Risk evaluation of carriers with chromosome reciprocal translocation t(7;13)(q34;q13) and concomitant meiotic segregation analyzed by FISH on ejaculated spermatozoa

� 2006 Wiley-Liss, Inc. American Journal of Medical Genetics 140A:245–256 (2006)

Risk Evaluation of Carriers With ChromosomeReciprocal Translocation t(7;13)(q34;q13)

and Concomitant Meiotic Segregation Analyzedby FISH on Ejaculated Spermatozoa

Alina T. Midro,1* Ewa Wiland,2 Barbara Panasiuk,1 Ryszard Lesniewicz,1 and Maciej Kurpisz21Department of Clinical Genetics, Medical University Białystok, Białystok, Poland

2Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland

Received 22 June 2005; Accepted 7 November 2005

We performed the segregation analysis of a relatively largepedigree of t(7;13)(q34;q13) carriers together with the spermkaryotype analysis of the one carrier using a tri-colorfluorescence in situ hybridization (FISH) method. The riskassessments for unfavorable pregnancy outcomes in a seriesof 36 pregnancies in eight reciprocal chromosome transloca-tion (RCT) couples of carriers were estimated directly from apedigree after ascertainment correction. The individualprobability rate for unbalanced child was predicted accord-ing to Stengel-Rutkowski and co-workers. The unbalancedkaryotypes in the form of monosomy 7q34!qter andtrisomy 13q13!qter were detected among stillborn/earlydeath newborns with holoprosencephaly (HPE), cyclopiaand other malformations. Based on clinical description ofunkaryotyped stillbirth progeny, it can be assumed that thephenotype distinctions were connected with the unbalancedkaryotype from 2:2 segregation (monosomy 7q with trisomy13q) and 3:1 segregation as interchange trisomy 13 (Patausyndrome). Probability rates for miscarriages, stillbirth/earlydeath were 12.9� 6% (4/31) and 29� 8.2% (9/31), respec-

tively. The results of the meiotic segregation patternindicated the rate of unbalanced spermatozoa for about60%, with the unusual high rate (29.4%) of 3:1 segregant (i.e.,13.4% of the tertiary segregation and 16% of the interchangesegregation). Adjacent-1 segregation followed with 23.5%and adjacent-2 followed with 7.2% of analyzed spermatozoa.The high rate of unbalanced gametes in comparison to thenumber of stillborn/early death and miscarriages detected inpedigree suggests a strong selection against unbalancedchromosomal constitutions during fetal development. Itcorresponds to a very small probability rate (about 0.3%)of viable unbalanced progeny from 3:1 meiotic segregationpredicted for maternal carriers. This knowledge can be usedin genetic counseling of families with similar RCT ascertainedin a different way. � 2006 Wiley-Liss, Inc.

Key words: partial distal monosomy 7q with partial prox-imal trisomy 13 reciprocal translocation 7;13; pedigreeanalysis; spermatozoa; FISH; risk assesment

INTRODUCTION

Reciprocal chromosomal translocations (RCT) arethe most common structural rearrangements inhumans. Their incidence in population of newbornswas estimated as 1:712 [Nielsen and Wohlert, 1991]and their frequency at the time of prenatal diagnosisby amniocentesis was about 1:250 [Van Dyke et al.,1983]. During meiosis, the chromosomes of RCTcarriers produce a quadrivalent configuration givinga reason for the origin of chromosomally unbalancedgametes. In aparticular carrier of RCT, theproportionof sperm karyotypes from different types of meioticsegregation constitutes the so-called segregationpattern. Normal or genetically balanced gametesoriginate only as the result of the alternate segrega-tion, when the RCT chromosomes segregate to one

pole and the normal homologous to the other pole.In the results of 2:2 segregation (adjacent-1 oradjacent-2), 3:1 segregation (tertiary or interchange)and 4:0 segregation, only chromosomally unba-lanced gametes are being produced. In most RCTcarriers the frequency of alternative product segre-gation studied in spermatozoa ranged from 33% to

Grant sponsor: Medical University Białystok; Grant numbers: AMB No4-06 780, 4-06 760, KBN P05A 089 27; Grant sponsor: Institute of HumanGenetics, Pol. Acad. Sci., Poznan.

*Correspondence to: Prof.Dr. Alina T. Midro, Department of ClinicalGenetics, Medical University of Białystok, Waszyngton str. 13, 15-089Białystok 8, PO Box 22, Poland. E-mail: [email protected]

DOI 10.1002/ajmg.a.31083

57%, for adjacent-1 disjunction from 16% to 52%, foradjacent-2 disjunction from 0% to 39% and forproducts of 3:1 segregation, 0%–40% [Faraut et al.,2000; Shi and Martin, 2001].

