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DETECTION OF LOW GRADE MOSAICISM IN TURNER SYNDROME USING FLUORESCENCE IN-SITU HYBRIDIZATION
BY
ANURADHA POONEPALLI (M.B.B.S.)
A thesis submitted for the degree of Master of Science
Department of Obstetrics and Gynaecology National University of Singapore 2004
ACKNOWLEDGEMENTS
First, I would like to express my sincere thanks to Dr.Leena Gole, my supervisor, for her support, valuable time, patience and guidance throughout this study and during thesis writing. Thank you for giving me an opportunity to gain insight into the FISH technology and advice whenever I had problems.
I would also like to thank my co-supervisor, Dr.Annapoorna Venkat, for her constant effort in providing samples, unfailing guidance and valuable suggestions during this study. Her ideas, advice and constructive criticisms contributed to the overall caliber of the thesis.
I would also like to thank Dr.Vesna Dramusic from the Department of Obstetrics and
Gynaecology and A/P Loke Kah Yin from the Department of Paediatrics for providing the patient samples, without which this study would not be completed.
I am immensely thankful to Dr. M. Prakash Hande, for allowing me to continue my part- time Masters whilst working under him as a Research assistant.
Special thanks to all the staff in the Cytogenetic laboratory at National University
Hospital for providing their kind support, help and assistance.
I would, in addition, like to thank the lab mates in Genome stability lab, Physiology for their continued help.
I would also like to extend my thanks to Ms. Shen Liang for providing her expertise in helping me analyze the statistical data.
Finally, I would like to thank my husband and daughter for their constant support and encouragement. And my parents for their never-ending moral support.
i
TABLE OF CONTENTS
PAGE SUMMARY vi
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF GRAPHS xi
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: LITERATURE REVIEW 5
2.1: Turner syndrome 6
2.2: Numerical anomalies of X chromosome leading to Turner syndrome 6
2.2.1: X chromosome monosomy 6
2.2.2: Mosaic Turner syndrome 11
2.3: Structural anomalies of X chromosome 13
2.3.1: Isochromosome Xq [46,X,i(Xq)] 13
2.3.2: Ring X chromosome [45,X/46,X,r(X)] 13
2.3.3: Deletions of the ‘p’ or ‘q’ arms of the X chromosome
i.e.46,X,del(Xp)or46,X,del(Xq) 14
ii
2.3.4: Patients with residual Y chromosome material 16
2.4: Clinical presentation of Turner syndrome through the
different life stages 16
2.5: Fluorescence in-situ hybridization (FISH) 22
2.5.1: Probes in FISH 23
2.5.2: Commonly used fluorescent dyes 24
2.5.3: Applications of FISH 26
2.5.4: Advantages of FISH 26
2.5.5: Fluorescence in-situ hybridization (FISH) as a sensitive tool in
detecting mosaicism 27
2.6: Hypothesis of this study 28
2.7: GOALS OF THE STUDY 28
CHAPTER 3: PATIENTS AND METHODS 29
3.1: Patients 30
3.1.1: Group I: -Details of karyotypically normal patients
with Turner stigmata 30
3.1.2: Group II: -Details of karyotypically proven Turner patients 33
iii
3.1.3: Group III:-Details of the control females 36
3.2: Methods 42
3.2.1: Specimen collection 42
3.2.2: GTL (G-bands by trypsin using Leishman) banding 45
3.2.3: Dual color fluorescence in-situ hybridization 46
CHAPTER 4: RESULTS 50
4.1: Group I- Results of patients with Turner stigmata with a normal karyotype 51
4.2: Group II: Results of Turner patients with a 45,X cell line
by G-banding 54
4.3: Group III -Results of the control group 58
4.4: Statistical analysis 69
4.4.1: Definition of value for low grade mosaicism 70
iv CHAPTER 5: DISCUSSION 73
CHAPTER 6: CONCLUSION 84
CHAPTER 7: REFERENCES 86
CHAPTER 8: APPENDIX 96
v SUMMARY
Detection of low grade mosaicism in Turner syndrome using fluorescence in-situ hybridization (FISH)
Turner syndrome is a common sex chromosomal abnormality, typically presenting with premature gonadal dysgenesis and short stature. This phenotype is due to the absence of
X-chromosome (45,X) in some or all cells; or due to the presence of structurally abnormal X-chromosome resulting in the haploinsufficiency of X-Y homologous loci which escape X-inactivation. Sometimes, although the resulting phenotype is similar to those with 45,X (Hassold et al.,1988), the diagnosis of 46,XX karyotype invalidates the attempts of genotype-phenotype correlations (Ferguson-Smith,1965), which could be due to the presence of low grade mosaicism undetected by the standard cytogenetic techniques. Vice versa, the high in-utero lethality of 45,X condition has led to the hypothesis that most of the live born 45,X individuals have low frequency of normal cells which might be necessary for survival.
The aims of this study were to detect the presence of low levels of monosomic X cells in
karyotypically normal patients (study group I) presenting with Turner stigmata (n=11), to
detect the percentage of normal 46,XX cells in karyotypically proven Turner patients
(study group II) (n=17), and to investigate a control group (group III) of normal women
(n=25). Based on the levels of monosomy X cells in the control group, baseline value
was established and values exceeding this could then be classified as low grade
mosaicism. This was achieved using the fluorescence in- situ hybridization (FISH)
technique in addition to the standard cytogenetic techniques.
vi Results showed a low percentage of abnormal X cells (45,X and 47,XXX) in the first group of patients ranging from 0.1%-2.74% (Mean: 1.31%, SD 1.03), which was significantly higher than that observed in the normals (p=0.003, Mann-Whitney U test).
Using the ROC curve, a baseline cut-off value of 0.897% was obtained with a sensitivity of 63.6% and a specificity of 100%.The results showed that the recommended cut-off value was 1% which was obtained by rounding off the cut-off value obtained from the
ROC curve analysis.
Eighty-eight percent (15 out of 17) of the second group of patients (karyotypically proven
Turner patients) showed normal 46,XX cells ranging from 0-95% with G-banding and
FISH. The remaining 12% (2 out of 17) showed a low percent ranging from 0.06% to
0.1% of 46, XX cells only with FISH technique. Two cases remained apparent 45,X
Turner with G-banding, which may be explained by tissue-confined mosaicism
(Held,1993) necessitating the need to analyze cells from a different germ line or may be a consequence of a selective loss of a second cell line during embryonic development (Held et al., 1992).
FISH appeared to be a more sensitive technique compared to the conventional methods in detecting low grade mosaicism and hence the use of FISH technique is suggested in such patients to enhance the diagnosis and enable genotype-phenotype correlation.
vii LIST OF TABLES
TABLE TITLE PAGE
Table 1: Phenotypic features of X chromosome deletions
with ideogram of X chromosome on the right hand side 15
Table 2: Summary and percentage of occurrence of some
of the physical findings in Turner syndrome 21
Table 3: Excitation and emission maxima of various
fluorochromes used in multi-color FISH experiments 25
Table 4: Summary of FISH results in patients with Turner
stigmata with a normal karyotype 51
Table 5: Summary of FISH results of Turner patients with
a 45,X cell line by G-banding 54
Table 6: Summary of FISH results of controls 58
Table 7: Coordinates of the ROC Curve 71
viii LIST OF FIGURES
FIGURE TITLE PAGE
Figure 1: Classical karyotype of Turner syndrome 7
Figure 2: Normal female karyotype 7
Figure 3: Non-disjunction in meiosis 1 9
Figure 4: Non-disjunction in meiosis 2 9
Figure 5: Illustration of FISH 22
Figure 6: Xpter probe on the X chromosome 48
Figure 7: X chromosome showing the position of the Xp
and Xcen probes used in the FISH experiment 49
Figure 8: FISH on a metaphase showing two X chromosomes
with signals on the Xcentromere and the Xp-arm 65
Figure 9: FISH on interphase nuclei showing two X signals, for
both centromere and Xp-arm 65
Figure 10: FISH on a metaphase showing a single X chromosome 66
ix
Figure 11: FISH on an interphase cell showing a single X with one
signal each on X centromere and on Xp-arm 66
Figure 12: FISH on interphases cells showing three signals each
for the X centromere and Xp-arm 67
Figure 13: FISH on a metaphase showing an Xp deletion 67
Figure 14: FISH showing mosaicism in interphase nuclei 68
Figure 15: ROC curve 70
Figure 16: FISH on interphase nuclei showing split centromeres 81
Figure 17: FISH on interphase nucleus showing three X centromere signals 82
Figure 18: FISH on interphase nuclei showing three signals for Xp-arm and
two for X centromere 83
x LIST OF GRAPHS
GRAPH TITLE PAGE
Graph 1: Histogram showing the percentage of monosomy
X cells and trisomy X cells detected by FISH
in control females undetected by the standard
G- banding 62
Graph 2: Histogram showing the percentage of abnormal cells
detected by FISH in study group I (patients with Turner
stigmata with a normal karyotype) 63
Graph 3: Histogram showing percentage of normal and abnormal
cells by FISH in the study group II (Turner patients with
a 45,X cell line) 64
xi
CHAPTER 1
INTRODUCTION
1 1. Introduction
Turner syndrome (45,X) is a common sex chromosomal abnormality with an incidence of about 1 in 2500 liveborn female babies. Individuals with Turner Syndrome are phenotypically females with gonadal dysgenesis and somatic stigmata, like short stature, but have normal intelligence. This phenotype is due to the absence of the X-chromosome
(45,X) in some or all cells; or due to the presence of a structurally abnormal X- chromosome, resulting in the haploinsufficiency of X-Y homologous loci, which escape X- inactivation. It is estimated that 40-50% of patients with Turner syndrome demonstrate sex chromosome mosaicism (45,X/46,XX) (Magenis et al., 1980). Although the resulting phenotype is similar to those with 45,X sometimes (Hassold et al., 1988), the diagnosis of
46,XX karyotype by conventional cytogenetic technique invalidates the attempts of genotype-phenotype correlations (Ferguson-Smith, 1965). This is most probably due to the inability to detect low-grade mosaicism. Detection of low-grade mosaicism involves many factors, like the type and number of tissues analyzed, the number of cells studied and the sensitivity of the techniques applied (Hook, 1977; Procter et al., 1984; Held et al., 1992;
Jacobs et al., 1997). Hence, in this study the fluorescence in-situ hybridization technique
(FISH) was used in addition to the conventional cytogenetic methods, to detect the low percentage of abnormal cell lines in patients with Turner stigmata, but with a normal karyotype.
Vice-versa, the high in-utero lethality of the 45,X condition has led to the hypothesis that most of the live born 45,X individuals may have a low frequency of normal cells (46,XX),
2 which might be necessary for survival. Therefore, this study also included the detection of normal 46,XX cells in typical Turner patients with a 45,X karyotype.
