Chromosome Aberration on Growth and Developmental Disorder

Growth and development in humans begin at conception, which shows progress and interdependence. Normal children show the characteristic of growth and development. In children with growth and development disorder, chromosomal abnormality may be found. This study aimed to determine chromosomal abnormality in patients with growth and development disorder. The samples were taken from karyotype results of patients in the Medical Genetics Laboratory, Faculty of Medicine, Universitas Wijaya Kusuma Surabaya, from 2010-2020. The inclusion criteria were infants to adolescents aged 24 years and unmarried who experienced impaired growth and development, including sexual development. Chromosomal abnormalities were obtained from cytogenetic analysis using the G-banding method. From 75 samples with growth and development disorders, there were abnormalities in the number of autosomal chromosomes and sex chromosomes found in patients with Down syndrome, Turner syndrome, Klinefelter syndrome, and Edwards syndrome. Chromosomal structural abnormalities found were deletion, translocation, inversion, duplication, marker chromosome, and heteromorphism. This study showed the importance of karyotyping in children and adolescents with growth and developmental disorders.


INTRODUCTION
All life forms show growth and development that start at conception and end when they reach maturity. The terms growth and development are used together. In normal children, growth and development are progressing and interdependent. Growth is defined as the increase of length or height, body mass, or other measurements due to the number of cells that leads to an overall increase. These increases can be seen, valued, and accurately measured (1,2).
Growth and development are unique characteristics of children, and any inhibition at any stage of these processes can lead to growth and development impairment. Besides a 10% prevalence of developmental delay, it is important to emphasize that identifying growth and development disorders are still difficult. Although severe disorders can be recognized at infancy, diagnosing speech difficulty, hyperactivity, or emotional disturbance before 3-4 years old is not easy, and learning disorders are rarely recognized before they enter school (1).
Child development includes sexual development that is not only about sex but includes values, feelings, attitudes, interactions, and behaviors. Sexual development is a part of sexuality, and it begins earlier in life than puberty (3). Disorders of sex development (DSDs) are congenital disorders in which gonad and phenotype do not match the individual's karyotype. The term DSDs covers a broad clinical spectrum that can be diagnosed at any age range, from the neonatal until adulthood, which are due to infertility problems. The clinical conditions vary from external genitalia ambiguity recognized at birth to mild forms, such as mild hypospadias or unilateral cryptorchidism, postnatal virilization, delayed/absent puberty, or infertility (4,5). The development of sexspecific gonad starts with the formation of bipotential gonad, which then differentiates to either testicular or ovarium tissue (4).
Congenital abnormalities in DSDs include abnormalities in chromosomes (numerical or structural), genes involved in gonadal and/or genital development, and changes in gonadal and/or adrenal steroidogenesis (6). Therefore, the etiological diagnosis of DSDs generally requires endocrinological tests, radiological images, and genetic tests. Generally, the initial test is the chromosome examination for classifying the abnormalities (4). Disorders of sex development related to the atypical arrangement of sex chromosomes are the Turner syndrome and its variants, the Klinefelter syndrome and its variants, and mosaicism in sex chromosome (45,X/46,XY or 46,XX/46,XY) (7-9).
As growth and sexual development disorders might be caused by chromosome aberration, this study used data from chromosome analysis on children with growth disorder and sexual developmental disorder during 2010-2021 at the Medical Genetics laboratory, Medical Faculty, Universitas Wijaya Kusuma Surabaya.

Patients
This study involved 75 subjects who met the inclusion criteria that were children and adolescents with growth and/or developmental disorder, including sexual development disorder. Based on the Law of the Republic of Indonesia Number 23 Year 2002 article 1 paragraph 1 concerning child protection, a child is someone who has not reached the age of 18 (eighteen), including a fetus in the womb. According to the Regulation of the Minister of Health of the Republic of Indonesia number 25 Year 2014, an adolescent is someone from 10-18 years old; and, according to National Population and Family Planning the Agency (BKKBN), an adolescent is someone in the age range of 10-24 years and not married yet. Therefore, in this study, children and adolescents were from babies in the womb to the age of 24 years and were not married. All Note: Based on the Down syndrome growth chart (11), Based on the Turner syndrome growth chart (12) patients with growth and/or development disorders were grouped according to phenotype, chromosomal features, or karyotype, including chromosomal abnormalities, chromosomal structural abnormalities, and the syndrome suffered.

Cell Culture Method
Chromosome analysis was done using G-banding analysis (400 bands) (10). As much as 3 ml of peripheral venous blood was inserted into a venoject containing sodium heparin. A total of 0.4 ml of blood was then put into a Nunc tube or 15 ml tube containing culture media and rd incubated in a 5% CO incubator for 72 hours. On the 3 2 day before harvesting, colcemid was added to the culture media to stop the mitosis at metaphase. After centrifugation at 3500 rpm for 10 minutes and supernatant aspiration, hypotonic solution was then given to swell and lyse the cells. Fixatif solution (methanol:acetic acid=3:1) was added, followed by centrifugation and supernatant aspiration. This process was carried out repeatedly until clear solution was obtained, then the pellet at the bottom of the tube was dripped on an object glass and dried before staining using Giemsa.

