A Cytogenetic Study of Turkish Children with Global Developmental Delay

Abstract

Global developmental delay (GDD)/intellectual disability (ID) is common in children and its etiology is unknown in many cases. Chromosomal abnormalities are predominant genetic causes of GDD/ID. The aim of this study is to determine the genetic risk factors that may be involved in the etiology of GDD/ID. In this study, 810 children with moderate to severe, clinically unexplained GDD/ID for whom cytogenetic analysis were performed were retrospectively rescreened. The results showed that GDD/ID affected more females than males (2 girls:1 boy). A total of 54 children (6.7%) with GDD showed chromosomal aberrations (CAs): 59.3% of these CAs were structural aberrations, and the rest were numerical aberrations (40.7%). Specifically, inversions, deletions, and reciprocal and robertsonian translocations, which were detected in 1, 0.7, 0.8, and 0.4% of the children, respectively, constituted important categories of structural CAs. Among numerical CAs, classic Turner and mosaics were detected in 1.2% of all children. Trisomy 21 and mosaic trisomy 21 were detected in 1% of the children. Marker chromosomes and 47,XXY karyotypes were found in two children each. Our results suggest that female sex is more affected by CAs among GDD/ID cases, and cytogenetic analysis is useful in the etiological diagnosis of GDD/ID.

Introduction

Global developmental delay (GDD)/intellectual disability (ID) is a term used for clinical conditions that include impairment in two or more of the following areas: motor, cognition, speech, language, and social, mostly in children aged 5 or under. ^{1 2} The precise prevalence of GDD/ID is unknown. Particularly, ID, which is one of the defining features of GDD, is estimated to be seen in 1 to 3% of children younger than 5 years old. ³ In general, genetic defects, prenatal problems or complications arising during pregnancy, prenatal exposure to drugs and/or alcohol, birth trauma, childhood injuries, and infections can cause GDD/ID. ⁴ However, the precise etiology is unknown for a large number of patients. It is estimated that half of GDD/ID cases are caused by genetic factors including chromosome aberrations (CAs). ^{5 6 7} CAs causing GDD/ID can be categorized into microscopically visible CAs and submicroscopic copy-number variants, and these defects can be inherited in mendelian fashion. ^{8 9} Chromosome analysis is very important for finding CAs, if any, deciding on clinical treatment, estimating the risk of recurrence in future pregnancies, and providing appropriate genetic counseling. ¹⁰ This study aimed to rescreen and retrospectively evaluate the chromosomal analysis results of 810 children, which constitute an important sampling group in children with GDD/ID in Turkey, and to contribute better understanding of the etiology and pathogenesis of GDD/ID cases.

Materials and Methods

This retrospective study included children with GDD/ID who were referred by Pediatric Neurology Department to the Medical Biology and Genetics Laboratory at Çukurova University between the years 1987-2003. Cytogenetic analysis results and clinical findings were evaluated in 810 children who were diagnosed with GDD, multiple congenital anomalies, and associated disorders of ID including cognitive, motor, speech, and language delays. For chromosome testing (Is it G banded karyotyping?…please mention it), 3 mL of venous blood was taken from each patient. Lymphocyte culture and preparation of metaphase chromosomes were made according to standard cytogenetic protocols. The chromosomes were stained by using the Giemsa-TrypsinGiemsa (GTG) banding method, fifty metaphases were analyzed in each case. CytoVision software was used in chromosome analysis, and karyotypes were constructed according to the guidelines of the International System for Human Cytogenetic Nomenclature (2009).

Results

The age of the analyzed population ranged between 1 month and 18 years, and the average age was 3.1 years. The sex ratio (male-to-female ratio) was recorded to be 0.5. Karyotype results of patients were presented under two overall categories, structural and numerical CAs. Table 1 shows the frequencies of structural and numerical CAs and other subcategories. CAs were detected in 54 cases, which corresponds to 6.7% of all children. Also, 59.3% of overall CAs were structural aberrations, including translocations, deletions, inversions, isochromosome, fragilities, and breaks, and the remaining 40.7% were numerical CAs.

Table 1. Frequencies and distributions of the karyotypes in children with global developmental delay.

