Hum Genet (1993) 91:389-391 human .. genetics

�9 Springer-Verlag 1993

Molecular cytogenetic characterization of three familial cases of satellited Y chromosomes

Tracy Ann Wilkinson, John Anthony Crolla

Wessex Regional Genetics Laboratory, Salisbury District Hospital, Odstock, Salisbury SP2 8B J, UK

Received: 20 November 1992 / Revised: 6 January 1993

Abstract. Three famil ies in which a satel l i ted Y chromo- some (Yqs) was segregat ing without apparent phenotypic effect were re- inves t iga ted with non- iso topic in situ hy- br id iza t ion methods . Act ive nucleolus organizer regions were seen in the distal long arm region of all Yqs chromo- somes s tudied and in situ hybr id iza t ion with the probe D15Z1 showed that, in all three famil ies , the Yqs was the result o f a 15p;Yq translocat ion. In one case, an addi t ional focus o f D15Z1 hybr id iza t ion was seen on 21p.

Introduction

A satel l i ted Y ch romosome (Yqs) was first descr ibed in 1967 in a pat ient with D o w n ' s syndrome (Genest et al. 1967), and since then a number o f other cases have been repor ted (Schmid et al. 1984; Chandley et al. 1989). Of these only one was de novo (Turleau et al. 1978), and no phenotyp ic effects were at t r ibuted to the abnormal Y chromosome. In some pedigrees , the Yqs has been t raced through a large number o f generat ions (Genest 1972, 1973).

A Yqs, therefore, is a rare structural variant that, f rom both morpho log ica l and convent iona l cy togenet ic staining criteria, is p resumed to be the product o f a t ranslocat ion be tween the short arm of an acrocentr ic ch romosome and a region within the he terochromat ic segment of Yq. How- ever, the ev idence for this assumpt ion has, to date, been largely c i rcumstant ia l ( rev iewed in Schmid et al. 1984). We present three famil ia l cases o f Yqs in whcih in situ hy- br id iza t ion has been used to demonst ra te that all the Yqs s tudied arose f rom a t rans locat ion be tween the hetero- chromat ic regions of 15p and Yq.

Materials and methods

Case A

The proband was referred at the age of 4 years with features sug- gestive of Soto's syndrome. Conventional cytogenetic studies

Correspondence to: T. A.Wilkinson

showed a normal karyotype except for a Yqs that had been inher- ited from his father (Fig. 1A).

Case B

This family was ascertained when the proband was referred at the age of 8 months with growth retardation, low set ears and a poste- rior cleft palate. His chromosomes were normal except for a Yqs that was also found in his father and grandfather (Fig. 1B).

Case C

The proband was found to have a Yqs following an amniocentesis on his 35-year-old mother, referred for prenatal diagnosis with a serum increased risk of 1 : 120. The proband's father also carried the Yqs and the pregnancy is continuing to term (Fig. 1C).

A I

7

B

III

7

C

it 7

Fig. 1. A-C Pedigrees of cases A, B and C showing the Yqs males, with arrows indicating the probands. Individual BH was seen to have an additional focus of 15p hybridization on the short arm of one chromosome 21 (see Fig. 2A, B)

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Fig.2A, B. Metaphase from case BII (A) before and (B) alter in situ hybridization with the classical satellite probe, D I5ZI. A The chromosomes were banded using standard GTG techniques. B The short arms of both 15s are clearly labelled, as are the short arms of one chromosome 21 and the distal long arm region of the satellited Y. Signals were detected using an immunocytochemical staining technique with diaminobenzidine precipitation at the foci of hy- bridization; chromosomes were counterstained in Giemsa. Slight cross-hybridization with the beterochromatin of chromosome 9 was sometimes seen at the stringency at which the experiments were carried out

Cvtogenetic and in situ hybridization methods

Metaphase chromosome preparations were obtained from periph- eral blood lympbocytes or amniotic fluid cells using standard tech- niques. Conventional cytogenetic analyses were carried out using GTG-banding (Seabright 1971 ), C-banding (Sumner 1972), silver (nucleolus organizer region; NOR) staining (Howell and Black 1980) and methyl green/DAPI (4,6-diamidiono-2-phenylinolole) (MG/DAPI) fluorescence (Donlon and Magenis 1983).

The D15ZI probe (Higgins et al. 1985) was biotinylated and in situ hybridization was performed overnight at 42~ The slides were washed at a stringency of 0.2xSSC (1 x SSC = 150 mM NaC1/ 15 mM sodium citrate, pH 7.0) in 50% formamide for 40 rain at 37~ alter which standard avidin-fluorescence or streptavidin- horseradish peroxidase/diaminobenzidine (HRP/DAB) detection protocols were followed (Pinkel et al. 1988; Crolla 1992). Chromo- somes were counterstained with propidium iodide or Giemsa and examined under a Zeiss axiophot microscope using either epi- fluorescence and Zeiss FITC filter set. no. 9 or direct light for HRP/ DAB-stained slides. Photographs were taken with Kodak Techni- cal Pan 245(/film.

