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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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We accept this thesis as conformingUNIPARENTAL DISOMY AS A CAUSE FOR CONGENITAL MALFORMATIONS AND ORDEVELOPMENTAL DELAY IN INHERITED APPARENTLYBALANCED CHROMOSOMAL REARRANGEMENTSbyELENA LOPEZ-RANGELM.D. Universidad Anahuac, Mexico City 1990A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FORTHE DEGREE OF MASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Genetics Program)THE UNIVERSITY OF BRITISH COLUMBIAJANUARY 1993© Elena Lopez-Rangel, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of ^GeneticsThe University of British ColumbiaVancouver, CanadaDate DE-6 (2/88)ABSTRACTChromosomal translocations are said to be balanced if there is noapparent gain or loss of genetic material. Apparently balancedchromosomal rearrangements are usually associated with a normalphenotype [Therman 1986, Daniel 1988]. However the frequency ofmental retardation and congenital anomalies appears to be increasedamong individuals who carry a de novo or an inherited apparentlybalanced chromosomal rearrangement [Funderburk et. al. 1977, Aymeet. al. 1979, Fryns et. al. 1986, Howard-Peebles and Friedman1986].Uniparental disomy occurs when both chromosomes in a pair areinherited from one parent instead of one of the chromosomes beinginherited from each parent [Searle et. al. 1985, Cattanach et. al.1985, Lyon et. al. 1985]. Uniparental disomy has recently beenestablished as a cause for congenital anomalies and mentalretardation in humans [Spence et. al. 1988, Voss et. al. 1989,Nicholls et. al. 1989, Henry et. al. 1991, Knoll et. al. 1989, Wanget. al. 1991, Temple et. al. 1991, Pentao 1992].In mice, uniparental disomy for any chromosome may be produced bymating animals that carry certain chromosomal rearrangements whichalter normal meiotic segregation [Cattanach 1985, Cattanach 1986,Cattanach 1988, Cattanach 1989).We have investigated the possibility that uniparental disomy maycause mental retardation and malformations in carriers of anii.inherited apparently balanced chromosomal translocation. We studied7 families in which one or more childen with congenital anomaliesand mental retardation have inherited an apparently balancedtranslocation from a carrier parent. In order to establish theparental origin of the chromosomes, we collected blood from bothparents and the affected child. We determined the parental originof each of the chromosomes involved in the translocations using DNAprobes that detect highly polymorphic loci which have beenpreviously 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 etal. 1991, Ranum et al. 1991, Scharf et al. 1992, Litt et al. 1992].We were able to rule out uniparental disomy for each of thechromosomes involved in the translocation in every patient exceptone who had inherited an apparently balanced translocation from acarrier parent and who presented with malformations and/ordevelopmental delay. In one patient uniparental disomy was ruledout for one of the chromosomes in the translocation but the markerswas uninformative for the other chromosome.We conclude that uniparental disomy is not a common occurence incarriers of inherited apparently balanced reciprocal translocationswho present with congenital anomalies and/or developmental delay.iiiSIGNIFICANCEUniparental disomy in patients who have inherited an apparentlybalanced chromosomal translocation has been reported in a fewindividual cases (Spence et. al. 1988, Voss et. al. 1989, Nichollset. al. 1989, Henry et. al. 1991, Knoll et. al. 1989, Wang et. al.1991, Temple et. al. 1991, Pentao 1992]. Parents who carry abalanced chromosomal translocation are now counselled thatphenotypic abnormalities are unlikely in offspring found to havethe same balanced translocation as the parent. If uniparentaldisomy occurred frequently in carriers of inherited balancedtranslocations, the approach to genetic and prenatal diagnosiswould need to be revised, at least in some cases.In view of our findings, parents can be reassured thatmalformations and mental retardation resulting from uniparentaldisomy are not a common occurrence among carriers of an inheritedapparently balanced chromosomal rearrangement.ivTABLE OF CONTENTSAbstract ^  iiSignificance  ivTable of contents ^  vList of Tables  viList of Figures ^  viiAknowledgements  viiiDedicatoria ^  ixIntroduction  ^iChromosomal anomalies and birth defects  ^1Congenital anomalies in balanced rearrangements  ^7De novo balanced rearrangements ^  7Inherited balanced rearrangements  11Proposed mechanisms for mental retrdation and congenitalanomalies in carriers of inherited apparently balancedchromosomal rearrangements ^  14Submicroscopic gain or loss of chromatin ^ 14Gene disruption at a breakpoint ^  15Changes in the positions of genes  17Imprinting^  18Evidence for uniparental disomy ^  22Uniparental disomy as a cause for developmental delay andcongenital malformation in carriers of inherited apparentlybalanced chromosomal translocations ^  24Mechanisms for uniparental disomy  26vLIST OF TABLESTable Page1 Results by Funderburk et al.^[1977] 92 Results by Funderburk et al. on the mentallyretarded group.93 Karyotypes of idividuals with an inheritedapparently balanced translocation, mentalretardation and/or malformations 464 Markers used in DNA analysis of thetranslocations 475 Results of family 01 496 Results of family 02 507 Results of family 03 518 Results of family 04 529 Results of family 05 5310 Results of family 06 5511 Results of family 07 56vi iTesting for uniparental disomy ^  34Hypothesis ^  38Materials and Methods ^  39Procedure  36Blood lysates and DNA extraction ^ 39Southern blotting technique^  41Labelling ^  41PCR amplification ^  43Results ^  44Discussion  57References ^  63Appendix  64viLIST OF FIGURESFigure Page1 Pairing at meiosis 52 Results by Jacobs et al.^[1974] 83 Result by Fryns et al.^[1985] 104 Results by Fryns et al. [1985] 125 Results by Fryns et al. [1985] on further studiesof 75 families with translocations.126 Normal^conception 297 Postfertilization error. 308 Gamete complementation. 319 Monosomy to isodisomy. 3210 Trisomy to disomy. 3311 Autoradiography in a fully informative casein which uniparental disomy can be ruled out 3612 Autoradiography of Southern blot in whichuniparental disomy cannot be ruled out. 3613 Maternal uniparental disomy. 3714 Paternal uniparental disomy 3715 Patient database search 45viiiARNOWLEDGEMENTSThe direction and support of my thesis comittee duringthe 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 ofMedical Genetics and Dr. Diana Juriloff, Department of MedicalGenetics.I would especially like to thank my supervisor Dr. Jan M.Friedman and his indefatigable red pen, Dr. Sylvie Langlois for herpatience and understanding, Linda Kwong and Irene Yam who taught meall the laboratory techniques I needed to complete this project.ixA mis padres, Jose Luis y Elena,que han sido apoyo incondicional,inspiracion y motivo creador.1INTRODUCTIONCHROMOSOMAL ANOMALIES AND BIRTH DEFECTSChromosomal anomalies occur in an estimated 0.4% of live births[Jacobs et. al. 1974] and are an important cause of mentalretardation and congenital anomalies [Schinzel 1984, Therman 1986,Daniel 1988].The phenotypic anomalies that result from chromosomal aberrationsoccur mainly due to genetic imbalance. Chromosomal anomalies may bedue to abnormalities of chromosome number or alteration ofchromosome structure.The most common abnormalities of chromosome number are trisomies[Schinzel 1984]. These occur when there are three representativesof a particular chromosome instead of the usual two. In most casesthis results from meiotic non-disjunction. Most patients withtrisomies exhibit a very specific phenotype depending on thechromosome involved.The most frequent and best known trisomy in humans is Downsyndrome. It was first described in 1866 [Down 1966] but its causewas not known until 1959 when Lejeune and Turpin showed that thesepatients carried 47 chromosomes, the extra one being a chromosome21 [Lejeune et. al. 1959]. Trisomies of chromosome 18 [Edwards 1960] and 13 [Patau et. al. 1960], associated with mentalretardation and congenital malformations are also relativelycommon.2Monosomies occur when only one representative of a chromosome ispresent. They may be complete or partial. The best known instanceof monosomy for a whole chromosome in liveborn humans is Turnersyndrome in which one sex chromosome is missing [Turner et. al.1938]. Partial monosomies occur when a piece of a certainchromosome 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 ateach end of the chromosome. The "sticky" ends then join to form thering. The phenotype of ring chromosomes varies greatly ranging frommental retardation and congenital anomalies to normal or nearlynormal phenotypes [Therman 1986]. If the ring replaces a normalchromosome then the result is partial monosomy. The phenotype seenin these cases often overlaps that of comparable deletion syndromesof the same chromosome. If there is a ring in addition to thenormal chromosomes then the result is partial trisomy and thephenotype will reflect the trisomy for that chromosome [Therman1986, Daniel 1988].A deletion may occur as a pure deletion