It seems that the segregation pattern for thetranslocated chromosomes depends greatly on thechromosomes involved in the individual RCT, onthe location of the breakpoints on a particularchromosome and on the size of the imbalance [fora review, see Guttenbach et al., 1997; Faraut et al.,2000; Schinzel, 2001; Shi and Martin, 2001]. Astatistical analysis of data from different studiesdemonstrated that the frequency of adjacent-1segregants is inversely linked with the shortesttranslocated segment, while the frequency of adja-cent-2 segregants is inversely linked with the lengthof the shortest centric segment [Faraut et al., 2000].However, the meiotic mechanisms responsible forsegregation of structural rearrangements remainunknown.

Gametes carrying the unbalanced reciprocal trans-locations may have caused the unfavorable preg-nancy outcomes (spontaneousmiscarriage, stillbirth,early death of newborn, and congenital malforma-tions in liveborn progeny). The different types ofunfavorable pregnancy outcomes can reflect thedifferent survival rate of different types of unba-lanced embryo/fetuses. The frequency of unba-lanced progeny at birth depends on the size andthe genetic content of the rearranged chromosomalsegments [Davis et al., 1985; Stengel-Rutkowski et al.,1988]. The respective role of the meiotic segregationmechanism and of natural selection with respect tothe different features of the above mentionedoutcomes is not yet fully understood. Some authorssuggest that in most cases of balanced translocationsno general rules can be drawn, and each one must beconsidered as a particular case [Geneix et al., 2002].In general, the probability rate of having unbalancedprogeny changes from translocation to translocation.There are some approaches to determine theprobability of unbalanced progeny compatible tosurvival for individual chromosome translocationcarriers [reviewed by Gardner and Sutherland, 2004].The risk estimation proposed by Stengel-Rutkowskiet al. [1988] and discussed in a review [Stene andStengel-Rutkowski, 1988] is based on the evaluationempirical data from the pedigrees pooled accordingto the individual chromosome segments involved inthe chromosome translocation. This method wasdeveloped taking into consideration the specificity ofthe RCT families instead of the standard statisticalmethods from general computer programs [Stene,1989].

We collected empirical data from the relativelylarge pedigree with an increased incidence ofdifferent unfavorable pregnancy outcomes among36 pregnancies of eight carriers of the samet(7;13)(q34;q13) and performed the sperm karyo-

type analysis of one carrier by tri-color FISH.Holoprosencephaly (HPE), cyclopia and the unba-lanced karyotypes in the form of monosomy7q34!qter and trisomy13q13!qter were detectedamong stillborn/early death malformed newbornsgiving base to phenotype–karyotype correlation anddata concerning the survival rate. The risk assess-ment for unfavorable pregnancy outcomes obtainedfrom a relatively large pedigree and compared tomeiotic pattern of segregation of the carrier of thesame translocation may be useful to illustratethe natural selection of unbalanced fetuses and canbe proposed for the genetic counseling of familieswith similar RCT.

MATERIALS AND METHODS

Patients

A 26-year-old woman (III;6) was karyotypedbecause of one stillbirth (IV;6) and two earlymiscarriages (IV;7,8) (Fig. 1c). A reciprocal chromo-somal translocation t(7;13)(q34;q13) was found(Fig. 1a). After identification of this translocationshe had three further pregnancies, two of themresulted in a malformed female (IV;9) and male(IV;11); newborns died on the first day of their livesand one miscarriage occured (IV;10). The firstnewborn (IV;9) was delivered at term with birth-weight of 2,500 g. She was cyanotic with features ofholoprosencephaly, proboscis, cleft palate, low-set,small ears, and bilateral clubfoot. Chromosomeanalysis revealed elongated long arm of chromo-some7 andunbalancedkaryotypewas interpreted asmonosomy 7q34!qter and trisomy 13q13!qter.Shedied during first day of her life.Histopathologicalinvestigation was not performed. The second boy(IV;11) was delivered at 39th week of pregnancy andsimilar malformations have been observed. He diedat 15 min after delivery. Karyotype examination wasnot performed. The family history was followed andfurther progeny with malformations and miscar-riages in the relatives was observed. In the family of arelative (III;15), the first boy (IV;18) was born at 38thweek of pregnancy by Cesarean with birth weight2,550 g. He died after 15 min. Cyclopia, hydroce-phalus internus and externus, displastic kidneys, pesvalgus were noticed during autopsy. A second child,a girl (IV;19), was born at 38th week of pregnancy byCesarean with birth weight 2,100 g and height 48 cm.She was cyanotic and hypotonic, and had a 1 minApgar. She died during the first hour because ofsevere respiratory distress. Phenotype (Fig. 2a)evaluation revealed macrocephaly, wide-open fon-tanelles, broad cranial sutures, face of triangularshape, cyclopia, proboscis, anirhinia, narrow palate,prominent mandible, dysplastic, low ears, transver-sal line on the palms bilaterally, omphalocele, pesvalgus, lack of right kidney. Karyotype analysis