Results

In all three pedigrees , the analysis of G T G - b a n d e d meta- phases showed satell i tes present on the long arm of the Y chromosome. In each case, s i lver staining demonst ra ted the presence of active NORs within the satel l i ted stalks of

the Yqs; C-banding showed the presence of const i tut ive heterochromat in in the long arm of all the Yqs studied, al- though the size of the C-banding var ied among the fami- lies. Al l the Yqs were Q-band-negat ive, including the satel- lites, but in all cases the satell i tes and satelli te stalks were M G / D A P I - p o s i t i v e .

It has been demonst ra ted that the probe D I 5 Z I (Hig- gins et al. 1985) and M G / D A P I h y b r i d i z e / f l u o r e s c e on the same region on 15p (Smeets et al. 1991). We therefore carried out in situ hybridizat ion with D I5ZI to test whether the M G / D A P I - p o s i t i v e satell i te stalks and satell i tes on the Yqs ch romosomes were compr i sed of 15p material . In all individuals tested, with one except ion (see below), the in situ hybr idizat ion pattern with D15ZI showed two bright f luorescent signals on 15p and an addit ional signal in the distal region of the Yqs. In the father of case B, however , in addi t ion to the three signals descr ibed above, a bright focus of 15p hybr idizat ion signal was also seen on the short arm of one ch romosome 21 (F ig .2A, B). This vari- ant 21p had not been inheri ted by his son (case B).

Discussion

All the Yqs in this study carr ied active NORs and were broadly s imilar in appearance, al though exhibi t ing inter- famil ial differences in both the amount of Yq heterochro- matin and the size o f the satell i te region. These differ- ences in turn probably reflect the inherent var iabi l i ty in the amount of Yq const i tut ive heterochromat in found in normal Y ch romosomes and also the highly po lymorph ic appearance of satell i tes in acrocentr ic chromosomes .

The Yq heterochromat in is complex and is compr i sed of a small p roximal region that is Q-band-nega t ive but C- and M G / D A P I - p o s i t i v e , and a larger distal region that is Q-, C-, and M G / D A P I - p o s i t i v e . It is noteworthy that, in all three famil ies , the Yq breakpoin t was in the proximal heterochromat in , or at the border of the proximal and dis-

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tal regions as, in all the Yqs chromosomes , the "dis tal por- t ion" o f the t rans located Y ch romosome was Q-band-neg- ative. A compar i son o f the Q-, C-, and DAPI- s t a in ing pat- terns in our famil ies with those observed in other pub- l ished Yqs pedigrees (Schmid et al. 1984) indicates that the Yq breakpoin t occurs in p rox imal Yql 2 in 7 cases and distal Yq l2 in 6 cases. The dis t r ibut ion of the breakpoin ts within the Yq he terochromat in appears, therefore, to be r andomly dis tr ibuted.

Of cases involv ing t ranslocat ions be tween a Y chromo- some and an autosome, 70% are be tween the Yq hetero- chromat in and the short arm of an acrocentr ic . In such t ranslocat ions, c h r o m o s o m e 15 is the most f requent ly ob- served (52%), fo l lowed by ch romosome 22 (33%), chro- m o s o m e 21 (7%) and ch romosomes 13 and 14 (4% each) (Smith et al. 1979). This non- random distr ibut ion of Yq/ acrocentr ic short arm t ranslocat ions p robab ly reflects the differ ing degree of h o m o l o g y be tween sub-sets I - I V o f satel l i te D N A ( rev iewed by Vogt 1990), with the c losest h o m o l o g y being shared be tween 15p, 22p and Yq. Fur- thermore, during the p rophase o f male meiosis , the XY bi- valent is f requent ly seen in c lose p rox imi ty to an NOR; Stahl et al. (1984) have sugges ted that this may be the rea- son for the relat ive f requency with which Yq/acrocentr ic short a rm t ranslocat ions are observed.

The molecu la r cy togenet ic use o f the probe D15Z1 has shown unequivoca l ly that the Yqs in our cases all arose f rom a t rans locat ion be tween 15p and distal Yq. In addi- tion, one indiv idual had a D15Z1 signal on one chromo- some 2 lp , s imi lar to the addi t ional signal on ch romosome 13 or 14 recent ly descr ibed by Smeets et al. (1991) and Stergianou et al. (1992). Our observat ion o f an addi t ional D15Z1 signal on 21p suggests that this phenomenon rep- resents an inheri ted po lymorph i sm.

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