or as a deletion with aduplication. The latter are usually the result of an unbalancedreciprocal translocation. Deletions may be located in chromosomeends or interstitial segments and are usually associated withmental retardation and malformations. The most commonly seendeletions in humans are 4p-, 5p-,9p-,11p-,11q-,13q-,18p- and 18q-[Schinzel 1984]A duplication is the presence of extra genetic material in a3chromosome. Even though it is called a duplication, it is in facta triplication because there are three copies of a chromosomalsegment. Duplications may result from abnormal segregation incarriers of translocations or inversions. Duplications resultingfrom abnormal segregation are usually associated with deletions[Therman 1986, Daniel 1988]Insertions occur when a piece of a chromosome breaks and isincorporated in another part of a chromosome. This requires 3breakpoints and may occur between two chromosomes or within one[Therman 1986, Daniel 1988].Inversions require the chromosome to break at two points. Thebroken 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 achromosome and are usually discovered because they change theposition of the centromere. More than 146 different pericentricinversions involving every chromosome except 12, 17 and 20 [Kaiser1980, Borgaonkar 1992] have been documented in humans [Therman1986, Daniel 1988]. In contrast a paracentric inversion involvesonly one arm of a chromosome. Paracentric inversions have beenfound in chromosomes 1,3,5,6,7,8,12,13,14, and X [Kaiser 1980].Most inversions in humans have no clinical significance and unliketranslocations are not associated with infertility, spontaneousabortions or abnormal offspring [Therman 1986, Daniel 1988].Translocations may be Robertsonian or reciprocal and have an4estimated frequency of about 1 in 500 liveborn infants [Fryns etal. 1986]. They may be inherited from a parent or appear "de novo"in a patient.Robertsonian translocations involve two acrocentric chromosomesthat fuse near the centromeric region with subsequent loss of theshort arms. The translocation chromosome is made up of the longarms of two fused chromosomes hence the resulting balancedkaryotype has only 45 chromosomes. The loss of the short arms hasno known deleterious effect. Although carriers of a Robertsoniantranslocation are usually phenotypically normal, they are at riskof having unbalanced gametes resulting in abnormal offsprings ormiscarriages [Thompson 1991].Reciprocal translocations are the result of breaks in non-homologous chromosomes with reciprocal exchange of the brokensegments [Therman 1986, Daniel 1988]. When the translocatedchromosomes pair at meiosis, they form a quadriradial figure andthe chromosomes may segregate in a number of ways (fig 1). Theresulting gametes may be balanced, if there is no apparent loss orgain of genetic material, or unbalanced if they contain arearrangement associated with chromatin gain or loss. Most patientswith an unbalanced chromosomal rearrangement have seriouscongenital anomalies and mental retardation [Schinzel 1984].Individuals with balanced translocations usually have a normalphenotype.53:1 segregation producing tertiary aneuploidy 111010111 *Tertiary trisomy^Tertiary monosomy3:1 segregation producing interchange aneuploidy6NMI*Interchange trisomy^Interchange monosomy(usually non-viable)Figure represents chromosome pairing during meiosis in a balancedreciprocal translocation carrier and segregation of the chromosomesinto the daughter cells [Daniel 1988]7CONGENITAL ANOMALIES IN BALANCED REARRANGEMENTSBalanced translocations occur in 1 out of every 500 liveborninfants (Evans et al., 1978]. Even though these rearrangements areusually associated with a normal phenotype, some reports have shownthe frequency of mental retardation and congenital anomalies to beincreased among carriers of balanced translocations.DE NOVO BALANCED REARRANGEMENTSStudies have consistently shown a higher frequency of apparentlybalanced de novo reciprocal translocations among patients withmultiple congenital anomalies/mental retardation (MCA/MR)syndromes.Jacobs et. al. [1974] examined 33,533 karyotypes over a 13 yearperiod (1959 to 1972). The reason for the cytogenetic assesment wasdivided into: 1) mental subnormality excluding Down syndrome, 2)consecutive or random babies and 3) the remainder. Cytogeneticresults revealed 94 (.28%) structural rearrangements that included38 (40.4%) Robertsonian translocations, 47 (50%) reciprocaltranslocations and 9 (9.5%) pericentric inversions. 12 (12.76%) hadof these rearrangements had arisen de novo. The authors found thatthe proportion of de novo rearrangements among categories 2) and 3)was not significantly different but the proportion of de novorearrangements among the mentally subnormal was significantlygreater than among the other individuals. They concluded that there38 (40.4%)Robertsoniantranslocations 47 (50%)reciprocaltranslocations9 (9.5%)pericentricinversions33,439(97.7%) nostructuralrearrangement 94 (0.3%) structural rearrangements (12/9412.76%) de novo82 (87.2%)familial 12 (12.8%)de novo8was an excess of de novo structural rearrangements among thementally subnormal.Fig 2. Results by Jacobs et al. [1974]33,533 consecutive karyotypesIn 1977 Funderburk et al. carried out cytogenetic studies in 2,134consecutive patients at the UCLA Child Psychiatric and MentalRetardation 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%) autosomalrearrangements were found, 2 (28%) were de novo. In the psychiatricdisorder group only 4 (0.23%) rearrangements were found. 100% ofthe rearrangements in the mentally retarded group were balanced.Six (85%) were reciprocal translocations and one (15%) was aninversion. Among the psychiatric disorder group there were four2134 consecutive karyotypes Total455 (21.3%)patientswith mentalretardation1679 (78.6%)patients withpsychiatricdisorders2134consecutivekaryotypesautosomalrearrangements 7 (1.5%) 4 (0.23%) 11 (1.73%)9pericentric inversions. When pooled with previous reports theirresults showed a significant increase in de novo rearrangementsamong the mentally retarded.Table 1. Results by Funderburk et al. [1977]Table 2. Results by Funderburk et al. [1977] on the mentallyretarded group455(21.3%)patientstotal autosomalrearrangements reciprocaltranslocation inversions7^(1.5%)2/7 (28%) de novo* 6^(85%) 1^(15%)* The authors do not specify whether these were inversion ortranslocations. de novo rearrangements are included in the 7(1.5%) autosomal rearrangements.In 1985 Fryns et al. looked at 48,000 constitutional karyotypesthat had been carried out in the Division of Human Genetics inLeuven Belgium from 1970 to 1984 for a variety of genetic reasons.They evaluated all the de novo translocation carriers and foundthat out of 18 (0.03%) de novo apparently balanced translocationcarriers 13 (72.2%) patients had mental retardation and11 10malformations. They concluded that the incidence of mentalretardation and malformations is higher among de novo translocationcarriers.Fig 3. Results by Fryns et al. [1985]48,000 consecutive karyotypes18 (0.03%) de novo translocations13 (72.2%) patients with malformations and mental retardation11INHERITED BALANCED REARRANGEMENTSAlthough a patient who inherits an apparently balancedtranslocation from a normal carrier parent is generally expected tohave a normal phenotype, previous studies have shown that theincidence of mental retardation and malformations is increasedamong these individuals [Breg et al. 1972, Funderburk et al. 1977,Ayme et. al. 1979, Fryns et al. 1985, Howard-Peebles and Friedman1986].Breg et al. [1972] reported three cases with apparently balancedtranslocations in a population of 1000 seriously retarded adultindividuals. This incidence (0.3%) was reported to be two to sixtimes higher than that in general population (.05-.13%). Theysuggested that further studies were needed to substantiate theapparently increased incidence of reciprocal translocations seenamong the mentally retarded.Ayme et al. [1979] reported three patients with apparentlybalanced inherited autosomal rearrangements who were detectedbecause of mental retardation and malformations. They suggestedthat the presence of such a rearrangement in a phenotypicallyabnormal child is not merely coincidental, even though thechromosomal aberration has been transmitted from a healthy parent.Funderburk et al. [1977] reported that 5 out of 7 of theapparently balanced rearrangements they found among the mentallyretarded were familial. In an extensive review of the literatureFunderburk et al. (1977] also demonstrated a slight increase of12familial rearrangements among mentally retarded children.Fryns et al. [1985] found 153 reciprocal translocations in a totalof 48,000 consecutive patients. Of these 153, 75 (49%) werefamilial. Of the 75 familial, 18 (24%) patients with balancedreciprocal translocations presented with malformations and/ormental retardation.Fig 4. Results of Fryns et al. [1985]48,000 consecutive karyotypes153 (0.3%) reciprocal translocationsI^ 75 (49%) familial reciprocal translocations ^I 18 (24%) patients with malformations and mental retardationWhen these 75 families were further studied, other offsprings alsoappeared to have a MCA/MR syndrome and a balanced translocationbringing the number of such