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Just Accepted by The Journal of Maternal-Fetal & Neonatal Medicine

A time stamp comparative analysis of frequent chromosomal abnormalities in Romanian patients

Nicolae Suciu, Vasilica Plaiasu

doi: 10.3109/14767058.2013.794215

Abstract

Chromosome abnormalities represent the leading cause in many human genetic disorders. Gain or loss of genetic material can disrupt the normal expression of genes important in fetal development and result in abnormal phenotypes. Approximately 60% of first-trimester spontaneous abortions exhibit karyotype abnormalities. The majority of these abnormalities consist of numerical chromosomal changes, such as autosomal trisomy, monosomy X and polyploidy. In our current study, 411 cases were analyzed over a period of 5 years, which reflected the incidence of cytogenetic abnormalities in Romania. Down syndrome showed the highest frequency at 79%. At 2.6% structural chromosome abnormality syndromes and Turner syndrome followed suit. Next were the Edwards and Patau syndromes with an incidence of 1.2%. Klinefelter, Cri du chat,

and Wolf-Hirschhorn syndromes all had an incidence of 0.7%. Finally, the lowest frequencies were shown by Williams at 0.4% and only one case of Beckwith-Wiedemann syndrome with abnormal karyotype. The average maternal age at childbirth was 31.15 years (SD=6.96) and the average paternal age was 33.41 years (SD=7.17).

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Introduction

The incidence of chromosomal abnormalities, especially those related to Down syndrome (DS),

increases with advanced maternal age [1,2]. Chromosomal disorders include a total of

approximately 15-20% of conceptions. Nevertheless, because of the lethal nature of these

conditions, only about 0.6% of them come to term. Overall, genetic disorders have various

known causes. These causes range from refined mutations, such as point mutation,

insertions/deletions inside individual genes, or deletion/duplication of entire genes up to extra

or missing chromosomes.

The most common genetic cause for congenital malformations and learning disability is

represented by Down syndrome, characterized by an extra chromosome 21. Down syndrome is

occurring in the human population with an incidence of around 1/600 newborns [3]. Although

more than 50 years have passed since the genetic background of DS was identified, the causes

of this syndrome are not fully known [4,5]. However, some recent studies suggest that maternal

trisomy 21 ovarian mosaicism might be the major causative factor [6,7].

From a genomic perspective, chromosomal abnormalities may affect totally or partially the way

in which heterochromatin is organized inside the nucleus of somatic cells in G0 phase, thus,

affecting the spatial and temporal expression of genes [8,9]. The absence/presence of some

developmental genes close to the heterochromatin landscape in an asynchronous manner with

the development plan, can trigger abnormal phenotypes or an overall incorrect reorganization

of the cell nucleus, that might be incompatible with a further development of the embryo

[10,11]. For instance, gene-poor duplicated or deleted chromosomal segments may have

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equally severe phenotypic actions to those regions that contain genes. New studies confirm the

crucial role of repetitive DNA in embryogenesis. Constitutive heterochromatin includes

repetitive DNA sequences, such as pericentromeres and telomeres [12,13]. However, the

repacking of repetitive DNA sequences (the so-called "Junk DNA") after each division seems to

correctly position genes involved in embryonic development outside heterochromatin regions

where they can be properly expressed [14-20].

Perhaps the most unexpected effects of chromosomal abnormalities are those with a delayed

phenotypic effect, which may arise in a certain period during an individual's life. There is

substantial evidence that alterations of small chromosomal segments, such as reciprocal

translocations, inversions, and insertions, which pass with undetectable abnormal phenotypes

at birth, can be early initiating events in tumor genesis [21]. Therefore, the activation of

oncogenes may relate to chromosomal abnormalities due to an alteration of chromatin

structure that can erroneously unpack silent genes [22]. A more interesting question would be

whether these chromosomal abnormalities that are able to deceive cellular senescence are a

consequence of mosaicism or not. Various studies have already suggested the role of

chromosomal mosaicism in human diversity [23,24], but also in the aging process and in many

diseases [25,26]. The cytogenetic field will probably answer these questions in the coming years

by exploring this uncharted territory.

