I.

ABSTRACT

Down's syndrome occurs due to structural or numerical anomalies in the 21st chromosome. It is named after John Langdon Down, the British physician who described the syndrome in 1866. The incidence of Down syndrome is estimated at 1 per 700 births and increases with maternal age at delivery. In our study, two Downs cases were studied that were referred to the Department of Human Genetics, NIMHANS, Bangalore. The first case was diagnosed as translocation Downs with karyotype 45 XX t (14:21). The second case was diagnosed as free trisomy 21 (47 XX + 21). In our study out of 122 cases of Downs syndrome reported by the Department of Human Genetics, NIMHANS ( 1991-2011), trisomy 21 was most frequent (86%), followed by mosaicism (9%) and translocation (5%). In translocation (14:21) was most frequent. All of the translocation cases were Robertsonian translocation. Out of these, 42 cases were selected for comparative studies. Out of which 28 cases had trisomy 21, 3 cases mosaicism, 3 cases were translocation t(14:21) and in 7 cases diagnosis was not known. One peculiar case was found, who had the clinical features of Down syndrome but had normal karyotype. The details such as cyto no., name, age, sex, parents age at subjects birth, consanguinity, other family member affected, degree of mental retardation, clinical features, dermatoglyphic features, pedigree analysis, diagnosis, prenatal factors, peri and postnatal factors, medical history, risk of recurrence, occupation of father, address, mother, date and reference number were recorded. Under each factor, the frequency of each feature was calculated and tabulated in the decreasing order of frequency. Fathers age, occupation of father (economical condition of the family), mothers age, other family member affected, prenatal and consanguinity were the factors we considered which could have been responsible for the occurrence of Down syndrome child. Hence in each case, each of this factor was looked for which might have played a role in occurrence of Down syndrome. Later, the clinical features were compared between variants of Down syndrome free trisomy, mosaicism and translocation. In our study, we conclude that other than well known factor mothers age, several other factors such as fathers age, consanguinity, economical condition of family, prenatal factors and other family member affected should also be considered to provide better and accurate genetic counseling and to assess the risk factors.
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III. TABLE OF CONTENTS

S. No.
1 2 3

Contents
Chapter 1: Introduction Chapter 2: Objectives Chapter 3: Review of Literature 3.1. Introduction on Down Syndrome 3.2. Mosaic Downs 3.3. Translocation Downs 3.4. Parental age effects 3.5. Consanguinity 3.6. Aetiology of Down Syndrome 3.7. Recurrence risk Chapter 4: Materials and Methods 4.1. Materials required 4.2. Chemical required 4.3. Protocol 4.4. Karyotyping 4.5. Individual characterization of human chromosomes. 4.6. Case study 4.7. Comparative studies 4.8. DNA extraction from mammalian whole blood using Pharmacia biotech kit. Chapter 5: Results 5.1. Standardization 5.2. Spectrophotometric readings of isolated DNA 5.3. Case studies 5.4. Comparitive studies of variants of Down syndrome using past 20 years data from NIMHANS, Bangalore. Chapter 6: Discussion Chapter 7: Summary Chapter 8: References

Page No.
9 11 12 12 13 15 16 19 20 27 30 30 30 30 31 32 33 34 35 37 37 42 43 47 70 77 80

6 7 8

IV. LIST OF TABLES

Table No. 3.1 3.2 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 5.16 5.17 5.18 5.19 5.20 5.21 5.22 5.23 5.24 5.25 5.26 5.27 5.28 5.29 5.30 5.31 Name Chromosomal mosaicism in presumably normal human tissues. Frequency of trisomy 21 at delivery according to maternal age Culturing DNA sample readings Details of Case study 1 Details of case study 2 Parents age during subjects birth Consanguinity Patient details Trisomy 21 Patient details Normal karyotype with DS clinical features Patient details Mosaic cases Patient details Translocation cases Patient details Diagnosis not known Degree of mental retardation Dermatoglyphic features Clinical features Prenatal factors Peri and postnatal factor Medical history Major factors that might have been responsible for occurrence of Down syndrome. Frequency of clinical features in Trisomy 21 Frequency of clinical features in Translocation Frequency of clinical features in Mosaic Consanguinity among variants of Down syndrome Family members affected among variants of Down syndrome Degree of mental retardation among variants of Down syndrome Dermatoglyphic features among variants of Down syndrome Age group of parents with DS childrens with Trisomy 21 Age group of parents with DS childrens with Mosaicism Age group of parents with DS childrens with Translocation Prenatal factors among variants of Down syndrome Peri and post natal factors among variants of Down syndrome Medical history among variants of Down syndrome Page No. 14 18 37 42 43 45 47 47 48 54 55 56 57 59 59 60 61 61 62 62 63 63 64 64 64 65 65 66 66 67 67 68 69
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V. LIST OF FIGURES
Fig No. 3.1 3.2 3.3 3.4 3.5 3.6 3.7 4.1 5.1 5.2 5.3 5.4 Name Changes in human oocyte number during prenatal and postnatal development Current concepts in biology of chromosomal mosaicism: somaticgermline aneuploidization pathway. Birth rate of T21 in relation to maternal age Male and female physiological timeline Normal meiosis and maternal meiotic errors MI non-disjunctional errors manifesting in MII Cartoon illustrating the different types of secondary non-disjunction that may take place in a T21 oocyte Pedigree Chart Normal male Karyotype Normal Female Karyotype Karyotype of Case study 1 Karyotype of Case study 2 Page No. 13 15 18 21 22 23 26 34 40 41 44 46

Chapter: 1

INTRODUCTION
Chromosome anomaly is either due to an atypical number of chromosomes or a structural abnormality in one or more chromosomes. A karyotype (a full set of chromosomes from an individual) is compared to a "normal" karyotype using the method known as karyotyping. Chromosome anomalies occur due to an error in cell division following meiosis or mitosis. They are organized into two basic groups, numerical and structural anomalies. Numerical abnormalities (Aneuploidy) occur when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy, Tetrasomy, etc). Structural abnormalities occur due to deletions, duplications, translocations, inversions, rings and isochromosomes. Chromosome anomalies occur as an accident in the egg or sperm and not inherited. The anomaly is present in every cell of the body. Some anomalies can happen after conception, resulting in mosaicism (where some cells have the anomaly and some do not). Down's syndrome occurs due to structural or numerical anomalies in the 21st chromosome. It is named after John Langdon Down, the British physician who described the syndrome in 1866. The condition was identified as a chromosome 21 trisomy by Jrme Lejeune in 1959. The incidence of Down syndrome (DS) is estimated at 1 per 700 births and increases with maternal age at delivery. Individuals with Down syndrome tend to have a lower-than-average cognitive ability, often ranging from mild to moderate disabilities. Early childhood intervention, screening for common problems, medical treatment where indicated, a conducive family environment, and vocational training can improve the overall development of children with Down syndrome. Education and proper care will improve quality of life significantly, despite genetic limitations. (Roizen et al. 2003) The average IQ of children with Down syndrome is around 50, compared to normal children with an IQ of 100 (Liptak et al. 2008). Health concerns for individuals with Down syndrome include a higher risk for congenital heart defects, gastroesophageal reflux disease, recurrent infections, obstructive sleep apnea, thyroid dysfunctions, duodenal stenosis or atresia, imperforate anus, and Hirschsprung disease. Eventhough there are over 50 clinical symptoms of DS, it is rare to find all or most of them in one person (Stine 1989). The clinical features of Down syndrome are comprised of severe cognitive impairment, characteristic facial profile, short stature, speech and developmental
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delay, chronic ear infections and hearing loss, and hypotonia (Jones 2006). The diagnostic facial profile consists of epicanthal folds, flat nasal bridge, upslanting palpebral fissures, and protruding tongue (Jones 2006). Down syndrome patients can also develop congenital heart disease (4050%), have an increased risk for Alzheimer disease (especially after the fourth decade) (Roper and Reeves 2006), acute megakaryocytic leukemia or acute myeloid leukemia (AML) type M7, according to the French- American-British classification, Hirschsprung disease, and duodenal atresia (Jones 2006). Studies revealed three genetic mechanisms to cause DS viz: free trisomy 21 (9295%), mosaic trisomy 21 (24%) and translocation (34%) in general (Stoll et al. 1998). However higher frequency of mosaicism (6 -7 %) than translocation (3 4%) have been reported in International and Indian studies. (Azman et al. 2007; Jyothi et al. 2002) Though Down syndrome is very well studied since 1866, still its incidence is high and several complications are associated with it. Hence further detailed studies must be done in order to understand several other factors that have not been reported till date which plays a major role in the occurrence of Down syndrome. Efforts must be made to provide better and accurate genetic counseling and to improve the quality of life of Down syndrome patients.

Chapter: 2
6

OBJECTIVES
1.

Standardisation of Protocol for karyotyping, especially for scoring high index metaphases. To study the clinical and genetic basis of Downs syndrome. Study of genetic variations in terms of mosacism, translocation and parental origin of Down Syndrome.

2. 3.

4. Genotype and phenotype correlations.

5. Susceptibility to heart diseases and recurrent infections and longevity of these subjects. 6. Comparative studies of variants of Down syndrome using past 20 years data from

NIMHANS, Bangalore.
7. To isolate DNA from Downs patients for making the repository of genomic DNA.

Chapter: 3

REVIEW OF LITERATURE
3.1 Introduction on Down syndrome
In 1866, John Langdon Down made the first detailed description of all affected individuals (Down 1866). While the syndrome was largely called mongolism early on, due to this misleading connotation mongolism for this mental deficiency syndrome the eponym was discontinued and was widely called Down syndrome (Nomenclature 1975). Even though there are over 50 clinical symptoms of DS, it is rare to find all or most of them in one person (Stine 1989). The clinical features of Down syndrome are comprised of severe cognitive impairment, characteristic facial profile, short stature, speech and developmental delay, chronic ear infections and hearing loss, and hypotonia (Jones 2006). The diagnostic facial profile consists of epicanthal folds, flat nasal bridge, upslanting palpebral fissures, and protruding tongue (Jones 2006). Down syndrome patients can also develop congenital heart disease (4050%), have an increased risk for Alzheimer disease (especially after the fourth decade) (Roper and Reeves 2006), acute megakaryocytic leukemia or acute myeloid leukemia (AML) type M7, according to the French- American-British classification., Hirschsprung disease, and duodenal atresia (Jones 2006). Studies revealed three genetic mechanisms to cause DS viz: free trisomy 21 (9295%), mosaic trisomy 21 (24%) and translocation (34%) in general (Stoll et al. 1998). However higher frequency of mosaicism (6 -7 %) than translocation (3 4%) have been reported in International and Indian studies. (Azman et al. 2007; Jyothi et al. 2002) The occurrence of DS in other parts of the world is ranging from 0.92/1000 live births. In India the prevalence of DS is still not clear because of limited work. Survey in a few places indicates the prevalence to be in the range of 0.811.2/1000 live births (Isaac et al. 1985; Verma et al. 1998; Modi et al. 1998). Males and females are affected almost in the same ratio (1: 1.1). No significant difference is noticed even in the younger maternal age groups, except for the mothers in the less than 25 years age group, where females were more affected than males (Azman et al. 2007). Moreover, variability of clinical presentation within a Down syndrome cohort provides evidence for gene dosage thresholds for specific phenotypes, and the action of genetic and environmental modifiers (Antonarakis et al. 2004). Prenatal testing for Down syndrome is
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performed by chorionic villus sampling (in first trimester), amniocentesis (in second trimester), biochemical analysis (Quad screen for maternal serum markers and alpha feto-protein analysis) and by ultrasound (for nuchal translucency) (Neilson and Alfirevic 2006; Saller and Canick 2008). About 68% of free trisomy originates from maternal meiosis I errors, about 20% from maternal meiosis II errors, 67% from paternal meiosis errors, and 56% from mitotic errors, respectively. Paternal meiosis II errors are twice as frequent as paternal meiosis I errors. Almost half of translocation trisomies have 14/21 translocations. De novo derivative chromosomes 21 [(der(21;21)(q10;q10)] are rare, and most of them are isochromosomes (Antonarakis 1998; Shaffer et al. 1993).

