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Uniparental disomy as a cause for congenital malformations and or developmental delay in inherited apparently… Lopez-Rangel, Elena 1993

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UNIPARENTAL DISOMY AS A CAUSE FOR CONGENITAL MALFORMATIONS AND OR DEVELOPMENTAL DELAY IN INHERITED APPARENTLY BALANCED CHROMOSOMAL REARRANGEMENTS by ELENA LOPEZ-RANGEL  M.D. Universidad Anahuac, Mexico City 1990  A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Genetics Program)  We accept this thesis as conforming  THE UNIVERSITY OF BRITISH COLUMBIA JANUARY 1993 © Elena Lopez-Rangel, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  ^Genetics  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  ABSTRACT  Chromosomal translocations are said to be balanced if there is no apparent gain or loss of genetic material. Apparently balanced chromosomal rearrangements are usually associated with a normal phenotype [Therman 1986, Daniel 1988]. However the frequency of mental retardation and congenital anomalies appears to be increased among individuals who carry a de novo or an inherited apparently balanced chromosomal rearrangement [Funderburk et. al. 1977, Ayme et. al. 1979, Fryns et. al. 1986, Howard-Peebles and Friedman 1986]. Uniparental disomy occurs when both chromosomes in a pair are inherited from one parent instead of one of the chromosomes being inherited from each parent [Searle et. al. 1985, Cattanach et. al. 1985, Lyon et. al. 1985]. Uniparental disomy has recently been established as a cause for congenital anomalies and mental retardation in humans [Spence et. al. 1988, Voss et. al. 1989, Nicholls et. al. 1989, Henry et. al. 1991, Knoll et. al. 1989, Wang et. al. 1991, Temple et. al. 1991, Pentao 1992]. In mice, uniparental disomy for any chromosome may be produced by mating animals that carry certain chromosomal rearrangements which alter normal meiotic segregation [Cattanach 1985, Cattanach 1986, Cattanach 1988, Cattanach 1989). We have investigated the possibility that uniparental disomy may cause mental retardation and malformations in carriers of an  ii.  inherited apparently balanced chromosomal translocation. We studied 7 families in which one or more childen with congenital anomalies and mental retardation have inherited an apparently balanced translocation from a carrier parent. In order to establish the parental origin of the chromosomes, we collected blood from both parents and the affected child. We determined the parental origin of each of the chromosomes involved in the translocations using DNA probes that detect highly polymorphic loci which have been previously mapped to these chromosomes [Nakamura et al. 1987, Lathrop et al. 1988, Nakamura et al. 1988, Boerwinkle et al. 1989, Batanian 1990, Standen et al. 1990, Weber et al. 1991, Zoghbi et al. 1991, Ranum et al. 1991, Scharf et al. 1992, Litt et al. 1992]. We were able to rule out uniparental disomy for each of the chromosomes involved in the translocation in every patient except one who had inherited an apparently balanced translocation from a carrier parent and who presented with malformations and/or developmental delay. In one patient uniparental disomy was ruled out for one of the chromosomes in the translocation but the markers was uninformative for the other chromosome. We conclude that uniparental disomy is not a common occurence in carriers of inherited apparently balanced reciprocal translocations who present with congenital anomalies and/or developmental delay.  iii  SIGNIFICANCE  Uniparental disomy in patients who have inherited an apparently balanced chromosomal translocation has been reported in a few individual cases (Spence et. al. 1988, Voss et. al. 1989, Nicholls et. al. 1989, Henry et. al. 1991, Knoll et. al. 1989, Wang et. al. 1991, Temple et. al. 1991, Pentao 1992]. Parents who carry a balanced chromosomal translocation are now counselled that phenotypic abnormalities are unlikely in offspring found to have the same balanced translocation as the parent. If uniparental disomy occurred frequently in carriers of inherited balanced translocations, the approach to genetic and prenatal diagnosis would need to be revised, at least in some cases. In view of our findings, parents can be reassured that malformations and mental retardation resulting from uniparental disomy are not a common occurrence among carriers of an inherited apparently balanced chromosomal rearrangement.  iv  TABLE OF CONTENTS  Abstract ^  ii  Significance ^  iv  Table of contents ^  v  List of Tables ^  vi  List of Figures ^  vii  Aknowledgements ^  viii  Dedicatoria ^  ix  Introduction  ^i  Chromosomal anomalies and birth defects  ^1  Congenital anomalies in balanced rearrangements  ^7  De novo balanced rearrangements ^ Inherited balanced rearrangements ^  7 11  Proposed mechanisms for mental retrdation and congenital anomalies in carriers of inherited apparently balanced chromosomal rearrangements ^  14  Submicroscopic gain or loss of chromatin ^ 14 Gene disruption at a breakpoint ^  15  Changes in the positions of genes ^ 17 Imprinting ^ Evidence for uniparental disomy ^  18 22  Uniparental disomy as a cause for developmental delay and congenital malformation in carriers of inherited apparently balanced chromosomal translocations ^  24  Mechanisms for uniparental disomy ^  26  v  LIST OF TABLES Page  Table 1  Results by Funderburk et al.^[1977]  9  2  Results by Funderburk et al. on the mentally  9  retarded group. 3  Karyotypes of idividuals with an inherited apparently balanced translocation, mental retardation and/or malformations  4  46  Markers used in DNA analysis of the translocations  47  5  Results of family 01  49  6  Results of family 02  50  7  Results of family 03  51  8  Results of family 04  52  9  Results of family 05  53  10  Results of family 06  55  11  Results of family 07  56  vi i  Testing for uniparental disomy ^  34  Hypothesis ^  38  Materials and Methods ^  39  Procedure ^  36  Blood lysates and DNA extraction ^ 39 Southern blotting technique ^  41  Labelling ^  41  PCR amplification ^  43  Results ^  44  Discussion ^  57  References ^  63  Appendix ^  64  vi  LIST OF FIGURES  Figure  Page  1  Pairing at meiosis  5  2  Results by Jacobs et al.^[1974]  8  3  Result by Fryns et al.^[1985]  10  4  Results by Fryns et al. [1985]  12  5  Results by Fryns et al. [1985] on further studies  12  of 75 families with translocations. 6  Normal^conception  29  7  Postfertilization error.  30  8  Gamete complementation.  31  9  Monosomy to isodisomy.  32  10  Trisomy to disomy.  33  11  Autoradiography in a fully informative case in which uniparental disomy can be ruled out  12  36  Autoradiography of Southern blot in which uniparental disomy cannot be ruled out.  36  13  Maternal uniparental disomy.  37  14  Paternal uniparental disomy  37  15  Patient database search  45  viii  ARNOWLEDGEMENTS  The direction and support of my thesis comittee during the course of this project is greatly appreciated: Dr. Jan. M. Friedman, Department of Medical Genetics, Dr. Sylvie Langlois, Department of Medical Genetics, Dr. Dessa Sadovnick, Department of Medical Genetics and Dr. Diana Juriloff, Department of Medical Genetics.  I would especially like to thank my supervisor Dr. Jan M. Friedman and his indefatigable red pen, Dr. Sylvie Langlois for her patience and understanding, Linda Kwong and Irene Yam who taught me all the laboratory techniques I needed to complete this project.  ix  A mis padres, Jose Luis y Elena, que han sido apoyo incondicional, inspiracion y motivo creador.  1  INTRODUCTION  CHROMOSOMAL ANOMALIES AND BIRTH DEFECTS  Chromosomal anomalies occur in an estimated 0.4% of live births [Jacobs et. al. 1974] and are an important cause of mental retardation and congenital anomalies [Schinzel 1984, Therman 1986, Daniel 1988]. The phenotypic anomalies that result from chromosomal aberrations occur mainly due to genetic imbalance. Chromosomal anomalies may be due to abnormalities of chromosome number or alteration of chromosome structure. The most common abnormalities of chromosome number are trisomies [Schinzel 1984]. These occur when there are three representatives of a particular chromosome instead of the usual two. In most cases this results from meiotic non-disjunction. Most patients with trisomies exhibit a very specific phenotype depending on the chromosome involved. The most frequent and best known trisomy in humans is Down syndrome. It was first described in 1866 [Down 1966] but its cause was not known until 1959 when Lejeune and Turpin showed that these patients carried 47 chromosomes, the extra one being a chromosome 21 [Lejeune et. al. 1959]. Trisomies of chromosome 18 [Edwards et. al 1960] and 13 [Patau et. al. 1960], associated with mental retardation and congenital malformations are also relatively common.  