Mechanisms underlying chromosomal abnormalities are relatively unknown, but some models

and correlations have been proposed [27]. For instance, in the past, chromosomal

translocations have been considered random events. More recent evidence suggests that

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alternative DNA structures (non-B DNA) may be at the root of a wide range of genomic

rearrangements [28,29]. One of the most interesting correlations regarding human genetic

diseases is that predicted localizations of non-B DNA structures are found near, or overlapping

on, observed translocation breakpoints, or deletion endpoints [30].

Due to the low cost, preliminary analysis of chromosomal abnormalities is performed in most

clinics and hospitals using light microscopy to visually inspect the chromosomes.

However, the resolution of this method is limited from 4 Mb up to 2 Mb of DNA. This

conventional method remains reliable and sufficient to highlight the incidence of major genetic

disorders within populations. In this article, we present our observations on the incidence of

the most frequent chromosomal abnormalities in Romania over a period of 5 years (2007-

2011).

Materials and Methods

Our study was conducted over a period of 5 years. During this period we have analyzed a total

of 2694 patients referred to our Genetics Department that were suspected of genetic

conditions. Of these patients, the 411 cases that were identified with chromosomal

abnormalities and were confirmed by conventional cytogenetic evaluation, have entered in our

present study, with the following annual distribution: in 2007 there were 68 cases out of 396

patients, in 2008 there were 82 cases out of 432 patients and in 2009 there were 80 cases out

of 575 patients, with chromosomal abnormalities. In the year 2010 the number of referred

patients significantly increased to 695, of which 91cases were diagnosed with chromosomal

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abnormalities. Finally in 2011 an increased incidence of chromosomal abnormalities was

observed among our patients since 90 out of 596 patients were found with abnormal

cytogenetic results. For each case 31 variables were noted and subsequently processed. These

variables consisted of address, residence, diagnosis, gender, weight at birth, karyotype, age of

the parents and other important features.

The cytogenetic analysis has been performed by culturing peripheral blood lymphocytes using

two commercially available lymphocyte medium (PBMax (Invitrogen) and Quantum PBL (PAA))

for each case. After 72h of incubation at 37˚C the cultures were harvested by administering

colchicine and metaphases were obtained by applying the routine lab procedure. For each case

30 GTG-banded metaphases were karyotyped using IKAROS (Metasystems) and LUCIA

Cytogenetics Systems softwares Chromosome analysis was performed according to the

International System for Human Cytogenetic Nomenclature (ISCN 2009) guidelines.

Results

Our study was undertaken on 411 cases from a total of 2694 Romanian patients (Fig. 1D).

Throughout the study, the lowest incidence of cytogenetic abnormalities has been observed

between the years 2007 (68 cases) and 2009 (80 cases). The highest incidence of cytogenetic

abnormalities was observed in 2010 (91 cases) and 2011 (90 cases). At the time of the

examination, almost 76.47% subjects were between the ages of 2 days and 1 year, averaging at

the age of 3.6 months. Other age groups range between 1 and 2 years (8.08% with an average

age of 1.5 years) and 2 and 5 years (7.84% with an average age of 3.1 years). Children between

5 and 17 years (7.59% with an average age of 9.7 years) were also taken into account.

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By far the most common syndrome in the 411 cases was Down syndrome, with 328 cases

(79.56%), followed by structural chromosome abnormality syndromes (2.67%) and Turner

syndrome (2.67%) with 11 cases each (Fig. 2A). With 5 cases each (1.21%) Edwards and Patau

syndromes were next exhibiting the same occurrence rate. Klinefelter, Cri du chat, and Wolf-

Hirschhorn syndromes had an equal incidence with 3 cases each (0.72%). There were also 2

cases (0.48%) of Williams syndrome and one case of Beckwith-Wiedemann syndrome with an

abnormal karyotype (Fig. 2A). Finally, there were 39 cases of single occurrence (9.48%) out of

the 411 that could not be included in a high-frequency syndrome.

The average paternal age was higher than the average maternal age (Fig. 1B). The average

maternal age at childbirth, of the 411 cases, was 31.15 years (SD=6.96). Their ages range from

13 up to 47 years. In contrast, the average paternal age at childbirth was 33.41 years (SD=7.17),

with ages ranging between 16 and 56 years. The parents of the Down syndrome cases (328)

were of higher than average age. The average maternal age at childbirth was 31.58 years

(SD=7.07) and the average paternal age was 33.63 years (SD=7.28).