persons to 28 (16.5%) in a total of 169translocation carriers.Fig 5. Results of Fryns et al. [1985] on further studies of 75families with translocations.75 families ^1169 balanced translocation carriers28 (16.5%) patients with malformations and mental retardation I13To evaluate the extent to which an apparently balanced reciprocaltranslocation 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 malformationsversus 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 thetotal 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 allfamilial translocation carriers. This percentage was much higherthan the 2.3% found in the MR/CM/N group (2 MR/CM/N persons in atotal of 85 N). They concluded that the incidence of MR/CM ishigher than expected in the balanced familial translocationcarriers.Phenotypic and developmental abnormalities in carriers ofinherited apparently balanced chromosomal rearrangements were alsofound by Howard-Peebles and Friedman [1986]. They reported on 6children with unexplained developmental delay and malformations whohad inherited an apparently balanced chromosomal rearrangement froma normal carrier parent. The frequency of 6 out of 1419 (0.4%)consecutive cytogenetic studies was significantly greater than the0.2% that would be expected from studies of normal populations.14PROPOSED MECHANISMS FOR MENTAL RETARDATION AND CONGENITAL ANOMALIESIN CARRIERS OF INHERITED APPARENTLY BALANCED CHROMOSOMALREARRANGEMENTS.A number of hypotheses have been proposed for the phenotypicabnormalities in children with apparently balanced chromosomalrearrangements. These include submicroscopic gain or loss ofchromatin, gene disruption, position effect, imprinting anduniparental disomy.SUBMICROSCOPIC GAIN OR LOSS OF CHROMATINA few mental retardation/malformation syndromes that wereinitially reported with normal chromosomes have now been found tohave submicroscopic deletions or duplications and are now known asmicrodeletion syndromes [Schinzel 1988].Approximately 60% of patients with Prader-Willi syndrome and 50%of Angelman syndrome patients have a small deletion in the 15q11-12region [Butler et. al. 1986, Magenis et. al. 1987, Butler 1990,Trent et al. 1991]. Most patients with Miller-Dieker syndrome havean interstitial deletion of 17p13.3 [Dobyns et. al. 1983]. Aninterstitial deletion of 8q23.3/24.1 is seen most patients withLanger-Gideon syndrome [Turleau et al. 1982]. Wilms tumor-aniridia-gonadoblastoma-retardation (WAGR) syndrome is associated with a11p13 deletion. Alagille syndrome is associated with aninterstitial deletion of the short arm of chromosome 2015specifically the 20p11.23 to 12.3 band [Byrne et al. 1986,Desmazeet al. 1992]. A microdeletion of 22q11.2 has been found in somepatients with DiGeorge or Velo-Cardio-Facial syndrome [Emanuel 1992]. Just recently Beckwith-Wiedeman has been associated withan 11p15.5 duplication [Newsham . 1991] and Brachmann deLangesyndrome has been associated with a duplication of 3q26.3 in somecases [Ireland et. al. 1991].In summmary this data suggests that although some patients mayappear to be cytogenetically normal, a submicroscopic deletion orduplication may be present and may in fact be the cause for themalformations and mental retardation. Inherited translocations evenwhen initially believed to be balanced can in fact be associatedwith gain or loss of chromatin. This is believed to be theconsequence of unequal exchange after misalignment between tandemrepeats during chromosome pairing at meiosis [Chandley 1989]. Inthese cases the carrier of the translocation is in fact unbalancedbut the alteration is so small that it is cytogeneticallyundetectable [Ricardi et al. 1982, Winsor et al. 1983, Hasegawa etal. 1984, Moore et al. 1986]GENE DISRUPTION AT A BREAKPOINTPhysical gene disruption caused by an apparently balancedchromosomal rearrangement without any loss of chromosome materialmay produce loss of function of the genes located at thebreakpoints [Edwards 1982].16Certain Mendelian diseases have been seen in patients who carrya translocation with a breakpoint involving the gene locus. Forexample, the Duchenne muscular dystrophy locus was assigned to bandXp21 by means of X/autosome translocations seen in several patientsin 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 Xp11.4region based on reports of a family who carries a pericentricinversion of chromosome X at bands p11.4 and q22. [McMahan et. al.1992]. Neurofibromatosis type I (NF1) has been mapped to theproximal long arm of chromosome 17 [Barker et. al. 1987, Seizingeret. al. 1987]. A female with a 17;22 (q11.2;q11.2) translocation[Ledbetter et. al. 1989] and a mother and her two children whocarried 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 balancedrearrangements usually appear to be the same in the normal carrierand affected offspring, gene disruption at a breakpoint is anunlikely mechanism. However if the breakpoints differ at themolecular level but this difference is cytogeneticallyundetectable, gene disruption may still be a cause for the abnormalphenotype.17CHANGES IN THE POSITION OF GENESChromosomal rearrangements change the position of genes relativeto each other. Such a change may cause genes to lie within theprovince of controlling regions which cause abnormal geneactivation or suppression without any obvious gain or loss ofgenetic material.An example of a translocation affecting the function of genes isseen in Burkitt lymphoma where the long arm of chromosome 8, whichcontains the c-myc oncogene, is translocated onto the long arm ofchromosome 22 causing inappropriate expression of the c-myconcogene. This translocation acts by juxtaposing c-myc to one ofthe three immunoglobulin loci with the result that the oncogene isconstitutively activated [Klein and Klein 1985].In chronic granulomatous leukaemia (CGL), a translocation betweenchromosomes 9 and 22 gives rise to what is known as thePhiladelphia chromosome. In this translocation, the c-abl oncogenelocated in the tip of chromosome 9 is transposed to the bcr regionof the deleted 22q- chromosome. This causes inappropriateactivation and transcription of the c-abl oncogene [Chan et al.1987].This mechanism for malformations and mental retardation may beexpected in cases of de novo apparently balanced translocations.But it would be unlikely to produce phenotypic abnormalities ininherited apparently balanced rearrangements in which the carrierparent is normal.18IMPRINTINGImprinting refers to the differential expression of geneticmaterial depending on whether it is inherited from the mother orthe father. The proportion of human genome that is imprinted is notknown, but studies in mice have shown that at least seven mousechromosome segments may have major differential effects on growth,behaviour and survival depending on whether inheritance is from themother or the father. Evidence for imprinting in mammals is derivedfrom pronuclear transplantation experiments, transgene expression,human triploids, chromosome deficiency syndromes, expression ofcertain specific genes and uniparental disomy.Pronuclear transplantation experiments in mice have clearlydemonstrated that the maternal and paternal genomes are notfunctionally equivalent. McGrath and Solter [1984] and Surani[1984] transplanted the pronuclei from one cell stage mouse embryosin order to create embryos with two female pronuclei (gynogenetic)and embryos with two male pronuclei (androgenetic). Gynogeneticmice had relatively good embryonic development but very poordevelopment of membranes and placenta. Androgenetic mice, on theother hand, had relatively normal development of membranes andplacenta but very poor embryonic development. McGrath and Solter[1984] and Surani [1984] concluded that the maternal and paternalcontribution to the embryonic genome in mammals is not equivalentand that a diploid genome derived only from one parent is incapableof supporting complete embryogenesis.19Transgene expression experiments have shown that when a specificforeign gene or "transgene" is inserted into a mouse embryo at avery early stage it is incorporated into the genome of the cells[Surani et al. 1988]. If this gene is incorporated into the germcells, it may be passed on to future generations. It has beenobserved that in about one fourth of the transgenes studied theirexpression in future generations depends on the parent transmittingthe gene. When the gene is transmitted from a transgenic male mouseit is expressed in the appropriate tissues, but when his expressingdaughter transmits the same gene to her offspring they do notexpress it. It has been suggested that when transgenes areimprinted they are somehow "silenced" or "turned off" when they areinherited from one particular parent [Surani et al. 1988].Human triploids are derived from twice the normal geneticcontribution from one parent and a normal contribution from theother [Lawler 1984]. Triploids with two paternal and one maternalcomplement (android) have very large cystic placentas with partialmolar changes. The fetuses are appropriately grown with normal heador microcephaly. Triploids with two maternal and one paternal(gynoid) complement have a very small underdeveloped placenta andthe fetuses show severe intrauterine growth retardation andrelative macrocephaly [McFadden et al. 1991]. It has been suggestedthat the paternal genetic information plays a critical role in thedevelopment and maintenance of the placenta and membranes [Hall1990].Some chromosome deficiency syndromes clearly show that maternal20and paternal genetic contributions are not always equivalent inhumans [Knoll et al. 1989, Magenis et al. 1987]. Deletions in theq11-13 region of chromosome 15, for example, produce a differentphenotype depending on their parental origin. If the deletion is onthe paternally derived chromosome [Butler et. al. 1986, . 