Cytogenetic category	Karyotypes	No. of cases	Frequency in anomalies (%)	Frequency in all cases (%)
Normal	46,XX or 46,XY	756		93.3
Abnormal	Numerical and structural chromosome abnormalities	54		6.7
	Abnormal karyotypes
Numerical chromosome abnormalities
Pure Turner (monosomy X)	45,X	3	5.6
Mosaic Turner	45,X/46,XX	3	5.6
Mosaic Turner with isochromosome X short arm abnormalities	45,X/46 X i(Xp) (18%)	1
Mosaic Turner with isochromosome X long arm abnormalities	45,X/46,Xi(Xq) (10%)	2
Mosaic Turner with chromosome X short arm abnormalities	45,X/46,XXp+ (30%)	1
	Total	10	18.5	1.2
Marker chromosome	47,XX, + mar	2	3.7
Free trisomy 21 (Down syndrome)	47,XX, + 21	4	11.1	1.0
	47,XY, + 21	2
Mosaic trisomy 21	46,XX/47,XX, + 21	2
	Total	10	18.5	1.2
Klinefelter syndrome	47,XXY	2	3.7	0.2
	Total	22	40.7	2.7
Structural chromosome abnormalities
Robertsonian translocations	45,XX,t(14;21)	2
	45,XY,t(14;21)	1
Reciprocal translocations	46,XX,t(8;7)(p22;q36)	1
	46,XX,t(1:2)(p32;q37)	1
	45,XY,t(9;14))(p24;q32.3)	1
	46,XX,t(2:14)(q27;q22)	1
	46,XY,t(7q;14q)	2
	Total	9	16.7	1.1
Deletions	46,XY,del(5)(p15.1)	2
	46,XY,del(5p-)	1
	46,XX/46,XX,del(18)(p13) 46,XX/46,X,del(Xp) (40%)	11
	46,X,del(X)(p11;qter)	1
	Total	6	11.1	0.7
İnversions	46,XX,inv(14)(q13;q24)	1
	46,XY, inv(9)(p11;q13)	3
	46,XX,inv(9)(p11;q12)	4
	Total	8	14.8	1.0
Isochromosome	46,X,i(Xq)	1 2
Other variants	46,XX,fra(Xq27.3)	1
	46,XY,fra(5q24) 46,XY, fra(13q32)	1
	46,XX,fra(5q,10q)	1
	46,XX,CA (50%)	1
	46,XX,CA (20%)	1
	46,XY,CA (8%)	1
	Total	8	14.8	1.0
	General total	31	59.3	3.8

Among the structural CAs, reciprocal and robertsonian translocations were the most common findings, with six and three cases, respectively, constituting 16.7% of overall CAs. Reciprocal translocation 46,XY,t(7q;14q) and robertsonian translocation 45,XY,t(14;21) each were represented by two cases. Besides, each karyotype of 45,XX,t(14;21); 46,XX,t(1:2)(p32:q37) and 45,XY,t(9;14); 46,XX,t(2:14)(q27:q22) was represented by one case. The inversions and other variants comprised 14.8% each of the overall CAs and they included 46,XX,inv(14)(q13;q24); 46,XY, inv(9)(p11;q13); 46,XX,inv(9)(p11;q12; 46,XX,fra(Xq27.3); 46,XY,fra(5q24); 46,XY, fra(13q32); 46,XX,fra(5q,10q); 46,XX,CA (50%); 46,XX,CA (20%), and 46,XY,CA (8%) karyotypes. The deletions comprised 11.1% of overall CAs and they included 46,XY,del(5)(p1.51); 46,XY,del(5p-); 46,XX/46,XX,del(18)(p13); 46,XX/46,X,del(Xp)(40%); 46,X,del(X)(p11;qter) karyotypes. Among numerical CAs, pure Turner (monosomy X) and mosaic Turner were detected in a total of 10 cases, which comprised 18.5% of all CAs, and they included 45,X; 45,X/46,XX; 45,X/46 X i(Xp) (18%); 45,X/46,Xi(Xq) (10%), and 45,X/46,XXp + (30%) karyotypes. Regular trisomy 21 and mosaic trisomy 21 comprised 13% of overall CAs. Klinefelter syndrome and marker chromosome were detected in two cases each ( Table 1 ). The incidence of abnormal karyotypes was higher in females than males (the male-to-female ratio was 0.5).