The age of parents for all other syndromes besides Down syndrome decreases significantly. The

average maternal age was 29.17 (SD=6.14) and the average paternal age was 32.35 years

(SD=6.58). Antenatal events for 20 women from 411 were unknown. Nevertheless, only 16 out

of 375 women had antenatal events. Furthermore, 389 women reported that they had not

been exposed to any teratogenic agents and 22 women stated they did not know.

Most fetuses with chromosomal abnormalities exhibit external or internal defects which can be

detected in some cases by detailed ultrasonographic examination during first and second

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trimester of pregnancy. Routine prenatal ultrasonographic assessment was performed in 390

patients. Abnormalities were detected in only 21 out of the 390 cases. The average gestational

age at the time of delivery was 8.75 months (SD=1.4). From 388 mothers, 142 gave birth by

caesarean and 246 by normal vaginal delivery. The average age of the subjects at the time of

the examination was 15.3 months (1.27 years). From the total number of cases, 276 were

located in urban areas and 135 within rural areas (Fig. 1C).

Phenotypic abnormalities after birth were primarily observed at the cranial level in 360 cases, in

pelvic area in 14 cases and in other areas for the remaining cases. The average newborn weight

for all 411 cases was 2830.71 grams (SD=559.41). The newborn weight by gender shows an

average of 2814.35 g (SD= 582.13) for females and an average of 2847.42 g (SD=536.38) for

males.

From the main group of 411 cases, 356 cases were used for plotting parents' ages (Fig. 1A). The

remaining 55 cases were excluded, because the age of the father, the age of the mother, both,

due to abandonment, were unknown. From the age distribution of the parents we have noticed

two trend changes in cytogenetic abnormalities. The first trend change seems to occur around

22 years for women and 25 years for men. The second trend change seems to occur around 37

years for women and 39 years for men (Fig. 1A).

The most common karyotype (47,XY,+21) is of the male expressed Down syndrome and

represents 39% of all cases, followed by the female expressed karyotype (47,XX,+21) of the

same syndrome with an incidence of 33% (Fig. 3). In the third place was 45,X karyotype of

Turner syndrome, representing 2% of the total. The sum of unique karyotypes (<2 cases)

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represents 18% from the total number of cases. In this percentage of 18%, uncommon karyotype

variants of high frequency syndromes are also included (i.e. mos

47,XY,+21[39]/48,XXY,+21[12] of Down syndrome). The incidence of Down syndrome is

higher in males than in females, nevertheless, other cytogenetic abnormalities affect females to a

greater extent (Fig. 2B). Also, urban areas seem to increase the frequency of cytogenetic

abnormalities. Moreover, it is worth mentioning that Klinefelter syndrome appeared only in

urban areas (Fig. 2C).

Discussion

By far the highest incidence was obtained by Down Syndrome (79.56%), caused by an additional

chromosome 21 (trisomy 21). The average age of the parents in the case of Down syndrome

was 32.6 years whereas the average age of the parents in other syndromes was 30.7 years.

Down Syndrome affects almost every organ in the body. Nevertheless, a proportion of these

patients exhibit Down's Syndrome mosaicism (some cells have an extra chromosome 21, but

others are normal). Thus, the specific distribution in different tissues influences the severity of

the condition. In our 411 group we have encountered a total of 15 cases of mosaicism, of which

5 cases belong to Down syndrome. New studies show that early embryonic chromosome

instability results in stable mosaic pattern in human tissues [31].

It seems that mitotic changes in copy number variations (CNV) regions may happen early during

embryogenesis and perhaps occur only once, after which the stable mosaic ratio is maintained

throughout the differentiated tissues. Changes in gene expression patterns due CNV plays an

important role in the etiology of common diseases such as diabetes, cancer, and heart disease

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[32,33]. Nevertheless, chromosomal abnormalities are a part of the evolutionary process.