In this study, both conventional cytogenetic techniques and fluorescence in-situ hybridization (FISH) techniques were used to analyze the chromosomes from the patient’s peripheral lymphocytes. The FISH technique has an advantage over the conventional cytogenetic methods in detecting low-grade mosaicism, as a large number of cells can be counted, and both the interphase as well as the metaphase nuclei can be analyzed.
It is known that as a consequence of aging, errors occur in cell divisions, leading to the loss of the inactivated X-chromosome (Surralles et al., 1999). So a low percent of abnormal cells could also be present in normal females. To verify whether the low-grade mosaicism observed is a significant cause for the phenotype, a group of age matched fertile females (25 controls) was also studied using the same test parameters. With these controls, a baseline value can be set and the values exceeding this could then be classified as low-grade mosaicism.
In general, a mosaic level of lower than 5% is considered to be low-level mosaicism
(Schinzel, 1974). Conventional cytogenetic analysis needs 60 cells to be scored to exclude a 5% mosaicism at a 0.95 confidence interval (Hook, 1977). In our study we scored 100 metaphases by standard G-banding. This excluded a 3% mosaicism at 95% confidence interval. For the detection of mosaicism less than 1%, analysis of at least 500 metaphases is necessary to prove the presence of low percent abnormal clones. This is
3 very difficult to do with conventional cytogenetic methods. Therefore the same samples were analyzed by the fluorescence in-situ hybridization technique to solve this problem.
The X chromosome was labeled with a dual probe containing the X centromere (DXZ1) as well as Xp terminal end (LSI STS Xp) probe. X centromeric probe (DXZ1) labeled in spectrum green and locus specific probes spanning the steroid sulfatase region (LSI STS
Xp) on the Xpter labeled in spectrum orange (as an internal control) from Vysis were used. DAPI was used as a counterstain and slides were visualized under a fluorescence microscope with the appropriate filters. Evaluating 5000 cells (both metaphases and interphases) will exclude exclude mosaicism at 95% confidence interval.
4
CHAPTER 2
LITERATURE REVIEW
5 2. Literature review
2.1: Turner syndrome
Dr.Henry Turner first described Turner syndrome in 1938 (Turner, 1938). It is a common sex chromosomal abnormality with an incidence of about 1 in 2500 liveborn female babies. Individuals with Turner syndrome display a female phenotype with typical features which include short stature, sexual infantilism due to rudimentary ovaries; a variety of somatic features will include micrognathia, prominent epicanthic folds, low set ears, cubitus valgus and a short and broad neck with webbing. Turner patients tend to have a high frequency of certain cardiovascular and renal abnormalities. The mental intelligence is usually normal (Lippe, 1991). This above phenotype is due to the absence of one X-chromosome leading to X-chromosome monosomy (45,X) in some or all cells or due to the presence of structurally abnormal X-chromosome resulting in the haploinsufficiency of X-Y homologous loci situated at the level of the pseudoautosomal region of the gonosomes which escape X-inactivation (Ogata et al., 1995).
2.2: Numerical anomalies of X chromosome leading to Turner syndrome:
2.2.1: X chromosome monosomy
The most common karyotype in Turner syndrome is the 45,X karyotype (45 chromosomes per cell, with only one sex chromosome) that represents 40 to 50% of cases
(Fig 1), whereas the normal female karyotype is 46,XX (Fig 2).
6 Figure 1: Classical karyotype of Turner syndrome
Figure 2: Normal female karyotype
7 There are two theories that try to explain this chromosomal monosomy (the loss of one of the sexual chromosomes).
According to the meiotic theory, during the formation of the ovule or sperms
(gametogenesis), some of them could have suffered an error and for this reason they carry one chromosome less. If the ovule or the sperm have suffered this chromosomal loss, the embryo formed from the fertilization will carry this chromosomal error. Monosomal aneuploidy is due to non-disjunction or failure of normal separation of a chromosome pair when the eggs or sperms are formed during meiosis. Normally the 46 chromosomes present in a cell are copied (replication) and paired up. The pairs of chromosomes are separated (segregation) during meiosis 1. During meiosis 2, a second division of the chromosomes occurs resulting in the formation of four sperms, or one egg and three polar bodies, each with 23 chromosomes. In the normal situation, the mature eggs and sperms are monosomic (one copy) for each chromosome. This leads to disomy (two copies of each chromosome) following fertilization.
Nondisjunction can occur in meiosis 1 or meiosis 2 (Fig 3 & 4). Nondisjunction leads to the formation of two chromosomally different eggs or sperms; one has a pair of chromosomes (disomic), and the other has no chromosome (nullisomic). The former, when fertilized by a normal egg or sperm, with one copy of each chromosome
(monosomic), leads to a trisomic fetus and the latter leads to a monosomic fetus.
8 Figure 3: Non-disjunction in meiosis 1
Figure 4: Non-disjunction in meiosis 2
In the mitotic theory, the loss of one of the chromosomes in the gametes (ovule or sperm) originates later, during the first period of the embryonic growth (in the first gestation
9 weeks). Anaphase lag is the mechanism where one chromosome simply fails to get incorporated into the nucleus of a daughter cell; or, a malfunction in chromosome sorting may find two identical chromosomes in the same daughter cell. This will result in mosaic
Turner syndrome, which is discussed in 2.2.2.
It has now been proven that the Turner syndrome is not necessarily due to the absence of entire X chromosome but is a result of haploinsufficiency of X-Y homologous loci that escape X-inactivation. Dosage compensation in mammals has been achieved by X- chromosome inactivation to allow the female to have the same amount of X-chromosome material as the average male. Lyon hypothesized that early in the development of a normal female embryo, random inactivation of one of the two X-chromosomes in each cell occurs. This inactivation of an X chromosome requires a gene on that chromosome called XIST. XIST encodes a large molecule of RNA which accumulates along the X chromosome containing the active XIST gene and proceeds to inactivate all (or almost all) of the other hundreds of genes on that chromosome. XIST RNA does not cross over to any other X chromosome in the nucleus. Barr bodies are the inactive X chromosomes
"painted" with XIST RNA (Bohorfoush et al., 1972).
During the early stages of embryonic development of a normal female, the XIST locus on each of her two X chromosomes is expressed. Transcription continues on one of the X chromosomes, leading to an accumulation of XIST RNA and converting that chromosome into an inactive Barr body. Transcription of XIST ceases on the other X chromosome allowing the hundreds of other genes to be expressed. The shut-down of the XIST locus on the active X chromosome is done by methylating XIST regulatory sequences. DNA
10 methylation usually results in gene repression, so methylation permanently blocks XIST expression and permits the continued expression of all the other X-linked genes (Gartler et al., 1983). However, some genes on the X chromosome escape inactivation. These are present on the pseudoautosomal regions (PAR) of both the X and Y-chromosomes. There are about 18 genes that are identical on both X and Y chromosomes and these genes escape inactivation in females to maintain a balance with the situation in males. In addition, there are other genes on the X chromosome that are not regulated by X inactivation whose expression is thus altered in Turner syndrome as compared to normal females (Brown et al., 1990; Fisher et al., 1990). In this way, the normal female has functioning genes from one complete X-chromosome along with functioning genes from the inactivated X-chromosome. On the other hand, Turner syndrome females, with X- chromosome aneuploidy lack the genes that would normally have remained active.
2.2.2: Mosaic Turner syndrome:
Fifteen to twenty percent of cases of Turner syndrome are cytogenetic mosaics with 45,X cells and clones of other cells with either 46,XX, 46,XY, 47,XXX or aberrant sex- chromosome complements. These result in variants like 45,X/46,XX,45,X/47,XXX,
45,X/46,XY etc (Abulhasan et al., 1999).
Chromosomal mosaicism is defined as the occurrence of 2 or more cell lines with different chromosomal make-up in an individual, developed from a single fertilized egg.
Turner syndrome mosaicism is an example of monosomy mosaicism specifically for the
X chromosome where, along with the normal diploid cell line, there is another cell line which has only one X chromosome instead of two (Kao et al., 1991). The cells with
11 abnormal chromosomes may be found in multiple tissues, or in just one tissue. Changes in the number or structure of chromosomes in different cells of the body can have variable impact on the proper functioning of the human body (Amiel et al., 1996). If only a tiny fraction of some tissues were involved, the aneuploidy would likely to have little effect on growth and development. However, a very minor degree of mosaicism could still be important if a crucial tissue carries the abnormal cells. As a general principle, an individual with a chromosome mosaicism in some of his or her tissues is likely to have less severe but qualitatively similar clinical features to that of someone with the non- mosaic form of the same chromosome abnormality.
The mosaic pattern depends on many factors.
• Mosaicism originating from an error, either in the first or second division of the
fertilized egg, leads to generalized mosaicism, since most tissues of the baby are
affected, often in a "patchy" way.
• An error that occurs at a later stage, for example at the 64-celled blastocyst stage, will
affect a smaller proportion of the cells in the baby. "Later errors" may lead to an
abnormal line of cells confined to a specific area or tissue in the developing
individual.
Age related mosaicism:
It is noted that 45,X cells are increasingly common in female blood cells as they age, but appear to have no harmful effect.
12 2.3: Structural anomalies of X chromosome:
Twenty five percent of cases with 46,XX karyotype have a structural alteration of one of the X chromosomes i.e., deletions, duplications or isochromosomes. In rare cases a ring
X chromosome complement can be identified. Structural X chromosome abnormalities are not unusual and occur as a result of breakages in the X chromosome with subsequent reunion of X chromosome sequences. These karyotypes include 46,X,i(Xq),
46,X,del(Xp), 46,X,del(Xq) and 45,X/46,X,r(X). The clinician must be aware of the differing susceptibilities of these various karyotypes, as the phenotype may be attributable to the limited amount of genetic material in these abnormal chromosomes
(Hook et al., 1983).
2.3.1: Isochromosome Xq [46,X,i(Xq)]:
This consists of the two long arms of the X-chromosome but no short distal arm (Fraser et al., 1989). It is the most common structural abnormality occurring in the Turner syndrome. The phenotype in these patients is similar to the phenotype of 45,X with perhaps an increased risk of autoimmune disorders (diabetes and thyroid disease) and is associated with deafness, but congenital abnormalities are conspicuously absent (Stratakis et al., 1994).
2.3.2: Ring X chromosome [45,X/46,X,r(X)]:
Intelligence is average or above average in Turner syndrome patients, except in rare cases of tiny ring X chromosomes. Mental retardation may be present in some cases due to the inability of these abnormal chromosomes to undergo X inactivation (Atkins et al, 1966;
13 Van Duke et al 1992). Recently, some cases in which the ring is small and does not contain the X-inactivation center have been described; the phenotype is abnormal with atypical Turner syndrome stigmata and severe mental retardation, possibly due to lack of dosage compensation (Dennis et al., 1993).
