INTRODUCTION

Down syndrome is caused by a total or partial trisomy of chromosome 21^1,2. It is considered the most common genetic cause of congenital malformations and intellectual disability, also comprising a complex set of pathologies in practically all organs and systems¹. The incidence of Down Syndrome worldwide is 1 per 1,000 to 1,100 live births³, which may vary according to the distribution of maternal age and the possibility of prenatal diagnosis⁴. In the Latin American Collaborative Study of Congenital Malformations, a prevalence of 1.88 per 1000 births was reported in the period 1998-2005 for 9 South American countries⁵. The diagnosis of Down syndrome is clinical and is confirmed with cytogenetic analysis¹, the latter is performed from the culture of peripheral blood lymphocytes in a liquid medium, which allows their proliferation and subsequent interruption in the metaphase stage in which the chromosomes achieve their maximum condensation. There are three most common cytogenetic findings in this syndrome, and they include free trisomy 21 (total trisomy), Robertsonian translocations involving chromosome 21, and mosaicisms; likewise, other cytogenetic variants related to the clinical picture of Down syndrome have been identified⁶. Free trisomy 21 is an autosomal aneuploidy characterized by an extra chromosome 21, showing 47 chromosomes in the karyotype. The Robertsonian translocation is the product of the fusion of chromosome 21 with an acrocentric chromosome. Mosaicism characterizes individuals with two or more distinct cell lines, one with trisomy 21. In studies carried out in Mexico, Algeria, Australia, Egypt and other countries in different periods of time, free trisomy 21 has been reported in frequencies that vary from 60% to 96%^2,7-10and, to a lesser extent, percentage, Robertsonian translocation and mosaicisms^11-14. In Peru, no epidemiological studies determine the incidence or prevalence of Down syndrome¹⁵. However, in 2016 the National Council for the Integration of Persons with Disabilities published that it had registered 8,800 people with Down Syndrome between the ages of 0 and over 60¹⁶. However, few reports have been found regarding cytogenetic alterations in patients with Down syndrome and its relationship with maternal age. The objective of this study was to describe the cytogenetic findings in patients with Down syndrome and their relationship with maternal age, as it would contribute to strategies to reduce its incidence and warn parents if there is a need for genetic counseling.

POPULATION AND METHODS

Cross-sectional, descriptive-analytical study. The study population consisted of 991 patients diagnosed with Down Syndrome admitted to the Instituto Nacional de Salud del Niño (INSN) in Lima (Peru), during the period 2017-2019. The cytogenetic finding and maternal age data were collected from the INSN Genetics and Inborn Errors of Metabolism Service records. To this aim, a data collection format was used whose content was uploaded to an electronic database.

Variables:

Cytogenetic alteration and maternal age.

RESULTS

During the study period, 991 patients with a clinical diagnosis of Down Syndrome were registered, of which 436 underwent chromosome study with the GTG technique (G bands, trypsin digestion, and Giemsa staining) and the cytogenetic findings were described according to the nomenclature according to The International System for Human Cytogenomic Nomenclature 2016. It was found that 99.3% of cases presented some type of cytogenetic alteration, and only three patients presented a normal karyotype. The age of the patients at the time of sampling was between 0.03 and 17 years, the male/female ratio was 1.2:1. Free trisomy 21 was the most frequent cytogenetic alteration (94.7%, n=410), followed by Robertsonian translocation (n=16) and finally mosaicism (n=6). Two patients with free trisomy 21 also presented another cytogenetic alteration in the same clone, which are described as: ?dup(10)(q21q22) and t(1;8)(q21;p22). A case was found with a non-classical cytogenetic alteration in Down syndrome represented by reciprocal translocation. t(5;21). Robertsonian translocations were mostly rob(14;21) (50%, n=8), but were also found: rob(21;21)(37.5%) and rob(15;21)(12.5%)(table 1), being 11 female patients and five male patients. In the cases of mosaicisms, between 15 and 82 metaphases were analyzed, observing that the normal cell line was the most predominant (83.3%), likewise, there was equal distribution by sex.

The maternal age of the population did not present a normal distribution, so the median was calculated, obtaining a value of 37 years (range: 13 to 47) (figure 1a). A greater number of patients with Down syndrome was found in the age group of 36 to 40 years (30.7%, n=134). Considering the most common cytogenetic alterations, it was found that between 36 and 46 there were more cases of free trisomy (n=132), 31 to 35 years for Robertsonian translocation (n=6) and 21 to 25 for mosaicism (n= 3). The data on maternal age were organized into three groups in order to analyze the relationship between the variables maternal age and cytogenetic alteration. Thus we have: under 30 years in group 1, from 30 to under 40 years in group 2 and, from 40 years and over in group 3. On the other hand, the cytogenetic alterations were grouped into: free trisomy 21, Robertsonian translocation and mosaicism (Table 2). Figure 1b shows the interquartile ranges, where the values ​​corresponding to the medians of each group can also be seen: 25, 28.5, and 37, respectively. Although free trisomy 21 was the most frequent finding in the three age groups, a greater number of cases of Robertsonian translocation (n=8) and mosaicism were observed in group 1 (n=4). Finally, it was found that there is a relationship between maternal age and the most common cytogenetic alterations in Down Syndrome (p=0.002).