2 Monosomies occur when only one representative of a chromosome is present. They may be complete or partial. The best known instance of monosomy for a whole chromosome in liveborn humans is Turner syndrome in which one sex chromosome is missing [Turner et. al. 1938]. Partial monosomies occur when a piece of a certain chromosome is missing. These are often referred to as deletions. Ring chromosomes have been found for all human chromosomes [Schinzel 1984]. The formation of a ring involves a deletion at each end of the chromosome. The "sticky" ends then join to form the ring. The phenotype of ring chromosomes varies greatly ranging from mental retardation and congenital anomalies to normal or nearly normal phenotypes [Therman 1986]. If the ring replaces a normal chromosome then the result is partial monosomy. The phenotype seen in these cases often overlaps that of comparable deletion syndromes of the same chromosome. If there is a ring in addition to the normal chromosomes then the result is partial trisomy and the phenotype will reflect the trisomy for that chromosome [Therman 1986, Daniel 1988]. A deletion may occur as a pure deletion or as a deletion with a duplication. The latter are usually the result of an unbalanced reciprocal translocation. Deletions may be located in chromosome ends or interstitial segments and are usually associated with mental retardation and malformations. The most commonly seen deletions in humans are 4p-, 5p-,9p-,11p-,11q-,13q-,18p- and 18q[Schinzel 1984] A duplication is the presence of extra genetic material in a  3  chromosome. Even though it is called a duplication, it is in fact a triplication because there are three copies of a chromosomal segment. Duplications may result from abnormal segregation in carriers of translocations or inversions. Duplications resulting from abnormal segregation are usually associated with deletions [Therman 1986, Daniel 1988] Insertions occur when a piece of a chromosome breaks and is incorporated in another part of a chromosome. This requires 3 breakpoints and may occur between two chromosomes or within one [Therman 1986, Daniel 1988]. Inversions require the chromosome to break at two points. The broken piece is then inverted and joined into the chromosome again. Inversions have a frequency of 1 in every 100 liveborns [Kleczkowska et al. 1987] and may be pericentric or paracentric. In pericentric inversions the breaks are on opposite arms of a chromosome and are usually discovered because they change the position of the centromere. More than 146 different pericentric inversions involving every chromosome except 12, 17 and 20 [Kaiser 1980, Borgaonkar 1992] have been documented in humans [Therman 1986, Daniel 1988]. In contrast a paracentric inversion involves only one arm of a chromosome. Paracentric inversions have been found in chromosomes 1,3,5,6,7,8,12,13,14, and X [Kaiser 1980]. Most inversions in humans have no clinical significance and unlike translocations are not associated with infertility, spontaneous abortions or abnormal offspring [Therman 1986, Daniel 1988]. Translocations may be Robertsonian or reciprocal and have an  4  estimated frequency of about 1 in 500 liveborn infants [Fryns et al. 1986]. They may be inherited from a parent or appear "de novo" in a patient. Robertsonian translocations involve two acrocentric chromosomes that fuse near the centromeric region with subsequent loss of the short arms. The translocation chromosome is made up of the long arms of two fused chromosomes hence the resulting balanced karyotype has only 45 chromosomes. The loss of the short arms has no known deleterious effect. Although carriers of a Robertsonian translocation are usually phenotypically normal, they are at risk of having unbalanced gametes resulting in abnormal offsprings or miscarriages [Thompson 1991]. Reciprocal translocations are the result of breaks in nonhomologous chromosomes with reciprocal exchange of the broken segments [Therman 1986, Daniel 1988]. When the translocated chromosomes pair at meiosis, they form a quadriradial figure and the chromosomes may segregate in a number of ways (fig 1). The resulting gametes may be balanced, if there is no apparent loss or gain of genetic material, or unbalanced if they contain a rearrangement associated with chromatin gain or loss. Most patients with an unbalanced chromosomal rearrangement have serious congenital anomalies and mental retardation [Schinzel 1984]. Individuals with balanced translocations usually have a normal phenotype.  5  6  3:1 segregation producing tertiary aneuploidy  111010111  *  Tertiary trisomy^Tertiary monosomy  3:1 segregation producing interchange aneuploidy  NMI  *  Interchange trisomy^Interchange monosomy (usually non-viable) Figure represents chromosome pairing during meiosis in a balanced reciprocal translocation carrier and segregation of the chromosomes into the daughter cells [Daniel 1988]  7 CONGENITAL ANOMALIES IN BALANCED REARRANGEMENTS  Balanced translocations occur in 1 out of every 500 liveborn infants (Evans et al., 1978]. Even though these rearrangements are usually associated with a normal phenotype, some reports have shown the frequency of mental retardation and congenital anomalies to be increased among carriers of balanced translocations.  DE NOVO BALANCED REARRANGEMENTS  Studies have consistently shown a higher frequency of apparently balanced de novo reciprocal translocations among patients with multiple congenital anomalies/mental retardation (MCA/MR) syndromes. Jacobs et. al. [1974] examined 33,533 karyotypes over a 13 year period (1959 to 1972). The reason for the cytogenetic assesment was divided into: 1) mental subnormality excluding Down syndrome, 2) consecutive or random babies and 3) the remainder. Cytogenetic results revealed 94 (.28%) structural rearrangements that included 38 (40.4%) Robertsonian translocations, 47 (50%) reciprocal translocations and 9 (9.5%) pericentric inversions. 12 (12.76%) had of these rearrangements had arisen de novo. The authors found that the proportion of de novo rearrangements among categories 2) and 3) was not significantly different but the proportion of de novo rearrangements among the mentally subnormal was significantly greater than among the other individuals. They concluded that there  8 was an excess of de novo structural rearrangements among the mentally subnormal. Fig 2. Results by Jacobs et al. [1974] 33,533 consecutive karyotypes  33,439 (97.7%) no structural rearrangement  94 (0.3%) structural rearrangements (12/94 12.76%) de novo  82 (87.2%) familial  38 (40.4%) Robertsonian translocations  12 (12.8%) de novo  47 (50%) reciprocal translocations  9 (9.5%) pericentric inversions  In 1977 Funderburk et al. carried out cytogenetic studies in 2,134 consecutive patients at the UCLA Child Psychiatric and Mental Retardation Clinic. Mental retardation was seen in 455 (21.3%) patients. Psychiatric disorders were seen in the remaining 1,679 (78.6%). In the mentally retarded group 7 (1.5%) autosomal rearrangements were found, 2 (28%) were de novo. In the psychiatric disorder group only 4 (0.23%) rearrangements were found. 100% of the rearrangements in the mentally retarded group were balanced. Six (85%) were reciprocal translocations and one (15%) was an inversion. Among the psychiatric disorder group there were four  9 pericentric inversions. When pooled with previous reports their results showed a significant increase in de novo rearrangements among the mentally retarded. Table 1. Results by Funderburk et al. [1977]  autosomal rearrangements  2134 consecutive karyotypes  Total  455 (21.3%) patients with mental retardation  1679 (78.6%) patients with psychiatric disorders  2134 consecutive karyotypes  7 (1.5%)  4 (0.23%)  11 (1.73%)  Table 2. Results by Funderburk et al. [1977] on the mentally retarded group 455 (21.3%) patients  total autosomal rearrangements  reciprocal translocation  inversions  7^(1.5%) 2/7 (28%) de novo*  6^(85%)  1^(15%)  * The authors do not specify whether these were inversion or translocations. de novo rearrangements are included in the 7 (1.5%) autosomal rearrangements.  In 1985 Fryns et al. looked at 48,000 constitutional karyotypes that had been carried out in the Division of Human Genetics in Leuven Belgium from 1970 to 1984 for a variety of genetic reasons. They evaluated all the de novo translocation carriers and found that out of 18 (0.03%) de novo apparently balanced translocation carriers 13 (72.2%) patients had mental retardation and  10 malformations. They concluded that the incidence of mental retardation and malformations is higher among de novo translocation carriers. Fig 3. Results by Fryns et al. [1985]  11  48,000 consecutive karyotypes  18 (0.03%) de novo translocations  13 (72.2%) patients with malformations and mental retardation  11  INHERITED BALANCED REARRANGEMENTS  Although a patient who inherits an apparently balanced translocation from a normal carrier parent is generally expected to have a normal phenotype, previous studies have shown that the incidence of mental retardation and malformations is increased among these individuals [Breg et al. 1972, Funderburk et al. 1977, Ayme et. al. 1979, Fryns et al. 1985, Howard-Peebles and Friedman 1986]. Breg et al. [1972] reported three cases with apparently balanced translocations in a population of 1000 seriously retarded adult individuals. This incidence (0.3%) was reported to be two to six times higher than that in general population (.05-.13%). They suggested that further studies were needed to substantiate the apparently increased incidence of reciprocal translocations seen among the mentally retarded. Ayme et al. [1979] reported three patients with apparently balanced inherited autosomal rearrangements who were detected because of mental retardation and malformations. They suggested that the presence of such a rearrangement in a phenotypically abnormal child is not merely coincidental, even though the chromosomal aberration has been transmitted from a healthy parent. Funderburk et al. [1977] reported that 5 out of 7 of the apparently balanced rearrangements they found among the mentally retarded were familial. In an extensive review of the literature Funderburk et al. (1977] also demonstrated a slight increase of  12  familial rearrangements among mentally retarded children. Fryns et al. [1985] found 153 reciprocal translocations in a total of 48,000 consecutive patients. Of these 153, 75 (49%) were familial. Of the 75 familial, 18 (24%) patients with balanced reciprocal translocations presented with malformations and/or mental retardation. Fig 4. Results of Fryns et al. [1985] 48,000 consecutive karyotypes 153 (0.3%) reciprocal translocations  I^  ^I  75 (49%) familial reciprocal translocations 18 (24%) patients with malformations and mental retardation  When these 75 families were further studied, other offsprings also appeared to have a MCA/MR syndrome and a balanced translocation bringing the number of such persons to 28 (16.5%) in a total of 169 translocation carriers. Fig 5. Results of Fryns et al. [1985] on further studies of 75 families with translocations. 