Isochromosome of long arm of X chromosome i(Xq), which occurs due to abnormal transverse cleavage of the centromere during cell division, found in some cases of Turner Syndrome. The aim of this study is to estimate the prevalence of isochromosome X in patients with short stature with or without primary (PA) and secondary amenorrhea (SA) in our population. An observational study was conducted at Vivekananda Institute of Medical Sciences (VIMS), Kolkata, India between 2013 to December 2014. Female patients aged between 12-25yrs who were referred to Cytogenetic outdoor, VIMS for chromosomal analysis with history of short stature and with or without Primary Amenorrhea were included in this study. Lymphocyte culture followed by Karyotyping was done for all the patients. We have documented 4 such cases of IsochromosomeXq among 50 female patients presented mainly with short stature, with or without Primary amenorrhea. Our study emphasizes the importance of chromosomal analysis of short stature patients with or without PA or SA to eliminate diagnostic dilemma for the children.
Isochromosome, Karyotyping, short stature
Short stature is defined as a standing height more than 2 standard deviations (SDs) below the mean (or below the 2.5 percentile) for sex according to standard growth chart (Cohen et al. 2007).
Many genotype phenotype analyses have proved that short stature has a strong genetic component. Either autosomal or sex chromosomal abnormality or both the abnormalities can contribute its effect in growth. Turner syndrome (TS) is considered to be the important genetic abnormalities leading to short stature in females affecting 1 in every 2000 girls. (Donaldson et al., 2006) TS is characterized cytogenetically either by X chromosome monosomy (45, X), the presence of an abnormal X chromosome (Isochromosome/ring chromosome/deletion in short arm), or mosaicism of a 45, X cell line with another cell line, which might be 46,XX, 46,XY or have an abnormal sex chromosome rearrangement like isochromosome or ring chromosome etc. (Jacobs et al., 1997)
Isochromosome is defined as the structurally abnormal chromosome consisting of either two short or long arms, because of the abnormal transverse middivision of the centromere (centric fission), resulting in unbalanced chromosomal constitution, and monosomy for the missing and trisomy for the duplicated arms (Young 2005). The formation may also be because of the more complex U-type exchange resulting in acentric or dicentric products. The process of isochromosome may occur in pre meiotic gamete, during meiotic cell divisions or in postzygotic cell divisions of a normal or trisomicconceptus (Gardner and Sutherland 2004). Manifestations of classical TS usually include short stature,webbed neck, broad chest with widely spaced nipples, cubitus valgus, congenital lymphedema, lack of spontaneous pubertal development resulting from ovarian sex hormone insufficiency, amenorrhea, low-posterior hairline. but the X isochromosome patients present with short stature with absence or minor Turner stigmata thus creating really a challenge to the clinicians.
This study is done to find out the prevalence of isochromosome X in female patients with short stature in population of West-Bengal.
Materials and Methods:
This study was carried out at Department of Genetics, Vivekananda Institute of Medical Sciences (VIMS), Kolkata,West Bengal and is approved by the ethical committee of VIMS. Informed consent was obtained from the patients after the procedure was fully explained to them. The female patients having short stature with or without amenorrhea between the age group of 12-25 years were selected who were referred to the cytogenetic outdoor for chromosomal analysis from different hospitals of West-Bengal. A total of 50 patients were included in the study. The physical examination included the accurate measurement of the height and weight and thorough inspection and palpation of the external genitalia, the primary and secondary sexual characters and a search for the other congenital anomalies. Tanner-Whitehouse growth charts were used for monitoring the growth and assessment of heights among the patients. After taking brief clinical and family history peripheral blood karyotyping was done. The blood sample was collected from the patients in a completely sterile heparinized vacutainer tube and mixed well. The cultures were set up with RPMI 1640 (Rosewell Park Memorial Institute) culture medium. Peripheral blood lymphocytes induced with 2% phytohaemagglutinin (PHA) were incubated at 37.5°C for 72 h. One and a half hours prior to harvest, the cultures were arrested with colchicine and treated with 0.75 M KCl (potassium chloride) for 30 min and fixed in fixative (3:1 ratio of methanol : glacial acetic acid). After air drying, conventional GTG banding was performed to identify the chromosomes. After banding, 50 metaphases were scanned under low power for each case on OLYMPUS BX51 microscope and then 10 metaphases were analyzed by automated karyotyping system (CYTOVISION software). In cases of mosaics 30 metaphases were analyzed.
Fifty females ranging from 10 to 35 years having short stature with primary or secondary amenorrhea were considered for chromosome examination. Karyotype analysis was carried out by standard methods in all patients. Among 50 patients, 21 patients were diagnosed as having chromosomal abnormalities and 29 patients were with normal karyotype, having some other causes rather than chromosomal defect. The patients under study were categorized into five groups on the basis of karyotype obtained; 11 patients were with classical Turner syndrome, 3 patients were having mosaic TS, 29 patients were with normal 46, XX chromosomal complement and 4 were with isochromosome X (variant TS). Patients with isochromosome X have no other gross morphological phenotypic stigmata of TS except short stature and their short histories are given in Table.