2.3.3: Deletions of the ‘p’ or ‘q’ arms of the X chromosome i.e. 46,X,del(Xp) or
46,X,del(Xq):
The tip of the Xp forms the meiotic pairing region and crossing over takes place in the pseudoautosomal region (PAR), which always stays active on both the chromosomes
(Burgoyne, 1983). The region adjacent to this PAR on Xp22.3 contains the SHOX gene important for long bone growth, deletion of which leads to short stature. The loss of
SHOX may also explain some of the skeletal features found in Turner syndrome, such as short fingers and toes, and irregular rotations of the wrist and elbow joints (Morizio et al.,
2003; Ogata et al 2001). Generally loss of the entire short arm from Xp11 to Xpter leads to a full-blown Turner syndrome.
The X centromere to the Xp11 region has been referred to as the active "b" region
(Therman et al., 1990) and may contain genes for gonadal development (Simpson et al.,
1987). The region from the X centromere to Xq13, which interestingly has never been found to be missing, contains the XIST gene that is always active on the inactive X chromosome.
In long arm deletions, Madan et al., (1981) postulated the so called critical region in the
Xq arm, for gonadal dysgenesis consisting of two segments, Xq13-q22 and Xq22-26,
14 separated by a short region in Xq22. Whereas loss of the Xp tip results in short stature, the tip of Xq has been postulated to have genes, loss of which lead to premature ovarian failure (Fitch et al., 1982). Surveys on various X chromosomal deletions, apart from the above mentioned characteristics show a surprising similarity in all the presenting symptoms. To explain this, a hypothesis was proposed that in Xq deleted patients the X- inactivation spreads to tip of Xp, thus inactivating the normally active X regions, hence it is the extent of X inactivation that causes the symptoms and not specific breakpoints
(Sharpe et al., 2002).
Table 1: Phenotypic features of X chromosome deletions with ideogram of X chromosome on the right hand side
Deleted regions Somatic stigmata Ovaries Stature of Turner syndrome Short arm Xpter~p21 Normal Short None All of Xp Dysgenesis Short Most or all Long arm Xqter~q22 Dysgenesis Normal None to few Xqter~q13 Dysgenesis Normal None to some All of Xq Dysgenesis Normal Several
Source: Modified from Table 13.3 of Epstein, 1986.
15 2.3.4: Patients with residual Y chromosome material:
Recent molecular studies done on peripheral blood cells have shown that some individuals with 45,X and even mosaic 45,X/46,XX have residual cytogenetically undetectable Y-chromosome material by cytogenetic methods (Muller et al., 1987;
Koncova et al., 1993; Shankman et al., 1995). The residual Y-chromosome material may not be present in the peripheral blood cells (Koncova et al, 1993). Thus fibroblasts and gonadal cells need to be studied if mosaicism for Y-chromosome DNA sequences is present; these patients are at increased risk for excessive virilization and increased risk of gonadoblastoma.
2.4: Clinical presentation of Turner syndrome through the different life stages:
Turner syndrome embraces a broad spectrum of features with almost all patients having ovarian dysfunction, short stature, somatic and visceral abnormalities; the severity of symptoms varies considerably amongst the individuals. The phenotype is complex and multiple (Judith et al., 1995). Female phenotype is due to the absence of Y-chromosome, the testis determining gene.
The clinical and presenting features of Turner syndrome change with age and can be divided into stages: embryonic period, newborn period, childhood period, adolescent period and adulthood (Hall, 1990).
Embryonic and fetal life:
Nearly 10% of spontaneously aborted fetuses have a 45,X karyotype and the incidence
16 has been estimated as 0.8% in zygotes, making it possibly the most common chromosomal disorder. Only 1% of human Turner zygotes survive to term. More than 95-
99% of 45,X conceptuses die during gestation (Simpson 1976, Hook et al., 1983).
It was observed that the early mortality is much less in 46,X,i(Xq) than in 45,X cases suggesting that the loss of loci on Xp may be lethal (Hook et al., 1983). Although the period of death may extend throughout the gestation, the vast majority of 45,X conceptuses die in the first trimester with a mean developmental age of 6 weeks (Boue et al., 1976).
Two explanations were considered for early death during gestation, which were not dissimilar to those regarding the lethality of autosomal aneuploidy.
• One explanation was that most 45,X abortuses have such severe congenital defects
that further viability is precluded (Burgoyne et al., 1983).
• The other explanation is that the problem is not with the 45,X embryos and fetuses
themselves but with their placentas. The aneuploid state probably interferes with the
placental growth and function so that the placenta is unable to sustain a normally
functioning embryo or fetus. This was suggested to be due to the compromised
placental steroidogenesis, thereby leading to an inability to maintain an otherwise
viable embryo and consequent spontaneous abortion (Burgoyne et al., 1983).
Is mosaicism necessary for survival in Turner syndrome?
The high percentage of fetal and embryonic miscarriage for karyotype 45,X points to the necessity of mosaicism for survival (Held et al., 1992, 1993). Natural selection does not
17 prevail when mosaicism is operative (Hook et al., 1983; Hassold et al., 1988), though the resulting phenotype is similar. Current hypothesis argues for the existence of a feto- protective effect (Porter et al., 1969; Held et al., 1992) of one or more genes on the X or
Y chromosome.
Newborn period:
The newborn may present with edema of the hands and feet, thick nuchal folds, cardiovascular malformations, like coarctation of aorta or hypoplastic heart, bicuspid aortic valve, aortic aneurysms etc., Genito-urinary abnormalities include horse-shoe kidney, silent hydronephrosis, malrotation etc., and auto-immune disorders like hypothyroidism, diabetes mellitus, inflammatory bowel diseases, rheumatoid arthritis may also be seen. A variety of somatic abnormalities like short neck with webbed appearance, low hairline at the back of the neck, micrognathia and low set ears are typical features of Turner syndrome. Weight and height at birth are below the mean for normal infants. Turner syndrome may be suspected in the newborn period because of a congenital heart defect that can be life threatening. Puffiness of hands and feet at birth are attributed to lymphatic obstruction and multiple pigmented nevi may be seen.
Childhood period:
The usual presenting feature in childhood is unexplained short stature, an invariant sign.
Skeletal maturation is normal or only slightly delayed during childhood, but lags in adolescence due to sex steroid deficiency. They may also present with some of the skeletal features such as short fingers and toes, irregular rotations of the wrist and elbow
18 joints. Linear growth is attenuated in utero and statural growth lags during childhood and adolescence. Developmental problems such as speech delay and neuromotor deficits as well as learning disabilities of variable severity are common, though mental retardation
(IQ<70) is rare. Cardiac anomalies that were previously not detected may also become apparent during childhood.
Adolescent period:
In adolescence failure to initiate puberty along with primary or secondary amenorrhea and absence of breast development is the common feature in Turner individuals. The short stature may also be marked in adolescence.
Adult period:
As in adolescents, the presenting features of adult women with Turner syndrome are also related to hormonal failure, which include amenorrhea, infertility and premature menopause with raised levels of luteinizing and follicle stimulating hormones.
In rare cases, enough ovarian development persists that the Turner syndrome patient undergoes normal pubertal maturation (with modest growth spurt) and menarche, and then goes into premature menopause. This was first reported in a 141/2 year old Turner girl who entered puberty at 10 years of age, experienced menarchy at 11 years, and had regular menses thereafter (Weiss et al., 1971).
In spite of the previous concept that “the gonad does not develop in Turner syndrome”
(Jaffe, 1986), Carr and associates recognized that the ovaries are normal in early fetal life.
19 Nevertheless at birth most of them have recognizable ovaries (Carr et al., 1968). In vast majority of cases the high rate of follicular atresia into dysgenetic streak gonads typical of Turner syndrome occurs in the early first decade of life. However, in some cases follicular loss is more gradual, and varying numbers of follicles persist longer.
The number of follicles is sometimes sufficiently adequate that antral follicles are capable of secreting estrogen that results in initiation of feminization in perhaps 5 to 10% of
Turner syndrome. If a major number of antral follicles are at a menopausal level at this time, puberty does not progress to menarche.
Philip and Sele reported a similar ovarian picture in a fertile patient with 45,X Turner syndrome (Philip et al., 1976). The duration of menstrual periods in between is variable and ranges from nil to decades. If menstruation persists long enough, fertility is possible
(Abir et al., 2001) However, fertile patients apparently have a relatively high incidence of fetal wastage and chromosomal non-disjunctional abnormalities, including Turner and
Down syndrome (King et al., 1978). Spontaneous pregnancies have also been recorded.
An interesting hypothesis is that mosaicism may be present in these patients at very low levels.
20 Table 2: Summary and percentage of occurrence of some of the physical findings in
Turner syndrome
(Goldman et al., 1982; Hall et al., 1990; Gotzsche et al., 1994; Hulcrantz et al., 1994;
Haddad et al., 1999).
Finding Incidence (%)
Short stature 98-100
Gonadal dysgenesis 95-98
Short neck 80
Broad chest 75
Hearing impairment 50-70
Cubitus valgus 45-75
Renal abnormalities 37-60
Nevi 25-70
Cardiovascular malformations 20-55
Lymphoedema 21-70
Diabetes mellitus 40
Low posterior hairline 40
Short metacarpals 35-65
Thyroiditis 35
Genu valgum 30
Webbed neck 23
Extracted from: Turner syndrome in a life span perspective by Albertsson-Wikland and Ranke, research and clinical aspects : proceedings of the 4th International Symposium on Turner Syndrome, Gothenburg, Sweden, 18-21 May, 1995 / editors, Kerstin Albertsson- Wikland, Michael B. Ranke. Amsterdam ; New York : Elsevier, 1995.
21 2.5: Fluorescence in-situ hybridization:
Fluorescence in-situ hybridization (FISH) is a technology utilizing fluorescently labeled
DNA probes (with a fluorescent dye or a hapten, usually in the form of fluor-dUTP or hapten-dUTP) to detect or confirm gene or chromosome abnormalities that are generally beyond the resolution of routine cytogenetics. The sample DNA (metaphase chromosomes or interphase nuclei) is first denatured a process that separates the complimentary strands within the DNA double helix structure. The fluorescently labeled probe of interest (also single stranded) is then added to the denatured sample mixture and hybridizes with the sample DNA at the complimentary sites as it reanneals (or reforms itself) back into a double helix. This attachment can be seen as a fluorescent probe signal by a fluorescence microscope and the sample DNA can be scored for the presence or absence of the signal.
Figure 5: Illustration of FISH
From NHGRI website. http://www.genome.gov/page.cfm.pageID=10000552
22 2.5.1: Probes in FISH:
There are different types of FISH probes used for various purposes.
Gene-specific probes or locus specific probes:
These probes target specific genes or specific nucleic acid sequences within the chromosomes approximately ranging from 110-200 kbs. These sequences of genes of interest are inserted into plasmids, cosmids, BACs and YACs. These probes have proven particularly useful in the study of microdeletion syndromes, where the absence of a gene often goes undetected by conventional banding methods. Gene-specific probes are also useful for mapping genes on chromosomes.