Fig: 3.1. Changes in human oocyte number during prenatal and postnatal development. Courtesy: Hulten et al. 2008 There is a very rapid increase in human female germ cell (oocyte) number early during fetal development with a peak at 7 months gestational age, followed by a relatively rapid decline before birth and postnatally before puberty, but a slower depletion during reproductive years until menopause (Fig 3.1) (Hulten et al. 2008).

3.2 Mosaic Downs

It has been suggested on the basis of dermatoglyphics that 10% of parents of children with Down syndrome are mosaic (Priest et al. 1973). Parental mosaicism, to be of reproductive significance, would have to involve germinal cells. A parent could be mosaic in either the germinal line or in both germinal and somatic cell lines. Better success from fibroblasts have been observed in the detection of somatic mosaicism. Studying chromosome 21 in ovarian cells of normal female foetuses, Prof. Maj Hulten and her
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colleagues were able to give experimental support for their original hypothesis suggesting meiotic aneuploidy in human conceptuses to be the result of ovarian germline mosaicism that is produced during the normal prenatal development (Hulten et al. 2008). Firstly, it has been recently noticed that chromosomal mosaicism is frequent among human foetuses, achieving the rate of 25% in spontaneous abortions (Vorsanova et al. 2005). Additionally, the confinement of chromosomal mosaicism to the specific tissue is a known phenomenon. As early as 1983, Kalousek and Dill have described the existence of chromosomal mosaicism exclusively confined to the placenta (confined placental mosaicism). About a year ago, there have been shown that somatic chromosomal mosaicism confines to the developing human brain in a significant proportion of normal human conceptions. Furthermore, it has been established that increase of mosaic aneuploidy in the developing human brain is an integral component of the human prenatal central nervous system development (Table 3.1) (Yurov et al. 2007). Therefore, one can conclude: (i) chromosomal mosaicism is extremely frequent in human foetuses; (ii) chromosomal mosaicism confines as to extraembryonic tissues (placenta) as to embryonic tissues (central nervous system and ovarian tissue). It is reasonable to suspect, that the later could be one of the major source for human tissue specific pathology or multi-system diseases (including those that arise due to meiotic errors). (Table 3.2) Table. 3.1. Chromosomal mosaicism in presumably normal human tissues.

Courtesy: Ivan et al. 2008.

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Fig: 3.2 Current concepts in biology of chromosomal mosaicism: somatic-germline aneuploidization pathway. Normal prenatal and postnatal development is hypothesized to be a matter of balance between two progressive processes: aneuploidization and "antianeuploidization" (the latter is arbitrarily covered by such term because it is still not completely clear what processes underlie the clearance of aneuploid cells in humans). Germline aneuploidzation results into prenatal death of aneuploid embryos or into chromosomal syndromes in newborns. Aneuploidization is observed in fetal germline tissues and in the fetal brain. This, if not cleared, has the potential to produce tissue-specific chromosomal mosaicism that can underlie the pathogenesis of brain diseases either in childhood or in adulthood. It also can be the reason of germline aneuploidization (mentioned earlier). Aneuploidization in adulthood (in some cases, in childhood) is suggested to be a key process of tumorigenesis and aging. This probably originates from the age-/environmentdependant inhibition of "antianeuploidization" processes. Courtesy: Ivan et al. 2008.

3.3 Translocation Downs

Translocation in Down syndrome is usually of Robertsonian type with the fusion of chromosome 21 to d or G group chromosomes. Most frequent forms are t (21;21) and t(14;21) (Mutton et al. 1996). The other less frequent translocations are t (13:21), t (15:21) and t(21;22). The occurrence of these can be sporadic or secondary if one of the parent happens to be of the t (14:21) type (43.47%), followed by t (21:21) type (36.95%). The frequency of other translocation Down syndrome like t (13:21, t(15;21), t(21;22) and translocated mosaics are
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below 5%. The frequency of translocation ranges from 3.6% to 5.1 % of Stoll et al. 1998 and Kim et al. 1999. Frenny et al. in 2007 reported that translocation is parentally inherited (in 26.5% cases) and maternal transmission was twice as common as paternal. Males were more pronounced to be affected than females. Familial inheritance in RT is seen in one quarter whereas in remaining it arises as a de-novo (Shaffer et al. 1992). An interesting observation was seen in one case where karyotypic analysis showed a mirror duplication of #21 which confirmed to be of paternal origin. This confirms that most de-novo rearrangements (21q21q) are isochromosomes derived from a single parental #21 and only small proportion is consistent with true RT (Shaffer et al. 1993). Robertsonian translocations have been suggested to occur during oogonial / spermatogonial mitosis (Ohno et al. 1961) or meiotic prophase I (Mirre et al. 1980; Stahl et al. 1983). Cytogenetic analysis using chromosomal heteromorphisms has suggested that the de novo t(14q;21q) chromosome has originated from the mother in 11 of 12 cases in which the parental origin has been determined (Robinson 1973; Magenis and Chamberlin 1981; Mikkelsen et al. 1989). Analysis of chromosome 21-specific DNA polymorphisms permitted the determination of the parental origin of the extra chromosome 21q and suggested that in the majority of cases the extra 21q was probably due to chromatid translocation in maternal meiosis I and to normal crossover and segregation in meiosis I and meiosis II. The origin was maternal in all families (Peterson et al. 1991). Previous cytogenetic analyses of 13 published families with de novo t(14q;21q) determined the origin of the t(14q;21q) to be maternal in 11 cases, and paternal in one case, and unknown in one case. In this latter analysis chromosomal heteromorphisms of the short arm of the free chromosomes 14 and 21 have been compared with those of the parental karyotypes. The origin of the t(14q;21q) has been determined because the free chromosome 14 had the same chromosomal heteromorphism pattern as did one of the parental chromosomes 14 (Robinson 1973; Magenis and Chamberlin 1981; Mikkelsen et al. 1989). However, the reliability of cytogenetic heteromorphisms in determining the parental origin of acrocentric chromosomes has been disputed (Antonarakis et al. 1998).

3.4 Parental age effects.

Lionel Penrose (1933) suggested a partial correlation between advanced maternal age and the risk of having a child with Down syndrome (Penrose 1933). Mantel and Stark (1967) suggested
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that a high correlation between the paternal and maternal age masked the actual paternal age effect and reduced the power of correlation studies. While increased paternal age association was identified in some epidemiological studies (Erickson and Bjerkedal 1981; Stene et al. 1981, 1987), no significant effect was detected in several other studies on Down syndrome (Erickson 1978; Hook et al. 1981; Hook 1987; Fisch et al. 2003; Dzurova and Pikhart 2005). Discrepancies in these reports were mainly due to different methods of statistical analysis and, possibly, the small sample sizes analysed (Fisch et al. 2003). A more recent study suggested a significant paternal age effect on Down syndrome only with mothers aged 35 years or older (Fisch et al. 2003). Fisch et al. (2003) also showed that the paternal age effect was the greatest in couples older than 40 years with the risk for Down syndrome increasing to six times the rate in couples younger than 35 years (Fisch et al. 2003). Interestingly, in familial cases of Down syndrome with more than one affected sibling, the mean maternal age for affected children was lower than the general maternal age estimates for Down syndrome (Penrose 1954). A similar reduction of mean maternal age was also observed in individuals when a maternal relative was also affected. No reduction of mean maternal age was observed when a paternal relative was affected. Further, a two to three times increased risk of Down syndrome in the siblings of affected individuals was observed, after accounting for selection bias (Penrose 1954). A recent study by Arbuzova and colleagues on familial cases of Down syndrome showed that while there is a greater recurrence risk for younger women, by the age of 40, the recurrence risk is not significantly different from non-familial cases (Arbuzova et al. 2001). Notably, a history of Down syndrome miscarriage also increases the risk of other fetal aneuploidies in subsequent pregnancies (Bianco et al. 2006). The mean grand maternal age at the birth of the mosaic parent of 30.1 years and the mean grand paternal age of 32.6 years suggest a possible age-dependent factor in the genesis of mosaics (Harris et al. 1982). Avramopoulos et al. in 1997 reported a case of apparent trisomy 21 without the DS phenotype. Because of the very mild phenotype it was concluded that the patient might be mosaic possibly with a high percentage of euploid cells. Earlier workers strongly advocated that the advanced maternal age is a major risk factor for trisomy 21. The likelihood that a woman under 25 and 30 years who becomes pregnant will have a baby with DS is less than 1 in 1,400 and 1,000 respectively. Chance of having a baby with DS increases to 1 in 350 for women who become pregnant at age 35 and continues to
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increase as the woman ages, so that by age 42, and by age 49, the chance is 1 in 60 and 1 in 12 respectively (Fig.3.3) (Table 3.2) (Antonarakis et al. 1998).

Fig: 3.3 Birth rate of T21 in relation to maternal age. The socalled maternal age effect was first recognized by Penrose in 1934, and has since been seen without much variation in different countries around the world.Courtesy: Hulten et al. 2008. Table. 3.2. Frequency of trisomy 21 at delivery according to maternal age.

