doi:10.1086/513907


Letters to the Editor

Am. J. Hum. Genet. 61:228 – 231, 1997

Inter- and Intrachromosomal Rearrangements Are priate informed consent, from the patient, both parents,
and paternal grandparents. DNA was isolated by use ofBoth Involved in the Origin of 15q11-q13 Deletions

in Prader-Willi Syndrome a QIAamp blood kit (Qiagen). We employed markers
D15S541 and D15S542, both mapping to YAC

To the Editor:
A124A3, proximal to the deletion region, and markers

Prader-Willi syndrome (PWS) is due to an interstitial de D15S165 and D15S1048, both mapping distal to the
novo deletion at 15q11-q13 in Ç70% of cases. The common deletion region (Hudson et al. 1995). In addi-
deletion spans a region of Ç4 Mb and invariably in- tion, marker ATC3C11, mapping õ1 Mb from the dis-
volves the paternally derived homologue (Robinson et tal deletion breakpoint (S. L. Christian and D. H. Led-
al. 1991). For most patients the distal breakpoint ap- better, unpublished data), was used. PCR assays were
pears to be located within a single YAC (Kuwano et al. performed as described elsewhere (Christian et al. 1995;
1992), whereas two consistent breakpoint hot spots Hudson et al. 1995). The results of the microsatellite
have been identified on the proximal side; one (class I) analysis are shown in table 1, and examples of the analy-
lies in the region between the centromere and D15S541/ sis performed on two independent families are shown
D15S542, and the other (class II) lies between in figure 1.
D15S541/D15S542 and D15S543, with each account- Three patients were deleted for the D15S541/D15S542
ing for approximately half of the deletions (Christian markers, thus being classified as class I – deletion patients.
et al. 1995). This finding was consistent with the known frequency

The relatively high frequency of deletions, significant of the class I breakpoints among PWS patients. The lack
clustering of the breakpoints in PWS deletion patients, of a proximal marker on the deleted allele in these pa-
and the finding of a similar location for the breakpoints tients precluded assessment of the haplotype for the re-
in small inv dup(15) has led to the hypothesis that small gion comprising the deletion. Of the seven class II pa-
regions in proximal 15q may contain sequences leading tients, five demonstrated a paternal recombination event
to instability (Knoll et al. 1993; Huang et al. 1997). between the markers flanking the common deletion re-
Recently, preliminary data for a low-copy repeat associ- gion. The genetic distance between marker D15S541 and
ated with a novel evolutionarily conserved gene family marker D15S165 has been estimated as 17.2 cM in males
spanning the proximal and distal breakpoint regions has (Robinson and Lalande 1995). Marker D15S1048 maps
been reported (Ji et al. 1996). 2 cM proximal to D15S165 (Hudson et al. 1995). There-

In order to analyze the mechanism underlying dele- fore, when the genetic distance between D15S541 and
tions in PWS, we genotyped 10 three-generation families D15S165 in male meiosis is taken into account, the iden-
of PWS-deletion patients, using microsatellite markers tification, in five of seven cases, of a different grandparen-
flanking the common deletion region. Each patient was tal origin for the alleles flanking the deletion is signifi-
known to be deleted for the interval from D15S11 to cantly different from the expected frequency (x2

GABRB3, by FISH and/or other molecular techniques. Å 12.438, P Å .0004). This finding is highly suggestive
of an unequal crossover occurring in the paternal meio-Peripheral blood samples were obtained, with appro-

228

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229Letters to the Editor

Table 1

Microsatellite Analysis of Chromosome 15 Markers in 7 PWS Families

PROXIMAL MARKERSa DISTAL MARKERSa

GRANDPARENTAL- GRANDPARENTAL-
ALLELE ALLELE MECHANISM OF

FAMILY D15S541 D15S542 INHERITANCE D15S1048 D15S165 INHERITANCE REARRANGEMENT

1 n.t. GF (ab), GM (cd), GM GF (be), GM (ad), n.t. GF Interchromosomal
F (ad), P(bd), F (bd), P (bc),
M (bc) M (bc)

2 n.t. GM (cd), F (bd), GF GM (ab), F (bd), GM (bb), F (ab), GM Interchromosomal
P (ab), M (aa) P (bc), M (bc) P (bb), M (ab)