[Normal hormone levels: FSH- 3.50-12.50 mIU/mL, LH-2.40-12.60 mIU/Ml, oestradiol- 24.50-195.00 pg/ml, Free thyroxine (FT4)- 0.7-1.9 ng/dl,TSH-0.27-4.20 µIU/ml, Insulin like growth factor(IGF-1)- 182 to 780 ng/ml for ages16 to 24 & 114 to 492 ng/ml for ages 25 to 39, Random growth hormone-Women: < 10 ng/ml Children: 0-20 ng/ml]
|Sl.no||Age(yrs)||Physical features||Hormonal profile||USG findings||Karyotype|
|1.||16||height - 129 cm, weight- 28 kg, high arched palate, clinodactyly Breast development : Tanner stage -2, sparse pubic hair normal intelligence average school performance. Primary Amenorrhea||LH-20.98mIU/mL; FSH -111.68mIU/mL; estradiol-15.12pg/mL TSH- 3.92uIU/Ml GH-normal||ovaries not visualised,uterus hypoplastic (48x23x14mm)||45,X/46,X,i(Xq)|
|2.||10||height -117.5 cm weight- 21 kg poor school performance behavioural disorder (temper tantrum)||TSH-24 mIU/ml(7)||hypoplastic uterus with normal sized ovaries||46,X,i(X)(q10)|
|3.||32||Height-132 cm weight -36 kg H/O secondary amenorrhea Breast well developed, no axillary hair but scanty pubic hair.||FSH-47.93mIU/mL LH-42.20 mIU/Ml FT4-1.7ng/dl,TSH-2.06 mIU/ml||Infantile Uterus (47.5x 20.8x21.6mm) small sized ovary||46,X,i(Xq)/45,X/46XX|
|4.||14||Height-115cm Weight-22kg Short neck, academic performance good||TSH-2.16 mIU/ml FT4-1.6ng/dl, GH- 12 ng/mL, IGF- 160 ng/mL||uterus with bilateral ovaries present.||45,X/46,X,i(Xq).|
There are numerous variant karyotypes seen in TS other than the classic monosomy X. Isochromosome X that is 46, X, i(Xq) syndrome one of those variant. There are a lot of cytogenetic and clinical differences than classical Turner syndrome. This isochromosome X consists of the two long arms of the X chromosome but no short distal arm. Lyon (1961) hypothesized that early in the development of a normal female embryo, random inactivation of one of the two X-chromosomes in each cell occurs which allows the female to have the same amount of Xchromosome material as the average male has. There are certain genes that escape this X inactivation like homeobox gene (SHOX), XIST gene etc. (Santana et al., 1977) These are located predominantly in the small regions of homology and pairing that persist on the sex chromosomes called the pseudoautosomal regions (PAR) present on the short distal arm. (Raoet al., 1997) In this way, the normal female has functioning genes from one complete X-chromosome plus functioning genes from the still active short distal arm of the mostly inactivated X-chromosome. The short stature phenotype is a result of haploinsufficiency of SHOX (short stature homeobox-containing gene located at Xp22.33) which encodes a transcription factor implicated in skeletal development. Thus, Patients with monosomy X or deletion in Xp or isochromosome of one of the long arm of X chromosome (i(Xq)) causes skeletal abnormality like short stature. Some reports (Sönmezet al., 1997; Garcíaet al., 1991; Zinmanet al., 1984) have indicated that patients with the 46,X, i(Xq) karyotype have characteristics similar to those observed in classical TS but in a milder form. Sybert and McCauley (2004) have reported the 46,X,i(Xq) karyotype in 7% of patients with TS. In our study we have found pure IsochromosomeXq in 1 patients and in 3 cases it was mosaic IsochromosomeXq. Patient 1 with mosaicism showed few phenotypical features of TS like clinodactyly and high arched palate, but others isochromosome-Xq didn't show any morphological stigmata of TS except short stature. Most of the patient with TS shows normal intelligence though 70% patients have learning disabilities affecting nonverbal perceptual motor and visuo-spatial skills (Ross et al., 2000). The patients with i(X) are at a higher risk of mental retardation, learning difficulties, autistic spectrum disorders, and structural brain abnormalities due to loss of X-inactive specific transcript (XIST) region. (Skuseet al., 2006) We observed no mental retardation in either of these patients but patient no.2 showed very poor school performances and temper tantrum disorders for which she needed psychological counseling by professionals. In our study we did not find any congenital malformations found in classical TS. Hypothyroidism is common in 15-30% of patients with TS. There are reported cases of more incidence of autoimmune thyroiditis in isochromosome X (Garcíaet al., 1991; Chiovatoet al., 1996; Medeiros et al., 2000). In our study patient 2 presented with hypothyroidism. She had a history of alopeciatotalis, an autoimmune disorder, thus she was advised to have thyroid scan to rule out autoimmune thyroiditis but didn't turn up with the report. Normal gonadal development needs the zinc finger protein, X-linked (ZFX) gene in the X chromosome short arm. Although oocyte development requires only a single X chromosome, oocyte maintenance requires two X chromosomes. In absence of a second X chromosome, therefore, oocytes in fetuses and neonates with TS degenerate and their ovaries atrophied into streaks of fibrous tissue. Women with an X monosomy, a X long arm isochromosome and short arm deletions commonly present with gonadal dysgenesis due to haploinsufficiency of this gene. (Ogata et al., 2001). Alteration in some regions of the X chromosome longarm results in ovarian failure. Presence of an IsochromosomeXq even in mosaic form with other cell lines, do not present spontaneous menarche. In our study we have found only growth retardation as constant feature with primary amenorrhoea in 1case. We found that the i(Xq) form of TS was generally milder than classic TS. A female with short stature, but without typical clinical findings of TS, should be evaluated for this chromosomal form, because 45, X karyotype can be diagnosed at birth due to typical dysmorphic features or cardiac abnormalities, but in isochromosome-X diagnosis may be delayed until childhood, adolescence or unfortunately until adulthood while evaluation for short stature,pubertal delay, primary amenorrhea and infertility. Early diagnosis is an important aspect of ideal treatment for these variant type of TS patients because growth hormone supplementation in proper time can help in achieving normal height at per age followed by sex hormone supplementation for overcoming ovarian dysfunction.