Discussion

In the present study, cytogenetic analysis of 810 children revealed CAs in 6.7% of all cases. Our results show a low incidence rate of CAs when compared with other studies, which have shown an incidence rate of CAs ranging between 16.5 and 50.6% of all cases. ^{11 12 13 14 15 16 17 18} However, some studies have reported the incidence rate of CAs to be between 4 and 28%. ¹⁹ The difference in prevalence of CAs obtained from various studies may have stemmed from the difference in clinical criteria applied for patient selection and the cytogenetic method used. It can also be presumed that there may be GDD-related micro-CAs that cannot be detected by conventional cytogenetic analysis. ²⁰ It is also estimated that approximately 5% of unexplained GDD/ID cases may be due to changes in subtelomeric regions. ^{21 22 23} In this study, the frequency of children with GDD differed significantly by gender. It was found that GDD/ID was observed twice as often in girls than in boys (2:1). This suggests that females are more susceptible to abnormal karyotype and growth retardation in the intrauterine period. It is suggested that the expression of the genes of the maternal X chromosome in male embryos supports a more stable development during early embryogenesis compared with female embryos. Normal development in female embryos is attributed to dose compensation of X-linked genes. ²⁴

In the present study, we found that structural CAs accounted for 3.8% of all studied children and 59.3% of overall CAs. Balanced structural rearrangements, which included reciprocal and robertsonian translocations, constituted an important part of structural CAs and overall CAs (16.7%). Inversions, other structural irregularities, and deletions were found in 14.8, 14.8, and 11.1% among overall CAs, respectively. Generally, balanced reciprocal translocations are the most common chromosomal rearrangements seen in humans and are known to occur in 0.16 to 0.20% of live births. ²⁵ In this study, reciprocal translocations were found in 0.7% of all GDD cases, a far higher ratio compared with the ratio found in the general population (live births), which could be attributed to low sample size of this study. It has been reported that 75% of all translocation cases are de novo and 25% can be inherited from a carrier parent, but more frequently by the mother. ²⁶ Thus, de novo balanced translocations pose a low risk of severe congenital anomalies in carriers and majority of cases carrying balanced structural rearrangements exhibit a normal phenotype. ²⁷ However, balanced translocations carriers may produce unbalanced gametes resulting from nondisjunction and other errors in meiosis, which develop during gamete production. Therefore, the parents carrying balanced translocations may experience reduced fertility or recurrent spontaneous abortions or have children with complex congenital conditions or malformations. ²⁸ Moreover, if the breakpoints of any balanced rearrangement disrupt important gene or genes, which may be autosomal dosage-sensitive genes, X-linked recessive genes, genes unmasking recessive mutations in normal alleles, or genes having long-range position effects, the presence of such balanced chromosomal rearrangements may still cause disorders or malformations. ²⁷ In the present study, robertsonian translocation between chromosomes 21 and 14 has been detected in three children. These cases showed that chromosome 14 most frequently translocated with chromosomes 21, 7, and 9. Interestingly, the chromosome regions of 7q36, 14q32.3, and 14q22 were involved in these translocations. It has been reported that the 7q36.3 region contains seven genes responsible for the development of the human brain during the embryonic period, and deletions in this region are generally associated with some facial dysmorphisms such as GDD/ID, low birth weight, growth retardation, abnormal skull, nasal malformation, hypertelorism, and ear malformation. ²⁹

We also detected balanced translocations between chromosomal regions of 8p22 and 7q36. Distal 8p deletions are known to be associated with growth and mental retardation (MR), minor facial anomalies, heart defects, and behavioral problems. Confirming these findings, a study reported that a very small deletion in the 8p23.3 region was associated with GDD, microcephaly, and minor facial dysmorphism. ³⁰ In two cases, we identified balanced translocations between the 14q32.3 and 14q22, and common clinical features associated with 14qter deletion have been reported to be mild-to-moderate GDD/ID, hypotonia, microcephaly, single palmar wrinkle, and craniofacial malformations, which include high forehead with lateral hypertrichosis, wide nasal bridge, long and wide philtrum, thin upper lip, and high arched palate. ³¹ The long arm of chromosome 14 contains about 15 genes involved in the regulation of cellular transcription, cell proliferation, and intercellular signal transduction, which are particularly relevant to human brain development ³² ( Table 2 ).

Table 2. Distribution of chromosomal abnormalities according to clinical features.