Centromeric probes:
These probes bind to repetitive sequences that are specific to the centromeric regions and are used specially for enumeration. Centromeres frequently contain A–T-rich tandem repeats, several hundred to thousand times in the centromeric regions each about 106 to
108 bp in size. They belong mainly to the alpha satellite (50-52) or satellite III families.
Alpha satellite sequences comprise of 171 bp monomers, whereas satellite III is made of
5 bp monomers. The beta satellite probes (Oncor) hybridize to the heterochromatic regions on chromosome 1 and the acrocentric chromosomes 13, 14, 15, 21 and 22 (Gole et al., 1997). Centromeric probes have applications in the identification of marker chromosomes and numerical chromosome abnormalities in interphase nuclei and sex determination in prenatal diagnosis.
23 Telomeric probes:
They recognize the repetitive sequence TTAGGG, and can be used to visualize all telomeres simultaneously. Telomeric probes and subtelomere-specific probes are used to identify cryptic chromosomal translocations such as those occurring in cases of unknown mental retardation and for telomere length measurement.
Whole chromosome probes or Chromosome-painting probes:
DNA pools from each human chromosome are directly labeled with one or more of 5 fluorophores in a combinatorial fashion resulting in a unique color signature of each of the 24 chromosomes. This technique is useful for identifying chromosome arms that are involved in translocations, as well as for marker chromosomes and ring chromosomes
(Carter et al., 1992).
2.5.2: Commonly used fluorescent dyes:
The most frequently used fluorochromes for the detection of the FISH signals are FITC
(fluorescein isothiocyanate) that emits a green fluorescence and dyes with orange or red fluorescence, such as Cy3, or TexasRed. Another commonly used fluorochrome in FISH experiments is Cy5 that emits in the far red/close infrared. As this fluorescence is not visible to the naked eye it would need detection by an infrared sensitive camera mounted on the microscope.
Since the emission spectra of some of the dyes partly overlap the selection of fluorochromes in multi-color experiments is often a compromise. To achieve a good
24 separation of dyes filters which do not fit 100% the emission maximum of the particular dye have to be used. Thus, only part of the actual emission spectrum of the fluorochrome is utilized to avoid "cross talk" with the emission of another fluorochrome (Table 3).
Table 3: Excitation and emission maxima of various fluorochromes used in FISH experiments
Blue Turquoise Green Orange Red
Dyes DAPI DEAC FITC/R110 TAMRA/Cy3 TexRed/Cy3.5
ExMax. 358 426 494/500 552/550 590/581 EmMax. 461 480 517/525 575/570 612/596 Extracted from Chambrios molecular cytogenetics http://www.chrombios.com/WebFinals/AboutFISH
Counter staining chromosomes and nuclei
For FISH experiments, chromosomes and cell nuclei are generally counter-stained using a fluorescent dye that is specific for DNA. The most common dye is DAPI (4´, 6-
Diamidino-2-phenyl-indol) that stains the chromosomes and cell nuclei resulting in a bright blue fluorescence. Another common counter stain is propidium iodide which shows a red fluorescence.
25 2.5.3: Applications of FISH:
FISH can be used in interphase as well as metaphase for analysis of chromosomal anomalies. In the interphase it is mainly used for aneuploidy detection. In the metaphase it can be used for detection of not only aneuploidies, but also structural rearrangements e.g. deletions, duplications, translocations etc. Based on this, it is utilized in diagnosis as well as prognosis in fields of
• Prenatal diagnosis
• Cancer cytogenetics
• Genetic diagnosis of preimplantation embryos
• Adolescent menstrual disorders, infertility in adults, pediatric disorders
2.5.4: Advantages of FISH:
Fluorescence in situ hybridization (FISH) is a rapid reliable technique in molecular cytogenetics. It supplements conventional karyotyping and in some cases provides additional information. The primary advantage of interphase FISH is that it can be performed very rapidly, if necessary within 24 hours, as cell growth is not required.
Hence, it permits analysis of cells in interphases as well as metaphase. FISH is a sensitive and useful adjunct to cytogenetic testing for the detection of abnormalities of chromosomal structure or numbers e.g. deletions, translocations, duplications, and aneuploidy. It is often the method of choice for detection of microdeletions. A large number of cells are available for quantitative analysis by FISH and it helps in detection of minimal residual disease and disease recurrence, as a very small percentage of abnormal
26 cells can also be identified. The distinct hybridization signals seen in metaphase as well as interphase nuclei help in rapid diagnosis, thereby shortening the reporting time. It also aids the confirmation of chromosomal anomalies in standard karyotyping for prenatal and pre-implantation genetic diagnosis and cancer cytogenetics.
2.5.5: Fluorescence in-situ hybridization (FISH) as a sensitive tool in detecting mosaicism:
FISH can diagnose chromosomal mosaicism as early as the preimplantation stage by studying polar bodies or later in blastomeres. This is followed by analysis at early gestational ages by testing chorionic villi cells and later by amniotic fluid or fetal blood analysis. Peripheral blood lymphocytes or skin fibroblasts are utilized for analysis in the postnatal period. Held et al., (1992) demonstrated the power of FISH to detect low-level mosaicism in peripheral blood cells. In their studies using conventional cytogenetic methods alone, the prevalence rate of 45,X in 45,X mosaics was 54.3%, a figure in good agreement with the 55-61.2% range reported in four major studies (Palmer et al., 1976;
Hall et al., 1982; Ranke et al., 1983; Park et al., 1993). However, on re-investigation by both conventional cytogenetic methods and FISH the prevalence rate dropped to 30% leading to a decrease in the ratio 45,X mosaics from 1.6 to 0.5. According to them, the sensitivity of detecting micro mosaics can be further improved by using FISH and PCR and RT-PCR techniques (Hernandez-Herrera et al., 2003).
27 2.6: Hypothesis of this study:
In many instances there are features of the Turner syndrome, but the resulting karyotype is normal by conventional method. When karyotype analysis does not correlate with the clinical phenotype, it is very puzzling for the clinicians as well as distressful to the patients. One of the reasons for this discrepancy could be that some of the cases may have a low-grade mosaicism i.e. a small minority of cells may be monosomic for the X chromosome.
Vice versa, the high percentage of fetal and embryonic miscarriage for karyotype 45,X points to the possibility of presence of a cell line with 46,XX for survival either in the fetus or extra-embryonic tissues of live born 45,X Turner syndrome females (Held et al.,1992, 1993).
2.7: GOALS OF THE STUDY:
1) Detection of the percentage of monosomic X cells in karyotypically normal patients
with Turner stigmata by FISH technique (Group I).
2) Detection of the percentage of normal 46,XX cells in karyotypically proven Turner
patients (Group II).
3) Proposition of a cut-off value for abnormal X cell incidence beyond which low grade
mosaicism can be classified as significant. This was achieved by analyzing a control
group of women, age matched to the patients (Group III).
28
CHAPTER 3
PATIENTS & METHODS
29 3.1: Patients
Study groups included patients with Turner stigmata who were referred by clinicians for
G-banded chromosomal karyotype analysis to confirm the diagnosis of Turner syndrome.
After analysis, those patients who showed a normal karyotype were grouped as study group I and FISH was done to exclude low-grade X chromosome mosaicism. Patients with 45,X cells on G-banding and confirmed as having Turner syndrome were grouped as study group II and the percentage of normal 46,XX cells was investigated.
Study group I: Patients with Turner stigmata but normal karyotype (n=11).
Study group II: Turner patients with a 45,X cell line (n=17).
Control group III: This group included the control group of normal fertile females (n=25).
An information leaflet about the procedures and implications was explained to the patients. Informed consent was obtained from all the participants in this study.
A form (Appendix 2) with details of the clinical features was given to the consultants and the clinical features for referral are presented below.
3.1.1: Group I: - Details of karyotypically normal patients with Turner stigmata :
Case 1:
A 15-years-old Chinese girl presented with short stature, webbed neck, widely spaced nipples, shield type of chest and primary amenorrhea. Other features included slant eyes, mild proptosis with frontal bossing, low-set ears, high arched palate, increased carrying
30 angle of the elbow, finger and toenail deformities, multiple nevi, low posterior hairline, clinodactyly and mild prolapse of atrial and mitral valves in the heart. Her bone age was less than her chronological age. Her thyroid hormone profile was normal [T4:15.5 pmol/l
(11-22), TSH: 1.09 mU/l (0.5-4)].
Case 2:
A 15-years-old Chinese girl presented with short stature (139.5cms), low-set ears and high arched palate. There was no history of primary or secondary amenorrhea.
Case 3:
A 31-years-old Chinese lady presented with secondary amenorrhea of 14 years duration.
Other features included short stature, thick nuchal fold and wide carrying angle of the elbow.
Case 4:
A 17-years-old Indian girl presented with primary amenorrhea, short stature, microcephaly and low IQ.
Case 5:
A 13-years-old Malay female presented with short stature, hirsutism and elevated
Testosterone levels, primary amenorrhea.
31 Case 6:
A 21-years-old Chinese female presented with Primary amenorrhea. She was of average height.
Case 7:
A 15-years-old Chinese girl presented with short stature, prepubertal ovarian failure
(FSH: 3.9IU/l, LH :< 0.1IU/l) and primary amenorrhea.
Case 8:
A 16-years-old Chinese girl presented with short stature, short neck, cubitus valgus, high arched palate and amenorrhea.
Case 9:
A 33-years-old Indonesian female presented with secondary amenorrhea and was diagnosed as premature ovarian failure.
Case 10:
A 19-years-old Chinese girl presented with features of Turner syndrome which included amenorrhea, short stature, webbed neck, cubitus valgus, high arched palate and delayed bone age. Her breast development was at stage 1. She was also diagnosed with hypothalamic pituitary failure by the clinician because of hypogonadotrophism.
32 Case 11:
A 17-years-old Indian girl presented with secondary amenorrhea. She attained menarche at 16 years of age. Subsequently, she was diagnosed with premature ovarian failure and follicle stimulating hormone level was high.
3.1.2: Group II: -Details of karyotypically proven Turner patients:
Case 1:
A 16-years-old girl presented with short stature, high arched palate, multiple pigmented nevi and finger and toenail deformities (hyperconvex nails).
Case 2:
A 20-years-old Chinese lady diagnosed as Turner syndrome presented with amenorrhea, short stature, webbed neck, high arched palate etc. She was on hormone replacement therapy.
Case 3:
A 9-years-old Chinese girl presented with short stature, high arched palate, low-set ears, multiple pigmented nevi, finger nail and toenail deformities (hyperconvex nails).
Case 4:
A 16-years-old Malay female presented with short stature, short neck and high arched palate.
33 Case 5:
A short 13-years-old Chinese girl presented with delayed puberty (delayed breast development), short stature and multiple pigmented nevi.
Case 6:
A 32-years-old Chinese female presented with secondary amenorrhea diagnosed as premature menopause (increased FSH: 100IU/L; decreased estradiol: 19 pmol/L).