Courtesy: American College of Obstetricians and Gynecologists. ACOG. On the contrary there are reports that 80% of DS babies are born to young women of age less than 30 years (James et al. 1999; Cooley and Graham 1991). Frenny et al. in 2007 reported that 91.6% of DS babies were born to younger mothers (20-35 yr) compared to 8.4% in elderly mothers (>35 yr). It has been reported that the mean maternal age of the DS children is around 30 years in Hyderabad, Mumbai and Punjab (Jyothi et al. 2000, 2001; Kothare et al. 2002; Rao 1999). It has been reported that most of the translocation DS cases were also born to younger mothers (Sayee and Thomas 1996; Yurov et al. 2007). . The findings also revealed that 75% of DS children were born to young mothers whose age ranged from 1829 years. In the control group remarkable difference in the number of children born at different age range of mothers
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and fathers establishes that more children were born to the young mothers (1824 years) and father of advanced age (3035 years). The number of children born to young mothers is also more when compared to fathers of the similar age. This is probably due to women getting married at young age (1829 years) and produces more children in Indian families. This kind of situation is not found in western families (Malini et al. 2007). However, there are a very few earlier reports indicating the influence of grand maternal age, on the risk of their grandchild being born with DS (Aagesen et al. 1984; Mikkelson 1985; Papp et al. 1977). On pedigree analysis of these families, it is clear that whenever the daughter was born to aged mother the chances of this daughter giving birth to DS children are increased (Malini et al. 2007). Further Jyothi et al. in 2002 presented data that 80.7% pure trisomy Down syndrome children were born to mother's <30 years age and 49.41 % to father's <30 years. In case of translocation 90.24% were born to mother <30 years age and 70.74% to father <30 years. Among 41 translocation cases 39.02% (n=16) were born to mother's age <20 years, 36.58% (n=15) to mother's age between 20-25 years, 14.63% (n=6) born to mother's age between 25-30 years and only 4 (9.75%) to mother's age >30 years. The mean maternal age of translocation Down syndrome cases was estimated to be 23.17 years. Most of the translocation DS children (n=31) were born to mother's <25 years of age and were first born in the birth order. This can be explained by the fact that most of the women were primigravidas (n=18) and the incidence of miscarriage is less in women conceiving at first pregnancy for unknown reasons. Parental karyotypes are therefore essential for all patients with a translocation to be sure that the rearrangement was not inherited. When either parent is found to be a balanced carrier of any chromosome rearrangement, the siblings of both the patient and the affected partner are at risk.

3.5 Consanguinity
An increased incidence of Down syndrome has been reported in a highly consanguineous population (Alfi et al. 1980). A major gene producing a greater risk of non-disjunction should be detectable from a study of pedigrees and populations. If this gene were inherited as an autosomal dominant, then there should be families with multiplex sibships in several generations. If it were recessive, one would expect multiplex families to be more prevalent in a population with a high inbreeding coefficient (Harris et al. 1982). In one interesting case of t(14;21), reported by Jyothi et al. in 2002 parental study revealed carrier status in both the parents. Mother had translocated chromosome in 100% of her cells
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and father showed translocated chromo some in only 45% of his cells. History of consanguinity revealed first cousin marriage. Karyotype of siblings showed mosaic translocation in 2 male sibs and normal chromosomal constitution in 2 female sibs. Chromosomal investigations, family history, pedigree analysis, parental ages and parental karyotypes are essential factors in ferreting genetic counselling, estimating the risk for the next conception.

3.6 Aetiology of Down Syndrome

In his 1954 report, Penrose indicated several plausible aetiological factors for Down syndrome: advanced maternal age resulting in altered rate of crossing over between closely linked genes, chromosomal translocations and endocrine imbalances, a strong hereditary component in familial cases with reduced mean maternal age, and maternal-fetal genotype-specific susceptibility (Penrose 1954). Additionally, in 1964 Penrose suggested that the Down syndrome cases influenced by advanced maternal age could be due to chromosomal nondisjunction, a failure of chromosomes to separate properly during meiosis (Penrose 1964; Sherman et al. 2007). Analysis of chromosome heteromorphisms and highly informative DNA polymorphic markers from parents and Down syndrome offspring helped to determine the parental origin of the extra chromosome and stage of the meiotic error (Hassold and Jacobs 1984; Antonarakis 1991; Petersen et al. 1991). The maternal versus paternal basis for meiotic errors are well illustrated by their physiological timeline (Hassold and Hunt 2001). In male fetal testis, after initial mitotic arrest, a longer mitotic proliferation is resumed (from prenatal period to puberty) until the onset of puberty when meiotic events are initiated and completed. Sperm production is continued throughout the lifetime. In contrast, in the female ovary, after a smaller mitotic proliferation event, the germ cells enter into protracted meiosis I and II periods. After homologous recombination, MI is arrested at prophase, and is completed only after ovulation, at the onset of puberty. MII is then initiated only to be arrested again, in metaphase II; MII is completed after fertilization. Possible advanced

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Fig: 3.4. Male and female physiological timeline. Male and female germ cells have different timelines for mitotic and meiotic events in the developing testis and ovary. maternal age-associated events are also shown. The figure is modified from Hassold and Hunt (2001). The prolonged meiotic arrest phase likely allows accumulation of toxic effects including environmental insults, degradation of meiotic machinery causing MI and MII errors, and suboptimal ovarian functioning likely resulting in hormonal imbalance (Sherman et al. 2007). Male meiosis, however, begins at puberty and all events are sequentially completed without interruptions, in the adult testis (Fig. 3.4) (Hassold and Hunt 2001). Research from the laboratories of Stephanie Sherman and Terry Hassold has provided insights into the origin of human aneuploidy, including those specific to trisomy 21. While maternal non-disjunction (majority being MI error) has accounted for >90% of trisomy 21 cases, 5% 10% were due to paternal non-disjunction (MI and MII errors), and <5% were due to mitotic errors (Hassold and Hunt 2001). However, only maternal meiotic non-disjunctions are associated with advanced maternal age (Lamb et al. 2005). MI errors are identified by the presence of a parental heterozygosity in the trisomic offspring and in MII error, the parental heterozygosity is converted to homozygosity in the offspring. However, if the parental heterozygosity is reduced to homozygosity in all informative loci (proximal, medial, and distal chromosome-21 regions) then a postzygotic, mitotic error is inferred (Fig. 3.5) (Savage et al.
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1998).

Figure 3.5. Normal meiosis and maternal meiotic errors. (a) The schematic shows the normal series of meiotic events. Briefly, MI ends in the separation of chromosomal homologs and MII results in the separation of sister chromatids. (b & c) Nondisjunction due to MI and MII errors is shown. Genotyping using informative microsatellite markers (a, b and x) in an individual with Down syndrome with MI error will show parental heterozygosity, while MII errors are characterized by marker homozygosity for one of the parental chromosome. Note that an excess of distal crossovers lead to MI errors and increased proximal crossover causes MII errors (modified after Lamb et al.2005). For maternal meiosis, location and number of crossovers in trisomic offspring compared to
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controls have shown significant differences and have indicated that: (a) increased distal (telomeric) crossovers (with shorter linkage maps) contribute to MI errors in younger women (aged <29 yr) but not so in older women (aged >34 yr), (b) increased proximal (pericentromeric) exchanges (with longer linkage maps) associated with MII errors are more common in older women and not in younger women, and finally, (c) absence of recombination (with no chiasmata formation) is associated with MI error in both age groups (Lamb et al. 2005). As recombination occurs during the prophase of MI, at least some MII errors are probably initiated early during MI. (Fig 3.6)

Figure 3.6 MI non-disjunctional errors manifesting in MII. Neil Lamb and colleagues suggested that the consequence of increased proximal crossovers contributing to MII errors may be due to (a) chromosomal entanglement atMI, wherein the bivalents are not separated until MII, or (b) premature separation of sister chromatids at MI, due to loss of sister chromatid cohesion. Here, there is separation of the whole chromosome and a single chromatid to each pole of the meiotic spindle (Lamb et al. 1997). Notably, there are several other possible patterns of MI and MII errors (refer to Hassold and Hunt 2001). It is therefore apparent that, apart from advanced maternal age, the only other factor that is consistent with maternal meiotic non-disjunction is altered recombination pattern (Sherman et al. 2007). Evaluation of paternal non-disjunction cases revealed a reduction in recombination
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in MI cases and an excess of MII errors associated with a slight increase in the amount of proximal-medial exchange compared to exchange at telomeres (Savage et al. 1998). Interestingly, there was an altered sex-ratio among the paternally derived MII cases with an overall increase in male probands (Savage et al. 1998; Petersen et al. 1993). The skewed sexratio was consistent with proximal-medial exchange in MII and was hypothesized to result from imbalanced non-disjunction products segregating with the Y chromosome during anaphase (Savage et al. 1998). It has been suggested that increased biological ageing of the ovaries is a major factor for aneuploidy conditions in females - a limited oocytes pool hypothesis (Warburton 1989). According to this hypothesis, the ageing of the ovary is associated with availability of limited and less optimal oocytes for fertilization. While several correlations with biological ageing of the ovary, including cigarette smoking, history of unilateral ovariectomy, and hormonal levels are still being evaluated, the median age for menopause was consistently found to be 0.91 year earlier in women with trisomic pregnancies compared to controls (Kline and Levin 1992; Kline et al. 2000). Kline et al. (2004) suggested that changes in follicular development, unrelated to the size of the oocyte pool, likely influence abnormal chromosome segregation. A preferential survival in older mothers of fetuses with Down syndrome prompted a relaxed selection hypothesis (Erickson 1978; Ayme and Lippman-Hand 1982). Accordingly, a significant component of maternal age association in Down syndrome live births was suggested to be caused by relaxed selection against aneuploid fetuses (Erickson 1978; Ayme and Lippman-Hand 1982). This hypothesis was not supported by maternal age-specific prevalence of trisomic spontaneous abortions and live births, which showed that the chance for miscarriage is, in fact, much higher in older women (Hook 1983; Hassold et al. 1984; Hook et al. 1989). The maternal age effect may be due to differential selection and accumulation of T21 oocytes in the ovarian reserve of older women. This type of ovarian mosaicism might also explain the maternal age effect (Hulten et al. 2008). It has been demonstrated by analysis of chromosome behavior in cases of foetal T21 that there is a substantial delay in foetal oocyte maturation in comparison to that seen in cases with a normal karyotype (Barlow et al. 2002; Jagiello et al. 1987; Luciani et al. 1976; Robles et al. 2007; Speed 1984; Zheng and Byers 1992). It seems reasonable to conclude that T21 foetal oocytes may lag behind the normal during foetal development, when there is a dramatic reduction in numbers by apoptosis from age 20 weeks
20

until birth and then postnatally until puberty. It is also possible that there is a further selection against T21 oocytes leading up to the total 300400 maturing to ovulation between puberty and menopause as discussed with respect to the oocyte selection model proposed by Zeng and co-workers. (Zheng et al. 2000; Zheng and Byers 1992). The net effect of this situation is that any T21 oocytes in the original pool will comprise a larger proportion of the ovarian reserve at later maternal ages. The low paternal origin of T21 may be due to more effective selection against T21 germ cells during spermatogenesis than oogenesis. The underlying reason might be for the relatively low frequency (around 10%) of T21 DS, where the extra chromosome 21 is paternally inherited could be the existence of similar degrees of mosaicism in foetal testes, but a more efficient selection against aberrant cells during spermatogenesis than oogenesis (Hamer et al. 2008; Hunt and Hassold 2002; Roeder and Bailis 2000). An indication that some T21 spermatocytes may nevertheless progress to reach first meiotic metaphase in the mature testes originates from studies of DS males post puberty (Johannisson et al. 1983; Hulten and Lindsten 1973; Kjessler and Chapelle 1971). These cases are characterized by substantially reduced numbers of spermatocytes reaching the first meiotic metaphase. Interestingly, the three chromosomes 21 then show the same principal pairing, crossing-over, chiasma formation and recombination abnormality as described above for T21 oocytes, which also concords with that expected from Drosophila and mouse experimentation (Kouznetsova et al. 2007; Voet et al. 2003; Xiang and Hawley 2006). Two types of T21 spermatocytes are seen at the metaphase I stage, either containing a bivalent plus a univalent 21 or a trivalent 21. Trivalents show chiasmata in aberrant positions, and univalents are expected to be liable to precocious anaphase I separation, leading to extra or missing chromatids in daughter cells. (Fig 3.8).