Referral diagnosis or clinical manifestations	No. of children	Karyotype
Global development delay	10	46,XX,t(6;11)(q25;q23); 46,XY,t(16;19)(q24;p11)
		46,XX,t(16;19)(q24;p11); 46,XX,t(2:14)(q27:q22)
		46,XY,t(5;12)(q34;p12); 46,XX,t(8;7); 46,XX,t(2;22); 46,XX/del(q18-p13); 46,XX/46,XX,del(11q24); 46,XX,del(5p13); 45,XY,robt(14;21); 46,XY,inv(9); 46,XX,inv(14)(q13;q24)
Developmental delay with multiple congenital anomalies	1	47,XX, + 22,del(22q12)
Developmental delay with inability to walk, sit, and speak	3	47,XX, + mar; 46,XX,del(5p12;p13); 46,XY,22p + +
Developmental delay with inability to walk and mental retardation	1	46,XY,del(7q34)
Delayed development and menstrual irregularity	10	45,X; 46,XX/45,X
Delayed development with inability to hold head upright	1	47,XX, + 18
Intellectual disability	7	46,XX/47,XY, + mar; 46,XY,t(5;12)(q34;p12); 46,XY,del(21q); 46,XY,t(6p;15q); 46,XX,t(D/G), + 21; 46,XY,inv(11)(q23;q13); 46,XY,inv(9)(p12;q21)
Developmental delay with mental retardation	1	46,XY,t(5;12)(q34;p12)
Delayed development with microcephaly/hydrocephalus and congenital anomalies	4	47,XY, + 13; 47,XX, + 21,inv(9)(p11;q13)
Moderate and mild mental retardation (Down, Klinefelter, and fragile X syndrome)	13	47,XX/XY, + 21; 47,XXY; 46,XX/XY,fraX(q27.3)
Level of intellectual disability and facial dysmorphism;	1	46,XX,CA(20%)
Epilepsy;	2	46,XYq + , 46,XX,hypodiploidy

We identified a rare balanced translocation between the chromosome 1p32 and 2q37 regions in one child. Chromosome 2q37 deletion syndrome is a rare chromosomal disorder characterized by mild-to-moderate developmental delay. In previous studies, the 1p32 and 2q37 regions have been reported to harbor susceptibility genes for GDD. ³³ We believe that the translocations detected in this case may play a role in phenotypic expression of GDD.

Chromosomal deletions or duplications can cause abnormal expression of certain genes that cause adjacent gene syndromes. ³⁴ Deletion or duplication of even the same gene can have different effects. We found six deletions at 5p15.1, 5p-, 18p13, Xp, and Xp11 regions. The deletions in these chromosomal regions cause diverse phenotypes, depending on both the size and location of the deletion, but almost invariably associated with GDD. Deletion of the short arm of chromosome 5 (5p-) is associated with cri du chat syndrome, which is a well-defined clinical entity, and individuals with 5p deletion may show phenotypic and cytogenetic variability. The most important clinical features are a high-pitched cat-like cry, microcephaly, MR, severe psychomotor retardation, and distinct facial dysmorphism. Deletions can vary in size, and larger deletions were reported to be associated with more severe phenotypes and cognitive deficits. ³⁵ Similarly, we detected deletions on short arm of chromosome X (Xp-) in three children. The presence of three genes related to brain development in the Xp11 region has been reported. ³⁶ Mutations in any of these genes hinder normal brain development and function, and they are potential causes of the X-linked MR. In addition, deletions of genes on the short arm of the X chromosome that escapes X inactivation are known to be responsible for most of the phenotypic features associated with Turner syndrome. ³⁷ X-linked forms of MR are estimated to account for 10 to 20% of all hereditary GDD/ID cases. Fragile X syndrome (FXS) is a disorder characterized by ID and typical physical/behavioral abnormalities. FXS is the second most common cause of ID after Down syndrome (DS) and is due to the massive expansion of CGG triplet repeats encoded by a mutated fragile XMR (FMR1) gene located in the Xq27.3 region. ³⁸ Confirming the literature data, we reported fragility in the Xq27.3 region in two children who showed significant developmental delay. Hence, FXS should be considered an important part of first-line investigation in patients with GDD/ID.

