A high percentage of genetic diseases manifest themselves in the first 28 days after birth, but the full clinical symptoms may not be evident in newborns. Conventional diagnostic tests are with low resolution. They are also time-consuming, labor-intensive and are difficult to automate, therefore, the etiology of congenital anomalies (CAs) in 40.0-60.0% of the cases remains unclear. Molecular and molecular cytogenetic techniques such as FISH (fluorescent
At present, great interest has arisen in a newly discovered variation known as copy number variations (CNVs), which, in 2007, was indicated to be the “breakthrough” of the year [1]. Currently, CNVs are defined as a ubiquitous segment of DNA longer than 1 kb, presented at a variable copy number, compared to the reference genome. The great interest in CNVs was aroused by the observation that they can affect transcriptional activity and translational levels of adjacent genes [2-8]. An association was found between certain CNVs and predisposition to multifactorial diseases [3,9-15]. Copy number variations differ in type and frequency in different populations and can be used as a valuable source of information for the study of genetic characteristics of the nations [16-20]. With the accumulation of huge amounts of data for different CNVs, certain phenotype-genotype correlations have been defined [21].
It is therefore possible for CNVs that have been initially considered benign to later be proved as increasing sensitivity to multifactorial disease or causing genetic disease with late onset or incomplete penetrance. Therefore, the term CNVs is generally used to describe the CNVs both in patients and in healthy controls, as well as to describe the genomic imbalances, causing known microdeletion/micro-duplication syndromes. Consequently, CNVs can be divided into benign, pathogenic and CNVs of unknown clinical significance [22-25] and can be polymorphic with frequency greater than 1.0% or rare, less than 1.0%. They can also arise as
Caramaschi
The present study focuses on 81 patients of both sexes with dysmorphic features, with or without intellectual disability, behavioral problems, failure to thrive, neurological disorders. All of them presented a normal karyotype. The blood was taken in compliance with the standard procedures for good laboratory practice. The genomic screening, array-based comparative genomic hybridization (aCGH) was performed by standard protocol using the CytoChip ISCA oligo microarray, 4 × 44 k (BlueGnome Ltd., Cambridge, Cambridgeshire, UK), resolution 70 kb. Data were analyzed with the BluefuseMulti v.4.2 software (BlueGnome Ltd.).
In the investigated group, pathogenic CNVs were found in 31 patients. In addition, in the majority of all surveyed individuals, we identified benign CNVs (58/81 patients) and variations of unknown clinical significance (66/81 patients). There were 280 CNVs detected, of which 41 were pathogenic (28 deletions, 13 duplications), 118 benign (91 deletions, 27 duplications) and 121 of unknown clinical significance (50 deletions, 71 duplications). Copy number variations have not been established in six of the patients. The size of all identified CNVs was from 100,021 to 13,881,527 bp. There were 169 deletions and 111 duplications. The largest duplication covered 13,881,527 bp, and the smallest included 100,021 bp. The region of the smallest deletion covered 102,202 bp, and the size of the largest in four (q34.3q35.2) region: arr 4q34.3q35.2 (178,213,959-190,896,645) ×1, was 12,682,687 bp. We revealed eight pathogenic variations associated with the phenotype in six patients from the total group. Variations were distributed over eight chromosomes (chromosomes 2, 4, 5, 10, 12, 15, 17, 22). In two of the analyzed patients, a combination of two pathogenic CNVs on different chromosomes was found, and four of the patients had a single pathogenic variation. Six of the aberrations were deletions and two of them were duplications. The amount of detected genomic pathogenic changes ranges from 300 kb to 13 Mb. In five patients of the total group, we discovered five pathogenic CNVs that were not associated with the observed phenotype (Table 1).
Summarized results with pathogenic findings identified after microarray comparative genomic hybridization and associated with the phenotype of the patient.