1989, Butler 1990, Trent et al. 1991] the phenotype is oneof Prader-Willi syndrome. If the deletion is in the maternallyderived 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 bothpaternal and maternal genome is essential for the normaldevelopment of fetal and extrafetal tissues [Engel and DeLozier-Blanchet 1991]. This concept is crucial to understandinguniparental disomy. In cases of inherited apparently balancedtranslocations if both the translocated segment and the normalchromosome are inherited from only one parent and there is nohomologous contribution from the other parent the result isuniparental disomy. If the involved chromosomes contain imprintedregions, uniparental disomy would be expected to produce anabnormal phenotype because some genetic material would not beexpressed normally even though there is no additional or missingchromatin.It is important to remember that imprinting does not affect allchromosomes 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 and21more specifically the 15q11-13 region involved in Prader-Willi andAngelman syndrome [Knoll et al. 1989, Magenis et al. 1987) and the11p5 region involved in Beckwith-Wiedemann syndrome [Viljeon et al.1992] are undoubtedly known to be imprinted. Although otherchromosomes and chromosomal regions may also be imprinted, anabnormal phenotype with uniparental disomy would only be expectedfor those known to be imprinted.Imprinting without uniparental disomy may also be a cause formalformations and/or mental retardation. The actual mechanisminvolved in imprinting is unknown but it has been suggested thatmethylation plays an important part in it [Hall 1990]. Studies oftransgenes have shown that all but one transgene showing parentalorigin effect are undermethylated when paternally derived [Solter1988). Abnormal expression of the methylated segments or genes thatare dependent on the parent of origin may also play an importantrole in the development of an abnormal phenotype in cases ofbalanced reciprocal translocations. In these cases and especiallyin those in which chromosomes or chromosomal regions are known tobe imprinted, the abnormal phenotype would be expected when thebalanced rearrangement is inherited from a parent of one sex butnot the other.22EVIDENCE FOR UNIPARENTAL DISOMY AS A CAUSE FOR CONGENITAL ANOMALIESIN HUMANS.Uniparental disomy (UPD) occurs when both chromosomes in a pairare inherited from one parent rather than one of the chromosomesbeing inherited from each parent [Searle et. al. 1985, Cattanachet. al. 1985, Lyon et. al. 1985] . Individuals with uniparentaldisomy 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. Maternaluniparental disomy for chromosome 15 has been described in patientswith Prader-Willi syndrome [Nicholls et al. 1989, Butler 1990]Angelman syndrome is characterized by severe mental retardation,microcephaly, mild hypotonia, seizures, prognathism, eyeabnormalities and distinctive facial features [Knoll et. al.1989].In contrast to Prader-Willi syndrome paternal uniparental disomyfor chromosome 15 has been demonstrated in some cases of Angelmansyndrome [Kaplan et al 1987, Magenis et al. 1987, Malcolm et al.1990]. Paternal uniparental disomy has also been described in onepatient with Angelman syndrome and a 15;15 balanced translocation[Freeman et al. 1991]Beckwith-Wiedemann syndrome is another multiple congenitalanomaly/mental retardation syndrome that has been associated withuniparental disomy. The syndrome is characterized by macroglossia,gigantism, earlobe creases, abdominal wall defects and an increased23risk for the development of tumours, especially Wilms tumour,rhabdomyosarcoma, hepatoblastoma or adrenal carcinoma [Irving 1967,Filippi et al. 1970]. Three cases of sporadic Beckwith-Wiedemannsyndrome have been reported to have uniparental paternal disomyspanning the 11p15.5 region [Henry et. al. 1991].Severe growth retardation has been reported in two cases ofmaternal uniparental disomy for chromosome 7. Spence et. al. [1988]described a 16 year old patient with cystic fibrosis and very shortstature. This girl had a height of 52 inches, normal intelligenceand growth hormone deficiency. High resolution cytogenetic analysiswas normal. DNA molecular studies revealed a lack of paternalinheritance for markers at the midpoint of the long arm ofchromosome 7 and the centromere. This data was consistent withmaternal uniparental disomy for at least the segment from thecentromere to q22 of chromosome 7.In 1988 Voss et al. reported another case of uniparental disomyfor chromosome 7 in a 4 year old cystic fibrosis patient withsevere growth retardation. 11 markers detecting DNA polymorphismsspanning the entire length of chromosome 7 were tested. No paternalcontribution could be shown in seven informative loci, this datawas consistent with maternal uniparental disomy for chromosome 7.Uniparental disomy for chromosome 10 was recently reported in achild with a tracheoesophageal fistula and a subaortic ventricularseptal defect [Kousseff et al. 1992]24UNIPARENTAL DISOMY AS A CAUSE FOR DEVELOPMENTAL DELAY ANDCONGENITAL MALFORMATIONS IN CARRIERS OF AN INHERITED APPARENTLYBALANCED CHROMOSOMAL TRANSLOCATIONUniparental disomy has only recently been demonstrated as aprobable cause for congenital malformations and mental retardationin a carrier of an inherited balanced chromosomal rearrangement.Wang et. al. [1990] studied a 9 year old mentally retarded femalewith a balanced 13;14 Robertsonian translocation inherited from herfather. Her mother carried a 1;14 reciprocal translocation. Thisgirl presented with mental retardation and multiple congenitalanomalies including short webbed neck, small thoracic cage withmarked angulation of the ribs, bilateral simian creases, and facialdysmorphism. To determine the parental origin of the chromosomes 14in the proband, two chromosome 14 probes, D14S13 [Nakamura et al.1987] and D14S22 [Nakamura et al. 1988] each detecting a VNTRpolymorphism on the long arm were used. Results showed that thepatient had inherited both chromosomes 14 from her father.Another case of maternal uniparental disomy for chromosome 14 wasrecently reported by Temple et al. [1991]. They described a 17 yearold male with hydrocephaly, bifid uvula, premature puberty, shortstature, small testes and normal intelligence who had inherited a13;14 Robertsonian translocation from his mother. Molecular studiesusing six probes that recognise loci in chromosome 14 and 10 probesthat recognise loci on chromosome 13 revealed that he had inheritedboth chromosomes 14 from his mother.25Mice can be bred so that they have uniparental disomy for aparticular chromosome, this is usually produced by translocation[Cattanach 1986]. It is not clear whether the major phenotypiceffects in mice are due to the duplication (i.e., presence of bothchromosome from one parent) or to deficiency (i.e., lack of achromosome from a parent). What is clear is that mice withuniparental disomy have severe growth retardation and somemalformations. All this evidence suggests that uniparental disomymay be the cause of the abnormal phenotype in at least someapparently balanced inherited translocation carriers.26MECHANISMS FOR UNIPARENTAL DISOMYThere are a few mechanisms which could cause uniparental disomy.These include 1) post-fertilization error in chromosomesegregation, 2) gamete complementation, 3) conversion of monosomyto isodisomy and 4) conversion of trisomy to disomy [Spence et al.1988].1) Postfertilization errors in which there was non-disjunctionwith duplication leading to trisomy and subsequent loss of aspecific chromosome would ultimately lead to uniparental disomy(Fig 3).2) Gamete complementation would involve fertilization between onenullisomic gamete and a disomic gamete for the same chromosome. Theresulting conceptus would have uniparental disomy derived from theoriginal disomic gamete (Fig 4) [Engel 1980, Spence et al 1988].3) If a normal haploid gamete was fertilized by a gametenullisomic for a single chromosome, the result would be monosomyfor that chromosome in the conceptus. If there was duplication ofthis single chromosome, the conceptus would end up with a doublecopy of that chromosome from only one parent or isodisomy for thatchromosome (Fig 5). This mechanism has been called monosomy toisodisomy [Engel 1980, Spence et al. 1988,]4) If an haploid gamete had two copies of a single chromosome dueto non-disjunction and was then fertilized by a normal haploidgamete, the conceptus would be trisomic for that chromosome. If oneof these trisomic chromosomes was lost due to a postzygotic error,27disomy would result. The zygote would thus have either uniparentaldisomy or with a normal set of chromosomes (Fig 6). This has beendesignated trisomy to disomy [Engel 1980, Spence et al. 1988].In cases of translocated chromosomes, the mechanisms by whichuniparental disomy may occur are somewhat similar to thosedescribed above. Good evidence exists to show that chromosomal non-disjunction occurs with higher frequency in gametes heterozygousfor trans locations.Lyon (1983] and Cattanach et. al. (1985] studied a large numberof mouse strains heterozygous for translocations. They found a highfrequency of chromosomal non-disjunction occurring spontaneously inthese mice. This condition usually leads to trisomic or monosomicconceptuses which die in utero unless uniparental disomy occurs asa result of the loss of one of the trisomic chromosomes orduplication of the monosomic chromosome [Cattanach et. al., 1986,Cattanach et. al. 1988].Non-disjunction is also common among human carriers of certaintranslocations [Engel 1980]. Some translocation carriers arepredisposed to 3:1 meiotic non-segregation, making the gametespotentially disomic by keeping a normal homologue along with thetranslocated chromosome. As a result, disomic gametes should occurmore frequently in such translocation carriers than in individualswith normal chromosomes [Engel and DeLozier-Blanchet 1991]. Adisomic gamete would usually be fertilized by a normal haploidgamete, resulting in a trisomic conception. Through subsequent lossof a chromosome, the conceptus could end up with normal biparental28contribution or with uniparental disomy.Gamete complementation is another possible mechanism. The samesituation described previously of non-disjunction of thetranslocated chromosomes would have to occur in the gamete, butwith fertilization by a nullisomic gamete for the homologue. Theoutcome would be a balanced translocation with uniparental disomy[Engel 1980].The frequency with which aneuploid gametes might complement eachother so that uniparental disomy may occur is directly related tothe frequency of aneuploidy in gametes. Cytogenetic studies ofspontaneous abortions have shown that at least 50% of firsttrimester losses result from chromosome aberrations. Half of thesecytogenetically abnormal conceptuses have autosomal trisomies.Autosomal monosomies are rarely observed and are presumably lethalbefore implantation [Hassold et al. 1979].Other studies have shown that at least 5% of male sperm havecytogenetic numerical abnormalitites [Martin et al. 1983], andthere is sufficient information to say that at least 20-30% ofoocytes are chromosomally abnormal [Martin et al. 1986].With the assumption that 20% of recognized conceptions end inmiscarriage and taking into account only the five most frequentsingle pair abnormalitites, Engel [1980] calculated the expectedincidence of uniparental disomy would be 2.8 in every 10,000conceptions.29Figure represents the division of normal chromosomes in gametes,zygote and somatic tissue.Non-disjunction duplication^Mitotic recombination or geneand loss^ conversion30Figure represents the division of the chromosomes in a post-fertilization error leading to UPD.31Figures represent the division of chromosomes in gametecomplementation leading to UPD.32Figures represent the division of the chromosomes in a conversionof monosomy to isodisomy leading to UPD.33Figures represent the division of the chromosomes in a conversionof trisomy to disomy leading to UPD.34TESTING FOR UNIPARENTAL DISOMYIt is now possible to test for uniparental disomy using highlypolymorphic genetic markers such as variable number of tandemrepeats (VNTR) or CA repeats [Wang et. al. 1991, Malcolm et. al.1990, Newsham et. al. 1991]. The value of any marker to test foruniparental disomy depends on how many variants it displays in thegeneral population.At many sites on human DNA, a single sequence that does not codefor a protein is repeated many times. These are called variablenumber of tandem repeats (VNTR). VNTR's are specially importantbecause the number of repeats at a given locus can vary from a fewto hundreds of copies. These loci are characterized by manyalleles. Restriction fragments created by cutting on both sides ofthese tandem repeats vary in length. The most informative markershave many different alleles. In a heterozygous individual theSouthern blot will reveal two distinct fragments of differentlength, one from each homologous chromosome. [White and Lalouel1988, Nakamura et. al. 1987, Litt and Luty 1989].CA repeats are a type of VNTR based on a series of dinucleotiderepeats and are highly polymorphic. This form of polymorphism canbe detected by amplifying the region containing the repeat andrunning the amplification product on a sequencing gel. Inheterozygotes the sequencing gel will reveal two differentfragments of different length, one from each chromosome [Litt andLuty 1989].35To test for uniparental disomy, it is essential to determine theparental origin of the two chromosomes inherited by an individual.Thus a useful genetic marker in cases of uniparental disomy is onethat can detect more than two alleles.A marker based on VNTR's or CA repeats can be highly informativewhen it exhibits many alleles. Since alleles at a certain locus aredistributed by chance, the frequency of heterozygosity orinformativeness at a locus is directly related to the number ofcommon alleles present in the population. The more alleles a locushas, the more likely it is that an individual will be heterozygous.Testing for uniparental disomy is largely dependent on highlypolymorphic loci [Reeders et. al. 1985, White et. al. 1985]. Torule out UPD it is not essential for the parents to have no allelesin common but it is mandatory that the offspring inherits thealleles that are not shared by the parents (fig 11). If the parentshave an allele in common and the allele is also found in theoffspring, it is impossible to determine the parental origin ofthat allele (fig 12). When fully informative, testing foruniparental disomy can determine whether the offspring has bothmaternal and paternal genetic contributions, only maternal (fig 13)or only paternal (fig 14). Patients with only maternal or onlypaternal contributions have UPD for the chromosome regioncontaining the loci studied.Parents are heterozygous but share one allele.36Uniparental disomy cannot be ruled out if child inherits the alleleshared in common.3738HYPOTHESISWe hypothesize that uniparental disomy is responsible for thephenotypic abnormalities in some children who have an inheritedapparently balanced chromosomal translocation.39MATERIALS AND METHODSPROCEDUREA computer database search was done at the Department of MedicalGenetics, University Hospital-Shaughnessy Site to identify alleligible patients to participate in the study. Approval for contactwas first obtained from the medical geneticist involved in the careof each patient. Approval to contact the family was then obtainedfrom either the referring physician or the family physician. Aninitial letter of contact was sent to the parents informing them ofour study and asking for their participation (see appendix forletters). This letter was followed up by a phone call. All sevenfamilies with one or more living children with an inheritedapprently balanced translocation and mental retardation/multiplecongenital anomalies agreed to participate.Both parents and the affected children in each family were seenat the Department of Medical Genetics, University Hospital,Shaughnessy Site. Audiovisual aids were used to explain the purposeof our research. Since the affected children were unable to giveconsent because of their ages and mental condition, both parentswere asked to sign a consent form for blood withdrawal andparticipation in the study.BLOOD LYSATES AND DNA EXTRACTION40DNA from the parents and offspring of each of the 7 families inour study was obtained by standard phenol-chloroform extraction andethanol precipitation according to the technique described byKunkel et al. (1977]. Tris-NH 4C1 was prewarmed at 37 C. One 50 mlFalcon tube was filled with 10 cc of EDTA blood and 40 ml ofprewarmed Tris NH4C1 and incubated at 37 C for five minutes. Afterincubation the tubes were centrifuged at 2600 rpm for 7 minutes andthe supernatant was aspirated. The pellet was resuspended in 20 mlof 85% saline solution and centrifuged at 2600 rpm for 7 minutes.The supernatant was aspirated. The pellet was resuspended in 5 mlof Tris-EDTA (TE) solution and 5 ml of lysis buffer solution wasinjected through an 19 gauge needle. 100 AL of proteinase K wereadded. 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 andcentrifuged at 3000 rpm for 5 minutes. The top layer including theinterface was removed to a new 50 ml Falcon tube. 2.5 ml of 6M NaC1solution were added and the tubes were manually shaken. 12.5 ml of(24:1) Chloroform:Isoamylalcohol solution were added and the tubeswere shaken mechanically at 80 for 30-60 minutes. The tubes werecentrifuged at 3000 rpm for 5 minutes. The top layer was carefullyremoved with a Pasteur pipette and transferred to a new 50 mlfalcon tube. 30 ml of 95% ethanol were added and the tubes shakengently in the mechanical shaker. The DNA precipitate was removedwith a curved pipette and washed with a few drops of 70% ethanol.The extracted DNA was resuspended in 500 AL of TE.41SOUTHERN BLOTTING TECHNIQUEDNA was digested, electrophoresed and blotted according to thetechnique described by Southern (1975). 