246 MIDRO ET AL.

American Journal of Medical Genetics: DOI 10.1002/ajmg.a

FIG. 1. Risk estimation for genetic counseling of the t(7;13)(q34;q13) carrier family. a: Cytogenetic results Left: partial karyotype demonstrating the breakpointposition localisation on chromosomes involved in translocation studied by GTG (upper line) and RBG (lower line) methods Right: schematic representation of thebreakpoint positions according to ISCN b: scheme of meiotic quadrivalent with visualisation of predicted form of imbalance compatible with survival from 3:1segregation (arrows) c: investigated pedigree with indication of ascertainment Legends: –Unkaryotyped malformed stillbirth/early death newborn; –Carrier of translocation; –Unbalanced malformed newborn; –Miscarriage; –Normal karyotype; d: Risk assessment for genetically unbalanced offspringmiscarriages and stillbirth/early died newborn by direct segregation analysis of pedigree and indirect method by Stengel-Rutkowski et al. [1988] (St-R).


performed during prenatal diagnosis in the2nd trimester revealed unbalanced translocation46,XX,der(7)t(7;13)(q34;q13)pat (Fig. 2b) as a resultof family reciprocal translocation t(7;13)(q34;q13)(Fig. 1a) after adjacent-1 and 2:2 segregation ofpaternal chromosomes.

Cytogenetic Examinations

Cytogenetic studies were performed by classicGTG and RBG banding methods with resolution 550bands/haploid set. Breakpoint position identifica-tion was performed according to ISCN [1995].

Family History (Fig. 1c)

Pedigree was constructed and supplementedduring long-term observation. Finally, 45 relativeswith 16 different pregnancy outcomes were identi-fied. Carriership of balanced RCT has been found bycytogenetic examinations in proband (III;6) and hersiblings (III;8,15), her mother (II;3) and maternalsiblings (II;2,5,8). Attention should be paid to thethree previous pregnancies that ended in twospontaneous miscarriages (IV;7,8) and unkaryo-typed stillbirth at 6 months with proboscis andholoprosencephaly (IV;6), other miscarriage (IV;10),one stillbirth (IV;11), andearly deathnewborn (IV;9).Early deaths of newborns were detected also infurther family members (II;1), (III;3,12,19,25) aswell as miscarriages (III,13,14). Karyotype of theproband’s early deceased sibling has not beenexamined. The unbalanced translocation in theform of monosomy 7q34!qter together withtrisomy13q13!qter have been detected in twoearly deceased offsprings (IV;9,19).

Risk Assessment

Probability rates for unfavorable pregnancy out-comes have been calculated on the basis of thedirect pedigree analysis according to Stene andStengel-Rutkowski [1988] and Stengel-Rutkowskiet al. [1988]. All types of abnormal pregnancies,which remained after ascertainment correction, havebeen counted and compared with the total numberof pregnancies. Three types of pregnancy outcomeshave been considered: genetically unbalanced childat birth, unkaryotyped stillbirth or early death andmiscarriage. Ascertainment correction has been per-formed by eliminating index cases. The probabilityrate for unbalanced liveborn progeny could not beestimated directly from the pedigree (as any unba-lanced progeny was observed as compatible withsurvival in this pedigree) but indirectly from thecollected empirical data of Stengel-Rutkowski et al.[1988].

The probability rate estimates for unfavorablepregnancieswere calculated according to the follow-ing formula:

p ¼ a

nS ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiaðn � aÞ

n3

r

where p, risk value; a, number of unfavorablepregnancies after ascertainment correction; n, num-ber of all pregnancies after ascertainment correction;and S, standard deviation.