Case 7:
A 17-years-old Chinese female presented with features of Turner syndrome such as amenorrhea and short stature.
Case 8:
A 6-years-old Malay girl presented with short stature, low-set ears, and low posterior hairline. Her karyotype showed 45,X.
Case 9:
A 9-years-old Malay girl presented with short stature, low posterior hair line and low-set ears.
Case 10:
A 25-years-old Chinese female presented with short stature and primary amenorrhea.
34 Case 11:
A 21-years-old Chinese lady presented with primary amenorrhea and low-set ears. She was of average height. She had no other physical abnormalities. She was on hormonal therapy.
Case 12:
A 34-years-old Chinese female presented with secondary amenorrhea. She attained menarche at the age of 12 years after which her periods were irregular and stopped subsequently. She was short and had low-set ears, high arched palate and low posterior hairline. She was on hormonal therapy.
Case 13:
A 20-years-old Chinese female presented with primary amenorrhea and short stature. She had no other stigmata. She was on hormonal therapy.
Case 14:
A 16-years-old Chinese lady, a known case of Turner syndrome on hormonal therapy, presented with short stature and amenorrhea.
Case 15:
A 21-years-old girl presented with primary amenorrhea and short stature. She is on hormonal treatment.
35 Case 16:
A 30-years-old lady presented with amenorrhea and short stature and she was on hormonal treatment.
Case 17:
A 16-years-old female presenting with features of Turner syndrome like primary amenorrhea and short stature.
3.1.3: Group III:-Details of the control females:
Control 1:
A 25-years-old Indian female presented with normal menstrual history and hormone profile. She was of average height with no abnormal physical findings.
Control 2:
A 26-years-old normal Indian female with regular menstrual history. Physical examination and hormonal profile were normal.
Control 3:
A 29-years-old healthy Indian female with normal menstrual history. She was of average height. Her hormonal profile was normal. Physical examination could not detect any abnormalities.
36 Control 4:
A 27-years-old Indian pregnant female with an uneventful previous pregnancy which ended in a full term normal delivery. Her hormonal profile was normal before pregnancy.
She was 155cms tall.
Control 5:
A 26-years-old tall (160cms), healthy Chinese had regular menstrual history. Her hormonal profile was normal.
Control 6:
A 23-years-old healthy Burmese female had normal menstrual history and hormone profile. She was 152 cms tall.
Control 7:
A 29-years-old healthy, tall (169cms) Indian female with a previous full term normal delivery. She attained menarche at 13years of age with regular menstrual history (4/24).
Physical examination was normal.
Control 8:
A 35-years-old normal, healthy Chinese female with regular menstrual history had normal hormonal profile. Physical examination was also normal. She was of average height (155cms).
37 Control 9:
A 33-years-old healthy, tall (162cms) Indonesian pregnant female had a previous full term normal delivery. She attained menarche at about 12years of age and had regular menses. Physical examination was normal.
Control 10:
A 31-years-old healthy Indian primigravida with normal menstrual history. She was of average height (157.5cms).
Control 11:
A 35-years-old normal Malaysian pregnant female with two previous uneventful full term normal deliveries. She attained menarche at 10 years of age and had regular menstrual history.
Control 12:
A 26-years-old pregnant Indian female with a previous full term normal delivery, had a normal menstrual history. She attained menarche at about 13 years of age.
Control 13:
A 38-years-old tall (168cms), healthy Indian female with two previous full term uneventful normal deliveries had a regular menstrual history. Her physical examination was normal.
38 Control 14:
A 31-years-old tall (165cms), healthy pregnant Indian female had regular menstrual history. Her previous pregnancy ended in a full term normal delivery. She attained menarche at the age of 12 years. Physical examination was normal.
Control 15:
A 30-years-old pregnant Indian female had normal menstrual history. Her previous pregnancy was uneventful and ended in a full term normal delivery. She was 160 cms tall. Her physical examination was normal.
Control 16:
A 34-years-old healthy Indian lady had two previous uneventful pregnancies and full term normal deliveries. Her menstrual history was normal and she was of average height
(158cms). Physical examination and hormonal profile was normal.
Control 17:
A 32-years-old healthy pregnant Indian lady had a previous full term normal delivery.
She attained menarche at the age of 12 years and had regular periods. Physical examination was normal. She was of average height (155cms).
Control 18:
A 28-years-old healthy pregnant Chinese female had a previous uneventful pregnancy
39 and a full term normal delivery. She attained menarche at the age of 11 years and had a regular menstrual history. Her physical examination was normal. She was 160cms tall.
Control 19:
A 27-years-old healthy primigravida of Indian origin had a regular menstrual history. She was 165 cms tall. She attained menarche at the age of 10 years. Her physical examination was normal.
Control 20:
A 32-years-old healthy pregnant Chinese lady had a previous full term normal delivery.
She attained menarche at the age of 12 years and had a regular menstrual history. She was
160 cms tall and her physical examination was normal.
Control 21:
A 27-years-old healthy primigravida of Indian origin had a regular menstrual history. She was 167 cms tall and had a normal physical examination.
Control 22:
A 26-years-old Indian primigravida had a regular menstrual history. She attained menarche at the age of 12 years. Her physical examination was normal. She was 165 cms tall.
40 Control 23:
A 32-years-old healthy pregnant Indian female had a previous uneventful pregnancy and a full term normal vaginal delivery. She attained menarche at 12 years of age and had a regular menstrual history. She was 160 cms tall and had a normal physical examination.
Control 24:
A 30-years-old pregnant Indian female had a normal, regular menstrual history. Her previous pregnancy was uneventful and she had a full term normal vaginal delivery. She was 155 cms tall. Her physical examination was normal.
Control 25:
A 28-years-old healthy Indian primigravida had a regular menstrual history. She attained menarche at the age of 13 years and was 160 cms tall. Her physical examination was normal.
41 3.2: Methods
In all the groups, mitogen stimulated short-term peripheral blood lymphocyte cultures were carried out and metaphase spreads were treated with trypsin-Leishman to obtain G- bands using the standard techniques. 5 metaphases were analyzed and 100 G-banded metaphase spreads were scored with special reference to the X chromosomes.
FISH analysis using X centromeric probes (DXZ1) labeled in spectrum green and LSI
STS Xp region probes labeled in spectrum orange (as an internal control) from Vysis was performed. DAPI was used as a counterstain and slides were visualized under a fluorescence microscope using the appropriate filters. 5000 cells (both metaphases and interphases) were scored.
3.2.1: Specimen collection
Peripheral blood was collected from two study groups and one control group of human volunteer donors in lithium heparin Vacutainer tubes. All collection and research protocols were approved by the local ethical review boards. The blood samples were kept at room temperature and processed within 3-4 hrs on the same day.
Lymphocyte culture
Mitogen (phytohaemagglutinin) stimulated short-term peripheral blood lymphocyte culture was done using PB Max karyotyping medium (Gibco cat no. 12557-013).
42 Set-up:
• To 5mls of complete media about 0.4mls (approximately 9 drops) of heparinised
blood was added and incubated at 37˚C for 70-71 hrs.
Mitotic arrest:
The lymphocytes were arrested at metaphase stage because the chromosomes are maximally condensed and can be visualized well under the microscope. Colcemid, an analog of colchicine that is less toxic to cells is used for this process. Three parameters: mitotic index, spreading and chromosome condensation are affected depending on
Colcemid concentration and time of exposure. Colcemid continues to be active until the addition of fixative. Higher concentrations and longer exposure times result in good mitotic index but shorter chromosomes.
• 100µl of Colcemid (Sigma Cat no.9754) 0.6µg/ml was added to T25flask after 70-
71hrs and incubated for about 60 minutes at 37˚C.
• After the Colcemid incubation, the blood cultures were centrifuged at 1200 rpm for 5
minutes in a Beckman centrifuge.
Hypotonic treatment:
The addition of the hypotonic solution, potassium chloride (KCl), will result in water rushing into the cells due to the concentration gradient caused by the less-than- physiological concentration outside of the cells. This results in swelling of cells and further separation of the chromosomes.
43 • Prewarmed 0.075 M KCl (0.56%) was added to the cells and incubated at 37˚C for
about 15 minutes. Cell suspension was centrifuged at 1200rpm for 5 minutes.
Fixative treatment:
With the addition of Carnoy’s fixative (3:1:: absolute methanol: glacial acetic acid), each cell is preserved in its swollen shape by extracting the surrounding material, removing lipids and denaturing proteins thus making the cell membrane very fragile, which helps chromosome spreading and fixing the chromosomes. Chemical changes make the cell hard (not as fragile as in hypotonic), but remaining a swollen sac of suspended chromosomes.
• Supernatant was discarded (buffy coat seen at this stage) and 7.5mls of cold freshly
prepared fixative was added slowly while vortexing. The cell suspension was chilled
at 4˚C for about 30-45 minutes or overnight. The cells were centrifuged at 1200rpm
for 5 minutes in the Beckman centrifuge.
• Supernatant was discarded (contains lysed red blood cells and cellular debris) and the
cells were resuspended in about 5mls of Carnoy’s fixative and washed 2-3 times till
the supernatant was clear (until all red cells were lysed and a white cell pellet was
observed). The pellet was stored at 4˚C until chromosome preparation.
• Metaphases were prepared on moist slides (by dropping cell suspension) and then
checking under phase contrast microscope for the quality of metaphase spread.
44 Slides were aged by
• Baking in the oven at 90˚C for 1 hour.
• Treating with ultra violet exposure for 55-60 seconds.
• Treating with methanol for 5 minutes.
• Treating with prewarmed 25% H2O2 for 1.5-2 minutes
3.2.2: GTL (G-bands by trypsin using Leishman) banding:
Giemsa bands are obtained by digesting the chromosomes with proteolytic enzyme trypsin. With G-banding chromosomes exhibit light and dark stained regions along their length. The mechanism is not clear but it is hypothesized that the differences between positive and negative bands may be due to the distribution of chromosomal proteins and
DNA (dark bands are relatively rich in disulfides and the light bands are rich in sulfhydryls). Further the dark bands are gene poor and the light bands are gene rich regions.
• Slides were treated with Trypsin (1:40:: Trypsin: 0.9% NaCl) for 20-30 seconds. The
slides were then stained with Leishman’s dye (1:5:: Leishman stain: PBS) for 3.5-4
minutes and washed with phosphate buffered saline (PBS).
• In this study 5 metaphases were analyzed in detail, counted 25 entire metaphases and
scored an additional 70 metaphases for the X chromosomes using the bright field
microscope.
45 3.2.3: Dual color fluorescence in-situ hybridization:
The Vysis LSI Steroid Sulfatase (STS) probe, a dual color probe containing the Spectrum
Orange STS (Xp22.3) probe and the Spectrum Green CEP X control probe (Xp11.1- q11.1) was used (Vysis-32-191004) (Fig.6). Two independent observers did FISH analysis. The supervisor or senior staff of the cytogenetic lab, Department of Obstetrics and Gynecology, always analyzed 500 cells out of 5000. The probe used from Vysis was
100% efficient (100% sensitive and 100% specific) as stated by the Quality Assurance certificates on each batch of probe used (Appendix 3).