10 AL of genomic DNA werecut with the appropiate enzyme and buffer, according to theconditions provided by the supplierThe digest was incubated in a 37 C water bath for 4 hours. Afterincubation, 5 Al of dye were added to the enzyme digest.1500 ml of Tris-Boric Acid-EDTA solution was poured into the gelbox and the agarose gel was placed in it. The digested DNA wasloaded into wells in the agarose gel. Electrophoresis was performedfor 12 to 24 hours depending on the size of the fragments to beseparated. The DNA was transferred onto a nylon membrane bySouthern blotting.LABELLING100 ng of each DNA probe (see VNTR- Table 4) was resuspended in32.5 AL of water in a 1 ml Eppendorf tube. The tube was boiled for5 minutes and quenched in ice for 3 minutes. 10 AL oligonucleotidelabelling reagent (OLB), 2 AL acetylated bovine serum albumin(BSA), 5 gL 32P dCTP and 0.5 AL large fragment polymerase wereadded for a total volume of 50 ML. The tube was then left at roomtemperature for 4 hrs.The barrel of a 1 ml syringe was stuffed with silaconized glasswool. A mixed slurry of SEPHADEX was poured into the barrel of the42syringe until it reached .8 ml. The syringe was centrifuged at 2500rpm for 3 minutes. 50 gL of TE were added to the previouslymentioned priming reaction tubes. The contents of the primingreaction tubes was loaded into the syringe barrel and centrifugedat 2500 rpm for 3 minutes. The contents of the syringe wastransfered into Eppendorf tubes which were then capped.The hybond n-plus nylon membrane used for blotting was sealed ina plastic bag. The Church's buffer was prewarmed in 65 C oven. 8-10ml of prewarmed Church's buffer were added into the sealed bag witha 10 ml syringe and 18 gauge needle. The bag was sealed again andshaken in a 65 C water bath for 2 hrs. The contents of the cappedEppendorf tubes containing the radiolabelled probe was boiled for5 minutes and quenched in ice for 3 minutes.0.5 ml of prewarmed Church's buffer was loaded into a 1 mlsyringe. The contents were injected into the capped Eppendorf tubecontaining the radiolabelled probe. The entire contents of the tubewas aspirated with the syringe. The sealed bag with blot wasremoved from the 65 C water bath and the contents of the syringewere injected into the bag. The bag was sealed again and submergedin a 65 C water bath and shaken overnight. The blots were removedfrom the sealed bags and washed as follows: with 2x sodiumchloride-sodium phosphate-EDTA solution (SSPE) 1% sodium doedecylsulphate (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 30minutes once, with preheated 0.2X SSPE + 0.1% SDS in 65 C water43bath for 30 minutes once. The moist filters were wrapped in saranwrap and exposed to KODAK XAR-5 films for 3-7 days.POLYMERASE CHAIN REACTION (PCR) AMPLIFICATIONPCR amplifications were carried out in a total volume of 25 uLcontaining 100 ng of genomic DNA, 1 AL of each primer, 2.5 AL of10x Taq polymerase buffer, 2 AL of dITP mix, 1.25 of 5mMspermidine, 0.05 of -32PdCTP and 0.2 AL of 5A/AL taq polymerase.The PCR conditions and primers varied depending on the markerinvolved and according to the protocol previously published forthat marker (Table 4). Products were resolved on an 8%polyacrylamide gel and visualized under UV light or on a DNAsequencing gel and visualized by autoradiography.44RESULTSA computer search was done on the patient database at theDepartment of Medical Genetics, University Hospital ShaughnessySite, looking for chromosomal abnormalities. This search showedthat between February 1966 and June 1991, 3906 patients were seenwith a diagnosis of a chromosomal abnormality. Of these 3906, 207(5.2%) patients had documented chromosomal rearrangements. These207 charts were reviewed and formed the initial basis for thepresent study.Of the 207, 45 (21.7%) families carried an inherited apparentlybalanced reciprocal translocation. Of these 45, 12 (26%) familieshad one or more offspring who carried an inherited apparentlybalanced reciprocal translocation and who presented with congenitalanomalies and developmental delay (Fig.15). Seven (58.3%) of these12 families were available to study. Two of these families each hadtwo affected children for a total of 9 cases. In five families, theaffected offspring was deceased.Complete karyotypes with high resolution banding for both parentsand the affected offspring in each of the seven families werereviewed. The chromosome breakpoints are listed in Table 3.The markers used to test for uniparental disomy (table 4) werechosen because of their capacity to detect polymorphisms andbecause they were previously available in the lab.45Fig 15. Figure illustrates the search of the patient databaseUBC-Department of Medical GeneticsPatient Database; 39,000 families3906 families with a diagnosis of chromosomal abnormality207 families with documented chromosomal rearrangements166 families with reciprocal translocations45 inherited reciprocal translocations12 families with inherited apparently balancedtranslocations and malformations and/or developmental delay5 families excluded due deathof affected child  7 families included in thestudy9 cases from 7 families with inheritedapparently balanced translocations and withmalformations and/or developmental delay 4 6Table 3. Karyotypes of individuals with an inherited apparentlybalanced translocation, mental retardation and/or malformations.Case 1 (Family 01) 46,XX,t(10;16)(p11.2;q23)patCase 2 (Family 02) 46,XY,t(6;17)(p11.2;q21.1)patCase 3 (Family 03) 46,XY,t(13;14)(p11;q11)patCase 4 (Family 04) 46,XY,t(2:12)(p14.2;q13.1)patCase 5 (Family 05) 46,XY,t(4;7)(p15.1;q36)patCase 6 (Family 06) 46,XY,t(1;5)(p13.1;q33.1)patCase 6b (Family 06) 46,XX,t(1;5)(p13.1;q33.1)patCase 7 (Family 07) 46,XY,t(11;14)(q12;p13)matCase 7b (Family 07) 46,XY,t(11;14)(q12;p13)mat47Table 4. Markers used in DNA analysis of the translocations.CHROMOSOME MARKER TYPE REFERENCE FAMILYSTUDIEDCHROMOSOME 1 D1S81 VNTR* Nakamura etal.^1987Family 03CHROMOSOME 2 APO B VNTR Boerwinkle etal.^1989Family 05CHROMOSOME 4 D4S227 CArepeatWeber et al.1991Family 06CHROMOSOME 5 D5S107 CArepeatWeber et,a1.1990 Family 03-CHROMOSOME 6 D6S89 CArepeatZoghbi et al.1991Ranum et al.1991Family 02CHROMOSOME 7 D7S396 VNTR* Nakamura etal.^1987Lathrop etal.^1985Family 06CHROMOSOME 10 D1OS15D10S28STCL-2VNTR*VNTR*CArepeatNakamura etal.^1987Lathrop etal.^1985Lairmore etal.^(inpress)Family 01CHROMOSOME 11 D11S35 CArepeatLitt et al.1991 Family 07CHROMOSOME 12 D12VWF VNTR Standen etal. 1990Family 05CHROMOSOME 13 RBD13 VNTR Scharf et al.1992Family 04CHROMOSOME 14 D14S13 VNTR* Nakamura etal. 1988Family 04Family 07CHROMOSOME 16 D16S291 CArepeatThompson etal.^1992Family 01CHROMOSOME 17 IYNZ22^IVNTR Batanian 1990 (Family 02All markers were tested by PCR except for the ones marked (*) whichtested by Southern blots.48The families have been numbered arbitrarily from 01 to 07. In thefollowing 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 eachof the chromosomes involved in the translocation are at the top ofeach column in the tables. Alleles were designated 1 to 4, with 1being the biggest molecular weight and 4 being the lowest.Key to pedigree symbols0 Female carrierMale carrier0 Translocation carrier with malformations and/ordevelopmental delayTranslocation carrier with malformations and/ordevelopmental delayO Normal female non-carrierNormal male non-carrier491,4Clinical reportCase 1: P.E. was a 7 year old female. She was born at 36 weeksafter a twin pregnancy, birth weight was 1.5 kg. Anomalies presentat birth were choanal atresia and Tetralogy of Fallot. Infancy wascomplicated by recurrent abdominal pain, vomiting and dehydrationwith ileus requiring multiple hospital admissions. At the age of 4years she was diagnosed as having a gallstone and cholecystectomywas performed. On physical examination her weight and height wereat the 3rd centile. She had a round face with mild malarflattening, hypertelorism, upslanting palpebral fissures, a verythin upper lip and bifid uvula. She had clinodactyly of the fifthfinger bilaterally. She had 3/6 systolic ejection murmur.Table 5. Results of family 01 t(10;16)(p11.2;q23)patFamily 01 Chromosome 16^I,D16S29101-1 1,201-2 3,401-3 1,4This family was uninformative for 2 VNTR markers on chromosome 10,D10S25 [Nakamura et al. 1987], D10S28 [Lathrop et al. 1985], and 1CA repeat sTCL-2 [Lairmore et al. 1991]. Uniparental disomy forchromosome 16 has been ruled out.501,2 2,3Clinical reportCase 2: D.J. was a 4 year old male. He was born at 42 weeks, birthweight was 4.4 kg. The neonatal period was uneventful. He sat at 6months, crawled at 10 months, walked at 11 months and spoke at 4years. He had language and developmental delay and bizarrre"autistic" behavior. On examinaton his weight was 19.5 kg (90thcentile), height was 107 cm (between 90 and 97th centile) and OFCwas 53.3 (greater than the 98th centile). He had no obviousdysmorphic features except for relative macrocephaly, a veryprominent nasal bridge and strabismus.Table 6. Results of family 02 t(6;17)(g11;q21.1)patFamily 02 Chromosome 6 Chromosome 17D6S89 YNZ2202-1 2,3 1,302-2 1,4 2,402-3 1,2 2,3Uniparental disomy for chromosomes 6 and 17 has been ruled out.511,2Clinical reportCase 3: B.S. was a 7 year old male. He was born at term after anuncomplicated