Cytogenetic evaluation of patients with clinical spectrum of Turner syndrome.

AIM: The objective of this study was to correlate the genotype, of female patients, withshort stature and primary amenorrhea.
MATERIALS AND METHODS: One hundred and forty-six subjects were recruited during 2005-2012. Microscopic and automated karyotyping analyses were done by using chromosomes isolated from the lymphocytes using Giemsa banding (GTG) to identify chromosome abnormalities.
RESULTS: A total of 146 clinically suspected Turner syndrome (TS) subjects were recruited for the study, of which, 61 patients were identified to have chromosome abnormalities. The chromosomal abnormalities detected were as follows: Monosomy X (n = 19, 13.01%), triple X syndrome (n = 4, 2.7%), mosaic TS (n = 12, 8.21%), XY gonadal dysgenesis (n = 13, 8.9%), and structural abnormalities including X chromosome (n = 15, 10.27%) and one patient each with autosomal changes involving 9qh inversion and translocation of chromosomes 12 and 14.
CONCLUSION: Karyotype abnormalities accounting for 46% in this study emphasize the need for karyotype testing in cases of short stature with primary amenorrhea.

INTRODUCTION

Turner syndrome (TS) is a common, but little known chromosomal aneuploidy that affects 1 in every 2000 girls.[1] Clinical features of TS are due to complete or partial absence of an X chromosome. Characteristics are short stature (average height of a full grown woman with TS is 4’8”) and primary amenorrhea,[2] with renal and heart structural abnormalities. TS is clinically suspected when patient has lymphedema of the hands and feet, excessive skin at the nape of the neck, cardiac anomaly in newborns, short stature, and delayed puberty in girls and also in cases of infertility and secondary amenorrhea in adult females.[3] Stature is considered short when height is less than 3 rd or 4 th percentile for that age.[4] Puberty begins with gonadal maturation and increasing production of sex steroids. Puberty is considered delayed if there are no secondary sexual characteristics by 13 years of age.[5] However, confirmation by karyotype and presence of Y chromosome will hint the need for gonadectomy to prevent gonadoblastoma. The clinicians may order hormone tests that can suggest TS, but karyotype is the only way to diagnose the condition with certainty. In the present study, the clinical data were recorded by physical examination and ultrasound. GTG banding was done for 146 patients with short stature and amenorrhea to facilitate individual chromosome identification.

MATERIALS AND METHODS

Automated karyotyping analysis was done by using IKAROS software in 146 females (between 6 and 33 years of age) who were suspected to have TS either due to short stature and primary amenorrhea, secondary amenorrhea, premature ovarian failure, or testicular feminization. Peripheral blood (5 mL) was collected in a heparinized vacutainer. Culture was setup as per the laboratory standardized protocol;[6] 1 mL of peripheral blood was added to 10 mL of culture media [RPMI 1640 (10 mL) with fetal bovine serum (20%), phytohemagglutinin (0.2%), 1,0000 units of penicillin and 1,0000 μg of streptomycin per ml]. The culture (T25) flasks were kept in CO2 incubator at 37°C for 72 h. The cultured cells were arrested at metaphase stage using colchicine (1 μg/mL), 30 min prior to the harvest of the cells. Harvesting was done by centrifuging the contents of the flask at 1000 rpm for 10 min, followed by hypotonic (0.075M KCl) treatment for 20 min. The cells were fixed with fixative (3:1 methanol and acetic acid) and after three changes of fixative; the metaphase slides were prepared by dropping method and were incubated for ageing at RT for 3 days. The slides were treated for GTG banding[7] with 0.05% of trypsin solution and stained with 4% giemsa stain. The karyotypes were constructed and analyzed as per International System for Chromosome Nomenclature (2009) guidelines. Further confirmation was done with laboratory standardized CBG and QFQ banding techniques as per the requirement.