After the UV treatment of the slides for 40 seconds, probe along with the hybridization buffer was applied on the slide and covered with a coverslip and sealed with rubber cement.
• The chromosomes were co-denatured along with the probe at 75˚C for 1 minute in
the Vysis Hybrite machine.
• Hybridization was carried out for 16-24 hours at 37˚C.
• After the hybridization was complete, the slides were washed using the rapid wash
procedure to wash off non-specific bound probe.
Rapid Wash procedure:
Wash solution 1: 0.4X SSC/0.3% NP-40
Wash solution 2: 2XSSC/0.1% NP-40
• Slides were kept in the first wash solution at 73±1˚C for 2 minutes in a water bath.
46 • Slides were gently rinsed in the second wash solution for 5minutes at room
temperature.
Counterstaining and visualizing the hybridization:
• The chromosomes were counterstained using DAPI (4',6’-Diamidino-2-
phenylindole) from Vectashield.
• The slides were visualized under a fluorescence microscope with the appropriate
filters.
The Applied Imaging FISH software was used for image analysis. About 5000 cells
(both metaphases and interphases) were scored for the X chromosome. Signals were classified as individual signals when the distance between them was greater than the diameter of a single signal. As the X centromeric probe was larger (thousands of kilobases), it had a tendency to split.
47 Figure 6: Xpter probe on the X chromosome
48 Figure 7: X-Chromosome showing the position of the Xp and Xcen probes used in the
FISH experiment
49
CHAPTER 4
RESULTS
50 4. RESULTS
4.1: Group I- Results of patients with Turner stigmata with a normal karyotype:
In this group FISH analysis showed a low percentage of monosomy X cells not detected by the routine karyotyping.
Table 4: Summary of FISH results in patients with Turner stigmata with a normal karyotype
Case Age/sex Race Phenotype Karyotype FISH-Cultured No.
Total: 5469 cells
1 15/F Chinese PA,SS,WN,CV,LSE,LPH XX : 5458 (99.8%) 46,XX[100] X : 11 ( 0.2%)
Total: 6291 cells 2 15/F Chinese SS.LSE,HAP 46,XX[100] XX : 6285 (99.85%) X : 6 ( 0.15%)
Total: 3301 cells 3 31/F Chinese SA,SS,WN, 46,XX[100] XX : 3220 (97.39%) X : 81 ( 2.51%)
51
Total: 5000 cells 4 17/F Indian PA,M,TNF 46,XX[100] XX: 4935 ( 98.70%) X: 49 (0.97%) XXX: 16 ( 0.33%) 1.3%
Total: 5000 cells XX: 4922 (98.45%) 5 13/F Malay PA, SS 46,XX[100] X: 54 ( 1.07%) 1.55% XXX: 24 ( 0.48%) Total: 5613 cells 46,XX[120] XX: 5460 (97.28%) 6 21/F Chinese MA /47,XXX[10] X: 53 ( 0.94%) 2.72% XXX: 100 ( 1.78%) Total: 4586 cells XX: 4509 (98.33%) 7 14/F Chinese SS,POF 46,XX[100] X : 76 ( 1.65%) XXX: 1 ( 0.02%) 1.67% Total: 5441 cells XX: 5412 (99.47%) 8 16/F Chinese SN,HAP, 46,XX[100] X : 25 ( 0.46%) XXX : 4 ( 0.07%) 0.53% Total: 5328 cells XX: 5270 (98.91%) 9 19/F Chinese PA,SS,WN,CV ,SN 46,XX[100] X : 46 ( 0.86%) 1.09% XXX : 12 ( 0.23%)
52 Total: 4942 cells XX: 4806 (97.36%) 10 33/F Chinese POF 46,XX[100] X: 86 ( 1.74%) 2.74% XXX : 50 ( 1 %)
Total: 5000 cells XX : 5000 (100%) 11 20/F Indian SA 46,XX[]100] X: 0 ( 0%)
CV(Cubitus Valgus); HAP(High arched palate); LPH(Low posterior hairline); LSE (Low Set Ears); M(Microcephaly);MA(Mullerian agenesis);MPN(Multiple pigmented nevi); PA(PrimaryAmenorrhoea); POF(Premature ovarian failure); SA(Secondary Amenorrhea);
SN(Short neck); SS(Short stature); TNF(Thick nuchal fold); WN(Webbed Neck);
53 4.2: Group II: Results of Turner patients with a 45,X cell line by G-banding:
Table 5: Summary of FISH results of Turner patients with a 45,X cell line by G-banding
Case number Age/Sex Race Phenotype Karyotype FISH Total: 6923 cells XX: 4494 (64.9%) X : 2429 (35.1%) 1 16/F Chinese SS,HAP,MPN 45,X[47]/46,XX[53] Total: 3181cells, XX: 16 ( 0.5%) X: 3000 (94.3%) 99.5% 2 20/F Chinese PA,SS 45,X[94]/47,XXX[6] XXX: 165 ( 5.2%) Total: 2209 cells XX: 92 ( 4.2%) 3 9/F Chinese SS,HAP,MPN,LSE 45,X[94]/46,X,+r[6] X: 2117 (95.8%) Total: 2008 cells XX: 1596 (79.5%) 4 16/F Malay SN,SS,HAP 45,X[9]/ 46,X,der(X) [51] X : 412 (20.5%)
54
Total: 4516 cells XX: 61 ( 1.35%) X: 4425 (97.99%) 5 13/F Chinese DP,SS, 45,X[241]/46,XX[9] XXX: 30 ( 0.66%) 98.65%
Total: 5316 cells XX: 167 ( 3.14%) X: 3437 (64.66%) 96.86% 6 32/F Chinese SA,POF 45,X[72]/47,XXX[28] XXX: 1712 (32.2%) Total: 5798 cells XX: 11 ( 0.21%) X : 5749 (99.13%) 99.79% Xp-: 38 ( 0.66%) 7 17/F Malay PA 45,X[98]/46,X,del(X)[2] Total: 4995 cells XX : 3 ( 0.06%) X: 4990 (99.9%) 99.79% Xp-: 2 (0.04%) 8 6/F Malay SS,LSE,LPH,CV 45,X[100]
Total: 3001 cells SS,LPH,LSE XX: 2665 (88.8%) 9 9/F Malay 45,X[10]/46,XX[90] X: 336 (11.2%)
Total: 5005 cells XX : 5 ( 0.1%) 10 16/F Chinese PA,SS 45,X [100] X: 5000 (99.9%)
55 Total : 5000 cells XX : 3223 (64.46%) 11 21/F Chinese PA,LSE 45,X[30]/46,XX[70] X 1777 (35.54%)
Total: 4349 cells XX: 378 ( 8.69%) X: 3500 (80.48%) 91.31% SA,SS,HAP,LSE, XXX: 471 (10.83%) 12 34/F Chinese LPH 45,X[85] / 46,XX[15]
Total: 5000 cells 45,X[5]/46,XX[95] XX: 4750 (95 % ) 13 20/F Chinese SS,PA X : 250 ( 5% )
Total: 5000 cells XX: 97 (1.95%) 14 16/F Chinese SS,PA 45,X[99]/46,XX[1] X : 4903 (98.05%)
Total: 6270 cells XX : 2660 (42.42%) X : 3600 (57.42%) 57.58% 15 21/F Chinese PA 45,X[55]/46,XX[45] XXX: 10 ( 0.16%)
Total: 5001 cells XX: 2400 (47.99%) X : 2600 (51.99%) 52.01% 16 30/F Chinese PA,SS 45,X[50]/46,X,r(X) [50] XXX: 1 ( 0.02%)
56
Total: 5000 cells XX: 2000 (40%) 17 16/F Chinese PA 45,X[60]/46,XX[40] X: 3000 (60%)
CV(Cubitus Valgus); HAP(High arched palate); LPH(Low posterior hairline); LSE (Low Set Ears);
M(Microcephaly);MA(Mullerian agenesis);MPN(Multiple pigmented nevi); PA(PrimaryAmenorrhoea); POF(Premature ovarian failure); SA(Secondary Amenorrhea); SN(Short neck); SS(Short stature); TNF(Thick nuchal fold); WN(Webbed Neck)
57 4.3: Group III -Results of the control group:
Table 6: Summary of FISH results of controls
25 normal fertile females age-matched to the patients were also studied as the controls.