75 families 169 balanced translocation carriers 28 (16.5%) patients with malformations and mental retardation I  ^1  13 To evaluate the extent to which an apparently balanced reciprocal translocation may influence the phenotype, Fryns et al. [1985] calculated the number of translocation carrier offspring (MR/CM/T) with mental retardation and/or with congenital malformations versus the total number of balanced translocation carrier offspring (T). The same was done for the offspring with mental retardation, congenital malformations and normal karyotype (MR/CM/N) versus the total number of normal karyotype offspring (N). The number of MR/CM/T offspring was 28, the total number of (T) offspring was 169. MR/CM/T patients constituted 16.5% of all familial translocation carriers. This percentage was much higher than the 2.3% found in the MR/CM/N group (2 MR/CM/N persons in a total of 85 N). They concluded that the incidence of MR/CM is higher than expected in the balanced familial translocation carriers. Phenotypic and developmental abnormalities in carriers of inherited apparently balanced chromosomal rearrangements were also found by Howard-Peebles and Friedman [1986]. They reported on 6 children with unexplained developmental delay and malformations who had inherited an apparently balanced chromosomal rearrangement from a normal carrier parent. The frequency of 6 out of 1419 (0.4%) consecutive cytogenetic studies was significantly greater than the 0.2% that would be expected from studies of normal populations.  14 PROPOSED MECHANISMS FOR MENTAL RETARDATION AND CONGENITAL ANOMALIES IN CARRIERS OF INHERITED APPARENTLY BALANCED CHROMOSOMAL REARRANGEMENTS.  A number of hypotheses have been proposed for the phenotypic abnormalities in children with apparently balanced chromosomal rearrangements. These include submicroscopic gain or loss of chromatin, gene disruption, position effect, imprinting and uniparental disomy.  SUBMICROSCOPIC GAIN OR LOSS OF CHROMATIN  A few mental retardation/malformation syndromes that were initially reported with normal chromosomes have now been found to have submicroscopic deletions or duplications and are now known as microdeletion syndromes [Schinzel 1988]. Approximately 60% of patients with Prader-Willi syndrome and 50% of Angelman syndrome patients have a small deletion in the 15q11-12 region [Butler et. al. 1986, Magenis et. al. 1987, Butler 1990, Trent et al. 1991]. Most patients with Miller-Dieker syndrome have an interstitial deletion of 17p13.3 [Dobyns et. al. 1983]. An interstitial deletion of 8q23.3/24.1 is seen most patients with Langer-Gideon syndrome [Turleau et al. 1982]. Wilms tumor-aniridiagonadoblastoma-retardation (WAGR) syndrome is associated with a 11p13 deletion. Alagille syndrome is associated with an interstitial deletion of the short arm of chromosome 20  15 specifically the 20p11.23 to 12.3 band [Byrne et al. 1986,Desmaze et al. 1992]. A microdeletion of 22q11.2 has been found in some patients with DiGeorge or Velo-Cardio-Facial syndrome [Emanuel et. al. 1992]. Just recently Beckwith-Wiedeman has been associated with an 11p15.5 duplication [Newsham et.al . 1991] and Brachmann deLange syndrome has been associated with a duplication of 3q26.3 in some cases [Ireland et. al. 1991]. In summmary this data suggests that although some patients may appear to be cytogenetically normal, a submicroscopic deletion or duplication may be present and may in fact be the cause for the malformations and mental retardation. Inherited translocations even when initially believed to be balanced can in fact be associated with gain or loss of chromatin. This is believed to be the consequence of unequal exchange after misalignment between tandem repeats during chromosome pairing at meiosis [Chandley 1989]. In these cases the carrier of the translocation is in fact unbalanced but the alteration is so small that it is cytogenetically undetectable [Ricardi et al. 1982, Winsor et al. 1983, Hasegawa et al. 1984, Moore et al. 1986]  GENE DISRUPTION AT A BREAKPOINT  Physical gene disruption caused by an apparently balanced chromosomal rearrangement without any loss of chromosome material may produce loss of function of the genes located at the breakpoints [Edwards 1982].  16  Certain Mendelian diseases have been seen in patients who carry a translocation with a breakpoint involving the gene locus. For example, the Duchenne muscular dystrophy locus was assigned to band Xp21 by means of X/autosome translocations seen in several patients in which the breakpoint disrupted the gene [Jacobs et al. 1981, Zatz et al. 1981, Boyd et al. 1987]. Similarly the Norrie disease locus was assigned to the Xp11.4 region based on reports of a family who carries a pericentric inversion of chromosome X at bands p11.4 and q22. [McMahan et. al. 1992]. Neurofibromatosis type I (NF1) has been mapped to the proximal long arm of chromosome 17 [Barker et. al. 1987, Seizinger et. al. 1987]. A female with a 17;22 (q11.2;q11.2) translocation [Ledbetter et. al. 1989] and a mother and her two children who carried a 1;17 (p34.3;q11.2) translocation [Schmidt et. al. 1987] provided evidence for the location of the NF1 locus. In both cases, the breakpoints disrupted the NF1 gene. Since the breakpoints in inherited apparently balanced rearrangements usually appear to be the same in the normal carrier and affected offspring, gene disruption at a breakpoint is an unlikely mechanism. However if the breakpoints differ at the molecular level but this difference is cytogenetically undetectable, gene disruption may still be a cause for the abnormal phenotype.  17 CHANGES IN THE POSITION OF GENES  Chromosomal rearrangements change the position of genes relative to each other. Such a change may cause genes to lie within the province of controlling regions which cause abnormal gene activation or suppression without any obvious gain or loss of genetic material. An example of a translocation affecting the function of genes is seen in Burkitt lymphoma where the long arm of chromosome 8, which contains the c-myc oncogene, is translocated onto the long arm of chromosome 22 causing inappropriate expression of the c-myc oncogene. This translocation acts by juxtaposing c-myc to one of the three immunoglobulin loci with the result that the oncogene is constitutively activated [Klein and Klein 1985]. In chronic granulomatous leukaemia (CGL), a translocation between chromosomes 9 and 22 gives rise to what is known as the Philadelphia chromosome. In this translocation, the c-abl oncogene located in the tip of chromosome 9 is transposed to the bcr region of the deleted 22q- chromosome. This causes inappropriate activation and transcription of the c-abl oncogene [Chan et al. 1987]. This mechanism for malformations and mental retardation may be expected in cases of de novo apparently balanced translocations. But it would be unlikely to produce phenotypic abnormalities in inherited apparently balanced rearrangements in which the carrier parent is normal.  18  IMPRINTING  Imprinting refers to the differential expression of genetic material depending on whether it is inherited from the mother or the father. The proportion of human genome that is imprinted is not known, but studies in mice have shown that at least seven mouse chromosome segments may have major differential effects on growth, behaviour and survival depending on whether inheritance is from the mother or the father. Evidence for imprinting in mammals is derived from pronuclear transplantation experiments, transgene expression, human triploids, chromosome deficiency syndromes, expression of certain specific genes and uniparental disomy. Pronuclear transplantation experiments in mice have clearly demonstrated that the maternal and paternal genomes are not functionally equivalent. McGrath and Solter [1984] and Surani [1984] transplanted the pronuclei from one cell stage mouse embryos in order to create embryos with two female pronuclei (gynogenetic) and embryos with two male pronuclei (androgenetic). Gynogenetic mice had relatively good embryonic development but very poor development of membranes and placenta. Androgenetic mice, on the other hand, had relatively normal development of membranes and placenta but very poor embryonic development. McGrath and Solter [1984] and Surani [1984] concluded that the maternal and paternal contribution to the embryonic genome in mammals is not equivalent and that a diploid genome derived only from one parent is incapable  of supporting complete embryogenesis.  