G-banding Analysis
After staining with Giemsa, the chromosomes could be seen under a microscope at 1500 times magnification aided with immersion oil. The total number of cells counted was 30-50 for each sample.

RESULT
A total of 75 samples were grouped based on the type of growth and development disorder (phenotype), the syndromes, and the karyotype ( Table 1). The samples were grouped based on the type of growth and development disorder and the phenotype, as well as the chromosome structure aberration (Table 2).

DISCUSSION
Patients with growth and/or developmental delay, including sexual developmental delay, in this study, showed (either or both) numerical or structural chromosome aberration (translocation, deletion, inversion, duplication, and marker chromosome) as well as sex chromosome aneuploidy. Mosaicism (two or more groups of cells with different karyotypes in one individual) was found in 9 patients, 4 of them showed 45,X/46,XY mosaicism with phenotype either female or male.
Down syndrome or trisomy 21 was the most growth and developmental disorder found in this study. With the live birth incidence at approximately 1:800, Down syndrome was the most common cause of genetic cause of intellectual disability. Babies with Down Syndrome experience hypotonia or muscle weakness, resulting in delays in development, such as the ability to prone, sit, and walk. The delay also occurs in the ability to speak (14). Patients with Down syndrome show characteristic phenotypes that are upslanting palpebral fissure, flat nasal bridge, epicanthal folds, hypertelorism, flat occiput, simian crease, and strabismus (15).
Individuals with Down syndrome also show growth and sexual development delay. With an adult height of approximately 150 cm, they seem shorter than their peers. In this study, Down syndrome height was measured using a Down syndrome growth chart (11). Children with Down syndrome also experience a sexual developmental delay. Male with Down syndrome will become fully sexually mature but later than normal individuals (16). While in the females, their menarcheal age does not differ from the general population (17,18).
Aneuploidy in sex chromosomes found in this study were Turner (and its variation) and Klinefelter syndromes. Females with Turner syndrome also suffer from growth and developmental disorder. Their main characteristics are short stature (>90%) seen at birth, during childhood, and final height below the average female height in the specific population. The cause of short stature is not fully understood but may be caused by SHOX gene haploinsufficiency (on the Pseudoautosomal region/PAR) (18,19) rather than primary disorder in the GH-IGF axis or the absence of sex steroids. Patients with Turner syndrome also experience no secondary sexual development (no breast development and axilla and pubic hair) and ovarian failure that causes primary amenorrhea and infertility (19).
Percentage rather than half Turner syndrome individuals have 45,X karyotype. Some show variations, which in this study were in the form of mozaic: 46,X del(Xp), del(X)(qter-->p11.2) and del(X)(q22-->qter). Females with partial monosomy on the X chromosome show variation of their phenotype. Patients with Xp deletion present with short stature, which is around 2% among Turner syndrome patients. Genes on the short arm of the X chromosome (in PAR) in normal females (46,XX) do not experience X inactivation; therefore, one or more genes deletion in PAR can cause clinical implications. Since the ovarian function remains normal, terminal Xp deletion can be passed to the next generation (20). There were no examinations on the patient's mother in this study, so it was unknown whether partial Xp deletion in this patient was inherited.
In this study, there were 4 patients with mosaic Turner syndrome (45,X/46,XY). These sex chromosome differences are thought to occur in mitosis after fertilization of an embryo with a 46,XY karyotype. In the subsequent cell division, the Y chromosome became lost due to non-disjunction (postzygotic mitotic error), so that there are cells that lose their Y chromosome (45,X) (21). Individuals with a 45,X/46,XY karyotype exhibit varying phenotypes, ranging from a seemingly normal appearance to showing clinical signs of Turner syndrome with incomplete sexual differentiation. In general, 60% of cases with a 45,X/46,XY karyotype exhibit ambiguous genitalia (21). There were 4 patients with 45,X/46,XY karyotype: 3 patients had female phenotype, and the remaining had male phenotype. In patients with female phenotype, sexual developmental disorders experienced were lack of secondary sex development and primary amenorrhea (patient 1), ambiguous sex (clitoral enlargement) (in patient 2), and short stature and no secondary sex development (patient 3). In patients with a male phenotype, the developmental disorder found was undescended testis.
The other sex chromosome aneuploidy was Klinefelter syndrome. The incidence ranges between 1:581 and 1:917 of live male birth. However, only 25% of this population will be diagnosed in childhood or later in adulthood after they experience developmental delay, behavioral disruptions, hypogonadism, gynecomastia, or infertility. During the infancy and pre-school period, individuals with Klinefelter syndrome experience growth and developmental disorder that includes hormonal, motor, speech, and behavioral development. In the transition to school age, impairment occurs in language difficulty, disfunction in execution, behavior, and reading and learning deficits (23).