Semen Collection and Preparation

Semen of healthy 36-year-old man (III;15)(Fig. 1c) with normal phenotype and a constitutional

FIG. 2. a: Phenotype of early died newborn (IV;19) born at term with holoprosencephaly, andproboscis.b: Partial karyotype showing unbalanced karyotype, der(7)is seen giving monosomy 7q34!qter and trisomy 13q13! qter, GTG banding technique. [Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]

248 MIDRO ET AL.


karyotype 46,XY,t(7;13)(q34;q13) was collected bymasturbation after 5 days of sexual abstinence.Seminological analysis was performed accord-ing to the standard WHO criteria [World HealthOrganization, 1992] where normal parameters wereconsidered as >20� 106 sperm/ml of ejaculate, 50%of sperm with progressive motility (AþB category)and >30% sperm with normal morphology.The result was contained within these normalparameters.

The aliquots of fresh sperm after liquefaction atroom temperature were washed three times in BWWmedium [Biggers et al., 1971] and then fixed withmethanol: acetic acid (3:1) for 20 min at�208C. Aftertwo rinses with fresh fixative, the sperm pellets weredropped onto the slides, air-dried and stored at�208C until used. The semen slides were allowed tostay at room temperature and sperm nucleardecondensation was obtained by plunging the slidesinto a solution of 25 mM dithiothreitol (DTT; Sigma)in a 0.1 M Tris-HCl (pH 8.5) for 10 min at 438C. Theslides were rinsed in 2� sodium chloride/sodiumcitrate (2� SCC, pH 7.0), then dehydrated in ethanoldilutions from 70% to 100% and air-dried.

DNA Probes and FISH Procedure

Four directly labeled probes were used for the FISHstudy (CYTOCELL, England). Two centromericprobes of the chromosome 7 (LPE 007 c, locusD7Z1): 7c Green and 7c Red were used and theircombination produced a yellow color. We also usedtwo telomeric probes: green for the chromosome 7q(Tel 7q, 40 kb) and red for the chromosome 13q (Tel13q, 100 kb). A three-color FISH was performedcombining mixed green-labeled and red-labeled 7c(yellow signal), red 13q and green 7q. The FISH wasperformed according to the manufacturer’s instruc-tion. The hybridization mixture (2.5 ml of each probe,10 ml of hybridization buffer) was applied to eachslide, covered with 20� 40 mm coverslip and sealedwith rubber cement. The probes were denatured for2min at 758C.Hybridizationwas carriedout overnightin a moist chamber at 378C. After hybridization, theslides were washed for 3 min in the solution of 0.4�SCC at 738C and then for 30 sec in the solution of 2�SCC/0.5% Tween 20. Counterstaining was performedwithDAPI.Hybridizationsignalswereobservedusingthe Olympus Bx40 microscope fitted with a triple-band-pass filter for DAPI/FITC/Rhodamine.

Parallel to the sperm of t(7;13)(q34;13) carrier, thecontrol FISH (the same experimental conditions andthe same probes) were performed on sperm from afertile donor with normal karyotype. Only spermwith attached tail were scored. The efficiency ofhybridization both in case of the patient and thedonor was at least 98%. In the donor’s case, theefficiency of hybridization was calculated countingthe number of hybridized spermatozoa with three

signals (yellow, red, and green) in the randomlyscored 500 spermatozoa. Two signals of the samecolor were counted as such only if they wereseparated by at least one domain. A total of 5,000sperm nuclei were scored with the aim to analyze thesegregation pattern of the proband (Fig. 3).

The segregation patterns were determined by thepresence and/or absence of the FISH signalscorresponding to chromosomes 7, 13, der(7) andder(13) as depicted in Table I.

RESULTS

FISH Analysis of Sperm

The frequencies of different types of spermsegregation t(7;13)(q34;q13) carriers are listed inTable I. All possible 2:2 segregations were observed.Thirty-four percent of the analyzed sperm resultedfrom an alternate segregation, and this was the mostcommon segregation type. The tri-color FISH did notallow us to distinguish normal and balanced sperm,and we cannot provide data on the ratio of these twopatterns. Adjacent-1 segregants had a frequency of23.5%. Adjacent-2 segregants (without recombina-tion in the interstitial segments) were less common(7.2%) and had unequal rates between both typesof unbalanced segregants. 1.5% of sperm werethe result of recombination within the interstitialsegments or, alternatively, of nondisjunction atanaphases II (the observed FISH signals wereidentical in both events).

Much more spermatozoa (29.4%) were generatedby a 3:1 segregation (13.4%of the tertiary segregationand 16.0% of the interchange segregation). Allpossible phenotypes were observed but unequalrates for sperm with 22 or 24 chromosomes weredetected. The frequency of sperm with 22 chromo-somes (19.2%) was higher than the frequency ofsperm with 24 chromosomes (10.2%). The percen-tage of sperm with 24,-7,þder7,þder13 (4.9%) washighest among the sperm with 24 chromosomes.