19  Transgene expression experiments have shown that when a specific foreign gene or "transgene" is inserted into a mouse embryo at a very early stage it is incorporated into the genome of the cells [Surani et al. 1988]. If this gene is incorporated into the germ cells, it may be passed on to future generations. It has been observed that in about one fourth of the transgenes studied their expression in future generations depends on the parent transmitting the gene. When the gene is transmitted from a transgenic male mouse it is expressed in the appropriate tissues, but when his expressing daughter transmits the same gene to her offspring they do not express it. It has been suggested that when transgenes are imprinted they are somehow "silenced" or "turned off" when they are inherited from one particular parent [Surani et al. 1988]. Human triploids are derived from twice the normal genetic contribution from one parent and a normal contribution from the other [Lawler 1984]. Triploids with two paternal and one maternal complement (android) have very large cystic placentas with partial molar changes. The fetuses are appropriately grown with normal head or microcephaly. Triploids with two maternal and one paternal (gynoid) complement have a very small underdeveloped placenta and the fetuses show severe intrauterine growth retardation and relative macrocephaly [McFadden et al. 1991]. It has been suggested that the paternal genetic information plays a critical role in the development and maintenance of the placenta and membranes [Hall 1990]. Some chromosome deficiency syndromes clearly show that maternal  20  and paternal genetic contributions are not always equivalent in humans [Knoll et al. 1989, Magenis et al. 1987]. Deletions in the q11-13 region of chromosome 15, for example, produce a different phenotype depending on their parental origin. If the deletion is on the paternally derived chromosome [Butler et. al. 1986, Knoll et.al . 1989, Butler 1990, Trent et al. 1991] the phenotype is one of Prader-Willi syndrome. If the deletion is in the maternally derived chromosome, the phenotype is one of Angelman syndrome [Magenis et. al. 1987, Knoll et. al. 1989, Pembrey et. al. 1989]. This evidence demonstrates that the functional presence of both paternal and maternal genome is essential for the normal development of fetal and extrafetal tissues [Engel and DeLozierBlanchet 1991]. This concept is crucial to understanding uniparental disomy. In cases of inherited apparently balanced translocations if both the translocated segment and the normal chromosome are inherited from only one parent and there is no homologous contribution from the other parent the result is uniparental disomy. If the involved chromosomes contain imprinted regions, uniparental disomy would be expected to produce an abnormal phenotype because some genetic material would not be expressed normally even though there is no additional or missing chromatin. It is important to remember that imprinting does not affect all chromosomes or all chromosomal segments. To date chromosome 7 [Spence at al. 1988, Voss et al. 1989], chromosome 14 [Wang et al. 1990, Temple et al.1991, Pentao et al. 1992], chromosome 15 and  21  more specifically the 15q11-13 region involved in Prader-Willi and Angelman syndrome [Knoll et al. 1989, Magenis et al. 1987) and the 11p5 region involved in Beckwith-Wiedemann syndrome [Viljeon et al. 1992] are undoubtedly known to be imprinted. Although other chromosomes and chromosomal regions may also be imprinted, an abnormal phenotype with uniparental disomy would only be expected for those known to be imprinted. Imprinting without uniparental disomy may also be a cause for malformations and/or mental retardation. The actual mechanism involved in imprinting is unknown but it has been suggested that methylation plays an important part in it [Hall 1990]. Studies of transgenes have shown that all but one transgene showing parental origin effect are undermethylated when paternally derived [Solter 1988). Abnormal expression of the methylated segments or genes that are dependent on the parent of origin may also play an important role in the development of an abnormal phenotype in cases of balanced reciprocal translocations. In these cases and especially in those in which chromosomes or chromosomal regions are known to be imprinted, the abnormal phenotype would be expected when the balanced rearrangement is inherited from a parent of one sex but not the other.  22 EVIDENCE FOR UNIPARENTAL DISOMY AS A CAUSE FOR CONGENITAL ANOMALIES IN HUMANS.  Uniparental disomy (UPD) occurs when both chromosomes in a pair are inherited from one parent rather than one of the chromosomes being inherited from each parent [Searle et. al. 1985, Cattanach et. al. 1985, Lyon et. al. 1985] . Individuals with uniparental disomy usually have no detectable cytogenetic abnormality. Prader-Willi syndrome is a disorder characterized by hypotonia, obesity, hypogonadotropic hypogonadism, small hands and feet, mental retardation and characteristic facial features. Maternal uniparental disomy for chromosome 15 has been described in patients with Prader-Willi syndrome [Nicholls et al. 1989, Butler 1990] Angelman syndrome is characterized by severe mental retardation, microcephaly, mild hypotonia, seizures, prognathism, eye abnormalities and distinctive facial features [Knoll et. al.1989]. In contrast to Prader-Willi syndrome paternal uniparental disomy for chromosome 15 has been demonstrated in some cases of Angelman syndrome [Kaplan et al 1987, Magenis et al. 1987, Malcolm et al. 1990]. Paternal uniparental disomy has also been described in one patient with Angelman syndrome and a 15;15 balanced translocation [Freeman et al. 1991] Beckwith-Wiedemann syndrome is another multiple congenital anomaly/mental retardation syndrome that has been associated with uniparental disomy. The syndrome is characterized by macroglossia, gigantism, earlobe creases, abdominal wall defects and an increased  23  risk for the development of tumours, especially Wilms tumour, rhabdomyosarcoma, hepatoblastoma or adrenal carcinoma [Irving 1967, Filippi et al. 1970]. Three cases of sporadic Beckwith-Wiedemann syndrome have been reported to have uniparental paternal disomy spanning the 11p15.5 region [Henry et. al. 1991]. Severe growth retardation has been reported in two cases of maternal uniparental disomy for chromosome 7. Spence et. al. [1988] described a 16 year old patient with cystic fibrosis and very short stature. This girl had a height of 52 inches, normal intelligence and growth hormone deficiency. High resolution cytogenetic analysis was normal. DNA molecular studies revealed a lack of paternal inheritance for markers at the midpoint of the long arm of chromosome 7 and the centromere. This data was consistent with maternal uniparental disomy for at least the segment from the centromere to q22 of chromosome 7. In 1988 Voss et al. reported another case of uniparental disomy for chromosome 7 in a 4 year old cystic fibrosis patient with severe growth retardation. 11 markers detecting DNA polymorphisms spanning the entire length of chromosome 7 were tested. No paternal contribution could be shown in seven informative loci, this data was consistent with maternal uniparental disomy for chromosome 7. Uniparental disomy for chromosome 10 was recently reported in a child with a tracheoesophageal fistula and a subaortic ventricular septal defect [Kousseff et al. 1992]  24 UNIPARENTAL DISOMY AS A CAUSE FOR DEVELOPMENTAL DELAY AND CONGENITAL MALFORMATIONS IN CARRIERS OF AN INHERITED APPARENTLY BALANCED CHROMOSOMAL TRANSLOCATION  Uniparental disomy has only recently been demonstrated as a probable cause for congenital malformations and mental retardation in a carrier of an inherited balanced chromosomal rearrangement. Wang et. al. [1990] studied a 9 year old mentally retarded female with a balanced 13;14 Robertsonian translocation inherited from her father. Her mother carried a 1;14 reciprocal translocation. This girl presented with mental retardation and multiple congenital anomalies including short webbed neck, small thoracic cage with marked angulation of the ribs, bilateral simian creases, and facial dysmorphism. To determine the parental origin of the chromosomes 14 in the proband, two chromosome 14 probes, D14S13 [Nakamura et al. 1987] and D14S22 [Nakamura et al. 1988] each detecting a VNTR polymorphism on the long arm were used. Results showed that the patient had inherited both chromosomes 14 from her father. Another case of maternal uniparental disomy for chromosome 14 was recently reported by Temple et al. [1991]. They described a 17 year old male with hydrocephaly, bifid uvula, premature puberty, short stature, small testes and normal intelligence who had inherited a 13;14 Robertsonian translocation from his mother. Molecular studies using six probes that recognise loci in chromosome 14 and 10 probes that recognise loci on chromosome 13 revealed that he had inherited both chromosomes 14 from his mother.  25  Mice can be bred so that they have uniparental disomy for a particular chromosome, this is usually produced by translocation [Cattanach 1986]. It is not clear whether the major phenotypic effects in mice are due to the duplication (i.e., presence of both chromosome from one parent) or to deficiency (i.e., lack of a chromosome from a parent). What is clear is that mice with uniparental disomy have severe growth retardation and some malformations. All this evidence suggests that uniparental disomy may be the cause of the abnormal phenotype in at least some apparently balanced inherited translocation carriers.  26  MECHANISMS FOR UNIPARENTAL DISOMY  There are a few mechanisms which could cause uniparental disomy. These include 1) post-fertilization error in chromosome segregation, 2) gamete complementation, 3) conversion of monosomy to isodisomy and 4) conversion of trisomy to disomy [Spence et al. 1988]. 