3 n.t. GF (ab), GM (ac), GM GF (ac), GM (bc), GF (ac), GM (bc), GM Intrachromosomal
F (bc), P (cd), F (bc), P (bb), F (bc), P (bb),
M (bd) M (bb) M (bb)

4 n.t. GF (cd), GM (ab), GF GF (bd), GM (ad), GF (de), GM (ac), GM Interchromosomal
F (ad), P (bd), F (ad), P (ac), F (ad), P (ab),
M (bb) M (bc) M (bc)

5 n.t. GM (ab), F (bc), GF GM (aa), F (ac), GM (bb), F (bc), GF Intrachromosomal
P (cd), M (de) P (cc), M (bc) P (ac), M (ac)

6 GF (ad), GM (ce), GF (ab), GM (bb), GM GF (bd), GM (ce), GF (cd), GM (be), GF Interchromosomal
F (cd), P (bc), F (bb), P (bb), F (de), P (ad), F (bc), P (ac),
M (bb) M (bc) M (ab) M (ae)

7 n.t. GM (cd), F (bd), GM GM (bb), F (bc), GM (cd), F (cd), GF Interchromosomal
P (ad), M (ab) P (cc), M (ac) P (bc), M (ab)

a n.t. Å Not tested; GF Å grandfather; GM Å grandmother; F Å father; P Å patient; and M Å mother.

sis, at the breakpoint, as being the mechanism leading junction fragment suggests the occurrence of strand-ex-
change events mediated through the transposase activityto the deletion.

Asymmetrical exchanges between nonsister chroma- (Reiter et al. 1996). Duplications of 15q11-q13 have
been reported in only a few instances (reviewed in Clay-tids in meiosis I have previously been demonstrated in

humans and are the basis of a number of genetic dis- ton-Smith et al. 1993), and it is unclear whether any of
these represent the reciprocal event of deletion by un-eases. When the related sequences are part of tandemly

arrayed homologous genes, nonhomologous recombi- equal crossing-over. The paucity of duplication cases
compared with deletions of this region is interesting andnation may lead to the formation of chimeric genes,

such as the globin-chain variants in some hemoglobin- indicates either that duplications occur much less fre-
quently or that a milder phenotype causes them to beopathies (Weatherall et al. 1995) and the red-green

pigment genes involved in color-vision abnormalities ascertained much less often.
Conversely, our study also has shown that in two(Nathans et al. 1986). In other instances, the deletion/

duplication event may arise from the unequal recombi- PWS families the data were consistent with an intrachro-
mosomal mechanism being responsible for the deletion.nation between repetitive elements interspersed

throughout a genomic region. A misalignment between It cannot be ruled out that this observation might be due
to a classical crossover occurring, at meiosis I, betweenAlu-repetitive sequences has been demonstrated in du-

plications of the LDL-receptor gene (Lehrman et al. D15S541/D15S542 and D15S1048/D15S165, followed
or preceded by an unequal homologous recombination.1987) and the hypoxanthine phosphoribosyltransfer-

ase gene (Marcus et al. 1993). However, a recombination event occurring twice in such
a small interval would be unlikely. IntrachromosomalRecent studies have demonstrated the presence of two

copies of a large repetitive element (CMT1A-REP) rearrangements have been infrequently demonstrated as
a mechanism leading to human diseases. One exampleflanking the region duplicated in Charcot-Marie-Tooth

disease type IA (CMT1A) patients and deleted in pa- is the intrachromosomal recombination occurring at
Xq28, between gene A, a small intronless gene withintients with hereditary neuropathy with liability to pres-

sure palsies (HNPP), at 17p11 (Pentao et al 1992; intron 22 of the factor VIII gene, and one of the two
copies of gene A located on the same chromosome, 500Chance et al. 1994). A model of unequal crossing-over

between misaligned CMT1A-REP homologues has been kb telomeric to the factor VIII gene, a recombination
causing severe hemophilia (Lakich et al. 1993). Molecu-proposed for the generation of both the CMT1A dupli-

cation and the HNPP deletion. Interestingly, the pres- lar studies have demonstrated that this rearrangement
arises almost exclusively in male meioses (Rossiter et al.ence of a mariner transposon – like element at the