In 2.0% of spermatozoa no signals were present.The lack of signals could probably be explained by ahybridization failure.

Analysis of Meiotic SegregationPattern in Pedigree

Among 46 family members in a constructed pedi-gree, eight carriers of balanced reciprocal trans-location t(7;13)(q34;q13) (one carrier’s status ofthe relative, that is, I,1,2 was evident from pedigreeinformation and was not identified by karyo-type investigation) with 36 definite pregnancieswere found (Table II). In total, two unbalanced earlydeath offsprings were included, 10 unkaryotypedstillbirth/early death newborns and 6 spontaneous

RISK EVALUATION OF RCT CARRIERS 249


miscarriages among 36 pregnancies of 8 carrierswere found in this pedigree (Table II, Fig. 1c).

Ascertainment Correction

Three pregnancy outcomes (1 stillbirth/early deathand two miscarriages; (IV;6,7,8) belonging to indexsiblings were noticed. They were all omitted afterascertainment correction. In addition, carriers indirect line were omitted from the total number ofpregnancies (corrected) (Fig. 1c, Table II).

Risk Estimation (Fig. 1d)

Estimation by direct method. Finally, anyunbalanced liveborn out of total 31 pregnancies,nine stillbirth/early death (including oneunbalancedfetus observed at prenatal diagnosis and followed tothe birth), and 4 miscarriages out of 31 pregnancieswere accepted to make a risk estimate afterascertainment correction. Results of probability ratesfor stillbirth, early death of newborn and miscar-riages are presented on Table II. Probability of rate

forstillbirth/earlydeathwas29� 8.2%(9/31)(Table II,#10, rate (a) and for miscarriage �12.9� 6% (4/31)(Table II, #10, rate(b). No risk for unbalancedprogenyat birth was obtained (�/31).Estimation by the indirect method. The

analysis of the possible form of imbalance producedby meiotic malsegregation of parental chromosomesshowed that monosomy 7q34-qter, trisomy 7q34-qter, trisomy 13q13-qter, trisomy 13q13-cen-pter,and trisomy 13 have been recorded in empirical datafor single segment imbalance compatible withsurvival [Stengel-Rutkowski et al., 1988; Schinzel,2001]. Considering the risk for imbalance after 2:2segregation adjacent-1, we can see from directobservation within the family where monosomy7q34-qter together with trisomy 13q13-qter haslimited survival rate up to 38 weeks of pregnancyand no risk for MAT/PAT carriers are predicted. Inaddition, the very rare form of tertiary trisomy ofsegment 7q34-qter together with trisomy 13q13-cen-pter, as a double segment imbalance after 3:1segregation may arise. We presume the highersurvival rate for unbalanced progeny with trisomic

FIG. 3. Tetravalent figure at meiotic I pachytene of t(7;13)(q34;q13) reciprocal translocation carrier with marked position of tri-color FISH probes explaining thedisjunctional possibilities and derivative chromosome combinations. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

250 MIDRO ET AL.


form in comparison to monosomic of this samesegment. Comparing values obtained by Stengel-Rutkowski et al. [1988] we may find, that the value ofprobability assessment for single segment 7q34-qterwas below maximum value of 0.8% for maternalcarriers (and mean about 0.4%), and it was lower incomparison to the risk value for single segment13q13-cen-pter. Therefore the half of this value equalto 0.2% was calculated. No risk for paternal carrierswas observed on the basis of available data. Inaddition, the risk of having a child with Patausyndrome from an interchange trisomy 13 for femalecarriers is predicted as below 0.1% with no risk formale carriers. In conclusion, we propose a very smallcumulative risk of having viable offspring at birth for

female carriers (about 0.3% for two types ofunbalanced segregants) and no risk for male carriers(Fig. 1d).