1) Postfertilization errors in which there was non-disjunction with duplication leading to trisomy and subsequent loss of a specific chromosome would ultimately lead to uniparental disomy (Fig 3). 2) Gamete complementation would involve fertilization between one  nullisomic gamete and a disomic gamete for the same chromosome. The resulting conceptus would have uniparental disomy derived from the original disomic gamete (Fig 4) [Engel 1980, Spence et al 1988]. 3) If a normal haploid gamete was fertilized by a gamete nullisomic for a single chromosome, the result would be monosomy for that chromosome in the conceptus. If there was duplication of this single chromosome, the conceptus would end up with a double copy of that chromosome from only one parent or isodisomy for that chromosome (Fig 5). This mechanism has been called monosomy to isodisomy [Engel 1980, Spence et al. 1988,] 4) If an haploid gamete had two copies of a single chromosome due to non-disjunction and was then fertilized by a normal haploid gamete, the conceptus would be trisomic for that chromosome. If one of these trisomic chromosomes was lost due to a postzygotic error,  27  disomy would result. The zygote would thus have either uniparental disomy or with a normal set of chromosomes (Fig 6). This has been designated trisomy to disomy [Engel 1980, Spence et al. 1988]. In cases of translocated chromosomes, the mechanisms by which uniparental disomy may occur are somewhat similar to those described above. Good evidence exists to show that chromosomal nondisjunction occurs with higher frequency in gametes heterozygous for trans locations. Lyon (1983] and Cattanach et. al. (1985] studied a large number of mouse strains heterozygous for translocations. They found a high frequency of chromosomal non-disjunction occurring spontaneously in these mice. This condition usually leads to trisomic or monosomic conceptuses which die in utero unless uniparental disomy occurs as a result of the loss of one of the trisomic chromosomes or duplication of the monosomic chromosome [Cattanach et. al., 1986, Cattanach et. al. 1988]. Non-disjunction is also common among human carriers of certain translocations [Engel 1980]. Some translocation carriers are predisposed to 3:1 meiotic non-segregation, making the gametes potentially disomic by keeping a normal homologue along with the translocated chromosome. As a result, disomic gametes should occur more frequently in such translocation carriers than in individuals with normal chromosomes [Engel and DeLozier-Blanchet 1991]. A disomic gamete would usually be fertilized by a normal haploid gamete, resulting in a trisomic conception. Through subsequent loss of a chromosome, the conceptus could end up with normal biparental  28  contribution or with uniparental disomy. Gamete complementation is another possible mechanism. The same situation described previously of non-disjunction of the translocated chromosomes would have to occur in the gamete, but with fertilization by a nullisomic gamete for the homologue. The outcome would be a balanced translocation with uniparental disomy [Engel 1980]. The frequency with which aneuploid gametes might complement each other so that uniparental disomy may occur is directly related to the frequency of aneuploidy in gametes. Cytogenetic studies of spontaneous abortions have shown that at least 50% of first trimester losses result from chromosome aberrations. Half of these cytogenetically abnormal conceptuses have autosomal trisomies. Autosomal monosomies are rarely observed and are presumably lethal before implantation [Hassold et al. 1979]. Other studies have shown that at least 5% of male sperm have cytogenetic numerical abnormalitites [Martin et al. 1983], and there is sufficient information to say that at least 20-30% of oocytes are chromosomally abnormal [Martin et al. 1986]. With the assumption that 20% of recognized conceptions end in miscarriage and taking into account only the five most frequent single pair abnormalitites, Engel [1980] calculated the expected incidence of uniparental disomy would be 2.8 in every 10,000 conceptions.  29  Figure represents the division of normal chromosomes in gametes, zygote and somatic tissue.  30  Non-disjunction duplication^Mitotic recombination or gene and loss^ conversion  Figure represents the division of the chromosomes in a postfertilization error leading to UPD.  31  Figures represent the division of chromosomes in gamete complementation leading to UPD.  32  Figures represent the division of the chromosomes in a conversion of monosomy to isodisomy leading to UPD.  33  Figures represent the division of the chromosomes in a conversion of trisomy to disomy leading to UPD.  34  TESTING FOR UNIPARENTAL DISOMY  It is now possible to test for uniparental disomy using highly polymorphic genetic markers such as variable number of tandem repeats (VNTR) or CA repeats [Wang et. al. 1991, Malcolm et. al. 1990, Newsham et. al. 1991]. The value of any marker to test for uniparental disomy depends on how many variants it displays in the general population. At many sites on human DNA, a single sequence that does not code for a protein is repeated many times. These are called variable number of tandem repeats (VNTR). VNTR's are specially important because the number of repeats at a given locus can vary from a few to hundreds of copies. These loci are characterized by many alleles. Restriction fragments created by cutting on both sides of these tandem repeats vary in length. The most informative markers have many different alleles. In a heterozygous individual the Southern blot will reveal two distinct fragments of different length, one from each homologous chromosome. [White and Lalouel 1988, Nakamura et. al. 1987, Litt and Luty 1989]. CA repeats are a type of VNTR based on a series of dinucleotide repeats and are highly polymorphic. This form of polymorphism can be detected by amplifying the region containing the repeat and running the amplification product on a sequencing gel. In heterozygotes the sequencing gel will reveal two different fragments of different length, one from each chromosome [Litt and Luty 1989].  35 To test for uniparental disomy, it is essential to determine the parental origin of the two chromosomes inherited by an individual. Thus a useful genetic marker in cases of uniparental disomy is one that can detect more than two alleles. A marker based on VNTR's or CA repeats can be highly informative when it exhibits many alleles. Since alleles at a certain locus are distributed by chance, the frequency of heterozygosity or informativeness at a locus is directly related to the number of common alleles present in the population. The more alleles a locus has, the more likely it is that an individual will be heterozygous. Testing for uniparental disomy is largely dependent on highly polymorphic loci [Reeders et. al. 1985, White et. al. 1985]. To rule out UPD it is not essential for the parents to have no alleles in common but it is mandatory that the offspring inherits the alleles that are not shared by the parents (fig 11). If the parents have an allele in common and the allele is also found in the offspring, it is impossible to determine the parental origin of that allele (fig 12). When fully informative, testing for uniparental disomy can determine whether the offspring has both maternal and paternal genetic contributions, only maternal (fig 13) or only paternal (fig 14). Patients with only maternal or only paternal contributions have UPD for the chromosome region containing the loci studied.  36  Parents are heterozygous but share one allele. Uniparental disomy cannot be ruled out if child inherits the allele shared in common.  37  38  HYPOTHESIS  We hypothesize that uniparental disomy is responsible for the phenotypic abnormalities in some children who have an inherited apparently balanced chromosomal translocation.  39 MATERIALS AND METHODS  PROCEDURE  A computer database search was done at the Department of Medical Genetics, University Hospital-Shaughnessy Site to identify all eligible patients to participate in the study. Approval for contact was first obtained from the medical geneticist involved in the care of each patient. Approval to contact the family was then obtained from either the referring physician or the family physician. An initial letter of contact was sent to the parents informing them of our study and asking for their participation (see appendix for letters). This letter was followed up by a phone call. All seven families with one or more living children with an inherited apprently balanced translocation and mental retardation/multiple congenital anomalies agreed to participate. Both parents and the affected children in each family were seen at the Department of Medical Genetics, University Hospital, Shaughnessy Site. Audiovisual aids were used to explain the purpose of our research. Since the affected children were unable to give consent because of their ages and mental condition, both parents were asked to sign a consent form for blood withdrawal and participation in the study.  BLOOD LYSATES AND DNA EXTRACTION  40  DNA from the parents and offspring of each of the 7 families in our study was obtained by standard phenol-chloroform extraction and ethanol precipitation according to the technique described by Kunkel et al. (1977]. Tris-NH 4 C1 was prewarmed at 37 C. One 50 ml Falcon tube was filled with 10 cc of EDTA blood and 40 ml of prewarmed Tris NH 4 C1 and incubated at 37 C for five minutes. After incubation the tubes were centrifuged at 2600 rpm for 7 minutes and the supernatant was aspirated. The pellet was resuspended in 20 ml of 85% saline solution and centrifuged at 2600 rpm for 7 minutes. The supernatant was aspirated. The pellet was resuspended in 5 ml of Tris-EDTA (TE) solution and 5 ml of lysis buffer solution was injected through an 19 gauge needle. 