21

Figure 3.7 Cartoon illustrating the different types of secondary non-disjunction that may take place in a T21 oocyte. a)Formation of a bivalent plus a univalent, where the univalent is undergoing precocious disjunction leading to a chromosome 21plus a chromatid in each of the daughter cells. b) Formation of a trivalent with chiasmata in an aberrant position, i.e. in this example distally within the long arm. Secondary non-disjunction at anaphase 1 will lead to two chromosomes 21 traveling into one of the daughter cells and one chromosome 21 into the other. Courtesy: Hulten et al. 2008. In general, the incidence of aneuploidy in humans varies during development; for example, 1% 2% in sperms and 20% in oocytes during gametogenesis, 20% incidence at pre-implantation stage, 35% in spontaneous abortions, 4% in stillbirths, and 0.3% incidence in live births, suggesting a strong in utero negative selection (Hassold and Hunt 2001). The nondisjunction error is more frequent in first meiotic division (80%) rather than second meiotic division (20%) (Hassold and Hunt 2001). Malini et al. (2007) proposed that advanced age of grandmother is responsible to bring disturbance in the meiosis of her daughter when the grandmother conceived. At the advanced age the grandmother's reproductive system may fail to make the essential proteins like spindle associated proteins, factors responsible for resting of oocyte, chiasma-binding proteins, DNA repair enzymes, etc. which are needed for proper meiotic segregation in the germ cells of her daughter. The non-availability or non-functioning
22

of proteins leads to impairment in the meiotic process, which in turn results in nondisjunction of chromosome 21 in the oocyte of the daughter. This event takes place during the embryogenesis of the mothers of the DS children when she was in grandmother's womb. It is also possible that recombination is reduced in the oocytes, which brings about the nondisjunction of chromosome 21. Though the advance maternal age is an established risk factor for DS, present study has shown increased number of DS babies born to the young mothers as more number of pregnancies occurs in this reproductive age group. This could either be due to MTHFR gene polymorphism (James et al. 1999) and/or nutritional factor (Sheth and Sheth 2003). It is well known that aneuploidy can have major detrimental health consequences when it occurs in either germinal or somatic cells. Germinal aneuploidies, a major cause of pregnancy loss, aneuploid births and developmental defects (Wyrobek et al. 2000) are thought to arise de novo, through meiotic errors in germ cells of either parents, or mitotically shortly after fertilisation. Both age-dependent and age-independent factors appear to be operating simultaneously. It could be due to age-dependent decay in the spindle fibres or their components, a failure in nucleolar breakdown or an accumulation of the effects of radiation, hormonal imbalances and infection (Chandley 1985). On the other hand, clinical and experimental studies have shown that age-independent DNA hypomethylation is associated with chromosomal instability and abnormal segregation. Based on this, Christman et al suggested a link between dietary folate and methyl deficiency in vivo and DNA hypomethylation (Christman et al. 1993). On the basis of these cellular observations, James et al and Hobbs et al have postulated a link between abnormal folate metabolism and mutation of the methylenetetrahydrofolate reductase gene, hence as a risk factor for nondisjunction and Down syndrome in younger (< 35 years) mothers. Further research on the role of polymorphism of other genes involved in folate metabolism as a causative factor for maternal nondisjunction and high risk genetic factor for Down syndrome is warranted, in view of the high proportion of young mothers giving birth to Down syndrome children. (Hulten et al. 2008)

3.7 Recurrence risk

Recurrence risks depend upon the extent of gonadal mosaicism. There may be germinal without somatic mosaicism, which could account for the multiplex sibships observed by
23

Frohlich et al. 1979 and Dhadial and Pfeiffer 1972. Stene in 1970 using cytogenetically confirmed cases of trisomy 21 and excluding known parental mosaic cases, estimated the recurrence risk for women who had their first affected child before age 30 to be between 1% and 2%. The recurrence risk was not increased over the general population if the first affected child was born when the mother was over age 30. Uchida in 1970, in extensive family studies, observed a risk of recurrence for trisomy 21 of 1:57. Milunsky in 1973 discussed national data culled from 1,663 amniocenteses and reports a 1:97 risk of recurrence. Golbus et al.1979 found the risk 1:67 for mothers over age 35 (2,404 amniocenteses) and 1:240 for younger mothers with previous trisomy 21. Harris et al. 1982 estimated the recurrence risk to be between 0.3% and 4.6% from the segregation analysis and the survey. There is an increased recurrence in young women having a second child with T21 DS which may be due to high grade T21 ovarian mosaicism (Hulten et al. 2008). If the translocation is de-novo, the recurrence risk is <1%. In case of familial RT DS, the genetic risk for female carrier to have a live born child with translocation DS is about 10%, which increases to 15% at amniocentesis. For male carrier the recurrence risk to have a child with translocation DS is about 1% (Gardner, 1996). In case of t(21;21) if one of the parent is a carrier, the chance of recurrence is 100%. In case of t(21:22) the risk is below 5% if one of the parent is a carrier and in case of D/G translocation if mother is a carrier the recurrence risk is below 10% and if father is a carrier the risk is below 5%. Chromosomal investigations, family history, pedigree analysis, parental ages and parental karyotypes are essential factors in ferreting genetic counselling, estimating the risk for the next conception (Jyothi et al. 2002). In the case of familial robertsonian translocation with Down syndrome, the genetic risk of the female carrier having a live born child with translocation in Down syndrome is about 20% if the mother is a carrier and 5% if the father is a carrier (Gardner and Sutherland 1996). The geneticist will inform the couple of the possibilities of antenatal diagnosis, if desired. In rare cases, trisomy 21 by translocation involves two chromosomes 21. If one of the parents is carrying this translocation in a balanced state, the risk of recurrence is 100% (Jaouad et al. 2010). The potential liveborn unbalanced outcome of this D/G Robertsonian is translocation trisomy 21 resulting in Down's syndrome; for female carriers, the empirical risk of occurrence at second trimester prenatal diagnosis is 15%, with a 10% risk of liveborn trisomy 21 plus a small
24

risk of uniparental disomy (UPD) 14, as before. For male carriers, the second trimester risk of translocation trisomy 21 is <0.5% (Bou and Gallano, 1984; Gardner and Sutherland, 1996), possibly due to the selective disadvantage for spermatozoa carrying an extra homologue of chromosome 21. Other D/G Robertsonians which involve chromosome 21 may be expected to have similar reproductive risks to the der(14;21); those involving chromosome 22 have a lower risk since trisomy 22 has very limited potential to be viable (Scriven et al. 2003).

Chapter: 4

MATERIALS AND METHODS

25

4.1 Materials required

Cotton, hand belt, sterilized disposable syringes, laminar air flow, autoclaved culture vials, micropipettes and tips, spirit lamp, CO2 incubator, hot air oven, solution bottles, volumetric flask, dropper, 15ml falcon tubes, centrifuge, beaker, cooling fridge, blower, slides, slide warmer, tissue paper, coplin jar, weighing balance, timer, cover slips, glass rod, microscopes and computer.

4.2 Chemicals required

Ethanol (75%). Sodium heparin anticoagulant (Sigma). Culture media- Lymphogrow media (Cytogen). Serum (Gibco). Mitogen Phytohemagglutinin Human peripheral blood. Colchicine (Sigma or Lobal Chemie): 0.02 % (1mg/5ml). Hypotonic solution (KCl): 0.075 M (56mg for 10 ml). Fixative - Carnoys (Methanol: Acetic acid :: 3:1) Trypsin: 7.5 mg in 50ml saline (450 mg NaCl in 50 ml).
Phosphate buffer (For 100ml KCl 20mg, NaCl 800mg, Na2HPO4.2H2O 92mg

and KH2PO4 20mg). Giemsa stain (4%): in water or buffer. DPX mountant.

4.3 Protocol: (Manjunatha et al. 1997)

4.3.1. Initiation
26

25 drops of blood was added to 5ml of culture media ( Lymphogrow media) in autoclaved vial. Vials shaken and incubated at 37 C for 70 hours in CO2 incubator. 2 hrs.

About 6 drops of Colchicine (0.2mg/ml) was added and the vials were incubated for

4.3.2. Harvesting
Cells were harvested by centrifuging at 1500 RPM for 10 minutes.

Supernatant was removed and cells re-centrifuged with fixative (methanol:acetic acid 3:1).

The process was repeated for 2-3 times till clear pellet was observed.

Cells finally re-suspended in 2-3ml of fixative and left overnight at 4 C.

4.3.3 Preparation of slides

The cells were dropped on pre-chilled glass slide at an angle of 45 and blown gently to spread.

The slides were heat fixed and air dried overnight.

4.3.4 Banding
The slides were GTG banded. Trypsin treatment 8-10 seconds. Giemsa stain 20 30 minutes.

4.3.5 Screening of slides

Slides were then observed for good metaphase spreads. Image was captured.

4.4. Karyotyping.
Open software Leica CW 4000 karyo. Load image. Using search window select the region of spread. Start Analysis. Adjust brightness and contrast.

27

 Using threshold option, boundaries around the chromosomes were determined for further analysis.

Metaphase spreads were edited using options such as separate, overlap, trim/extend , join ,delete etc.

Click continue. Each chromosome is highlighted in different colours. Click continue. Karyogram is generated by the software.

Each chromosome can be edited using options such as rotate, trim, straightened/nonstraightened, scaling etc.

Further the karyogram can be arranged manually if it is not generated correctly.

Click done. Save the result along with patients details.

The results will be stored in the result data base. Take the print out of the result.

4.5. Individual characterization of human chromosomes

Group A (no. 1-3): Large metacentric and submetacentric chromosomes. Chromosome 1: Largest metacentric. centromere index (CI) is 48-49. Chromosome 2: Largest submetacentric. CI is 38-40.

Chromosome 3: 20% shorter than 1, metacentric . CI is 45-46.

Group B (no. 4 and 5): Chromosome 4 and 5 cannot be distinguished without autoradiography.

Chromosome 4: large submetacentric. CI is 24-30. Chromosome 4 is late replicating over the whole length. Chromosome 5: large submetacentric. CI is 24-30. Only short arm of chromosome 5 is late replicating over the whole length.