pregnancy. Birth weight was 3.9 kg. Severedevelopmental delay was noted shortly after birth. He pulls himselfto a stand but does not walk or talk. Over the past two years hehas had peculiar "spells" with marked hyperactivity, yelling andscreaming. At the age of 3 years, severe cortical visual impairmentwas diagnosed. On examination his weigth was 18.5 kg (5th centile),height was 49.1 cm (10th centile) and OFC was 49.2 cm (10thcentile). He has a round face and broad nasal root with mildanteversion of the nostrils. His fingers were tapered, not flexibleand he had camptodactyly of the fifth finger. Chest examination wasnormal.Table 7. Results of family 03 t(13;14)(p11;q11)patFamily 03 Chromosome 13^'Chromosome 14 ID13RBD D14S1304-1 1,2 1,304-2 2,3 2,304-3 1,3 1,2Uniparental disomy for 13 and 14 has been ruled out521,4Clinical reportCase 4: J.S was a 9 year old male born at term. The pregnancy wascomplicated by vaginal bleeding. Birth weight was 2.9 kg. At birtha foot deformity was noted and treated with splints. Developmentaland speech delay were noted at the age of 18 months. He had adevelopmental evaluation at age 8 years and was found to have mildmental retardation and a significant language disorder.On examination his weight was 25.5 kg (25th centile), height was126.9 cm (10th centile) and OFC was 53 cm (25th centile). He hadmicrognathia with an open bite, a slight upward slant of thepalpebral fissures and his ears had a posterior slope.Table 8. Results of family 04 t(2;12)(p14.2;q13.1)patFamily 04 Chromosome 2 Chromosome 12ApoB D12VWD05-1 1,3 1,205-2 2,4 3,405-3 3,4 1,4Uniparental disomy for 2 and 12 have been ruled out.532,4 3,4Clinical reportCase 5: W.C. was a 21 year old male who was born at term. Birthweight was 3.1 kg. The neonatal period was complicated by cyanoticspells. A heart murmur was noted. A ventricular septal defect wasdiagnosed at age 3 and he underwent cardiac cathetherization.Developmental delay was also noted at the age of 3. Mild mentalretardation was diagnosed at the age of 10. On examination hisweight 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 themean). He had macrocephaly, mid-face hypoplasia, down-slantedpalpebral fissures, a high-arched palate, prognathism and largeears. He had prominent thoracic kyphosis with mild left-sidedscoliosis of the thoracic spine. His hands had lateral deviation ofthe distal phalanx of the third digits bilaterally.Table 9. Results of family 05 t(4;7)(p15.1;q36)patFamily 05 Chromosome 4 'Chromosome 71D4S227 D7S39606-1 3,4 1,406-2 1,2 2,306-3 2,4 3,4Uniparental disomy for chromosomes 4 and 7 has been ruled out54Clinical reportsCase 6: D.C. was a 8 year old male. He was born at term. Birthweight was 3.2 kg. There was mild developmental delay. Speech wassignificantly delayed. On examination his height was 125 cm (25thcentile), his weight was 29 kg and OFC was 54 cm. He had a softsystolic ejection murmur at the left sternal border. There were noobvious dysmorphic features.Case 6b: C.C. was a 5 year old female. She was born at term aftervaginal delivery. The delivery was precipitous and the child hadrespiratory difficulties that required resuscitation. Shortly afterbirth she had a seizure and was placed on phenobarbital. There wereno further seizures and the child was taken off the phenobarbitalat 4 years of age. On examination she did not make eye contact, shemade whining noises but used no verbal language, she had continuousrepetitive hand clapping movements and her fingers were constantlyin her mouth. Her weight was 15 kg (5th centile), her height was102.7 cm (less than 5th centile), and OFC was 47.5 (less than 2ndcentile). She had deep set eyes, pronounced eyebrows and aprominent jaw. Her ears had a prominent antihelix and her ear lobeswere attached. She had a 1 x 1/2 cm hyperpigmented macular lesionon her right buttock, a slightly hypopigmented patch of skin overthe left knee and a 1 x 1/2 cm hyperpigmented macule on the dorsumof her left foot. A CT Scan revealed bilateral destructive areas ofcerebral cortex.55Table 10. Results of family 06 t(1;5)(p13.1;q33.1)patFamily 06 Chromosome 1 I Chromosome 5D1S81 D5S10703-1 1,3 1,303-2 2,4* 2,3*1 03-31 1,4 1,203-4 2,3 2,3* Deduced on the basis of unaffected children's genotypesWith results from two other offsprings we have been able to deducethe father's alleles. Molecular results show a maternalcontribution for chromosomes 1 and 5. Cytogenetic results revealthat both offsprings have inherited both translocation derivativesfrom their father. Molecular and cytogenetic results rule outuniparental disomy for chromosomes 1 and 5.56Clinical reportsCase 7: J.B was a 9 year old male born at term. Birth weight was3.6 kg. He had intermittent ataxia, mild mental retardation andseizures. On examination his height was 136 cm, his weight was 27.4cm 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 was3.6 kg. He had intermittent ataxia, mild mental retardation andseizures 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)matFamily 07 Chromosome 11 !Chromosome 14 'Chromosome 14D11S35 D14S13 D14S4207-1 2,4 2,3 1,207-207-3 1,4 1,2 2,307-4 1,2 1,2 2,3Cytogenetic results reveal the translocation was inherited from themother. Molecular analysis in each child reveals one allele thatdoes not come from the mother. Assuming this is the paternalcontribution, uniparental disomy for chromosomes 11 and 14 can beruled out.57DISCUSSIONDevelopmental delay and congenital anomalies have been observedin two patients with inherited apparently balanced chromosomaltranslocations of chromosomes 13 and 14 who were found to haveuniparental disomy [Wang et. al. 1991, Temple et al. 1991). Theauthors suggested that uniparental disomy was the most likely causefor their developmental delay and congenital anomalies.It appears, however, from the results in this study thatuniparental disomy is not a frequent occurrence among carriers ofinherited apparently balanced chromosomal translocations who havephenotype abnormalities. Other mechanisms must therefore accountfor the phenotypic abnormalities in most individuals with aninherited apparently balanced translocation who have congenitalanomalies and mental retardation.Imprinting, that is, the possibility that certain genes are markedso that they are expressed differently when they have beeninherited from the father rather than when they are inherited fromthe mother must be considered. In 6 out of 7 families (cases 1through 6b) in our study, the translocation was paternallyinherited. In 1 family (cases 7 and 7b) the translocation wasmaternally inherited. The origin of the translocation in 3 of these6 (families 01, 05 and 06) fathers was unavailable . In 1 (family04) the father's translocation appeared de novo and in the other 2(family 02 and 03), the fathers inherited the translocation fromtheir mothers.58An excess of paternally inherited apparently balancedtranslocations in children with mental retardation andmalformations was previously noted by Howard-Peebles and Friedman[1986]. In their report five out of six rearrangements werepaternally inherited. The authors however do not mention from whomthe fathers inherited their translocations.In the report by Jacobs et. al.[1974] in which they looked at33,533 consecutive karyotypes, they observed a total of 85Robertsonian and reciprocal translocations. In 40 (47%) cases bothparents were available for the study. 28 (70%) out of the 40translocations were inherited, 14 (50%) were paternal and 14 (50%)were maternal. However the authors do not report whether thecarriers of these inherited translocations had a normal or abnormalphenotype. A larger sample of patients with inherited apparentlybalanced translocations and abnormal phenotype is needed in orderto ascertain the importance of imprinting.The families we studied had translocations that involved some ofthe chromosomes or chromosomal regions that are known to beimprinted. Beckwith-Wiedemann is an overgrowth syndrome andevidence for paternal imprinting for the 11p15.5 region has beenreported in sporadic cases [Viljoen et. al.1992]. Weksber et al.