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230 Letters to the Editor

ROMEO CARROZZO,1,2,4 ELENA ROSSI,2

SUSAN L. CHRISTIAN,10 KIRK KITTIKAMRON,10

CHIARA LIVIERI,5 ANDREA CORRIAS,7

LUCIA PUCCI,8 ALBERTO FOIS,8 PAOLO SIMI,9

LAURA BOSIO,3 LUCIANO BECCARIA,3

ORSETTA ZUFFARDI,2,6 AND DAVID H. LEDBETTER10
1Servizio di Genetica Medica, 2Laboratorio di
Citogenetica, 3Clinica Pediatrica III, Universita degli
Studi Milano, Ospedale San Raffaele, and 4Telethon
Institute of Genetics and Medicine (TIGEM), San
Raffaele Biomedical Science Park, Milan; 5Clinica
Pediatrica, 6Biologia Generale e Genetica Medica,
Universita degli Studi, Pavia; 7Divisione di
Endocrinologia Pediatrica, Ospedale Infantile Regina
Margherita, Turin; 8Clinica Pediatrica Universita degli
Studi, Siena; 9U.O. Azienda Ospedaliera Pisana, Pisa;

Figure 1 Representative microsatellite analysis of two families, and 10Center for Medical Genetics, The University of
illustrating an interchromosomal deletion event and an intrachromo- Chicago, Chicago
somal deletion event. Data for one proximal and one distal marker
are presented. In each example an arrow indicates the inheritance of
the patient’s paternal chromosome that bears the PWS deletion. A,

AcknowledgmentsInterchromosomal mechanism is inferred where the proximal marker,
D15S542, shows grandpaternal inheritance whereas the distal marker, We wish to thank Drs. G. Casari and A. Ballabio for helpful
ATC3C11, shows grandmaternal inheritance of the deleted chromo-

discussion, and we wish to thank Nehal Bhatt for assistance
some. B, Intrachromosomal mechanism, demonstrated by the fact that

in preparation of the figures.both the proximal marker, D15S542, and the distal marker, D15S165,
show grandmaternal inheritance of the deleted chromosome. GF
Å grandfather; GM Å grandmother; F Å father; P Å patient; and M

ReferencesÅ mother.
Chance PF, Abbas N, Lensch MW, Pentao L, Roa BB, Patel

PI, Lupski JR (1994) Two autosomal dominant neuropa-
thies result from reciprocal DNA duplication/deletion of a1994). The in-cis mechanism leading to the deletions in
region on chromosome 17. Hum Mol Genet 3:223 – 228PWS patients can be related either to an exchange of

Christian SL, Robinson WP, Huang B, Mutirangura A, Linechromosomal material between sister chromatids or to
MR, Nakao M, Surti U, et al (1995) Molecular characteriza-the formation of an intrachromosomal loop, either dur-
tion of two proximal deletion breakpoint regions in bothing meiosis or as a somatic event, followed by an exci-
Prader-Willi and Angelman syndrome patients. Am J Humsion of the chromosomal material lying between the re-
Genet 57:40 – 48

combining regions.
Clayton-Smith J, Webb T, Cheng XJ, Pembrey ME, Malcolm

The overall findings of this study are similar to S (1993) Duplication of chromosome 15 in the region
those of Dutly and Schinzel (1996) for Williams syn- 15q11-13 in a patient with developmental delay and ataxia
drome; this latter syndrome is due to a 500-kb inter- with similarities to Angelman syndrome. J Med Genet 30:
stitial deletion at 7q11.23. Segregation analysis of 529 – 531
grandparental markers flanking the deleted region in Dutly F, Schinzel A (1996) Unequal interchromosomal rear-

rangements may result in elastin gene deletions causing the15 patients and their parents demonstrated a recom-
Williams-Beuren syndrome. Hum Mol Genet 5:1893 – 1898bination between grandmaternal and grandpaternal

Huang B, Crolla JA, Christian SL, Wolf-Ledbetter ME, Machamarkers on chromosome 7, at the site of the deletion
ME, Papenhausen PN, Ledbetter DH (1997) Refined molec-in two of the three cases, whereas an intrachromoso-
ular characterization of the breakpoints in small inv dup(15)mal recombination appeared to have occurred in the
chromosomes. Hum Genet 99:11 – 17remaining cases.