Group C (no. 6-12) Medium sized submetacentric chromosomes. They are easily distinguished by their banding patterns. Chromosome 6: Medium sized submetacentric. CI is 27-35
28

 Chromosome 7: Medium sized submetacentric. CI is 27-35 Chromosome 8: Medium sized submetacentric. CI is 27-35

Chromosome 9: Medium sized submetacentric. CI is 27-35. No.9 Shows a constriction in the proximal part of its long arm.

secondary Chromosome 10: Medium sized submetacentric. CI is 27-35 Chromosome 11: Medium sized submetacentric. CI is greater than CI of no. 12. Chromosome 12: Medium sized submetacentric. Group D (no. 13-15)

Chromosome 13, 14 and 15 are medium sized acrocentric chromosome. CI is 15, which is the lowest in the human karyotype. All the 3 pairs have satellites and their short arm regions show high inter-chromosomal variability.

The proximal short arms of all three D group chromosomes are clearly distinguished by Q or G banding. Group F (no. 19-20) Chromosome 19 and 20 are short metacentric. CI is 36-46. Group E (no.21-22)

Chromosome 21 and 22 are short acrocentric chromosomes. CI is 13-33.

They are easily distinguished by their banding patterns. X Chromosome Medium sized metacentric (group C) Y Chromosome Short acrocentric (group G).No satellites.

4.6. Case Study

The cases referred to the Department of Human Genetics were studied. The consent is obtained from the parents or guardian of the patient. The referral form is then used to note down the observed clinical features.
29

 Basic information like name, age, sex, reference number, address, fathers name and age, mothers name and age, education of the patient and the occupation are noted down. The age of the parents at the time of birth of subject is noted down.
The parents were enquired if their marriage was consanguineous. If yes, it was noted

down as how father is related to mother. They were also asked if any other family member was affected. Pedigree analysis was done up to three generations using symbols from the pedigree chart.

Fig 4.1. Pedigree Chart. Degree of mental retardation was noted down as whether it is mild, moderate, severe or profound. Important clinical features were taken down under 3 subheadings congenital anomalies, facial anomalies and physical anomalies.

30

 Dermatoglyphic features were then noted down. The hand was looked for the presence

of simian crease, sydney line, single crease on little finger and other features if present. The digits were then observed for loops, especially the frequency of ulnar loops. About 3 ml of blood is collected from the patient in a sterilized and heparinised syringe for diagnosis by karyotyping and for repository of DNA bank.

4.7. Comparative studies:

A list of patients name and Neuro number were made using the registers maintained by the Department of Human genetics, NIMHANS, Bangalore. The list was forwarded through our guide to the Medical Records Section of NIMHANS for obtaining the medical records of the patient. Using the record the patients details were recorded and tabulated using excel sheet.
The details contained cyto no., name, age, sex, parents age at subjects birth,

consanguinity, other family member affected, degree of mental retardation, clinical features, dermatoglyphic features, pedigree analysis, diagnosis, prenatal factors, peri and post natal factors, medical history, risk of recurrence, occupation of father, address, mother, date and reference no.
Under each factor the frequency of each feature was calculated and tabulated in the

decreasing order of frequency.

Fathers age, occupation of father (economical condition of the family), mothers age,

other family member affected, prenatal and consanguinity were the factors we considered which could have been responsible for the occurrence of Down syndrome child. Hence in each case, each of this factor was looked for which might have played a role in occurrence of Down syndrome. The frequency of these factors was then calculated and tabulated to observe which factor contributed more. Later the clinical features were compared between variants of Down syndrome free trisomy, mosaicism and translocation.

4.8. DNA extraction from mammalian whole blood using

31

Pharmacia biotech kit.

Extraction from 300 l sample:
300l of whole blood + 900l RBC lysis solution. Invert to mix and incubate for 10 mins at room temperature. Invert once during incubation. Centrifuge at 13000 16000 RCF for 20 secs. Remove supernatant, leaving behind cell pellet and 10 20 l residual liquid. Vortex vigorously Add 300 l of lysis solution and pipette up and down to lyse the cells. Incubate at 37C if clumps are seen. Add 1.5l of RNAase A solution .mix by inverting and incubate at 37C for 15 mins. Cool to room temperature Add 100l of protein precipitation and vortex for 20 mins. Centrifuge at 13000-16000 RCF for 3mins. Pour the supernatant containing DNA into a tube containing 300l of 100% isoproponol. Mix by inverting until white threads of DNA form a clump. Centrifuge at 13000 16000 RCF for 1 min. Pour off the supernatant and drain the tube.
32

 Add 300l of 70% ethanol Centrifuge at 13000 16000 RCF for 1 min and carefully pour off ethanol. Drain the tube and air dry for 15 mins. Add 100l of DNA hydration and rehydrate at 65C for 1hour. Tap the tube periodically. Store at 2 8 C

Chapter: 5

RESULTS
5.1. Standardisation
5.1.1. Cultures
Culturing was done four times in order to standardize our protocol and the alterations and results for each culture are tabulated in table 5.1. Table: 5.1. Culturing. Culture 1st culture Observation The culture (alkaline) turned 48 dark hours pink of

within

incubation i.e. the pH was not maintained neutral. Culture did not give normal cell population after harvesting and
33

contamination was observed. Few metaphases were seen and the chromosomes were small and morphology could not be studied. 2 culture Alterations: Due to culture turning alkaline few drops of 0.1N HCl was added to culture media prior initiation. Due to less cell population one set of vials were added with 5-6 drops of PHA. To check for contamination vials were incubated in two different incubators. Alterations were also done in banding. Trypsin (8-10 seconds) and Giemsa (30 minutes). 3rd Culture. Alterations: was added to one set. 4th Culture. Alterations:

nd

Banding was unseen. Culture still turned dark pink after incubation. The problem may be with the buffering action of the culture media.

The set with PHA showed increase in the normal cell population. PHA in the media is not effective.

Cells were not healthy. Problem might be with nutrients in the media.

The 3rd culture failed and very less cells were observed. No metaphase spread was

Different culture media were used and PHA seen. media: media and Cytogen Gibco

The set with the media Cytogen lymphogrow gave better results. Culture turned slightly alkaline. Very good cell population and good mitotic index. Cells were healthy. Chromosomes were bigger and pluffy. Morphology and banding could be observed.

Culture lymphogrow

RPMI 1640 media. Two sets each. Serum (Gibco): 1 ml was added to each vial. PHA: 220l. Blood : 30 drops KCl : 15mins

34

Centrifuge: 1000 1050 rpm for 8 10 mins. Trypsin: 15 20 seconds depending on temperature. Giemsa: 30 mins.

5.1.2. Standardised protocol:

The fourth culture gave us good results hence the protocol is standardized. The standardized protocol is as follows. Initiation

1ml serum (gibco), 200-220 l of Phytohaemagglutin and 30-35 drops of blood was added to 5-6ml of Lymphogrow media (Cytogen), in autoclaved vials and pH was maintained between 7-7.6. Vials shaken and incubated at 37 C for 70 hours in CO2 incubator. About 6 drops of Colchicine (0.2mg/ml) was added to arrest the cells in metaphase stage and the vials were incubated for 2 hours.

Harvesting Cells were harvested by centrifuging at 1000-1050 RPM for 8 minutes.

Supernatant decanted and sedimented cells are subjected to hypotonic treatment by adding 5-6ml of 0.075 M KCl. Tubes are incubated at 37 for about 15 mins.

The tubes are again centrifuged at 1000-1050RPM for 8-10mins. Supernatant decanted and the cells re-suspended in about 0.2ml of the left over volume.

About 3 ml of the fixative (methanol : acetic acid - 3:1) was slowly added along the sides of the tubes and the contents mixed gently to lyse RBC. The tubes are then refrigerated overnight at 4deg C.

The cells are repeatedly washed 2-3 times with freshly prepared fixative until a clear white pellet is obtained.

Finally 1-2ml of the fresh fixative is added to the cells and the volume is adjusted depending up on the density of the cell suspension.

Preparation of slides

35

The cells were dropped on pre-chilled glass slide at an angle of 45 and blown gently to spread. The slides were heat fixed on slide warmer at 50-55 for 2mins and air dried overnight.

Banding The slides were GTG banded.

Trypsin treatment -7.5 mg in 50ml saline (450 mg NaCl in 50 ml) for 15-20mins.

Rinse the slides with phosphate buffer (For 100ml KCl 20mg, NaCl 800mg, Na2HPO4.2H2O 92mg and KH2PO4 20mg).

Giemsa stain (4%)- 2ml in 48ml water or buffer 20-30 mins.

Leave the slides overnight at 37 . Mount the slides using DPX mountant and was kept overnight in room temperature.

Screening of slides Slides were then observed for good metaphase spreads. Pictures of good metaphase spreads were taken using Leica CW 4000 microscope. Karyotyping was done using Leica CW 4000 Karyo software. Figure 5.1. Normal male Karyotype.

36

Figure 5.2. Normal Female Karyotype.

37

5.2. Spectrophotometric readings of isolated DNA.

Table 5.2. DNA sample readings. Sample Control 1 Control 2 Control 3 Control 4 Kalpana Sumalatha OD @ 260nm 0.011 0.071 0.055 0.053 0.075 0.073 OD @ 280nm 0.006 0.036 0.028 0.026 0.038 0.037 1.83 1.97 1.96 2.04 1.97 1.97

DNA was extracted using kit method and purity was determined using spectrophotometer. DNA sample is said to be pure if its is between 1.8 and 2.0.

38

5.3. Case Studies.

In our study, two Downs cases were studied that were referred to the Department of Human Genetics, NIMHANS, Bangalore. One was diagnosed as free trisomy and the other as Translocation (t 14:21). The clinical details, pedigree analysis and the cytogenetic studies were carried out.

5.3.1. Case study 1:

Table 5.3. Details of Case study 1. Name Age Sex Kalpana 1 yr Female

Age at the time of birth of Father : 34 subject Mother : 24 Consanguinity No.

Any other family member No. affected

39

Pedigree analysis

Degree of Retardation Clinical features

Mental Mild. Small ears. Slanting palpebral fissure. : 1 whorl and 4 ulnar loops. : 1 whorl and 4 ulnar loops.

Dermatoglyphic features

Simian crease : Present. Right hand Left hand

Figure 5.3. Karyotype of Case study 1:

40

5.3.2. Case study 2:

Table 5.4. Details of Case study 2. Name Age Sex Age at the time of birth of subject Consanguinity Any other affected family Sumalatha 6 yrs. Female Father : not known. Mother : 29 years. Yes, father is maternal uncle of the subjects mother. member No.

41

Pedigree analysis

Degree of Mental Retardation Clinical features

Moderate to severe. Hypertelorism. Epicanthal folds. Long tongue. Short fifth finger in Hand. Short stature.

Dermatoglyphic features

Simian crease : Absent. Right hand : 2 whorls and 3 ulnar loops. Left hand : 5 ulnar loops.