DISCUSSION

We herein present the segregation pattern analysisof reciprocal translocation t(7;13)(q34;q13) in alarge four-generation family with eight carriers ofthis translocation (two paternal, five maternal, andone unknown sex) with a high incidence ofunfavorable pregnancy outcomes and with twounbalanced stillborn/newborn deaths included(Figs. 1 and 2). The study of the meiotic segregationpattern of the paternal t(7;13)(q34;q13) carrier

FIG. 3. (Continued )



showed that karyotypically unbalanced gametesproduced almost all theoretically possible forms ofimbalance (Table I). The results concerning 2:2segregation types were typical, since in most RCTcarriers alternate segregants are the most common

ones (34% in our case) and the frequency ofadjacent-1 segregants is higher than adjacent-2segregants (23.5% and 7.2%, respectively in ouranalysis) [Shi and Martin, 2001; Faraut et al., 2000,review]. The case of the t(7;13)(q34;q13) carrierexemplified that both reciprocal types of spermphenotypes that arise from adjacent-1 segregationwere found with a frequency similar to the expected1:1 ratio. However, the expected 1:1 ratio ofreciprocal adjacent-2 segregants was not observed,as 23,-7,þder13 was seen more frequently (4.8%)than 23,-13,þder7 (2.4%) (Table I). The otherauthors also noticed this distortion of adjacent-2segregants [Spriggs and Martin, 1994; Mercier et al.,1998; Estop et al., 1999], but it is difficult to explainthe significant excess of one sperm phenotype overthe complementary phenotype.

In the case of the presented t(7;13)(q34;q13)carrier, the high rate (29.4%) of 3:1 segregants wasfound (Table I). This type of segregant has beendescribed inonly few reports [Estopet al., 1995, 1999;Martini et al., 1998; Van Assche et al., 1999; Geneixet al., 2002]. The high rate of 3:1 segregants can beaccounted by the specificity of the particulartranslocation.

Although the models of meiotic segregation oftranslocations require that at 3:1 type, the gameteswith 22 or 24 chromosomes should be found in equalproportions for each phenotype of reciprocal segre-gants, this phenomenon was not documented. Thefrequency of 19.2% was derived from the sperm with22 chromosomes and 10.2% from the sperm with 24chromosomes (Table I). The other authors have alsoobserved similar unequal proportions [Estop et al.,1999; Geneix et al., 2002]. Estop et al. [1999]suggested, that the higher frequency of the spermwith 22 chromosomes may be due to an increasedability to survive a 3:1 segregation. Some authorspoint out the possibility of an overestimated fre-quency of sperm with 22 chromosomes because ofhybridization failure in sperm with 24 chromosomes[Armstrong et al., 2000].

A comparison of the risk determined by bothpedigree analysis and by FISH analysis of sperm hasbeen given sporadically [Cora et al., 2002]. In thethree generation family described by Trappeet al. [2002] with t(2;20)(p24.1;q13.1) no malformedchildren were born, but 7 of 12 pregnancies oftranslocation carriers resulted in spontaneousabortions prior to the 12th week of gestation.The abortions were not investigated cytogenetically.The authors concluded that in their case there was agood correlation of the risk determined by bothanalyses.

The clinical picture of the reported children withdouble segment imbalance in the form of monosomy7q34!qter together with trisomy 13q13!qterrevealed a set of different malformations withholoprosencephaly, cyclopia, and proboscis (Fig. 2).

TABLE I. Frequencies (%) of Different Types of Segregationand FISH Signals Corresponding to Segregation Sperm Products

of the t(7;13)(q34;q13) Carrier

252 MIDRO ET AL.


We considered a strong pathologic connectionbetween the observed malformations and theimbalance of their karyotypes. Holoprosencephaly(HPE) is a common developmental malformation ofthe human forebrain and midface. The prevalence isabout 1 to 11,000–20,000 live births and 1–250during embryogenesis. The etiology of HPE isextremely heterogeneous including both environ-mental factors (e.g., maternal diabetes) and geneticcauses. Approximately, 50% of HPE cases areassociated with a cytogenetic abnormality or amonogenic syndrome.At least 12 genetic loci containgenes implicated in the pathogenesis of HPE, such aslocus SHH at 7q36 and ZIC2 at 13q32 among others[Roessler et al., 1996; Brown et al., 1998; Nanni et al.,2001]. The form of HPE associated with chromoso-mal aberrations in the 7q36 region was designated asHPE type 3. A gene of Currarino triad disorder, withmidline embryonic defects, also maps to the same7q36 region. It has been suggested that haploinsuffi-ciency at locus 7q36 predisposes to both disorders[Lynch et al., 2000, a review]. The relevant literatureprovides many cases of HPE in patients withconfirmed loss of 7q34-q36 [Kleczkowska et al.,1990; Lurie et al., 1990; Benzacken et al., 1997;Nowaczyk et al., 2000]. The connection of HPE withtrisomy 13q as a consequence of family chromoso-mal translocation has been also described [Cohen,1989; Hatziioannou et al., 1991; Croen et al., 1996;Moog et al., 2001]. Cyclopia and cebocephaly wereconspicuous features in the cases of del(7q)[Schwartz et al., 1983; Schinzel, 2001]. Some casesof cyclopia have been also observed in associa-tion with trisomy 13. Cyclopia is a rare fetalmalformation characterized by a single palprebralfissure and a proboscis associated with severebrain malformation [Nowaczyk et al., 2000; Cannistraet al., 2001]. Approximately, 1.05–100,000 birthsincluding stillbirth are identified as cyclops. Cyclopiais a lethal syndrome. Cyclopia was also ob-served in our unbalanced children and, therefore,could be the crucial reason of stillbirth/new-

born death unbalanced progeny of t(7;13)(q34;q13)carriers.