Case Age/Sex Race Phenotype Karyotype FISH Total: 5297 cells XX: 5268 (99.45%) X: 20 ( 0.38%) 0.55% XXX: 9 ( 0.17%) 1 25/F Indian Normal 46,XX[100] Total: 5134 cells XX: 5116 (99.65%) X: 12 ( 0.23%) XXX : 6 ( 0.12%) 0.35% 2 26/F Indian Normal 46,XX[100] Total: 5084 cells XX: 5065 (99.63%) 3 29/F Indian Normal 46,XX[100] X : 19 ( 0.37%) Total: 5025 cells Normal, XX: 5010 (99.7%) G2,P1,L1 X: 13 ( 0.26%) 0.3% 4 27/F Indian Ht:155cms 46,XX([100] XXX: 2 ( 0.04%) Normal,Primigravida,Ht- Total: 5035 cells 5 26/F Chinese 160cms 46,XX([100] XX: 5035 (100%) Total: 5047 cells XX: 5024 (99.54%) X: 22 ( 0.44%) 6 23/F Myanmar Normal,Ht:152cms,unmarried 46,XX[100] XXX: 1 ( 0.02%) 0.46%
58 Total: 5020 cells XX: 5018 (99.96%) 7 29/F Indian Normal,Ht-169cms,P1L1 46,XX[100] X: 2 ( 0.04%) Total: 4950 cells XX: 4932 (99.64%) X: 13 ( 0.26%) 0.36% 8 36/F Chinese Normal,Ht-55cms 46,XX[100] XXX: 5 ( 0.10%)
Total: 5191 cells XX: 5173 (99.65%) X: 12 ( 0.23%) 0.35% 9 33/F Chinese Normal,G2P1L1,Ht-162cms 46,XX[100] XXX: 6 ( 0.12%)
Total: 4514 cells XX: 4510 ( 99.8%) Normal, Pregnant X: 3 ( 0.06%) 0.08% 10 31/F Indian Ht-157.5cms 46,XX[100] XXX: 1 ( 0.02%) Total: 5351 cells XX: 5348 (99.9%) 11 35/F Malay Normal,G3P2L2,Ht-168cms 46,XX[100] X: 3 ( 0.10%)
Total: 4616 cells XX: 4603 (99.72%) X: 9 ( 0.2%) 0.28% 12 28/F Indian Normal,G2P1L1,Ht-160cms 46,XX[100] XXX: 4 ( 0.08%)
Total: 6130 cells XX: 6120 (99.84%) 13 33/F Indian Normal,P2L2,Ht:168cms 46,XX[100] X: 10 ( 0.16%)
59 Total: 5669 cells XX: 5654 (99.73%) X: 14 ( 0.25%) 0.27% XXX: 1 ( 0.02%) 14 31/F Chinese Normal,G2P1L1Ht:165cms 46,XX[100] Total: 5191 cells XX: 5184 (99.86%) X: 6 (0.12%) 0.14% XXX: 1 (0.02%) 15 30/F Indian Normal,G2P1L1,Ht:160cms 46,XX[100]
Total: 5561cells XX: 5561 (99.8%) X: 9 ( 0.16%) 0.2% 16 34/F Indian Normal,P2L2,Ht:158cms 46,XX[100] XXX: 2 ( 0.04%)
Total: 5421 cells XX: 5399 (99.6%) X : 17 ( 0.3%) 0.4% 17 32/F Chinese Normal,G2P1L1,Ht:155cms 46,XX[100] XXX: 5 ( 0.1%)
Total: 4455 cells XX : 4426 (99.3%) X: 2 0.5%) 0.7% 18 28/F Chinese Normal,G2P1L1,Ht:160cms 46,XX[100] XXX: 8 ( 0.2%)
Total : 5000 cells XX: 5000 (100%) 19 27/F Indian Normal,G1P0L0,Ht:165cms 46,XX[100] X: 0 ( 0%)
60 Total: 5313 cells XX : 5299 (99.7%) X: 9 ( 0.2%) XXX : 5 ( 0.1%) 0.3% 20 32/F Chinese Normal,G2P1L1,Ht:160cms 46,XX[100] Total: 5221 cells XX: 5216 (99.9%) X: 4 ( 0.08%) 0.1% Normal, Primigravida, XXX: 1 ( 0.02%) 21 27/F Indian Ht-167cms 46,XX[100]
Total: 5152 cells XX: 5145 (99.86%) X: 2 ( 0.04%) 0.14% 22 26/F Indian Normal,G1P0,165cms 46,XX[100] XXX : 5 ( 0.1%) Total: 5014 cells XX: 5012 (99.96%) X: 2 ( 0.04%) 23 32/F Indian Normal,G2P1,Ht:160cms 46,XX[100] Total: 5039 cells XX : 5035 (99.92%) X : 3 ( 0.06%) 0.08% XXX : 1 ( 0.02%) 24 30/F Indian Normal,G2P1,Ht:155cms 46,XX[100] Total: 5025 cells XX: 5025 (100%) 25 28/F Indian Normal,G1P0,Ht:160cms 46,XX[100] X: 0 ( 0%)
61 Graph 1: Histogram showing the percentage of monosomy X cells and trisomy X cells detected by FISH in control females undetected by the standard G- banding
Percentage abnormal cells detected by FISH technique s l
l 3 e 2.5 c l a 2 XXX m 1.5 1 X bnor 0.5
% a 0 1 3 5 7 91113151719212325 Case numbers
62 Graph 2. Histogram showing the percentage of abnormal cells detected by FISH in study groupI (patients with Turner stigmata with a normal karyotype ):
Percentage abnormal cells detected by FISH in patients w ith Turner stigmata
s 3 l l
e 2.5 c l 2 a XXX 1.5 X 1 bnorm 0.5 a
% 0 123456789 1011 Ca se num be r
63 Graph 3: Histogram showing percentage of normal and abnormal cells by FISH in the study group II (Turner patients with a 45,X cell line):
Percentage of different cells in typical Turner syndrome by FISH
100% 80% Xp- 60% XXX ses a 40% X C 20% XX 0% 1 2 3 4 5 6 7 8 9 1011121314151617 Cell percentage
64 Figure 8: FISH on a metaphase showing two X chromosomes with signals on the X centromere and the Xp-arm
Xcen Xpter
Figure 9: FISH on interphase nuclei showing two X signals, for both centromere and
Xp-arm
Xcen Xpter
65 Figure 10: FISH on a metaphase showing a single X chromosome
Xcen Xpter
Figure 11: FISH on an interphase cell showing a single X with one signal each on X centromere and on Xp-arm
Xcen XXcenpte r Xpter
66 Figure 12: FISH on interphases cells showing three signals each for the X centromere and
Xp-arm
Xcen Xpter
Figure 13: FISH on a metaphase showing an Xp deletion.
67 Figure 14: FISH showing mosaicism in interphase nuclei
A
Xcen Xpter
B
Xcen Xpter
68 4.4: Statistical analysis:
Comparison of the statistics of frequency of abnormal X cells between study group I
(i.e., patients with Turner stigmata but a normal karyotype on G-banding) and the control group (group III):
Eleven patients were studied in this patient study group I and 25 females were studied in the control group III. All of them showed a normal 46,XX karyotype with the routine cytogenetic techniques. But with FISH, group I showed a percentage of abnormal cells which ranged from 0.0%-2.45% (mean: 1.31%, SD 1.03). However, the control females showed a lower percentage of abnormal X cells ranging from 0.0%-0.7% (mean: 0.23%;
SD: 0.185).
Mann-Whitney U test:
The main goal was to investigate if there was a significant difference between the percentages of abnormal X cells of the control group and the patient group. Mann
Whitney U test was selected to test this using the software SPSS 12.0. A significant p value (p=0.003) was obtained using this test. This test uses the comparison of median which is more robust than the mean, especially for this study, as the distribution of the samples was skewed. Hence t-test which compares the difference of the means could not be used. The incidence of Turner syndrome being very small, a larger sample size could not be obtained for t-test. The median of percentage abnormal cells in patient group is
1.3% (0%~2.74%), while the median of the same in the control group is 0.2%
(0%~0.7%).
69 4.4.1. Definition of value for low-grade mosaicism:
The next goal was to determine cut-off value for percentage of abnormal X cells above which low-grade mosaicism could be classified as significant. This was because the control group also had a low percentage of abnormal X cells, it was necessary to determine a value beyond which one can say it is definitely a significant low-grade mosaicism. This value was obtained using the Receiver Operating Characteristic curve
(ROC curve), to obtain a value with maximum sensitivity and specificity.
Figure 15: ROC curve
ROC Curve
1.0
0.8
0.6
Sensitivity 0.4
0.2
0.0 0.0 0.2 0.4 0.6 0.8 1.0 1 - Specificity
Diagonal segments are produced by ties.
The area under the ROC curve is 0.811(95% CI: 0.621 to 1).
70 Table 7: Coordinates of the ROC Curve
Positive if Greater Than or Equal To(a) Sensitivity 1 - Specificity -1.0000 1.000 1.000 .0200 .909 .880 .0600 .909 .800 .0900 .909 .720 .1200 .909 .640 .1450 .909 .560 .1550 .818 .560 .1800 .818 .520 .2350 .727 .480 .2750 .727 .440 .2900 .727 .400 .3250 .727 .320 .3550 .727 .240 .3650 .727 .200 .3850 .727 .160 .4300 .727 .120 .4950 .727 .080 .5400 .636 .080 .6250 .636 .040 .8950 .636 .000 1.1950 .545 .000 1.4250 .455 .000 1.6100 .364 .000 2.0900 .273 .000 2.6150 .182 .000 2.7300 .091 .000 3.7400 .000 .000
The smallest cutoff value is the minimum observed test value minus 1, and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the averages of two consecutive ordered observed test values.
From the ROC curve, using a cut-off value of 0.895% (percent abnormal X cells) gives a sensitivity of 63.6% and a specificity of 100% while a cut-off value of 0.497% gives
72.7% sensitivity and 92% specificity.
71 Percentage of normal cells in karyotypically proven Turner patients study group II:
The next goal was to detect the presence of normal 46,XX cells in typical Turner patients with 45,X on G-banding. Out of 17 patients studied, 15 showed normal 46,XX cells with routine cytogenetic methods. This percent of normal cells ranged from 0.2% to 95%. But two patients among the 17 were pure 45,X with G banding. However, these two patients showed a low percentage of normal 46,XX cells of 0.1% and 0.06% with the FISH technique.
72
CHAPTER 5
DISCUSSION
73 5: Discussion
Individuals with Turner Syndrome are phenotypically females with gonadal dysgenesis and somatic stigmata, especially short stature but have normal intelligence. Similar clinical features were observed in the study group of patients. Of the 28 patients referred for chromosomal analysis, 17 were proven to have a 45,X cell line, detected by conventional cytogenetic G-banding analysis (Group II). The remaining 11 patients who were found to be karyotypically normal (46,XX) formed our study group I. This small sample size was a result of stringent criteria for patient selection and exclusion of patients with other structural X chromosomal anomalies (e.g. mosaics of 45,X/46,XX with ring X, isodicentric X, Xp and Xq deletions).
In group I, 2 out of 11 patients presented with short stature as the primary clinical manifestation, 5 out of 11 presented with some form of gonadal dysgenesis and 4 had both these features. Similarly in group II, 6 out of 17 presented only with short stature, 5 out of 17 presented with gonadal dysgenesis and 6 had both the features. Though the clinical presentation was identical in both the groups, karyotypes differed.
The patients’ in group I whose clinical presentation suggested a diagnosis of Turner syndrome but proved to be karyotypically normal, could be due to the gonadal mosaicism reflected in only a small percentage in the peripheral blood, which might be missed by conventional karyotyping. Using the formula (Hook, 1977), pn+ (1-p)n=α, (where α is
0.05 at 95% confidence interval, p is the percent mosaicism excluded and n is the number of cells counted), at least 500 metaphases need to be analyzed to prove the presence of
74 low percent abnormal clones (<1%) which is very difficult to do with conventional cytogenetic methods. In this study, 5000 cells were scored for abnormal X cells using the
FISH technique that excludes absolutely all the mosaicism (p=0.000000…). Ten out of eleven patients showed a low percentage of 45, X cells ranging from 0.1% to 2.45% with
FISH technique, but one patient (case 11) did not show mosaicism. There are to date no references in literature, which provide us with the significance of this low level abnormal
X cells in patients with some of the Turner stigmata. There is no information or guideline for a baseline value, above which the presence of these abnormal X cells can be considered to be the cause for the phenotype. Also, 45,X mosaicism is not uncommon and probably a harmless finding in blood cultures from normal women. As a consequence of aging, errors occur in cell divisions, leading to the loss of the inactivated X- chromosome (Surralles et al., 1999). So a low percent of abnormal cells could also be present in normal females.
In this study, the control females (group III) showed a percentage of 45,X cells ranging from 0.0%-0.7%. Group I had a significantly higher percentage of abnormal X cells as compared to that of the controls (p=0.003, Mann-Whitney U test). To obtain a baseline value, beyond which low grade X-mosaicism could be classified as significant, the ROC curve was derived from the FISH results of group I and group III. Normally statistical tests aim for a value with maximum sensitivity and specificity, which in this case is
0.495% (sensitivity: 72.7%; specificity: 92%). Applying this value, 7 out of 11 patients were classified as low-grade mosaics. However, two of the controls (cases 1 and 18 of group III), which were absolutely normal were also classified as low-grade mosaics.