100 AL of proteinase K were added. The tube was incubated overnight at 37 C or 2-3 hrs at 65 C. After incubation, 10 ml of phenol were added to the blood lysate. The tubes were shaken in a mechanical shaker for 10-20 minutes and centrifuged at 3000 rpm for 5 minutes. The top layer including the interface was removed to a new 50 ml Falcon tube. 2.5 ml of 6M NaC1 solution were added and the tubes were manually shaken. 12.5 ml of (24:1) Chloroform:Isoamylalcohol solution were added and the tubes were shaken mechanically at 80 for 30-60 minutes. The tubes were centrifuged at 3000 rpm for 5 minutes. The top layer was carefully removed with a Pasteur pipette and transferred to a new 50 ml falcon tube. 30 ml of 95% ethanol were added and the tubes shaken gently in the mechanical shaker. The DNA precipitate was removed with a curved pipette and washed with a few drops of 70% ethanol. The extracted DNA was resuspended in 500 AL of TE.  41  SOUTHERN BLOTTING TECHNIQUE  DNA was digested, electrophoresed and blotted according to the technique described by Southern (1975). 10 AL of genomic DNA were cut with the appropiate enzyme and buffer, according to the conditions provided by the supplier The digest was incubated in a 37 C water bath for 4 hours. After incubation, 5 Al of dye were added to the enzyme digest. 1500 ml of Tris-Boric Acid-EDTA solution was poured into the gel box and the agarose gel was placed in it. The digested DNA was loaded into wells in the agarose gel. Electrophoresis was performed for 12 to 24 hours depending on the size of the fragments to be separated. The DNA was transferred onto a nylon membrane by Southern blotting.  LABELLING  100 ng of each DNA probe (see VNTR- Table 4) was resuspended in 32.5 AL of water in a 1 ml Eppendorf tube. The tube was boiled for 5 minutes and quenched in ice for 3 minutes. 10 AL oligonucleotide labelling reagent (OLB), 2 AL acetylated bovine serum albumin (BSA), 5 gL 32P dCTP and 0.5 AL large fragment polymerase were added for a total volume of 50 ML. The tube was then left at room temperature for 4 hrs. The barrel of a 1 ml syringe was stuffed with silaconized glass wool. A mixed slurry of SEPHADEX was poured into the barrel of the  42  syringe until it reached .8 ml. The syringe was centrifuged at 2500 rpm for 3 minutes. 50 gL of TE were added to the previously mentioned priming reaction tubes. The contents of the priming reaction tubes was loaded into the syringe barrel and centrifuged at 2500 rpm for 3 minutes. The contents of the syringe was transfered into Eppendorf tubes which were then capped. The hybond n-plus nylon membrane used for blotting was sealed in a plastic bag. The Church's buffer was prewarmed in 65 C oven. 8-10 ml of prewarmed Church's buffer were added into the sealed bag with a 10 ml syringe and 18 gauge needle. The bag was sealed again and shaken in a 65 C water bath for 2 hrs. The contents of the capped Eppendorf tubes containing the radiolabelled probe was boiled for 5 minutes and quenched in ice for 3 minutes. 0.5 ml of prewarmed Church's buffer was loaded into a 1 ml syringe. The contents were injected into the capped Eppendorf tube containing the radiolabelled probe. The entire contents of the tube was aspirated with the syringe. The sealed bag with blot was removed from the 65 C water bath and the contents of the syringe  were injected into the bag. The bag was sealed again and submerged in a 65 C water bath and shaken overnight. The blots were removed from the sealed bags and washed as follows: with 2x sodium chloride-sodium phosphate-EDTA solution (SSPE) 1% sodium doedecyl sulphate (SDS) solution for 10 minutes at room temperature twice, with 2X SSPE + 0.1% SDS for 10 minutes at room temperature twice, with preheated 0.5X SSPE + 0.1% SDS in a 65 C water bath for 30 minutes once, with preheated 0.2X SSPE + 0.1% SDS in 65 C water  43 bath for 30 minutes once. The moist filters were wrapped in saran wrap and exposed to KODAK XAR-5 films for 3-7 days.  POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION  PCR amplifications were carried out in a total volume of 25 uL containing 100 ng of genomic DNA, 1 AL of each primer, 2.5 AL of 10x Taq polymerase buffer, 2 AL of dITP mix, 1.25 of 5mM spermidine, 0.05 of -32PdCTP and 0.2 AL of 5A/AL taq polymerase. The PCR conditions and primers varied depending on the marker involved and according to the protocol previously published for that marker (Table 4). Products were resolved on an 8% polyacrylamide gel and visualized under UV light or on a DNA sequencing gel and visualized by autoradiography.  44  RESULTS  A computer search was done on the patient database at the  Department of Medical Genetics, University Hospital Shaughnessy Site, looking for chromosomal abnormalities. This search showed that between February 1966 and June 1991, 3906 patients were seen with a diagnosis of a chromosomal abnormality. Of these 3906, 207 (5.2%) patients had documented chromosomal rearrangements. These 207 charts were reviewed and formed the initial basis for the present study. Of the 207, 45 (21.7%) families carried an inherited apparently balanced reciprocal translocation. Of these 45, 12 (26%) families had one or more offspring who carried an inherited apparently balanced reciprocal translocation and who presented with congenital anomalies and developmental delay (Fig.15). Seven (58.3%) of these 12 families were available to study. Two of these families each had two affected children for a total of 9 cases. In five families, the affected offspring was deceased. Complete karyotypes with high resolution banding for both parents and the affected offspring in each of the seven families were reviewed. The chromosome breakpoints are listed in Table 3. The markers used to test for uniparental disomy (table 4) were chosen because of their capacity to detect polymorphisms and because they were previously available in the lab.  45  Fig 15. Figure illustrates the search of the patient database UBC-Department of Medical Genetics Patient Database; 39,000 families  3906 families with a diagnosis of chromosomal abnormality  207 families with documented chromosomal rearrangements  166 families with reciprocal translocations  45 inherited reciprocal translocations  12 families with inherited apparently balanced translocations and malformations and/or developmental delay  5 families excluded due death of affected child  7 families included in the study  9 cases from 7 families with inherited apparently balanced translocations and with malformations and/or developmental delay  46  Table 3. Karyotypes of individuals with an inherited apparently balanced translocation, mental retardation and/or malformations.  Case 1 (Family 01)  46,XX,t(10;16)(p11.2;q23)pat  Case 2 (Family 02)  46,XY,t(6;17)(p11.2;q21.1)pat  Case 3 (Family 03)  46,XY,t(13;14)(p11;q11)pat  Case 4 (Family 04)  46,XY,t(2:12)(p14.2;q13.1)pat  Case 5 (Family 05)  46,XY,t(4;7)(p15.1;q36)pat  Case 6 (Family 06)  46,XY,t(1;5)(p13.1;q33.1)pat  Case 6b (Family 06)  46,XX,t(1;5)(p13.1;q33.1)pat  Case 7 (Family 07)  46,XY,t(11;14)(q12;p13)mat  Case 7b (Family 07)  46,XY,t(11;14)(q12;p13)mat  47  Table 4. Markers used in DNA analysis of the translocations. CHROMOSOME  MARKER  TYPE  REFERENCE  FAMILY STUDIED  CHROMOSOME 1  D1S81  VNTR*  Nakamura et al.^1987  Family 03  CHROMOSOME 2  APO B  VNTR  Boerwinkle et al.^1989  Family 05  CHROMOSOME 4  D4S227  CA repeat  Weber et al. 1991  Family 06  CHROMOSOME 5  D5S107  CA Weber et repeat ,a1.1990  Family 03 -  CHROMOSOME 6  D6S89  CA repeat  Zoghbi et al. 1991 Ranum et al. 1991  Family 02  CHROMOSOME 7  D7S396  VNTR*  Nakamura et al.^1987 Lathrop et al.^1985  Family 06  CHROMOSOME 10  D1OS15  VNTR*  Family 01  D10S28  VNTR*  STCL-2  CA repeat  Nakamura et al.^1987 Lathrop et al.^1985 Lairmore et al.^(in press)  CHROMOSOME 11  D11S35  CA repeat  Litt et al. 1991  Family 07  CHROMOSOME 12  D12VWF  VNTR  Standen et al. 1990  Family 05  CHROMOSOME 13  RBD13  VNTR  Scharf et al. 1992  Family 04  CHROMOSOME 14  D14S13  VNTR*  Nakamura et al. 1988  Family 04 Family 07  CHROMOSOME 16  D16S291  CA repeat  Thompson et al.^1992  Family 01  CHROMOSOME 17 IYNZ22^IVNTR  Batanian 1990 (Family 02  All markers were tested by PCR except for the ones marked (*) which tested by Southern blots.  