RESULTS

The diagnosis was made after thorough clinical evaluation of phenotypic features including height and cytogenetic study was carried out in 146 patients, diagnosed as short stature (n = 27), primary amenorrhea (n = 92), secondary amenorrhea (n = 16), premature ovarian failure (n = 2), testicular feminization (n = 5), and short stature with sexual ambiguity (n = 2). Sixty-one (41.78%) females were found to have chromosome variations among the recruited patients. The chromosome analysis was done and the results shown in Table 1 and Figure 1a–h are as follows: 46,XXGD in 85 (58.2%) and 46,XYGD in 13 (8.96%) and 47,XXX in 4 (2.73%). Turner's phenotype including ovarian dysgenesis and genotype monosomy (45,X) were detected in 19(13.01%) and mosaic karyotypes in 14(9.58%) patients. The patients with mixed gonadal dysgenesis (45,X/46,XY) phenotypes range from ambiguous to female. A total of 17(11.64%) patients showed X chromosome structural abnormalities such as deletions in two patients with 46,X,del(X)(pter….<p21) and 46,X,del(X)(qter….patient each within v(X) chromosome and isodicentric (X) chromosome showed clinical manifestations of both short stature and primary amenorrhea. We also found one 12-year-old girl with short stature, primary amenorrhea and karyotype was autosomal translocation 45/46,XX,t(12;14). A heteromorphic variant; pericentric inversion of chromosome 9 (46,XX, inv(9qh)) was observed in another girl with gonadal dysgenesis.

Table 1

Chromosomal complements reported in patients with short stature and primary amenorrhea

Figure 1

Partial karyotypes in patients with short stature and primary amenorrhea (a) X,iso(X), (b) X,idic(X), (c) X,inv(X)(q21q28), (d) X,del(X)(q21.1-q28), (e) X,del(X)(p11.2-p22.3), (f) X,r(X), (g) X,r(X), (h) XX,t(12;14) (q23;q13)

DISCUSSION

Human chromosomes are gender-specific with female exhibiting 22 pairs of autosomes and a pair of X chromosomes. Prevalence of X chromosomal abnormalities like monsomy X, trisomy X, XY gonadal dysgenesis/XY female is 1 per 1000.[8910] Sex chromosome abnormalities are common and lead to various phenotypic features. The effects of X and Y chromosome abnormalities may not be as severe as those from autosomal abnormalities, but stature and pubertal development may be affected since sexual development is the result of numerous genes on the X chromosome and mutation in any of these genes can result in partial or complete failure of stature and pubertal development. Somatic growth and maturation in humans are influenced by several factors that act independently or in concert to modify an individual's genetic potential. Stature and puberty are dynamic periods of development marked by rapid changes in body size, shape, and composition, all of which are sexually dimorphic. TS is characterized cytogenetically by a 45, X or X deletion complement or mosaicism of 45, X cell line with either 46XX or 46XY complement and clinically by short stature, pubertal delay, and gonadal dysgenesis. Turner individuals are phenotypically female and suffer from premature ovarian failure and other phenotypes, the most severe being poor viability in utero. Over the last 30 years, cytogenetic studies have indicated that short stature and primary amenorrhea affects at least 95% of all individuals with TS. The distal X short arm (Xp) in particular has been implicated in somatic development. The short stature phenotype is a result of haploinsufficiency of SHOX (short stature homeobox-containing gene located at Xp22.33;Yp11.3) which encodes a transcription factor implicated in skeletal development.[1112] This was evidenced in these patients who had monosomy X and mosaic chromosome complements including X chromosome deletions/duplications; 46, X,del(Xp); 46, X, del(Xq); and 46, X.iso(X). However, 46, XYGD and 47, XXX had normal stature but with delayed puberty ovarian dysgenesis.