Figure 5.4. Karyotype of Case study 2:

42

5.4. Comparitive studies of variants of Down syndrome using past 20 years data from NIMHANS, Bangalore.
Out of 122 Downs cases reported by the Department of Human Genetics, NIMHANS, 105 (86%) were found to be free 21 trisomy, 11 mosaicism (9%) (two 20%, one 10%, one 60%, one 50%, one 75%, two 80%, one 30%) cases and 6 translocation (5%) (two t 21:21, four t 14:21) cases. Out of these 42 cases were taken for comparative studies. Out of which 28 cases had trisomy 21, 3 cases mosaicism, 3 cases were translocation (t 14:21) and in 7 cases diagnosis was not
43

known. One peculiar case was found who had the clinical features of Down syndrome but had normal karyotype. The parents age ranging from 16 to 45 years of age were divided in 6 groups. The frequencies in each group were determined and are tabulated below. Table 5.5. Parents age during subjects birth (Out of 42 cases) Age groups Father 16-20 21-25 26-30 31-35 36-40 41-45 Not known 0 5 5 18 8 1 5 % 0 11.90 11.90 42.85 19.04 2.38 11.90 Frequency Mother 8 11 12 4 0 1 6 % 19.04 26.19 28.57 9.52 0 2.38 14.28

The number of consanguineous marriages was determined and was classified as close and far. The frequency of each was determined and tabulated. Table 5.6. Consanguinity: 7 cases (out of 42 cases) Consanguinity Close Far Normal Not mentioned Frequency 5 1 35 1 % 11.90 2.38 83.33 2.38 The number of cases where family members were affected was noted down. Family members affected: 11 cases (26.19 %) Table 5.7. Patient details Trisomy 21.

44

45

46

47

48

49

Table 5.8. Patient details Normal karyotype with DS clinical features.

50

Table 5.9. Patient details Mosaic cases.

51

Table 5.10. Patient details Translocation cases.

52

Table 5.11. Patient details Diagnosis not known.

53

54

The degree of mental retardation was classified as mild, mild to moderate, moderate and moderate to severe. Table 5.12. Degree of mental retardation: Out of 42 cases. Degree of mental retardation Frequency 31 % 73.80 Mild Mild to moderate 2 4.76 3 7.14 Moderate Moderate to severe 1 2.38 3 7.14 2 4.76 Not known No

Dermatoglyphic features in the palm were noted down in each case and noted down. The frequency was then determined. The digits were then observed for Ulnar loops (loops towards ulnar bone). Table 5.13. Dermatoglyphic features (Out of 42 cases) Feature Frequency %
55

Normal Simian crease present Single crease on little finger Sydney line, Clinodactyly ,Palmar crease Middle phalanx on right hand little finger is absent High frequency of ulnar loops was observed.

17 16 3 2 1

40.47 38.09 7.14 4.76 2.38

Table 5.14. Clinical features: Out of 42 cases. The frequency of clinical features were recorded and tabulated. Features Epicanthal folds Depressed nasal bridge High arched palate Slanting palpebral fissure Hypertelorism, Microcephaly, short neck. Short stature. Short stubby fingers. Light grey hair, sparse hair, Protruding tongue. simplified ears, small ears, long forehead, eyebrow meeting in centre. Round face, malformed dentition, increased distance between 1st and 2nd toe, flat occipital, distended abdomen, posterior ears, bending eye brow. Frequency 29 20 18 13 9 8 7 6 4 3 2 % 69.04 47.61 42.85 30.95 21.42 19.04 16.66 14.28 9.52 7.14 4.76

56

Deformed 5th phalanx, Injured fifth finger, Mongoloid facial features, 1 low set ears, hypotonia of limbs, small chin, Dwarfism, obesity, webbing of neck, short neck, flaring nose, Brachocephaly, Bitemporal bosny, kyphoscoliosis, demoid sinus present, dimpliny sacenal, anteverted, clubbing, flat feet, short digits, mongoloid slant, coarse skin, hand abnormality, stunted growth, dry skin, asthenic body, scancy hair, anaemic, Cafau-lait spots on skin, distended abdomen, bushy eyebrows, eyebrows meeting in centre, open mouth, haemangioma of right leg (mid leg), posterior noselong philtrum, short limbs, short neck, Thick skin, partial hearing, dentate tongue, Thickened skin in the anterior aspect of neck, inguinal hernia of abdomen, small external genetalia, increased lamalia (vision), posterior offset malformed lop earsshort and web neck, loose skin on the back, fullness in subment, frontal gap not healed, decreased body hair, mild obesity.

2.38

Out of 42 cases, in 22 cases prenatal, peri and post natal factors and medical history could be studied. Each factor was noted and tabulated along with their frequency. Table 5.15. Prenatal factors (Out of 22 cases) Factors None Medication during pregnancy High BP at pregnancy, Antepartum bleeding Vomiting during pregnancy, Diastollic flow reverses, Abortion, Stomach crambs, Injections given, Fever in 5th month, Exposure to vitamins, Scanning Table 5.16. Peri and postnatal factors (Out of 22 cases) Factors Normal delivery. Neonatal jaundice. Premature delivery. Delayed or weak cry. Respiratory distress, incubator care, caesarian, Congenital anomalies, Normal home delivery and feeding problems. Low birth weight. Pareital encephalocele, respiratory stridor, laryngeo malasia, fetal Frequency 9 8 5 4 3 2 1 % 40.90 36.36 22.72 18.18 13.63 9.09 4.54
57

Frequency 7 3 2 1

% 31.81 13.63 9.09 4.54

distress, meconium stained amniotic fluid, kidney obstruction, cyanotic attacks and medication.

Table 5.17. Medical history (Out of 22 cases) Factors Recurrent respiratory infection. Behavioral disorder, convulsions and Congenital heart defects (CHD). Low grade fever, vomiting, delayed motor development, operated, epilepsy, fits, tempertantrum, recurrent fever, stereotypes, stuttering in patient and family member, constipation, delayed speech and language, no head control and standing, sinusitisis, mother tubectomised, loose motions, macrocephaly, failure to thrive. We considered six factors viz fathers age, occupation of father (Economical condition of family), mothers age, other family members affected, prenatal factors and consanguinity in each case. Frequency of each factor was calculated and tabulated in order to find which factor contributed in most cases. Table 5.18. Major factors that might have been responsible for occurrence of Down syndrome. Factors Fathers age (42 cases) Occupation of father (22 cases) Mothers age (42 cases) Other family member affected (42 cases) Prenatal (22 cases) Consanguinity (42 cases) Clinical features:
58

Frequency 9 2 1

% 40.90 9.09 4.54

Frequency >30 = 29 16 12 (<20 = 4 ; >30 = 8) 11 8 6

% 69.04 72.72 28.57 26.19 36.36 14.28

In order to observe how clinical features varied in each variant of Down syndrome, the frequency of each feature was calculated and tabulated for each variant and then compared.

Table 5.19. Frequency of clinical features in Trisomy 21 (28 cases). Features Epicanthal folds High arched palate Mongoloid facial features Depressed nasal bridge Palpabral fissures Low set ears Hypertelorism , microcephaly Short stature Protruding tongue , long forehead/sloping forehead Simplied ears , short neck , short stubby fingers, CHD Bending eyebrow , increased distance between 1st and 2nd toe Deformed 5th phalanx , hypotonia of limbs , small chin, round face , obesity , malformed dentition , flaring nose , branchocephaly , flat occipital , bitemporal bosny , kyphoscoliosis , demoid sinus , dimpliny sacenal , asthenic body , distended abdomen , increased lamalia , partial hearing , dentate tongue , thichened skin in the anterior aspect of neck , anteverted / posterior / small ears , caf-aulait , thick / coarse / dry skin , light grey / scancy / sparse hair , eyebrow meeting in center. Table 5.20. Frequency of clinical features in Translocation (2 cases) Features Frequency Epicanthal folds , Depressed nasal bridge , 2 Palpabral features High arched palate , mongoloid facial features 1 % 100 50 Frequency 20 12 11 10 8 7 6 5 4 3 2 1 % 71.42 42.85 39.28 35.71 28.57 25 21.42 17.85 14.28 10.71 7.14 3.57

59

Table 5.21. Frequency of clinical features in Mosaic (3 cases) Features Frequency Epicanthal folds , depressed nasal bridge 3 High arched palate , hypertelorism , 2 Microcephaly , posterior ears , posterior offset 1 malformed lop ears ,flat occipital ,short and web neck , distended abdomen , loose skin on the back , fullness in subment. In order to observe which variant of DS was most frequent in consanguineous marriage, consanguinity was compared between different variants. Table 5.22. Consanguinity among variants of Down syndrome. Clos Trisomy 21 (28cases) Mosaic (3 cases) Translocation e 4 % 14.28 0 0 Fa r 1 % 3.57 0 0 Normal 22 3 2 % 78.5 7 100 100 Not mentioned 1 % 3.57 0 0 % 100 66.66 33.33

(2 cases) In order to observe in which variant the family members are affected most, the frequency was calculated and observed. Table 5.23. Family members affected among variants of Down syndrome. Affected Trisomy 21 4 % 14.28 0 0 Not affected 23 3 2 % 82.14 100 100 Not known 1 % 3.57 0 0

(28 cases) Mosaic (3 cases) Translocation (2 cases) -

To see if degree of mental retardation varied between the variants of DS. Table 5.24. Degree of mental retardation among variants of Down syndrome. Mild % Mild % Mod% Mod% Not No
60

to moderate Trisomy 21 (28 3 100 0 0 0 cases) Mosaic (3 cases) Translocation (2 cases) 22 78.57 1 3.57

erate

erate to severe 1

known

7.14

3.57

7.14

0 50

0 0

0 50

0 0

To observe how dermatoglyphic features vary among different variants. Table 5.25. Dermatoglyphic features among variants of Down syndrome. Trisomy 21 (28) Features Normal Simian crease Single crease on little finger Palmar crease,clinodactyly Sydney line , middle phalanx on right hand little finger is absent Mosaic (3) Translocation (2) To compare which age group of parents have higher chances of giving birth to different variant of DS children. Table 5.26. Age group of parents with DS childrens with Trisomy 21(28 cases) Age groups 16 20 21 25 26 30 cy 2 4 Frequen 0 7.14 14.28 Father % cy 3 8 9 Frequen 10.71 28.57 32.14
61

Frequency 12 10 3 2 1

% 42.85 35.71 10.71 7.14 3.57

Normal simian crease Simian crease

2 1 2

66.66 33.33 100

Mother %

31 35 36 40 41 45 Not known

11 7 4

39.28 25 0 14.28

4 1 3

14.28 0 3.57 10.71

Table 5.27. Age group of parents with DS childrens with Mosaicism (3 cases) Age groups 16 20 21 25 26 30 31 35 36 40 41 45 cy 1 1 1 Frequen 0 33.33 33.33 0 33.33 0 Father % cy 2 1 Mother Frequen % 66.66 33.33 0 0 0 0

Table 5.28. Age group of parents with DS childrens with Translocation (2 cases) Age groups 16 20 21 25 26 30 31 35 36 40 41 45 cy 2 Frequen 0 0 0 100 0 0 Father % cy 1 1 Frequen Mother % 50 50 0 0 0 0