48  The families have been numbered arbitrarily from 01 to 07. In the following tables, the mother in each family is given the number 1, the father is 2 and the affected children are designated as 3 or 4, according to birth order. The markers or probes used to test for uniparental disomy in each of the chromosomes involved in the translocation are at the top of each column in the tables. Alleles were designated 1 to 4, with 1 being the biggest molecular weight and 4 being the lowest. Key to pedigree symbols  0  Female carrier  Male carrier  0 Translocation carrier with malformations and/or developmental delay  Translocation carrier with malformations and/or developmental delay  O  Normal female non-carrier  Normal male non-carrier  49  1,4 Clinical report Case 1: P.E. was a 7 year old female. She was born at 36 weeks after a twin pregnancy, birth weight was 1.5 kg. Anomalies present at birth were choanal atresia and Tetralogy of Fallot. Infancy was complicated by recurrent abdominal pain, vomiting and dehydration with ileus requiring multiple hospital admissions. At the age of 4 years she was diagnosed as having a gallstone and cholecystectomy was performed. On physical examination her weight and height were at the 3rd centile. She had a round face with mild malar flattening, hypertelorism, upslanting palpebral fissures, a very thin upper lip and bifid uvula. She had clinodactyly of the fifth finger bilaterally. She had 3/6 systolic ejection murmur. Table 5. Results of family 01 t(10;16)(p11.2;q23)pat  Family 01  Chromosome 16^I ,D16S291  01-1  1,2  01-2  3,4  01-3  1,4  This family was uninformative for 2 VNTR markers on chromosome 10, D10S25 [Nakamura et al. 1987], D10S28 [Lathrop et al. 1985], and 1 CA repeat sTCL-2 [Lairmore et al. 1991]. Uniparental disomy for  chromosome 16 has been ruled out.  50  1,2  2,3 Clinical report Case 2: D.J. was a 4 year old male. He was born at 42 weeks, birth weight was 4.4 kg. The neonatal period was uneventful. He sat at 6 months, crawled at 10 months, walked at 11 months and spoke at 4 years. He had language and developmental delay and bizarrre "autistic" behavior. On examinaton his weight was 19.5 kg (90th centile), height was 107 cm (between 90 and 97th centile) and OFC was 53.3 (greater than the 98th centile). He had no obvious dysmorphic features except for relative macrocephaly, a very prominent nasal bridge and strabismus. Table 6. Results of family 02 t(6;17)(g11;q21.1)pat  Chromosome 6  Chromosome 17  D6S89  YNZ22  02-1  2,3  1,3  02-2  1,4  2,4  02-3  1,2  2,3  Family 02  Uniparental disomy for chromosomes 6 and 17 has been ruled out.  51  1,2 Clinical report Case 3: B.S. was a 7 year old male. He was born at term after an uncomplicated pregnancy. Birth weight was 3.9 kg. Severe developmental delay was noted shortly after birth. He pulls himself to a stand but does not walk or talk. Over the past two years he has had peculiar "spells" with marked hyperactivity, yelling and screaming. At the age of 3 years, severe cortical visual impairment was diagnosed. On examination his weigth was 18.5 kg (5th centile), height was 49.1 cm (10th centile) and OFC was 49.2 cm (10th centile). He has a round face and broad nasal root with mild anteversion of the nostrils. His fingers were tapered, not flexible and he had camptodactyly of the fifth finger. Chest examination was normal. Table 7. Results of family 03 t(13;14)(p11;q11)pat Family 03  Chromosome 13^'Chromosome 14 I D13RBD  D14S13  04-1  1,2  1,3  04-2  2,3  2,3  04-3  1,3  1,2  Uniparental disomy for 13 and 14 has been ruled out  52  1,4 Clinical report Case 4: J.S was a 9 year old male born at term. The pregnancy was complicated by vaginal bleeding. Birth weight was 2.9 kg. At birth a foot deformity was noted and treated with splints. Developmental and speech delay were noted at the age of 18 months. He had a developmental evaluation at age 8 years and was found to have mild mental retardation and a significant language disorder. On examination his weight was 25.5 kg (25th centile), height was 126.9 cm (10th centile) and OFC was 53 cm (25th centile). He had micrognathia with an open bite, a slight upward slant of the palpebral fissures and his ears had a posterior slope. Table 8. Results of family 04 t(2;12)(p14.2;q13.1)pat  Chromosome 2  Chromosome 12  ApoB  D12VWD  05-1  1,3  1,2  05-2  2,4  3,4  05-3  3,4  1,4  Family 04  Uniparental disomy for 2 and 12 have been ruled out.  53  2,4  3,4  Clinical report Case 5: W.C. was a 21 year old male who was born at term. Birth weight was 3.1 kg. The neonatal period was complicated by cyanotic spells. A heart murmur was noted. A ventricular septal defect was diagnosed at age 3 and he underwent cardiac cathetherization. Developmental delay was also noted at the age of 3. Mild mental retardation was diagnosed at the age of 10. On examination his weight was 90 kg (above the 90th centile), his height was 185.5 cm (90th centile) and OFC was 60 cm (greater than two SD above the mean). He had macrocephaly, mid-face hypoplasia, down-slanted palpebral fissures, a high-arched palate, prognathism and large ears. He had prominent thoracic kyphosis with mild left-sided scoliosis of the thoracic spine. His hands had lateral deviation of the distal phalanx of the third digits bilaterally. Table 9. Results of family 05 t(4;7)(p15.1;q36)pat  Family 05  Chromosome 4 'Chromosome 71 D4S227  D7S396  06-1  3,4  1,4  06-2  1,2  2,3  06-3  2,4  3,4  Uniparental disomy for chromosomes 4 and 7 has been ruled out  54  Clinical reports Case 6: D.C. was a 8 year old male. He was born at term. Birth weight was 3.2 kg. There was mild developmental delay. Speech was significantly delayed. On examination his height was 125 cm (25th centile), his weight was 29 kg and OFC was 54 cm. He had a soft systolic ejection murmur at the left sternal border. There were no obvious dysmorphic features. Case 6b: C.C. was a 5 year old female. She was born at term after vaginal delivery. The delivery was precipitous and the child had respiratory difficulties that required resuscitation. Shortly after birth she had a seizure and was placed on phenobarbital. There were no further seizures and the child was taken off the phenobarbital at 4 years of age. On examination she did not make eye contact, she made whining noises but used no verbal language, she had continuous repetitive hand clapping movements and her fingers were constantly in her mouth. Her weight was 15 kg (5th centile), her height was 102.7 cm (less than 5th centile), and OFC was 47.5 (less than 2nd centile). She had deep set eyes, pronounced eyebrows and a prominent jaw. Her ears had a prominent antihelix and her ear lobes were attached. She had a 1 x 1/2 cm hyperpigmented macular lesion on her right buttock, a slightly hypopigmented patch of skin over the left knee and a 1 x 1/2 cm hyperpigmented macule on the dorsum of her left foot. A CT Scan revealed bilateral destructive areas of cerebral cortex.  55  Table 10. Results of family 06 t(1;5)(p13.1;q33.1)pat  Family 06  1  1  Chromosome 1  I Chromosome 5  D1S81  D5S107  03-1  1,3  1,3  03-2  2,4*  2,3*  03-3  1,4  1,2  03-4  2,3  2,3  * Deduced on the basis of unaffected children's genotypes With results from two other offsprings we have been able to deduce the father's alleles. Molecular results show a maternal contribution for chromosomes 1 and 5. Cytogenetic results reveal that both offsprings have inherited both translocation derivatives from their father. Molecular and cytogenetic results rule out uniparental disomy for chromosomes 1 and 5.  56  Clinical reports Case 7: J.B was a 9 year old male born at term. Birth weight was 3.6 kg. He had intermittent ataxia, mild mental retardation and seizures. On examination his height was 136 cm, his weight was 27.4 cm and his OFC was 53.4 cm He had constant choreiform movements. There were no obvious dysmorphic features. Case 7b: D.B was a 5 year old male born at term. Birth weight was 3.6 kg. He had intermittent ataxia, mild mental retardation and seizures since the age of 4. On examination height was 114 cm, weight was 19 kg and OFC was 52.2 cm. Table 11. Results of family 07 t(11,14)(q12;p13)mat Family 07  Chromosome 11 !Chromosome 14 'Chromosome 14 D11S35  D14S13  D14S42  2,4  2,3  1,2  07-3  1,4  1,2  2,3  07-4  1,2  1,2  2,3  07-1 07-2  Cytogenetic results reveal the translocation was inherited from the mother. Molecular analysis in each child reveals one allele that does not come from the mother. Assuming this is the paternal contribution, uniparental disomy for chromosomes 11 and 14 can be ruled out.  57 DISCUSSION  Developmental delay and congenital anomalies have been observed in two patients with inherited apparently balanced chromosomal translocations of chromosomes 13 and 14 who were found to have uniparental disomy [Wang et. al. 1991, Temple et al. 1991). The authors suggested that uniparental disomy was the most likely cause for their developmental delay and congenital anomalies. It appears, however, from the results in this study that uniparental disomy is not a frequent occurrence among carriers of inherited apparently balanced chromosomal translocations who have phenotype abnormalities. Other mechanisms must therefore account for the phenotypic abnormalities in most individuals with an inherited apparently balanced translocation who have congenital anomalies and mental retardation. Imprinting, that is, the possibility that certain genes are marked so that they are expressed differently when they have been inherited from the father rather than when they are inherited from the mother must be considered. In 6 out of 7 families (cases 1 through 6b) in our study, the translocation was paternally inherited. In 1 family (cases 7 and 7b) the translocation was maternally inherited. The origin of the translocation in 3 of these 6 (families 01, 05 and 06) fathers was unavailable . In 1 (family 04) the father's translocation appeared de novo and in the other 2 (family 02 and 03), the fathers inherited the translocation from their mothers.  58  An excess of paternally inherited apparently balanced translocations in children with mental retardation and malformations was previously noted by Howard-Peebles and Friedman [1986]. In their report five out of six rearrangements were paternally inherited. The authors however do not mention from whom the fathers inherited their translocations. In the report by Jacobs et. al.[1974] in which they looked at 33,533 consecutive karyotypes, they observed a total of 85 Robertsonian and reciprocal translocations. In 40 (47%) cases both parents were available for the study. 