Patient
Chromosome
Type of Aberration
Position (bp)
Size (bp)
Cytoband
Start
End
Start
End
79
2
deletion
50,982,143
51,314,401
332,259
2p16.3
2p16.3
5
deletion
175,470,501
177,136,261
1,665,761
5q35.2
5q35.3
52
10
deletion
122,804,780
135,434,149
12,629,370
10q26.12
10q26.3
46
4
deletion
178,213,959
190,896,645
12,682,687
4q34.3
4q35.2
12
duplication
230,451
14,111,977
13,881,527
12p13.33
12p13.1
41
17
deletion
34,450,435
36,248,889
1,798,455
17q12
17q12
35
22
deletion
21,561,492
22,905,039
1,343,548
22q11.21
22q11.22
30
15
duplication
22,765,658
29,030,488
6,264,831
15q11.2
15q13.1
As well as pathogenic variations, benign CNVs and CNVs of unknown clinical significance were found. There were 239 (an average of 2.9 per patient variations of the total group). These CNVs were distributed over 75 patients. All established benign and unknown CNVs ranged in size from 106,847 to 1,348,283 bp. From the total number of identified CNVs (239 without pathogenic CNVs), 179 (74.9%) covered 100-500 Kb, 52 (21.8%), from 500 Kb to 1 Mb and eight (3.3%) were larger than 1 Mb. There were 141 deletions and 98 duplications. One hundred and eighteen of the CNVs were benign. They were divided by size as follows: 90 (76.3%) covered 100-500 Kb; 21 (17.8%) were from 500 Kb to 1 Mb; seven (5.9%) were larger than 1 Mb. There were 27 duplications and 91 deletions (Table 2). The CNVs of unknown clinical significance numbered 121 and were of the following size: 89 (73.5%) from 100 to 500 Kb; 31 (25.6%) were from 500 Kb to 1 Mb; one (0.8%) was a size larger than 1 Mb. Copy number variations (excluding the pathogenic ones) were present in 74 patients. In 70 persons, the size of CNVs ranged from 100 to 500 Kb; in 39 CNVs were from 500 Kb to 1 Mb, and in seven larger than 1 Mb. In the majority of the cases, a combination of benign CNVs and CNVs of unknown clinical significance was defined. It is noteworthy that in our sample, CNVs ranging in size between 100 and 500 kb were of greater frequency.
Number and size of detected benign copy number variations and copy number variations of unknown clinical significance. CNVs: copy number variations.
Parameters
Total Number
100-500 kb
500 kb - 1 Mb
>1 Mb
Benign and CNVs of unknown clinical significance
239
179
52
8
Benign CNVs
118
90
21
7
CNVs of unknown clinical significance
121
89
31
1
In 31 patients, 41 pathogenic variations were found. In six of these patients, eight pathogenic aberrations associated with the corresponding phenotype were defined. The interpretation of other variations was made on the basis of the frequency in the studied group, the size of the variations, content of genes in the region and the type of the CNVs (deletion or duplication).
The CNVs with a 1.0-3.0% frequency in the population are accepted as benign polymorphisms. This gave us reason to consider as probably benign, those variations that occurred at frequencies higher than 1.2% in the studied group. Twenty-four of the CNVs conform with these criteria and were therefore considered to probably be benign. Abnormal variations with a frequency higher than 1.2%, were found in the following chromosomal loci: 2q13 (2.5%), 3q29 (2.5%), 7q11.23 (3.7%), 8p23.1 (8.6%) and Xp22.33 (18.5%).
The deletion in the (2)(p16.3) region, found in patient 79, was pathogenic and covered one
The pathogenic deletion in patient 46, del(4) (q34.3q35.2), included 49
Patient 41 was revealed to carry pathogenic deletion 17, del(17)(q12q12), covering 24
The detected deletion on chromosome 22, del(22) (q11.21q11.22), in patient 35, was also pathogenic covering 68
The last detected pathogenic aberration was a duplication in chromosome 15, dup(15)(q11.2q13.1), which included 111
The total number of established benign CNVs and CNVs of unknown clinical significance, amounted to 239, 141 of which were deletions and 98 were duplications. The total number of variations (with pathogenic ones) was 280, an average of 3.5 variations per patient. Two large studies in 2004 revealed that the genome of each person contains approximately 12 CNVs [27,28]. Later, in 2009, Itsara
In more than one patient, benign CNVs were identified in the following cytogenetic loci: 8p11.22, 6p25.3, 1q21.2, 2q37.3, 10q11.22, 14q11.2, 5q13.2, 10q26.3, 15q11.2, 19q13.31, and two different CNVs were detected in region 8p23.1. In six patients, six single variations in the loci were found: 14q32.33, 10q11.22, 5p15.33, 16p13.11, 15q14, 14q21.2 (Figure 1).