A relatively large number of unfavorable preg-nancy outcomes observed in the analyzed pedigree(Fig. 1c) could reflect the high rate of fetusesresulting from genetically unbalanced gametes. Itcorresponds with findings from meiotic studies thatmajority (i.e., about 60%) of the analyzed spermphenotypes from t(7;13)(q34;q13) carrier showedunbalanced forms of gametes (Table I). Twoobserved unbalanced offsprings with double seg-ment imbalance in the form monosomy 7q34!qterand trisomy 13q13!qter could not survive the firstday of their lives, so we suggest that this formrepresents the limited survival rate through 32–38weeks of pregnancy. Additionally, the presence ofeight unkaryotyped early deceased children withsimilar malformations presumably have been con-nected with their unbalanced karyotype derivedfrom 2:2 segregation (monosomy 7q with trisomy13q) and/or from 3:1 segregation. It is known, thatsurvival rate of a complete interchange trisomy 13Patau syndrome is limited in utero and/or after birth[Gardner and Sutherland, 2004] and this form ofimbalance can be expected in stillborn/newborndeaths as well as in miscarriages (Fig. 1c). Interest-ingly, a relatively high rate (16.0%) of 3:1 interchangesegregation was observed in the meiotic examina-tion of sperm chromosomes (Table I). Taking intoaccount the high rate of unkaryotyped early death ofnewborns in the studied pedigree, we have beenunable to exclude that segregants from 3:1 can beinvolved in the origin of unbalanced karyotype. Inaddition, we can expect that in the presence of themeiotic quadrivalent with unequal sizes of translo-cated segments and with the participation of anacrocentric chromosome, a 3:1 segregation mayoccur preferentially [Jalbert et al., 1988; Faraut et al.,2000]. It is the case with quadrivalent in actuallyconsidered chromosome translocation (Fig. 1b).Some other examples illustrating this point can befound [Midro et al., 1992].

TABLE II. The Probability Rates for Unkaryotyped Pregnancy Outcomes (Miscarriages, Stillbirth/Early Death) of Maternal (MAT), Paternal (PAT),and Unknown Sex (?) RCT Carriers With t(7;13) in Relation to Total Pregnancies After Ascertainment Correction

No Carrier Sex

Stillbirth/early died newborn* Miscarriages** Pregnancies

Ratea RatebT C T C T C

1 III;6 MAT 3 2 3 1 6 3 2/3 1/32 III;8 MAT - - 1 1 4 4 -/4 1/43 III;15 PAT 2 2 - - 2 2 2/2 -/24 II;2 MAT 1 1 - - 3 3 1/3 -/35 II;3 MAT - - - - 3 2 -/2 -/26 II;5 PAT 1 1 2 2 5 5 1/5 2/57 II;8 MAT 2 2 - - 8 8 2/8 -/88 I;1,2 ? 1 1 - - 5 4 1/4 -/4Total 10 9 6 4 36 31 9/31 4/31RISK 29� 8.2% 12.9� 6%

*Probability rate for stillbirth/early died newborn.**Probability rate for miscarriage.