How prenatal, peri and postnatal factors and medical history varied among different variants of Down syndrome were studied. Table 5.29. Prenatal factors among variants of Down syndrome Factors Frequen %
62

Trisomy (10)

cy 21 None 8 Medications during pregnancy 2 th Fever in the 5 month ,high BP at 1 pregnancy ,diastolic flow reverses,antepartum bleeding

80.0 20 10

Mosaic (2) Translocation (2)

None Medication ,scanning , none

2 1

100 50

Table 5.30. Peri and post natal factors among variants of Down syndrome Factors Trisomy 21 (10 Neonatal jaundice Normal delivery cases) Respiratory distress ,incubator care , Frequency % 6 60 4 40 caeserian 2 20 10

,premature delivery Low birth weight , parietal encephalocele , delayed/weak 1 cry, respiratory stridor,laryngeo malasia,fetal distress,meconium stained amniotic fluid , congenital anomalies , kidney obstruction , feeding problems , medications Normal Mosaic cases) Translocation (2cases) ( 2 Normal delivery Feeding problem Caeserian ,feeding problem, premature delivery 1 2 1 1

10 100 50 50

63

Table 5.31. Medical history among variants of Down syndrome % Factors Recurrent respiratory infection Behavioural / oppositional disorder , none Trisomy 21 (10 Low grade fever , vomiting , delayed motor cases) development , operation, convulsions ,tempertantrums , sluttering , sluttering (family members) ,constipation , delayed speech and language , no head control and standing , mother tubectomised . Mosaic cases) (2 None Recurrent respiratory infections Recurrent respiratory infections, failure to thrive 1 1 1 50 50 50 Frequency 4 40 2 20 1 10

Translocation (2 cases)

64

Chapter: 6

DISCUSSION
To study the clinical and genetic basis of Down syndrome, two cases of Downs were studied in detail, which were referred to the Department of Human Genetics, NIMHANS, Bangalore. The first case was a 1 year old female baby named Kalpana who had mild mental retardation. The marriage was non consanguineous and the no other family member was affected. Pedigree analysis revealed that the second girl child born to the couple had frequent vomiting and died. Karyotyping revealed that the child had Robertsonian translocation between 14th and 21st chromosome. In this case the mother is young aged 24 years. Father age was 34 years at the time of birth of child. This is similar to that reported by Malini et al. 2007 that translocation DS cases were born to the young mothers (1824 years) and father of advanced age (3035 years). The second case was a 6 year old female child named Sumalatha. Her fathers age at the time of her birth was not known but her mother was 29 years old. The couple marriage was consanguineous where the father is the maternal uncle of the subjects mother. The child was diagnosed as free trisomy 21 in karyotyping (47 XX + 21). In this case consanguinity might have played a role in the occurrence of DS as an increased incidence of Down syndrome has been reported in a highly consanguineous population (Alfi et al. 1980). In our study out of 122 cases of Downs syndrome reported by the Department of Human Genetics, NIMHANS, trisomy 21 was most frequent (86%), followed by mosaicism (9%) and translocation (5%). In translocation (14:21) was most frequent. All of the translocation cases
65

were robertsonian translocation. Our findings were similar to that of Jyothi et al., 2002 and Azman et al., 2007 where they reported higher frequency of mosaicism than translocation. 42 cases were taken for comparative studies. Out of which 28 cases had trisomy 21, 3 cases mosaicism, 3 cases were translocation (t 14:21) and in 7 cases diagnosis was not known. One peculiar case was found who had the clinical features of Down syndrome but had normal karyotype. It is known that the incidence of Downs syndrome increases with maternal age (>30) at the time of birth (Antonarakis et al. 1998). But in our study involving 42 cases greater incidence of Down syndrome was found in the maternal age between 20 and 30 i.e 23 (54.76%) cases. However, greater incidence was observed in paternal age 30 to 35(18 cases) (42.86%). This was similar to the several reports from India and outside India (James et al. 1999, Jyothi et al. 2000, 2001; Kothare et al. 2002; Rao 1999). In Indian the number of children born to young mothers is also more when compared to fathers of the similar age. This is probably due to women getting married at young age (1829 years) and produces more children. This kind of situation is not found in western families (Malini et al. 2007). Further paternal age has been reported to be significant (Fisch et al. 2003). Malini et al. 2007 had reported the influence of grand maternal age, on the risk of their grandchild being born with DS. An increased incidence of Down syndrome has been reported in a highly consanguineous population (Alfi et al. 1980). In our study consanguineous marriages were observed in 7 cases (16.66%). In 11 (26.19%) cases other family member was affected in our study. Majority of the cases in Down syndrome had mild mental retardation (73.8%). The most common clinical features were found to be epicanthal folds (69.04%), depressed nasal bridge (47.61%), high arched palate (42.85%), slanting palpebral fissures (30.95%), hypertelorism (21.42%), Microcephaly and short neck (19.04%), short stature (16.66) and short stubby fingers (14.28%). Our findings were similar to that of Jones 2006. In dermatoglyphic features, the most common feature observed was simian crease (38.09%). In the majority of the cases it was normal (40.07%). Single crease on little finger, Sydney line and clinodactyly were less frequent. Ulnar loops were most frequent on the digits. Penrose and Smith in 1966 described in detail on dermatoglyphic features in their book Downs Anomaly. Detailed information was obtained from old medical records in 22 cases. During prenatal period most of the cases were normal (31.81%). In 3 cases mother was exposed to medication
66

(13.63), in 2 cases mother had high BP and antepartum bleeding. The other problems reported were vomiting, diastollic flow reverses, abortion, stomach crambs and fever in 5th month. Some were exposed to injections, vitamins and scanning. During Peri and post natal period most of the cases had normal delivery (40.90%). Neonatal jaundice (36.36%) was the most reported problem followed by premature delivery in 22.72% cases and delayed or weak cry in 18.18% of cases. Respiratory distress, incubator care, caesarian, congenital anomalies and feeding problems were observed in 13.63% cases. In medical history, recurrent respiratory infection was the most frequent and was observed in 40.9% of the cases. Behavioral disorder, convulsions and congenital heart disease were observed in about 9 % of the cases. We considered six factors viz fathers age, occupation of father (Economical condition of family), mothers age, other family members affected, prenatal factors and consanguinity in each case. Frequency of each factor was calculated and tabulated in order to find which factor contributed in most cases for the occurrence of Down syndrome. In 29 cases (69.04%) the fathers age was above 30. About 67% of free trisomy originates from paternal meiosis errors. Paternal meiosis II errors are twice as frequent as paternal meiosis I errors. For male carriers, the second trimester risk of translocation trisomy 21 is <0.5% (Bou and Gallano, 1984; Gardner and Sutherland, 1996), possibly due to the selective disadvantage for spermatozoa carrying an extra homologue of chromosome 21. Research from the laboratories of Stephanie Sherman and Terry Hassold has provided insights into the origin of human aneuploidy, including those specific to trisomy 21. 5%10% of trisomy 21 was due to paternal nondisjunction (MI and MII errors). In general, the incidence of aneuploidy in humans varies during development; for example, 1% 2% in sperms. Robertsonian translocations have been suggested to occur during oogonial / spermatogonial mitosis (Ohno et al. 1961) or meiotic prophase I (Mirre et al. 1980; Stahl et al. 1983). The genetic risk of the male carrier having a live born child with translocation in Down syndrome is about 5% (Gardner and Sutherland 1996). Further an increase in fathers age result in less recombination increasing the chances of aneuploidy. Evaluation of paternal non-disjunction cases revealed a reduction in recombination in MI cases and an excess of MII errors associated with a slight increase in the amount of proximal-medial exchange compared to exchange at telomeres (Savage et al. 1998). Fisch et al. (2003) also showed that the paternal age effect was the greatest in couples older than 40 years with the risk for Down syndrome increasing to six times
67

the rate in couples younger than 35 years (Fisch et al. 2003). Hence increase in paternal age is a major contributing factor. In 4 cases the mothers age was below 20 and in 8 cases it was above 30. It is well known that incidence of Down syndrome increases with increased maternal age. But here more number of younger mothers is also seen. Hence the mechanism must be understood in detail. About 68% of free trisomy originates from maternal meiosis I errors, about 20% from maternal meiosis II errors. Increased proximal (pericentromeric) exchanges (with longer linkage maps) associated with MII errors are more common in older women. It has been suggested that increased biological ageing of the ovaries is a major factor for aneuploidy conditions in females - a limited oocytes pool hypothesis (Warburton 1989). According to this hypothesis, the ageing of the ovary is associated with availability of limited and less optimal oocytes for fertilization. Further selection against T21 oocytes leading up to the total 300400 maturing to ovulation between puberty and menopause as discussed with respect to the oocyte selection model proposed by Zeng and co-workers. (Zheng et al. 2000; Zheng and Byers 1992). The net effect of this situation is that any T21 oocytes in the original pool will comprise a larger proportion of the ovarian reserve at later maternal ages. The maternal age effect may be due to differential selection and accumulation of T21 oocytes in the ovarian reserve of older women. This type of ovarian mosaicism might also explain the maternal age effect (Hulten et al. 2008). Also the prolonged meiotic arrest phase likely allows accumulation of toxic effects including environmental insults, degradation of meiotic machinery causing MI and MII errors, and suboptimal ovarian functioning likely resulting in hormonal imbalance (Sherman et al. 2007). These findings help in understanding how increase in maternal age increases the occurrence of Down Syndrome. There are a very few earlier reports indicating the influence of grand maternal age, on the risk of their grandchild being born with DS (Aagesen et al. 1984; Mikkelson 1985; Papp et al. 1977). On pedigree analysis of these families, it is clear that whenever the daughter was born to aged mother the chances of this daughter giving birth to DS children are increased (Malini et al. 2007). The non-availability or non-functioning of proteins leads to impairment in the meiotic process, which in turn results in nondisjunction of chromosome 21 in the oocyte of the daughter. This event takes place during the embryogenesis of the mothers of the DS children when she was in grandmother's womb. It is also possible that recombination is reduced in the oocytes, which brings about the nondisjunction of chromosome 21. Increased number of DS
68