The most common form of mosaicism associated with short stature and gonadal dysgenesis is 45,X/46,XX. Somatic anomalies are less likely to exist in 45,X/46,XX than in 45X; in case of subjects in the present study, 45,X/46,XX/47,XXX is individuals. A female patient with 45,X/46,XY chromosome complement had primary amenorrhea, short stature, but no other typical features of Turner's syndrome. Trisomy X (47,XXX) is a sex chromosome aneuploidy condition in which females have an extra X chromosome, compared to the 46,XX karyotype in typical females. Significant facial dysmorphology or striking physical features are not commonly associated with 47,XXX; however, in our patients, minor physical features were observed-a 7-year-old girl with hypertelorism and two patients with normal height and breast with secondary amenorrhea and one with irregular periods exhibited minor phenotypes. Partial or complete X chromosome deficiency may affect stature and pubertal development.[13] However, clinical manifestations widely vary depending on the gender of the patient and the gene content of the deleted/duplicated segment. Partial deletions of the short arm of the X cause significant ovarian insufficiency with Turner's sign and gonadal dysgenesis.[14] The p11-p21 segment of X chromosome carries a gene essential for gonadal development and Xp21 seems to cause short stature.[15] Studies on the deletion of the Xq have revealed that in most of these cases the breaks occur within the critical Xq13q27 region.[16] The fact that a patient with an absence of long arm of X chromosome had normal stature suggests that the presence of short arm of X chromosome maintains the stature of the affected. Deletion involving the long arm of X chromosome generally results in ovarian failure if they involve the proposed critical region Xq13-q26.[17] In our study, it was evidenced that eight subjects had X chromosome structural abnormalities of partial monsomy/trisomy 46,X,del (Xp), 46,X, del(X)q, 46,X ring(X), and 46,X,iso(X) as shown in Figure 1a–h.

There are numerous variant karyotypes seen in TS other than the classic monosomy X. These involve a partial deletion/duplication of the second X such as 46,X,der(X). It is important not to confuse iso (X) chromosome that is 46,X,i(Xq) syndrome with the 45,X classical Turner's syndrome. There are profound cytogenetic and clinical differences between the two syndromes, which must be borne in mind in the differential diagnosis of amenorrhea and of infertility.[18] The perceptual representation of certain individual lines may drastically change by selective gaps and by loss of derived X chromosomes. The instability of derivative X chromosomes is a mitotic phenomenon, at the beginning of zygote formation, whereas aberration toward iso, ring, and isodicentric X chromosome may be a meiotic event. However, if the loss of an X chromosome [or preferential loss of an abnormal sex chromosome derivative such as iso(Xq) chromosome] occurs early in embryogenesis, this may lead to mosaicism (e.g. X/, X/i(Xq), which may also be associated with fewer (or no) features of TS. Most of the females with a ring chromosome X that is r(X) have appearance of somatic signs of TS, including short stature, peripheral edema, characteristic facial features, low posterior hairline, ovarian dysgenesis, and also endocrine disorders, and autoimmune conditions.[19] The patients with r(X) are at a higher risk of mental retardation, learning difficulties, autistic spectrum disorders, and structural brain abnormalities due to loss of XIST region.[20]

Possible submicroscopic duplications in active ring X chromosomes can go undetected by chromosomal analysis and may encompass dosage sensitive genes, leading to functional disomy or even trisomy, and resulting in clinically relevant phenotypes as has been demonstrated in autosomes.[2122] Usually, translocations involving X chromosome and an autosome are rather rare due to the associated infertility in men and subfertility in women. X-autosome translocations are frequently associated with primary or secondary ovarian failure and at times TS-like features.[23] Pericentric inversion in chromosome 9 is a common heteromorphism in general population. However, in this study, two girls were found to have 46,XX, inv(9qh), and one patient with autosomal translocation of chromosome 12 and 14 that is 45,X/46XX,t(12;14). Here, the breakpoint of chromosome 12 is may be PTPN11 gene which is associated with Noonan syndrome.

In conclusion, we strongly reiterate the importance of cytogenetic study and karyotype-phenotype correlations in patients with TS to obtain information about the genotype-phenotype correlations related to the X chromosome.