[1992] reviewed a cohort of patients with Beckwith-Wiedemannsyndrome. Two patients had a maternally inherited apparentlybalanced translocation. The Beckwith-Wiedemann syndrome phenotypewas not present in the mothers carrying the translocation. Theysuggest that these findings as well as the paternal uniparental59disomy seen in sporadic cases of Beckwith-Wiedemann are consistentwith an imprinting model with suppression of the maternallyinherited allele [Websker et al. 1992]. Two of the children in ourstudy (case 7 and 7b) had inherited a 11;14 (q12;p13) translocationfrom their mother. The marker we used D11S35 is located in the11q22 region so the 11p15 region associated with Beckwith-Wiedemannsyndrome was not tested. Nonetheless we know that in some cases ofsporadic Beckwith-Wiedemann syndrome, this region is imprinted soit may be possible for other regions of chromosome 11 to beimprinted as well.Evidence for imprinting can also be seen in the two cases ofcystic fibrosis and uniparental disomy for chromosome 7 reported bySpence et al. [1988] and Voss et al. [1989]. These reports clearlyshow there is differential function of the maternal and paternalchromosome 7. One of our patients (case 5) had inherited a (4;7)(p15.1;q36) translocation from his father. The two previous reportsof uniparental disomy for chromosome 7 and CF describe patientswith severe growth retardation. It is also important to note thatin mouse studies defining the phenotypes of uniparental disomies nomajor congenital anomalies have been noted, but variations ofgrowth, behaviour and survival are present [Hall 1990]. Case 5,however, had no growth failure. On the basis of this phenotypeuniparental disomy for chromosome 7 was highly unlikely.In 1990 Hall listed the mouse chromosome areas involved inimprinting, she then compared them to human chromosome areas andsuggested that the genes that have been mapped within those regions60in humans may also be imprinted. The families in our study havetranslocations that involve some of the chromosomes in her list.Family 04 had a (2;12) translocation, the 2p11-p13 region of thehuman chromosome is homologous to the 6C area of the mousechromosome which is imprinted. Family 02 had a (6;17)translocation, the 17A-D region of the mouse chromosome area isinvolved in imprinting and is homologous to the human 6pter-p12.Family 01 had a (10;16) translocation, the mouse chromosome 8involved in imprinting is homologous to the 16q22.1-q24 region.Family 05 had a (4;7) translocation, the 6B-C,11A and 6A-C mouseareas thought to be imprinted are homologous to the human 7p21-p14,7p14-p12 and 7q22-qter.If imprinting is occuring in cases of inherited balancedtranslocations and there is more than one affected family memberthe phenotype in both affected members would be expected to be thesame. Family 06 and family 07 have two affected children each. Thepattern of the abnormal phenotype in family 07 is similar. But infamily 06 they are completely different. However case 6b had acomplicated neonatal period with a precipituous delivery andrepiratory difficulties requiring resuscitation. It is possiblethat part of the abnormal phenotype is due to deliverycomplications.To corroborate our findings it is important to study a largernumber of individuals with an inherited balanced translocation andmental retardation and/or congenital anomalies and compare bychromosome the ones in which the mother is the carrier versus the61ones in which the father is the carrier. If there is an importantpaternal or maternal imprinting effect, a significant differencefor specific chromosomes between the two groups regarding thepresence of malformations and mental retardation should be seen.This is especially important if the translocation involves achromosomes or chromosomal region known to be imprinted.Although cytogenetically undetectable, a very small deletion orduplication present in the offspring but not in the carrier parentmay also be the cause for the mental retardation and themalformations seen in some individual carriers of an inheritedapparently balanced translocation. The development of molecularmarkers very close to or at the breakpoints of these translocationswill help further elucidate this possibility. It is also importantto remember that one locus per chromosome does not rule outuniparental disomy for the entire chromosome. It has been shown incases of Beckwith-Wiedemann [Viljoen et al. 1992] that there may beuniparental disomy for only a certain region of a chromosome. Thereis still a possibility of partial uniparental disomy that may bethe cause for the abnormal phenotypeWe believe that even though this was a "negative" study theinformation gained from it is important. The reports of Wang[1991], Temple et al. 1991 and Pentao et al. [1992] raised thepossibility that uniparental disomy might be a frequent cause ofmental retardation and multiple congential anomalies in childrenwith inherited apparently balanced translocations. If so,counselling families who are known to carry a translocation about62possible risks related to uniparental disomy is importantespecially when doing prenatal testing. We have learned from thisstudy that uniparental disomy in inherited apparently balancedtranslocations is not a usual occurence.63REFERENCESAldridge A. Kleczkowska A, Fryns JP. (1990) On the variable effectof mosaic balanced chromosomal rearrangements in man. J. Med.Genet.27;505-507Alfi OS, Chang R, Azen SP (1980) Evidence for genetic control ofnondisjunction in man. Am. J. Hum. Genet. 32;477-483Ayme S, Mattei AG (1979) Abnormal Childhood phenotypes associatedwith the same balanced chromosome rearrengements as in the parents.Hum. Genet. 48;7-12Batanian JR, Ledbetter SA„ Wolff RK, Nakamura Y, White R, DobynsW, Ledbetter DH (1990) Rapid diagnosis of Miller-Dieker syndromeand isolated lissencephaly sequence by the polymerase chainreaction. Hum. 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Am. J. Hum. Genet. 48;1069-1074Warburton D (1988) Editorial: Uniparental disomy: A rareconsequence of the high rate of aneuploidy in Human Gametes. Am. J.Hum. Genet. 42;215-216.Weber JL, Kwitek AE, May PE (1990) Dinucleotide repaetpolymorphisms at the D5S107, D5S108, D5S111, D5S117 and D5S118loci. Nucleic Acids Research 18;403578Weber JL, May PE (1989) Abundant class of human DNA polymorphismswhich can be typed using the polymerase chain reaction. Am. J. Hum.Genet. 44;388-396Weyerts LK, Jones MC (1992) The natural history ofthe DiGeorgesequence: Overlapping phenotype with Velocardiofacial syndrome.Procceding of the Greenwood Genetic Centre.Wiedemann HR (1964) Complexe malformatif familial avec hernieombilicale et macroglossie-un syndrome nuveau? J. Genet. Hum.13;223Wilmot PL, Shapiro LR (1990) Disomic balanced reciprocaltranslocation. Clin. Genet. 38;126-127Winsor E, Welch J (1983) Prader-Willi syndrome associated withinversion of chromosome 15. Clin. Genet. 24;456-461White R, Lalouel JM (1988) Chromosome mapping with DNA markers.Scientific Am. 258;40-48264APPENDIXTHE UNIVERSITY OF BRITISH COLUMBIA Department of Medical GeneticsUniversity Hospital—Shaughnessy Site4500 Oak StreetVancouver, B.C. Canada V6H 3N1Tel: (604) 875-2157^Fax: (604) 875-2376Dear Dr. January 13,1991.Recent publications suggest that patients who have inherited an apparentlybalanced chromosomal rearrangement and who present with congenital anomaliesand/or developmental delay may have inherited both the normal and the rearrangedchromosome from only one parent. This type of inheritance is called uniparentaldisomy and has also been described in cases of Prader Willi Syndrome.Dr. J.M. Friedman, Dr.L. Langlois and Dr. Lopez Rangel will be startingresearch at the Department of Medical Genetics, University Hospital, ShaughnessySite. We would like to contact patients who are known to carry a inheritedchromosomal rearrangement and have congenital anomalies and/or developmentaldelay. !  is a patient of yours who is eligible for our studyand was last since in our clinic on February 1987.Participation in our study would require a blood sample. The blood would beanalyzed at the DNA laboratory at the University Hospital, Shaughnessy Site. Wewould like to get information on the chromosomal rearrangement and look foruniparental disomy as a cause for the malformations and the developmental delay.I have been in touch with   and they are very interestedin participating in our study. We would like your help in obtaining the bloodsamples. Enclosed please find requisition forms for blood to be drawn on bothparents 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, 1Medical Genetics MSc Student.CONSENT FORMName of the study: Uniparental disomy as a cause for malformationsand/or developmental delay incases of inheritedapparently balanced translocations.Institute:PrincipalInvestigators:The University of British ColumbiaDr. J.M. Friedman, Dr. S. Langlois, Dr. E. LopezWe, ^to participate with our child in aDepartment of Medical Genetics,Columbia.agree / do not agreeresearch study conducted by theat the University of BritishThe aim of the projeCt is to try to determine at the cytogeneticand DNA level the change that could be responsible for thecondition affecting our child.To achieve this goal, we agree to undergo a blood test (20cc) andto have our child have a blood test (lOcc). We understand there maybe some discomfort associated with the placement of the intravenousneedle for blood withdrawal and that ocassionally, bruising mayresult.We understand that only the investigators involved will know thename of the participants in this research project. We are awarethat much of the information obtained from this study willeventually be used in scientific publications but the identity ofsubjects in the study will not be revealed in such publications orany other report.We understand that our participation in this study is entirelyvoluntary and that refusal to participate will in no way jeopardizethe care of our child in the Department of Medical Genetics at theUniversity Hospital.Our signature on this form signifies, that we have decided toparticipate in this study after reading the above information.We have been given the opportunity to discuss pertinent aspects ofthe research study and to ask questions, and hereby consent toparticipate in the study outlined above. We also have received acopy of this consent form for our own personal records.Subject's parents signatureWitnessDateDate


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