babies born to the young mothers could either be due to MTHFR gene polymorphism (Forester and Merz 2002) and/or nutritional factor (Sheth and Sheth 2003). Increased distal (telomeric) crossovers (with shorter linkage maps) contribute to MI errors in younger women (aged <29 yr) but not so in older women (aged >34 yr (Lamb et al. 2005). On the basis of these cellular observations, James et al and Hobbs et al have postulated a link between abnormal folate metabolism and mutation of the methylenetetrahydrofolate reductase gene, hence as a risk factor for nondisjunction and Down syndrome in younger (< 35 years) mothers. Further most of the younger women were primigravidas (n=18) and the incidence of miscarriage is less in women conceiving at first pregnancy for unknown reasons (Jyothi et al. 2002). These findings help in understanding why there is an increased incidence in younger mothers giving birth to DS children. In 16 cases (72.72%), the occupation of the father was related to agriculture work, handlooms etc. They belonged to the lower income group and further their wives were housewives. This reflects on the economical condition of the family. This can be correlated with the ability to meet their nutritional requirements. And hence if nutritionally deficient the chances of non disjunction is higher as a consequence of abnormal folate mechanism and other co factors. Hence the economical status of the family is an important factor to consider. In 8 cases (36.36%) prenatal factor has played a role in contributing to the occurrence of Down syndrome. Factors such as medication during pregnancy, diastolic flow reverses, injections, abortion, fever, exposure to vitamins and scanning were considered. In the female ovary, after a smaller mitotic proliferation event, the germ cells enter into protracted meiosis I and II periods. After homologous recombination, MI is arrested at prophase, and is completed only after ovulation, at the onset of puberty. MII is then initiated only to be arrested again, in metaphase II; MII is completed after fertilization. Both age-dependent and age-independent factors appear to be operating simultaneously during this process. It could be due to agedependent decay in the spindle fibres or their components, a failure in nucleolar breakdown or an accumulation of the effects of radiation, hormonal imbalances and infection (Chandley 1985). On the other hand, clinical and experimental studies have shown that age-independent DNA hypomethylation is associated with chromosomal instability and abnormal segregation. There is a very rapid increase in human female germ cell (oocyte) number early during fetal development with a peak at 7 months gestational age, followed by a relatively rapid decline before birth and postnatally before puberty, but a slower depletion during reproductive years
69

until menopause.( Courtesy: Hulten et al. 2008). Hence it can be seen that exposure to harmful radiations or infections or even sub standard medications during prenatal period can lead to the aneuploidy in the chromosomes as the oocytes number are the highest during this period. Prof. Maj Hulten and her colleagues were able to give experimental support for their original hypothesis suggesting meiotic aneuploidy in human conceptuses to be the result of ovarian germline mosaicism that is produced during the normal prenatal development (Hulten et al. 2008). Hence prenatal factor is another important factor to be considered. In 6 cases (14.28%), consanguinity was observed to have played an important role in the occurrence of Down syndrome. In 11 cases (26.19%), other family members were affected. An increased incidence of Down syndrome has been reported in a highly consanguineous population (Alfi et al. 1980). A major gene producing a greater risk of non-disjunction should be detectable from a study of pedigrees and populations. If this gene were inherited as an autosomal dominant, then there should be families with multiplex sibships in several generations. If it were recessive, one would expect multiplex families to be more prevalent in a population with a high inbreeding coefficient (Harris et al. 1982). In one interesting case of t(14;21), reported by Jyothi et al. in 2002 parental study revealed carrier status in both the parents. Mother had translocated chromosome in 100% of her cells and father showed translocated chromosome in only 45% of his cells. History of consanguinity revealed first cousin marriage. In familial cases of Down syndrome with more than one affected sibling reduction of mean maternal age was also observed in individuals when a maternal relative was affected. Further, a two to three times increased risk of Down syndrome in the siblings of affected individuals was observed. This shows that consanguinity and if other family member is affected plays an important role in the occurance of Down syndrome. In the comparative studies of clinical features among the variants of Down syndrome Epicanthal folds, High arched palate, Mongoloid facial features, depressed nasal bridge, Palpabral fissures and hypertelorism were the most common features observed among all variants. Congenital heart defects were seen in 10.71 % of the cases in trisomy 21. Out of 3 cases two cases had ventricular septal defect. The clinical features observed were numerous in trisomy 21 patients. The clinical features were slightly mild in case of mosaicism and further milder in case of translocations. Penrose and Smith in Downs anomaly also reported this pattern. In order to observe which variant of DS was most frequent in consanguineous marriage,
70

consanguinity was compared between different variants. Only Trisomy 21 (14.28%) was observed to be more frequent in consanguineous marriage according to our study. Similar pattern was observed even in case of other family members affected. Trisomy 21 had the highest frequency (14.28%). Mild mental retardation was the most frequent in both trisomy 21 and mosaic cases. Only in case of translocation mental retardation was moderate. In dermatoglyphic features simian crease was observed in all the variants of Down syndrome. Normal features were more frequent in free trisomy and mosaic cases. In case of free trisomy, 64.28% of cases had paternals age greater than 30 and 71.42% cases had maternals age below the age of 30. In case of mosaicism, 66.66 % cases had paternals age below 30. Maternals age was lower than 25 in all cases. In case of translocation, paternals age was found to be above 30 in all cases and in case of maternals age it was below 30 in all the cases. In prenatal factors, no problem was observed in mosaic cases. In case of trisomy most of the cases had no problem and in 20 % of cases mother was exposed to medications. In 10 % of cases, problems like fever in the 5th month, diastolic flow reverses, antepartum bleeding was observed. However in case of translocation the mother was exposed to medication and scanning. In case of peri and post natal factors, several problems were associated with trisomy 21 the most frequent being neonatal jaundice. Further feeding problem was observed in all the variants. In case of medical history trisomy 21 had the most problems and recurrent respiratory infection was observed in all the variants. Hence we conclude that other than well known factor mothers age, several other factors such as fathers age, consanguinity, economical condition of family, prenatal factors and if other family member is affected must be considered to provide better and accurate genetic counseling.

71

Chapter: 7

SUMMARY
Down's syndrome, though it is very well studied since 1866, still its incidence is high and several complications are associated with it. Hence further detailed studies must be done in order to understand several other factors that have not been reported till date which plays a major role in the occurrence of Down syndrome. Efforts must be made to provide better and accurate genetic counseling and to improve the quality of life of Down syndrome patients. For our study, the standardization of protocol was done especially for scoring high index metaphases. Image of karyotypes were taken using Leica CW 4000 microscope and karyotyping was done using Leica CW 4000 karyo software. In our study, two Downs cases were studied that were referred to the Department of Human Genetics, NIMHANS, Bangalore. The first case was a 1 year old female baby named Kalpana who had mild mental retardation. She was diagnosed as translocation Downs with karyotype 45 XX t (14:21). Her mothers age at the time of Kalpanas birth was 24 years. Father age was 34 years old. This case was similar to that reported in previous studies on translocation. The second case was a 6 year old female child named Sumalatha. The couples marriage was consanguineous where the father is the maternal uncle of the subjects mother. The child was diagnosed as free trisomy 21 in karyotyping (47 XX + 21). In this case consanguinity might have played a role in the occurrence of DS as an increased incidence of Down syndrome has been reported in a highly consanguineous population. In our study out of 122 cases of Downs syndrome reported by the Department of Human Genetics, NIMHANS, trisomy 21 was most frequent (86%), followed by mosaicism (9%) and translocation (5%). In translocation (14:21) was most frequent. All of the translocation cases were Robertsonian translocation. Out of these 42 cases were taken for comparative studies. Out
72

of which 28 cases had trisomy 21, 3 cases mosaicism, 3 cases were translocation (t 14:21) and in 7 cases diagnosis was not known. One peculiar case was found in which the subject had the clinical features of Down syndrome but had normal karyotype. From the patients medical record, details such as cyto no., name, age, sex, parents age at subjects birth, consanguinity, other family member affected, degree of mental retardation, clinical features, dermatoglyphic features, pedigree analysis, diagnosis, prenatal factors, peri and post natal factors, medical history, risk of recurrence, occupation of father, address, health condition of the mother, date and reference number were recorded. Under each factor, the frequency of each feature was calculated and tabulated in the decreasing order of frequency. In our study involving 42 cases greater incidence of Down syndrome was found in the maternal age between 20 and 30 i.e 23 (54.76%) cases. However, greater incidence was observed in paternal age 30 to 35(18 cases) (42.86%). Consanguineous marriages were observed in 7 cases (16.66%). In 11 (26.19%) cases other family member was affected in our study. The most common clinical features were found to be epicanthal folds (69.04%), depressed nasal bridge (47.61%), high arched palate (42.85%), slanting palpebral fissures (30.95%), hypertelorism (21.42%), Microcephaly and short neck (19.04%), short stature (16.66) and short stubby fingers (14.28%). In dermatoglyphic features the most common was simian crease (38.09%) or normal (40.07%). During prenatal period most of the cases were normal (31.81%). In 3 cases mother was exposed to medication (13.63%), in 2 cases mother had high BP and antepartum bleeding. The other problems reported were vomiting, diastollic flow reverses, abortion, stomach crambs and fever in 5th month. Some were exposed to injections, vitamins and scanning. During Peri and post natal period most of the cases had normal delivery (40.90%). Neonatal jaundice (36.36%) was the most frequently reported problem followed by premature delivery in 22.72% cases and delayed or weak cry in 18.18% of cases. Fathers age, occupation of father (economical condition of the family), mothers age, other family member affected, prenatal and consanguinity were the factors we considered which could have been responsible for the occurrence of Down syndrome child. Hence in each case, each of this factor was looked for which might have played a role in occurrence of Down syndrome. The frequency of these factors was then calculated and tabulated to observe which factor contributed more. Each of these factors might have contributed significantly according to our study. Later the clinical features were compared between variants of Down syndrome free trisomy,
73

mosaicism and translocation. Epicanthal folds, high arched palate, mongoloid facial features, depressed nasal bridge, palpabral fissures and hypertelorism were the most common features observed among all variants. The clinical features observed were numerous in trisomy 21 patients. The clinical features were slightly mild in case of mosaicism and further milder in case of translocations. Trisomy 21 (14.28%) was observed to be more frequent in consanguineous marriage. Similar pattern was observed even in case of other family members affected. Trisomy 21 had the highest frequency (14.28%). Mild mental retardation was the most frequent in both trisomy 21 and mosaic cases. Only in case of translocation it was moderate. In dermatoglyphic features simian crease was observed in all the variants of down syndrome. Normal features were more frequent in free trisomy and mosaic cases. In case of free trisomy, 64.28% of cases had paternals age greater than 30 and 71.42 cases had maternals age below the age of 30. In case of mosaicism, 66.66 % cases had paternals age below 30. Maternals age was lower than 25 in all cases. In case of translocation, paternals age was found to be above 30 in all cases and in case of maternals age it was below 30 in all the cases. In prenatal factors, no problem was observed in mosaic cases. In case of trisomy most of the cases had no problem and in 20 % of cases mother was exposed to medications. In 10 % of cases, problems like fever in the 5th month ,high BP at pregnancy ,diastolic flow reverses, antepartum bleeding were observed. However in case of translocation, mother was exposed to medication and scanning. In case of peri and post natal factors, several problems were associated with trisomy 21 the most frequent being neonatal jaundice. Further feeding problem was observed in all the variants. In case of medical history trisomy 21 had the most number of problems and recurrent respiratory infection was observed in all the variants. Hence we conclude that other than well known factor mothers age, several other factors such as fathers age, consanguinity, economical condition of family, prenatal factors and if other family member is affected must be considered to provide better and accurate genetic counseling and to assess the risk factors.

74

Chapter: 8

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