The most common benign CNVs were found in over 10.0% of the tested patients and were at the following locations: 8p11.22 and 1q21.2, and two different variations in the 8p23.1 locus. Deletions were identified in some of the cases, while in others, duplications, which indicated that these areas contained a significant number of variations. To establish the actual frequency of the detected unique benign CNVs, it was better to perform a genomic screening of a larger group of patients. According to Kooy [37], polymorphic CNVs are part of the natural genetic differences in humans, while rare variants can be related to certain disorders. A large study found that polymorphic CNVs are probably not closely related to the genetic etiology of multifactorial diseases, and it is possible that they are part of the benign genetic variations between individuals [38].
The number of variants of unknown clinical significance was 121. They were distributed by size as follows: 89 (73.6%) 100-500 kb; 31 (25.6%) 500 kb-1 Mb; one (0.8%) larger than 1 Mb. There were 50 deletions and 71 duplications. From the identified 121 variations of unknown clinical significance, in the following cytogenetic loci: 8p11.22, 8p23.1, 14q32.33, Xp22.33, 17q21.31, 16p11.2, 2q37.3, 14q11.2, 22q11.23, 11q25, 12p13.31, Xq22.3, 2p11.1, CNVs were found in more than one patient. There were 33 single CNVs detected in 27 patients.
Our results revealed a high rate of CNVs of unknown clinical significance. It is very likely that some of these variations are benign for the Bulgarian population. It is well known that the Bulgarian nation has a great genetic heterogeneity. Thirteen types of CNVs of unknown clinical significance were defined. They were located as follows: 8p11.22, 8p23.1, 14q32.33, Xp22.33, 17q21.31, 16p11.2, 2q37.3,14q11.2, 22q11.23, 11q25, 12p13.31, Xq22.3, 2p11.1 (88 of the total number), and occurred in over 1.2% of the patients. That gave us reason to assume that they had no pathogenic nature. The most common, probably benign CNVs in our study, were in loci 14q32.33, 17q21.31 and 16p11.12, occurring in over 15.0% of the patients. Thirteen of the CNVs detected in loci 14q32.33, 16p11.2, 16p12.3, 17q21.31, 1q21.2, 20p12.1, 22q11.22, 2q37.3, 4q35.2, 8p23.1 were found in patients with already defined pathogenic aberrations of major size, and associated with the corresponding phenotype. According to some sources, in the presence of large chromosomal aberrations associated with a particular phenotype, it is unlikely for the identified smaller rearrangement to be essential for the development of the disease [23]. Therefore we assumed that it was possible for these 13 variants to have no relation with the clinical phenotype. In patient 30 a duplication of unknown clinical significance was identified in the 1q21.2 region, which contained 15
Five types of the pathogenic CNVs, located in chromosomal loci 2q13, 3q29, 8p23.1, 7q11.23, Xp22.33 (24 variations) were found in a high percentage (over 1.2%) of the patients. Their high frequencies gave us reason to suppose that despite their pathogenic nature, these variations might be considered to be CNVs of unknown clinical significance or probably benign. In four patients, we identified four pathogenic variations with no clinical significance, in the following chromosomal loci: 22q11.22, 8p23.2, 2q13, 4q12. To determine participation in the variation genes, we made a reference to the Database of Genomic Variants (DGV). The
In the selected group of patients with congenital anomalies, 280 CNVs have been proven, respectively 41 pathogenic, 118 benign and 121 of unknown clinical significance. We found 13.6% incidence of pathogenic CNVs by aCGH. Eight pathogenic variations corresponding to the clinical phenotype of the patients, were identified. We revealed genotype-phenotype correlations between many genes and the manifested clinical features. During the interpretation of the CNVs of unknown clinical significance and the pathogenic ones, an additional category was defined, “probably benign variations.” It was concluded that there is a need to apply new genomic technologies with greater resolution, as a significant proportion of the patients were left with an unclear diagnosis. This would facilitate the detection and interpretation of the genomic aberrations in order to make an accurate diagnosis and to optimize therapeutic approaches.