In the case of RCT carriers a genetic counselingshould be offered and probability rates should beestimated for all particular types of pathology(unbalanced progeny at birth and at prenataldiagnosis, unkaryotyped miscarriages, stillbirth/early death newborns). This can be done on thebasis of segregation analysis of pedigrees, if avail-able [Stengel-Rutkowski et al., 1988; Midro et al.,1992, 2000; Barisic et al., 1996; Stasiewicz-Jarockaet al., 2004]. To assess the probability rate of thepresence of unbalanced progeny, the informationon given combinations of chromosome segments inthe karyotype of the offspring of the individual RCTcarrier, and the information about the number ofnormal offspring should be obtained directly fromthe analysis of a relative large pedigree consisted ofseveral generations and many members. In spiteof the relatively great number of cytogeneticallyexamined relatives in the presented pedigree(Fig. 1c), the number of observations was not largeenough in order to give a risk value concerning thepresence of unbalanced progeny compatible withsurvival rate. There were no unbalanced viableoffspring at birth and risk figure should be describedas �/31 (no risk ?-not enough data) (Fig. 1d). It mostlikely reflects the observation of many imbalancesproduced in the couples with this translocation,which have been most always lethal in utero whilethe survival to the term, if at all, must have been theexception. It corresponds to the high probabilityrates (about 30%) for stillbirth/early newborn deathsand for miscarriages (about 13%) (Fig. 1d). Compar-ing the probability rate for stillbirth/early death tothe high rate of about 60% for unbalanced gametes(Table I), we can see that this rate is twice diminishedwhen comparing to our pedigree. It can representthe reduced survival rate for unbalanced progenyfrom the onset up until 38 weeks of pregnancy.

Using the indirect method for risk estimates fromthe compiled empirical data elaborated by Stengel-Rutkowski et al. [1988] (Fig. 1d-2.2), we can expectthe survival progeny with the particular rare combi-nation of double trisomy, that is, the trisomy 13q13-cen-pter together with the trisomy 7q34-qter after 3:1segregation (tertiary trisomy). This expectancy isbasedon theobservation that both areon recordwithviability in the single segment state, namely withtrisomy of segment 7q34-qter [Vogel et al., 1973;Forabosco et al., 1999] and trisomy of the segment13q13-cen-pter [Tharapel et al., 1986]. Noteworthy,the double imbalance has not been described so farand a more severe clinical picture may result withregard to malformation, survival and growth thaneither of the two aberrations (the trisomy7q34-qterand the trisomy13q13-cen-pter) alone. We canpresume that survival rate is higher for this productof malsegregation than the observed in this familymonosomy 7q34!qter and trisomy 13q13!qterbecause it is generally known that the unbalanced

children with trisomic forms of the same segment areusually less affected in comparison to monosomicones [Schinzel, 2001].

The risk figure is predicted using the rule of thumb,that the risk will be half of the smaller out of the tworisk figures considering each segment separately andthis is proposed as being below 0.2% for femalecarriers (Fig. 1d-2.3). This risk figure is very small andeven this may be an overestimate. The risk of havinga child with Patau syndrome from an interchangetrisomy is also remarkably small and for femalecarriers is predicted as below 0.1%. In conclusion,we propose a very small cumulative risk of viableoffspring at birth for female carriers, about 0.3% fortwo types of unbalanced segregants and no risk formale carriers. However, the risk figures derived inthis way are speculative rather than definitive ones.To check this prediction we would have to followmore than a thousand members of the pedigree inprospective study, which is practically impossible.

The limited lifespan of unbalanced progeny ofcarriers in the presented pedigree was the reasonthat information on fetal karyotypes was missingand the direct comparison of distribution of thesperm karyotypes of balanced translocation carryingmen with postnatal distribution of karyotypes wasalmost not possible, despite the relatively largepedigree data. However, the relative high numberof unfavorable pregnancy outcomes amongprogeny of t(7;13)(q34;q13) carriers in observedpedigree together with the high rate of productionof unbalanced gametes with a particular patternof increased proportion of spermatozoa at 3:1segregation suggest indirectly the relationshipbetween both observations, making them useful ingenetic counseling.

As a probability rate for viable unbalanced childis low, an option for pre-implantation geneticdiagnosis should be discussed with parents as thesolution to avoid the frequently expected unfavor-able pregnancy outcome. However, independentlyof the risk values to give birth to a malformed child,the parental decisions concerning procreation, pre-implantation chromosome diagnosis, prenatal diag-nosis or acceptance of a possible handicapped childmay be very different in each case.

ACKNOWLEDGMENTS

We thank Prof. Dr. Sabine Stengel-Rutkowski fromthe Munchen University, Germany for providing theopportunity to use her knowledge about themethodology of RCT risk. We thank our colleagues,Dr. Beata Stasiewicz-Jarocka and mgr Anna Sawickafrom Cytogenetic Laboratory of Department ofClinical Genetics of Medical University Białystok forexcellent technical assistance, Dr. Krystyna Debekfrom Department of Neonatology for clinical evalua-tion case IV, 19 and all families for kind co-operation.

254 MIDRO ET AL.


This work was supported by Polish grant of MedicalUniversity Białystok. Grant number: AMB No 4-06780 and 4-06 760, and statuary of Institute of HumanGenetics, Pol. Acad. Sci., Poznan.

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