75 Since the X chromosome probe used in the FISH experiments had 100% efficiency, a cut- off value with 100% specificity should be selected to avoid misclassification of the controls as low grade mosaics. Hence the cut-off percentage of 0.895% i.e., 1% (rounding up to the nearest whole number) with a sensitivity of 63.6% and a specificity of 100% was selected to classify low grade mosaicism. A count of 500 cells should be sufficient to rule out <1% mosaicism. However, with a view to applying this in clinical settings, a more cautious approach would be to count 1000 cells by FISH (NB: This value would be applicable only if the probe used is with 100% efficiency). Analysis of more patients (>30) should also be done to use it in the clinical set-up.
Applying this value, none of the controls were classified as low-grade mosaics. In the study group I the classification remained the same i.e., 7 out of 11 patients were above the cut-off. The other three patients (cases 1, 2 and 8) in this group, who also showed a low percentage of monosomy X cells, could not be classified as low-grade mosaics, as they did not fall above the set cut-off level. This could be due to the proportions and distribution of the two cell lines present in the peripheral blood (Sergey et al., 1999).
Hence such patients with Turner stigmata necessitate the assessment of karyotypes in other tissues, notably the skin (Nazarenko et al., 1999). Therefore, the genetic diagnosis in such cases may also require the use of FISH analysis in cells from tissues of different germ layers. Another possible reason could be the possible selection that may result in the disappearance of cell lines (Procter et al., 1984; Held et al., 1992).
76 The high in-utero lethality of 45,X condition has led to the hypothesis that most of the liveborn 45,X individuals have low frequency of mosaicism. Most, if not all, liveborn
45,X Turner syndrome individuals are mosaic and have some normal cells in some tissues or possibly had normal cells in the placenta. Sex chromosomal mosaicism in karyotypically proven 45,X Turner patients has been comparatively studied in greater detail as opposed to the study group I. With the use of FISH, ‘hidden’ mosaicism has been detected in apparently non-mosaic 45,X patients (Held et al., 1992). In one study, it was shown that out of 22 Turner patients, 8 had similar results by FISH and karyotyping, whereas an additional 5 showed a second 46,XX cell line by FISH (Abulhasan et al.,
1999). In another study, 37 out of 47 Turner patients were classified as having hidden mosaicism with the 46,XX cell line by FISH technique (Fernandez-Garcia et al., 2000).
Similar results were reported, where out of 35 women with a clinical suspicion of Turner syndrome, 17 cases (48.6%) showed the same karyotype with FISH and routine cytogenetics; in 18 cases (51.4%) a new cell line was identified by FISH (Cortes-
Gutierrez et al., 2003).
In this study Group II, 17 patients who had typical Turner phenotype were analyzed; only two patients (cases 8 and 11) had the typical monosomy X karyotype. 15 out of 17 patients showed 45,X/46,XX mosaicism both with routine G-banding and the FISH technique. The percent of normal 46,XX cells ranged from 0.2% to 95% and there was no discrepancy in the values between the two techniques. The remaining two patients who did not show a second cell line with routine G-banding showed a low percentage of normal 46,XX cells ranging from 0.06% to 0.1% with the FISH technique. This
77 percentage of normal cells is relatively low as compared to other studies which show a range of 3-18%, with an average of 7% normal cells (Nazarenko et al., 1999).
These two cases remained apparent 45,X with G-banding, which may be explained by tissue-confined mosaicism (Held, 1993) or a consequence of a selective loss of a second cell line during embryonic development (Held et al., 1992). Tissue-confined mosaicism could again point to the necessity of analysis of tissue types other than the blood analysis only as in our study.
Another reason could be confined placental mosaicism, which refers to mosaicism that is only present in the placenta. The placenta is a very important organ for all mammals. It has often been overlooked when studying individuals with Turner syndrome. Careful examination of the placenta as well as placental cytogenetic and molecular studies is necessary to understand as to what allows individuals with Turner syndrome to survive.
From this study, the FISH technique proves to be a valuable tool in detecting low-grade mosaicism in contrast to the routine cytogenetic methods. In some patients, FISH application has highlighted the differences between the initial diagnosis based on the standard cytogenetic technique and the final diagnosis determined by the application of
DNA probes specific for the X-chromosomes. Other molecular methods like quantitative fluorescent polymerase chain reaction (QF-PCR) have been employed mainly for rapid detection of common aneuploidies. This technique consists of DNA amplification by polymerase chain reaction (PCR) using fluorescent labeled primers for the analysis of
78 chromosome specific small tandem repeats (STR) (Covone et al., 2004). However, the molecular detection of sex chromosomal mosaicism is still hindered by the sensitivity of
QF-PCR to detect low percentages of cell lines with X monosomy. This was first demonstrated in a trial performed on blood samples (Cirigliano et al., 1999), which showed that mosaics 46,XX (90%)/45,X (10%) are not detectable by the assay and was also confirmed on prenatal samples (Schmidt et al., 2000). Once again interphase FISH proves to be a better technique in diagnosing low percent mosaicism (~1%) compared to
QF-PCR.
Another experimental technique in addition to interphase FISH and QF-PCR which has been tried is the molecular detection of 45,X Turner patients based on the ability of
HpaII, a methylation sensitive endonuclease. HpaII induces the cleavage of non- methylated DNA in the active X-allele. Normal control females or mosaic patients, with a second methylated X-chromosome, escape from HpaII digestion, whereas 45,X patients who have just one active non-methylated X-chromosome, completely digested by HpaII
(Longui et al., 2002). However, by means of this technique it is highly impossible to detect low grade 46,XX/45,X mosaicism.
Dual-labeled X-chromosome probes were used in the FISH experiments in this study.
One probe was the FITC-labeled X centromeric probe and the other one was the spectrum orange labeled locus-specific Xp terminal probe. This enables the detection of Xp deletions very easily, which might be missed with the use of a single X centromeric probe
79 (Figure 13 & Figure 17 A and B). In addition, the Xp terminal probe serves as a good internal control. It was noted that some X chromosomes might have split centromeres and reflect as two green centromeric signals giving a false impression of two X chromosomes
(Figure 16 A and B). By using the internal control this error was avoided. Hence the use of both X centromeric as well as Xp terminal probe while scoring for X chromosomes is suggested. Cells with FISH signals which could not be reasonably explained were not scored in the study (Fig 18). However this was a rare occurrence.
Karyotyping and FISH technique both have their advantages and should ideally be carried out together. However, in serially repeated samples, karyotyping need not be repeated each time, as the change in percentage of normal and abnormal cells is better detected by FISH. The results of FISH are available in two days. The FISH test is thus rapidly gaining importance as a useful diagnostic tool, as more probes are now available for common chromosome abnormalities.
80 Figure 16: FISH on interphase nuclei showing split centromeres
A
Xcen Xpter
B
Xcen
Xpter
81 Figure 17: FISH on interphase nucleus showing three X centromere signals.
A
Xcen
B
Xcen
82 Figure 18: FISH on interphase nuclei showing three signals for Xp-arm and two for Xcentromere
Xcen Xpter
83
CHAPTER 6
CONCLUSION
84 6. Conclusion
Using a set of control patients and the highly sensitive and reliable technique of FISH, a cut-off level of 1% abnormal cells (on a count of 1000 interphase cells and 100% efficient probe) was selected, above which low grade mosaicism can be classified as significant. Analysis of more patients should be done before this could be offered as a clinical test to patients.
Using this value, 7 out of 11 patients were classified as low grade mosaics. However, with analysis of other additional tissues from a different germ line, a higher sensitivity may be achieved.
Eighty eight percent of group II patients (karyotypically proven Turner patients) showed normal 46,XX cells, both with G-banding and FISH whereas the remaining 12% showed a low percent of 46,XX cells only with FISH technique. No conclusion can be drawn regarding the significance of the XX cell line, as this study unfortunately has only two samples which are pure 45,X with G-banding.
However, standard metaphase analysis if supplemented with additional FISH studies of interphase nuclei to screen for the presence of ‘hidden’ mosaicism would enhance the diagnosis and enable proper genotype-phenotype correlation.
85
CHAPTER 7
REFERENCES
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95
CHAPTER 8
APPENDIX
8. Appendix
96 8.1: Appendix 1:
Colchicine working solution:
Material: Colchicine (Sigma Cat. No: 9754)
Method: Dissolve 0.04gms colchicine in 100ml phosphate buffered saline (pH7.2).
Filter sterilize using 0.22µ filter. Dilute 1:9 to give 0.004% working solution. Store stock and working solution at 4°C.
Phosphate buffered saline:
Material: pH 6.8 Cat no. 331992P. pH 7.2 Cat no.332012W
Method: Dissolve 1 PBS tablet in 1000ml distilled water.
Trypsin for banding:
Material: Difco-Bacto Trypsin (Difco Cat no. 0153-61-6)
Method: Reconstitute vial with 10ml sterile distilled water. Dissolve 1ml of stock with
39ml saline.
Normal Saline(0.9%):
97 Material: Analar grade Sodium chloride (Sigma Cat no. S-3014).
Method: Dissolve 9gms NaCl in 1 litre distilled water.
Hydrogen peroxide:
Material: H2O2 (Merck Cat no.1.07210.1000).
Stock solution is 30%.
Method: Dilute 1:3 with warm tap water for banding pretreatment.
Leishman’s stain:
Materials:
3gms Leishman’s powder (BDH 34042M)
1000ml absolute methanol
Method: Dissolve Leishman’s powder in absolute methanol slowly and stir well. Cover loosely with plastic film and leave stirring overnight. Filter leishman’s stain with No.1
Whatman filter paper.
0.075M potassium chloride(KCl):
Method: 0.56 gms potassium chloride in 100 ml distilled water.
20XSSC solution:
98 Mix thoroughly 132g of 20XSSC in 400ml purified water and adjust pH to 5.3 with HCl.
Add purified water to bring the final volume to 500ml.
Wash Solution 1(0.4X SSC/0.3% NP-40) for FISH:
Mix thoroughly 20ml 20XSSC (pH5.3) with 950ml purified water. Add 3ml of NP-40.
Mix thoroughly until NP-40 is better dissolved. Measure pH and adjust pH to 7.0-7.5 with NaOH. Add purified water to bring the final volume to 1 litre.
Wash solution 2 (2XSSC/0.1% NP-40) in FISH:
Mix thoroughly 100ml of 20XSSC (pH 5.3) with 850ml of purified water. Add 1ml NP-
40. Measure pH and adjust to pH 7.0±0.2 with NaOH. Add purified water to bring the final volume to 1 litre.
99 8.2: Appendix 2: The form with details of the clinical features for referral.
100 8.3: Appendix 3:
Quality Assurance certificate stating the 100% efficiency (100% sensitive and 100% specific) of the probe from Vysis.
101