Figure 1. Distribution according to age group and cytogenetic alteration. 1a: N=991, 1b: Most common cytogenetic alterations 

DISCUSSION

The cytogenetic study is a diagnostic tool for congenital disorders such as Down Syndrome. In the INSN, one of the most complex hospitals, which cares for children and adolescents from all over Peru, it was found that free trisomy 21 was the most frequent cytogenetic alteration in patients with Down syndrome (94.7%). A previous investigation carried out by Mansilla (2014) in another health institution in Peru found a similar value (98.6%)¹⁷⁾. In other regions, studies conducted in Egypt and India found free trisomy 21 in 96.1% and 93.75%, respectively^7,11, lower percentages were found in Bosnia and Herzegovina with 86.6%.¹⁸and 87.3% in Mexico⁸. This variation could be attributed to the population studied, maternal age, or the number of metaphases analyzed, although free trisomy 21 is always predominant in all reports. It has been described that the additional chromosome 21 is of maternal origin in about 95%, mainly in meiosis I¹⁹.

This study observed Robertsonian translocation in 3.7%; similarly, in Mexico, Kosovo, Cuba, Bosnia and Herzegovina, and India, percentages ranging from 4.3 to 15.2% were reported^{8,12,14,18,20}, this finding being the second in frequency, with the exception of Mexico⁸. Belmokhtar et al. (2016) compared the cytogenetic findings in patients with Down syndrome in various countries, finding that, in some, Robertsonian translocation is less frequent than mosaicism⁹. Since Robertsonian translocations are more common de novo⁸and of maternal origin²¹, the differences between these frequencies probably respond to genetic variability²²or factors that affect the mother, specific to each region. Nevertheless, since the Robertsonian translocations that cause Down syndrome can be de novo or inherited, it is necessary to perform a karyotype on the parents to detect a possible carrier and assess the risk of recurrence⁶, which is important in genetic counseling. In this study, the information on the karyotypes of the parents was not available, so its origin could not be known.

Regarding mosaics, these were observed in 1.4% and normal karyotypes were only 3 (0.7%). In other studies, no normal karyotypes were found, as in a previous report in Peru²³, or percentages such as 4%¹²and 7.4%²⁴were found in India and 4.8%¹³and 12.2%⁸in Mexico. The normal karyotypes in Down syndrome could actually be mosaics in which there were not a sufficient number of metaphases analyzed to determine them, or they are a consequence of the duplication of a "critical region" on chromosome 21¹³. This would be, in part, an explanation for the variable clinical manifestations in this syndrome, to which are added the effect of other genes not located in this region and which, as a whole, interact with each other and with the environment, giving it a high complexity to its underlying pathophysiology¹. Modi et al. (2003) showed the direct relationship between the percentage of trisomic cells and the degree of phenotypic manifestations in mosaic Down syndrome²⁵. Thus, it is essential to consider the number of metaphases that should be included in the cytogenetic analysis when mosaicism is suspected, since it depends on the proportion of trisomic cells²⁶and this, in turn, on the selected tissue²⁷. Even for certain clinical characteristics of Down syndrome, there would be a different affectation than mosaicism on tissues of the same embryonic origin²⁸. Therefore, to improve the ability to detect mosaicisms, the analysis of hundreds of interphase nuclei is established through the fluorescence in situ hybridization technique (FISH)²⁹due to its greater sensitivity.

Maternal age has been determined to be the most important risk factor for Down syndrome³⁰. This study had information on maternal age in the entire population, which did not present a normal distribution, verified with the Shapiro-Wilk test, determining a median of 37 years. The three age groups formed presented medians of: 25; 28.5 and 37, respectively. Most of the mothers of patients with Down syndrome presented in age group 2 (42.1%, n=182), followed by group 3, which reflects that the majority were elderly mothers (over 35 years, 59.6%); however, 3.9% (n=17) were adolescent mothers (under 20 years of age), a characteristic that has also been suggested as a risk factor³⁰. To analyze whether maternal age varies according to the type of cytogenetic alteration in Down Syndrome, our findings are represented in Figure 3, where it can be seen that there is a different distribution of Down Syndrome cases by translocation, in the age group 1:50. % (n=8), group 2: 43.8% (n=7) and group 3: 6.3% (n=1). Mosaics were more frequent in group 1 (66.7%, n=4). Free trisomy 21 was common in all three age groups, but translocation Down syndrome was more frequent in the offspring of mothers under 30 years of age. Several biological processes are likely to be affected by advanced maternal age, such as the accumulation of toxic effects and the degradation of the meiotic machinery during oocyte arrest, leading to a suboptimal resumption of meiosis. Chromosomal disjunction errors explain free trisomy 21 in Down syndrome, but there would be other risk factors beyond maternal age, such as environmental exposure and ethnicity²², that could also account for Down syndrome due to translocation or mosaicism which, as has been reviewed in other works, is mostly present in patients with this syndrome whose mothers are under 30 years old^{7,12,13,18,31}.

The limitations of this study include the difficulty of performing the cytogenetic analysis, both in the entire study population and in the parents of patients with Robertsonian translocations, in addition to the reduced number of metaphases examined in cases of mosaicisms (between 15 and 82).

CONCLUSIONS

It was found that free trisomy 21 was the most frequent cytogenetic alteration in patients with Down syndrome, representing 95%, followed by Robertsonian translocation, represented mostly by translocation rob(21;21). Free trisomy was more common in children of elderly mothers, but Robertsonian translocation and mosaicism in children under 35, suggesting that there are risk factors other than advanced maternal age involved in this age group.