013], at least in part, from our study, it could also be conclude that percentages of abnormal cell will affected the physical severity and similarity with the DS patient. In DS patients with mosaicisms, further research with large samples and tissue-specifics karyotyping still needed, in order to

confirm the phenotype related mosaicism event.

Conflict of interest

The authors report no conflicts of interest.

Acknowledgements

We would like to thanks to Special Elementary School for Disability in Malang area for the permission to taking research sample. Furthermore, we also thank to Syaiful Anwar Hospital for the assisting in sample preparation and member of LSIH-UB laboratory for the technical support.

Conflict of Interest

The authors report no conflicts of interest

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