Natural selection causes these changes in order to better adapt a species to a new

environment. Inversions, insertions or reciprocal translocations are common chromosomal

rearrangements. Depending upon mosaic patterns and which parts of chromosomes are

involved, the severity of symptoms varies within certain limits. Generally, loss or gain of genetic

material without any phenotypic consequences probably contributes to a delayed malignant

transformation by activation of oncogenes, or by reducing/increasing the expression of specific

genes in the affected chromosomal regions. However, many carriers of chromosomal

rearrangements will never experience any symptoms. Certain chromosomal abnormalities are

perfectly compatible with life and their carriers have a normal development, however they may

have problems in reproduction. Therefore, it is becoming increasingly clear that the genome,

through chromatin structure, performs multiple functions which intersect and synergistically

work across generations. Perhaps in the future the new sequencing technologies with almost

zero errors, will lead to an easier solution for finding new genetic disorders with imperceptible

or delayed phenotypic presentations [34]. Recent studies have already taken the first steps in

this direction and were able to detect fetal chromosomal abnormalities by massively parallel

DNA sequencing of cell-free fetal DNA from maternal blood [35].

The normal state of the nucleus in which cells exert their function inside the body, is

represented by G0 phase. Thus, new models that demonstrate how the cell nucleus functions in

G0 phase [36,37] or how different parts of the DNA sequence can influence gene expression

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[38], are essential for understanding the intricacies behind the pathology generated by

cytogenetic abnormalities.

Conclusions

This study has shown the incidence and distribution of chromosomal abnormalities among

Romanian couples over a period of 5 years (between 2007 and 2011). From the total of 411

cases, the average maternal age at childbirth was 31.15 years (SD=6.96) and the average

paternal age was 33.41 years (SD=7.17). As expected, in Down syndrome (79.5%) the average

age of parents was higher than average. From the age distribution of the parents we have

observed two trend changes in cytogenetic abnormalities. The first trend change seems to

occur around 22 years for women and 25 years for men. The second trend change seems to

occur around 37 years for women and 39 years for men. The average age of the subjects at the

time of the examination was 15.3 months (1.27 years). The average newborn weight of our 411

cases was 2830.71 grams (SD=559.41). The newborn weight by gender shows an average of

2814.35 g (SD= 582.13) for females and an average of 2847.42 g (SD=536.38) for males. From

the total number of cases, 276 were located in urban areas and 135 within rural areas.

The frequency of syndromes found over the 5 year period, displays Down syndrome on the first

place with an incidence of 79.5%, followed by structural chromosome abnormality syndromes

and Turner syndrome with 2.67% each. Next, Edwards’s and Patau syndromes apparently

exhibit the same incidence of 1.21%. These were followed by Klinefelter, Cri du chat and Wolf-

Hirschhorn syndromes, all three with an equal incidence of 0.72% each. Finally, on the last two

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places in this findings classification, were Williams syndrome with an incidence of 0.4% and

Beckwith-Wiedemann syndrome with abnormal karyotype (one case).

Acknowledgements

We thank the families for their participation and biologists Diana Ochiana and Gabriela Motei

from the Cytogenetics Laboratory. This work was supported by the Romanian Ministry of Health

National Programs.

Conflict of Interest Statement

The authors confirm that there are no conflicts of interest.

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Figure Legends

Figure 1. Distribution of ages in parents and the number of annual cases. (A) blue dots represent

the distribution of ages in parents (maternal age on X-axis and paternal age on Y-axis). The

curved orange line represents a polynomial trendline (order 3) value between points (y =

0.0013x3 - 0.1091x

2 + 3.819x-17.729, R² = 0.6129), (B) bars colored in burgundy show the

average age of the parents, (C) blue bars show the proportion of cases belonging to rural or urban

areas, (D) blue line shows the total number of patients (2694) analyzed each year between 2007

and 2011. The purple line shows the total number of cases (411) used in this study.

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Figure 2. Distribution of cytogenetic abnormalities in Romanian patients. (A) blue bars show the

number of cases for each syndrome separately, (B) each pie colored in burgundy shows the

gender distribution for each syndrome, (C) each pie colored in gray shows the distribution of

each syndrome in urban and rural areas.

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Figure 3. The karyotype frequency of the most common genetic conditions encountered in Romania. The

most common karyotype (47,XY,+21) it is shown in blue color and represents 39% of all cases, followed

by 47,XX,+21 karyotype of the same syndrome with an incidence of 33%. Individual cases (<2 cases) are

shown in olive green color and represent 18% from the total number of cases.

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