Males with Klinefelter syndrome show characteristic s y m p t o m s s u c h a s t y p i c a l p h y s i c a l a n d neurodevelopmental manifestation focused on growth, cognitive development, endocrine function, and reproduction. Based on a study, the optimum result depends on early detection combined with long-life c o n s i s t e n t a n d t a r g e t e d t r e a t m e n t o n neurodevelopmental (23). In this study, Klinefelter syndrome was accompanied by sexual developmental delay, and one patient showed hypospadias. There were no data about his cognitive ability. In this study, the number of Klinefelter syndrome was smaller than Turner syndrome since many males with Klinefelter syndrome may be undiagnosed due to the unspecific symptoms, variation in clinical presentation, and no dysmorphic appearance. Postnatal diagnosis is usually made due to physical abnormality in early childhood, such as hypospadias, small phallus, or cryptorchidism (23).
The abnormalities in chromosome structure found in this study were translocation, duplication, deletion, inversion, marker chromosome and heteromorphism. Duplication in the chromosome segment will double the DNA sequence and gene dosage on this segment, influencing the phenotype. In this study, two patients with karyotype 46,XY,dup (9) (q12;q13) showed sexual developmental disorder: hypospadias, scrotum bifidum, and microtestis. In contrast, another patient with the same karyotype showed hypertelorism, flat nose, and protruding tongue, similar to Down syndrome phenotype. The other d u p l i c a t i o n w a s f o u n d i n a p a t i e n t w i t h 47,XX,+21,dup(16)(q11.2) karyotype. Since the patient had trisomy 21, the patient showed a Down syndrome phenotype with growth and developmental disorder.
In this study, a patient with Karyotype 46,XY,dup (18) (q23) showed hypospadias, bilateral undescensus testis, and intellectual disability. Patient with trisomy 18 (Edwards syndrome) is purely intellectually disabled as well as other congenital abnormalities. Meanwhile, some patients with partial trisomy 18 are known to show variation in severity from relatively mild without any internal organ malformation to a classical characteristic of Edwards syndrome. Some researchers have tried to identify which region on chromosome 18 is the critical region (CR) for Edwards syndrome. Partial duplication of chromosome 18q, which involves the CR was reported in patients with Edwards syndrome phenotype. The literature stated that partial duplication of proximal chromosome 18q would be more severe than if the duplication occurs in the distal part (24).
Another chromosome structure abnormality in this study was the deletion or missing of segment chromosome, which means loss of genes on the deleted segment. This study found patients with Xp chromosome deletion, that are 2 patients with Xq deletion and 1 with Yq deletion.
Patients with X or Y partial deletion showed sexual developmental disorder, namely primary amenorrhea (partial X del), undescensus testes, and hypospadias (del Yq). All female patient with del X has short stature, even in mosaic monosomy X.
This study also found inversion or chromosome segment which is reversed in position. Of all patients with an inversion, all of them involved an inversion of the centromere of chromosome 9 (pericentric inversion). All patients were accompanied by mental retardation and developmental delay. One patient showed mosaicism: inversion and trisomy 21; therefore, the patient showed Down syndrome phenotype.
Translocation or segment chromosome transfer between non-homolog chromosomes was found in five patients. Transfer happens between non-acrocentric chromosomes (reciprocal translocation) and between acrocentric chromosomes (Robertsonian translocation). Reciprocal translocation is more common, and a translocation carrier usually does not show any abnormalities, but their offspring may exhibit abnormal phenotype or karyotype due to the addition of genetic material (25). There were two patients with reciprocal chromosomes between chromosome 3 and 18 who showed short stature and one patient with translocation between chromosome 13 and 13 showed multiple congenital anomalies. No patient with Robertsonian translocation.
In this study, a patient was found with Heteromorphism, a normal variant that can be detected using G-banding. Chromosome 9 showed the most morphology variant among non-acrocentric chromosomes in humans. These variants, such as 9qh+, 9cenh+, 9ph+, 9qh-or inv (9) (p11q13), are commonly detected in routine karyotyping, and the estimated frequency is 1.5% in the general population (26). The patient with heteromorphism (9q12h+) in this study also had trisomy 21, so the patient showed Down's syndrome phenotype.
Chromosome aberrations, either numerical or structural, are known to cause growth and developmental disorder, developmental delay, including sexual developmental disorder. This study showed that patients with growth and developmental disorders had numerical aberration (autosome and sex chromosomes) or chromosome structure aberration (translocation, deletion, inversion, duplication, heteromorphism, and marker chromosome).