28 (70%) out of the 40 translocations were inherited, 14 (50%) were paternal and 14 (50%) were maternal. However the authors do not report whether the carriers of these inherited translocations had a normal or abnormal phenotype. A larger sample of patients with inherited apparently balanced translocations and abnormal phenotype is needed in order to ascertain the importance of imprinting. The families we studied had translocations that involved some of the chromosomes or chromosomal regions that are known to be imprinted. Beckwith-Wiedemann is an overgrowth syndrome and evidence for paternal imprinting for the 11p15.5 region has been reported in sporadic cases [Viljoen et. al.1992]. Weksber et al. [1992] reviewed a cohort of patients with Beckwith-Wiedemann syndrome. Two patients had a maternally inherited apparently balanced translocation. The Beckwith-Wiedemann syndrome phenotype was not present in the mothers carrying the translocation. They suggest that these findings as well as the paternal uniparental  59  disomy seen in sporadic cases of Beckwith-Wiedemann are consistent with an imprinting model with suppression of the maternally inherited allele [Websker et al. 1992]. Two of the children in our study (case 7 and 7b) had inherited a 11;14 (q12;p13) translocation from their mother. The marker we used D11S35 is located in the 11q22 region so the 11p15 region associated with Beckwith-Wiedemann syndrome was not tested. Nonetheless we know that in some cases of sporadic Beckwith-Wiedemann syndrome, this region is imprinted so it may be possible for other regions of chromosome 11 to be imprinted as well. Evidence for imprinting can also be seen in the two cases of cystic fibrosis and uniparental disomy for chromosome 7 reported by Spence et al. [1988] and Voss et al. [1989]. These reports clearly show there is differential function of the maternal and paternal chromosome 7. One of our patients (case 5) had inherited a (4;7) (p15.1;q36) translocation from his father. The two previous reports of uniparental disomy for chromosome 7 and CF describe patients with severe growth retardation. It is also important to note that in mouse studies defining the phenotypes of uniparental disomies no major congenital anomalies have been noted, but variations of growth, behaviour and survival are present [Hall 1990]. Case 5, however, had no growth failure. On the basis of this phenotype uniparental disomy for chromosome 7 was highly unlikely. In 1990 Hall listed the mouse chromosome areas involved in imprinting, she then compared them to human chromosome areas and suggested that the genes that have been mapped within those regions  60  in humans may also be imprinted. The families in our study have translocations that involve some of the chromosomes in her list. Family 04 had a (2;12) translocation, the 2p11-p13 region of the human chromosome is homologous to the 6C area of the mouse chromosome which is imprinted. Family 02 had a (6;17) translocation, the 17A-D region of the mouse chromosome area is involved in imprinting and is homologous to the human 6pter-p12. Family 01 had a (10;16) translocation, the mouse chromosome 8 involved in imprinting is homologous to the 16q22.1-q24 region. Family 05 had a (4;7) translocation, the 6B-C,11A and 6A-C mouse areas thought to be imprinted are homologous to the human 7p21-p14, 7p14-p12 and 7q22-qter. If imprinting is occuring in cases of inherited balanced translocations and there is more than one affected family member the phenotype in both affected members would be expected to be the same. Family 06 and family 07 have two affected children each. The pattern of the abnormal phenotype in family 07 is similar. But in family 06 they are completely different. However case 6b had a complicated neonatal period with a precipituous delivery and repiratory difficulties requiring resuscitation. It is possible that part of the abnormal phenotype is due to delivery complications. To corroborate our findings it is important to study a larger number of individuals with an inherited balanced translocation and mental retardation and/or congenital anomalies and compare by chromosome the ones in which the mother is the carrier versus the  61  ones in which the father is the carrier. If there is an important paternal or maternal imprinting effect, a significant difference for specific chromosomes between the two groups regarding the presence of malformations and mental retardation should be seen. This is especially important if the translocation involves a chromosomes or chromosomal region known to be imprinted. Although cytogenetically undetectable, a very small deletion or duplication present in the offspring but not in the carrier parent may also be the cause for the mental retardation and the malformations seen in some individual carriers of an inherited apparently balanced translocation. The development of molecular markers very close to or at the breakpoints of these translocations will help further elucidate this possibility. It is also important to remember that one locus per chromosome does not rule out uniparental disomy for the entire chromosome. It has been shown in cases of Beckwith-Wiedemann [Viljoen et al. 1992] that there may be uniparental disomy for only a certain region of a chromosome. There is still a possibility of partial uniparental disomy that may be the cause for the abnormal phenotype We believe that even though this was a "negative" study the information gained from it is important. The reports of Wang et. al.[1991], Temple et al. 1991 and Pentao et al. [1992] raised the possibility that uniparental disomy might be a frequent cause of mental retardation and multiple congential anomalies in children with inherited apparently balanced translocations. 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Hum. 13;223  Wilmot PL, Shapiro LR (1990) Disomic balanced reciprocal translocation. Clin. Genet. 38;126-127  Winsor E, Welch J (1983) Prader-Willi syndrome associated with inversion of chromosome 15. Clin. Genet. 24;456-461  White R, Lalouel JM (1988) Chromosome mapping with DNA markers. Scientific Am. 258;40-482  64  APPENDIX  THE UNIVERSITY OF BRITISH COLUMBIA  Department of Medical Genetics  University Hospital—Shaughnessy Site 4500 Oak Street Vancouver, B.C. Canada V6H 3N1 Tel: (604) 875-2157^Fax: (604) 875-2376  Dear Dr.  January 13,1991.  Recent publications suggest that patients who have inherited an apparently balanced chromosomal rearrangement and who present with congenital anomalies and/or developmental delay may have inherited both the normal and the rearranged chromosome from only one parent. This type of inheritance is called uniparental disomy and has also been described in cases of Prader Willi Syndrome. Dr. J.M. Friedman, Dr.L. Langlois and Dr. Lopez Rangel will be starting research at the Department of Medical Genetics, University Hospital, Shaughnessy Site. We would like to contact patients who are known to carry a inherited chromosomal rearrangement and have congenital anomalies and/or developmental delay. is a patient of yours who is eligible for our study and was last since in our clinic on February 1987. !  Participation in our study would require a blood sample. The blood would be analyzed at the DNA laboratory at the University Hospital, Shaughnessy Site. We would like to get information on the chromosomal rearrangement and look for uniparental disomy as a cause for the malformations and the developmental delay. I have been in touch with and they are very interested in participating in our study. We would like your help in obtaining the blood samples. Enclosed please find requisition forms for blood to be drawn on both parents and child. Sincerely,  Jan. M. Friedman. MD. Ph.D. FAAP. FABMG. Professor and Acting Head. Department of Medical Genetics. University of British Columbia.  Elena Lopez Rangel M.D, 1 Medical Genetics MSc Student.  CONSENT FORM Name of the study: Uniparental disomy as a cause for malformations and/or developmental delay incases of inherited apparently balanced translocations. Institute: Principal Investigators:  The University of British Columbia Dr. J.M. Friedman, Dr. S. Langlois, Dr. E. Lopez  We, ^ agree / do not agree to participate with our child in a research study conducted by the Department of Medical Genetics, at the University of British Columbia. The aim of the projeCt is to try to determine at the cytogenetic and DNA level the change that could be responsible for the condition affecting our child.  To achieve this goal, we agree to undergo a blood test (20cc) and to have our child have a blood test (lOcc). We understand there may be some discomfort associated with the placement of the intravenous needle for blood withdrawal and that ocassionally, bruising may result. We understand that only the investigators involved will know the name of the participants in this research project. We are aware that much of the information obtained from this study will eventually be used in scientific publications but the identity of subjects in the study will not be revealed in such publications or any other report. We understand that our participation in this study is entirely voluntary and that refusal to participate will in no way jeopardize the care of our child in the Department of Medical Genetics at the University Hospital. Our signature on this form signifies, that we have decided to participate in this study after reading the above information. We have been given the opportunity to discuss pertinent aspects of the research study and to ask questions, and hereby consent to participate in the study outlined above. We also have received a copy of this consent form for our own personal records.  Subject's parents signature  Date  Witness  Date  

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