Pericentric inversion of chromosome 9 is the most common structural disorder in the general population and its prevalence varies by ethnicity. It is estimated that the incidence rate of inversion 9 in Asian populations is approximately 1.5%. The incidence of both paracentric and pericentric inversions of chromosome 9 among patients with ID is known to be 7.2%. ³⁹ In the current study, we found an inversion in eight children (1% of all children) including inversion 9 in seven patients. Majority of cases carrying inversion 9 in this study do not show any specific phenotypic abnormality. However, inversion 9 has been reported to be associated with infertility, recurrent fetal losses, congenital anomalies, and ID in some cases. Similarly, inversion 9 may possibly be considered a predisposing factor leading to nondisjunction and interchromosomal effect events. ^{40 41}

We also report a boy carrying a pericentric inversion of inv(14)(q13;q24). This child presented with severe developmental delay with muscular hypotonia and focal epilepsy with apneic episodes progressing to serial tonic seizures. Supporting our finding, a region of 2 Mb was mapped to the 14q13 region in which the presence of a gene responsible for holoprosencephaly development was suspected. ⁴²

Turner syndrome is the most common sex chromosomal aneuploidy in women. It is usually caused by the complete or partial absence of one of the two X chromosomes found in females. That is, it can also be caused by a structurally abnormal X chromosome, where deletion or duplication of genetic material occurs. It is usually diagnosed during adolescence in these patients because of the inability to mature sexually resulting from ovarian dysgenesis. However, these patients are recognized at birth or in childhood due to signs and symptoms such as peripheral lymphedema, growth retardation, short stature, webbed neck, raised chest, low posterior hairline, pigmented nevi, hypoplastic nails, short fourth metacarpals, and aortic coarctation. ⁴³ In this study, Turner syndrome karyotypes, including classical and mosaic forms, were detected in 10 patients, which constituted 18.5% of overall chromosomal abnormalities and 1.2% of all cases. Turner syndrome karyotypes included three classical 45,X monosomies, three mosaics, and four mosaics involving X chromosome structural defects. Our cytogenetic findings involving chromosome X were similar to the reported studies. ^{17 44} We found 47,XXY karyotype (Klinefelter syndrome) in two children. Adolescent and adult individuals with Klinefelter syndrome show typical features such as tall stature, hypergonadotropic hypogonadism, and fertility problems. Besides, Klinefelter syndrome is associated with language dysfunction, delayed auditory processing, autistic behaviors, and, in rare cases, with severe ID—symptoms that constitute important milestones of GDD. ⁴⁵ Our findings further confirm that a proportion of XXY boys display traits of GDD that may affect their daily lives.

DS (trisomy 21), which is the most common autosomal aneuploidy, is the most frequent reason for referral to cytogenetic analysis. DS presents with signs and symptoms that contain characteristic facial features, ID including significant speech problems and delayed syntactic development, and a range of other health problems. DS is most prominently associated with ID and contributes to about 30% of all moderate-to-severe cases of MR. ^{46 47} In the present study, eight (about 1% of all patients) children showed a DS karyotype, which included six cases presenting with a classical trisomy 21 karyotype and two cases presenting with mosaic karyotypes. Different DS karyotypes are associated with varying phenotypic expressions. Supernumerary marker chromosomes are small, extra abnormal chromosomes of unknown origin detected by cytogenetic analysis. We also detected two children with 47,XX, + mar, who developed GDD. We did not perform molecular analysis of this marker chromosomes. Indeed, it has been reported that marker chromosomes are associated with GDD, ID, craniofacial dysmorphism, and dysmorphic features. ^{48 49}

Conclusion

To our knowledge, this study is the first large-scale study on GDD/ID in a Turkish population collating data on cytogenetic abnormalities contributing to GDD/ID. CAs were detected in 6.7% of children showing clinical signs and symptoms of GDD. Our results confirm that cytogenetic evaluation is an important part of the investigation of causes of congenital anomalies, GDD/ID, or other developmental disabilities. Therefore, routine chromosome analysis should be performed in children with GDD, even in the absence of dysmorphic features or other clinical features or a positive family history. Recognition of specific phenotypes will assist in the clinical etiological diagnosis of GDD. It is also very useful in establishing associations between genotype and phenotype in the context of GDD or any other unexplained disorder. More importantly, precise mapping of genes in these chromosomal regions provides essential clues for localizing critical regions and provides a strategy for the identification of new candidate genes associated with GDD/ID. It is also critically important that understanding genetic risk factors will possibly contribute to reducing the incidence of GDD/ID and to alleviating the burden of GDD on society.

Funding Statement

Funding This study was conducted by means and allowances of Cytogenetic Laboratory of Medical Biology and Genetics Department at Çukurova University.