Hudson TJ, Stein LD, Gerety SS, Ma J, Castle AB, Silva J,
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Slonim DK, et al (1995) An STS-based map of the human
leading to PWS syndrome appears, therefore, to be due

genome. Science 270:1945 – 1954
to both inter- and intrachromosomal rearrangements. Ji Y, Buiting K, Walkowicz MJ, Johnson DK, Amos-Langraf
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Knoll JHM, Wagstaff J, Lalande M (1993) Cytogenetic andsimilarly responsible for that disorder.

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231Letters to the Editor

molecular studies in the Prader-Willi and Angelman syn- usually first evident in the pelvic girdle and then spread-
drome: an overview. Am J Med Genet 46:2 – 6 ing to the upper limbs while sparing facial muscles. On-

Kuwano A, Mutirangura A, Dittrich B, Buiting K, Horsthemke set of symptoms is variable (mean age 9 years), and
B, Saitoh S, Niikawa N, et al (1992) Molecular dissection creatine kinase (CK) levels are elevated from early in-
of the Prader-Willi/Angelman syndrome region (15q11-13) fancy and remain elevated until the individual is well
by YAC cloning and FISH analysis. Hum Mol Genet 1:417 –

past this age (Jackson and Strehler 1968). Affected indi-
425

viduals are often wheelchair-bound 20 – 30 years afterLakich D, Kazazian HH, Antonarakis S, Gitschier J (1993)
the onset of symptoms. There is variability in the age ofInversions disrupting the factor VIII gene are a common
death, and most individuals die in middle age.cause of severe haemophilia A. Nat Genet 5:236 – 241

The gene for LGMD2A was first linked to chromo-Lehrman MA, Goldstein JL, Russel DW, Brown MS (1987)
Duplication of seven exons in LDL receptor gene caused by some 15 by Beckmann et al. (1991). Allamand et al.
Alu-Alu recombinatiion in a subject with familial hypercho- (1995) narrowed the region to 15q15.1-q15.3, using
lesterolemia. Cell 48:827 – 835 large kindreds from the Isle of La Réunion and the

Marcus S, Hellegren D, Lambert B, Fallstrom SP, Wahlstrom northern Indiana Amish. The muscle-specific calcium-
J (1993) Duplication in the hypoxanthine phosphoribosyl- activated neutral protease 3 or calpain 3 (CANP3) gene,
transferase gene caused by Alu-Alu recombination in a pa- a possible candidate gene in the 15q15.1-q15.3 region,
tient with Lesch Nyhan syndrome. Hum Genet 90:477 – 482

was examined by Richard et al. (1995). Fifteen different
Nathans J, Piantanida TP, Eddy RL, Shows TB, Hogness DS

mutations, including missense, splice-site, frameshift,(1986) Molecular genetics of inherited variation in human
and nonsense mutations, were identified in LGMD2Acolor vision. Science 232:203 – 210
patients, and many others have subsequently been iden-Pentao L, Wise CA, Chinault AC, Patel PI, Lupski JR (1992)
tified. Since the affected patients in La Réunion belongCharcot-Marie-Tooth type 1A duplication appears to arise

from recombination at repeat sequences flanking the 1.5 Mb to a genetic isolate presumed to derive from a single
monomer unit. Nat Genet 2:292 – 300 ancestor who immigrated to the island during the late

Reiter LT, Murakami T, Koeuth T, Pentao L, Muzny D, Gibbs 1670s, it was expected that all affected patients from
RA, Lupski JR (1996) A recombination hotspot responsible La Réunion would have the same LGMD2A mutation.
for two inherited peripheral neuropathies is located near a Paradoxically, six different mutations were identified.
mariner transposon-like element. Nat Genet 12:288 – 297 This paradox led the investigators to propose digenic

Robinson WP, Bottani A, Yagang X, Balakrishnan J, Binkert
inheritance, in which the founder effect is due to an

F, Mächler M, Prader A, et al (1991) Molecular, cytogenetic,
as-yet-unidentified modulating gene (either nuclear orand clinical investigation of Prader-Willi syndrome patients.
mitochondrial) that permits mutations in CANP3 to ex-Am J Hum Genet 49:1219 – 1234
press LGMD2A. This hypothesis does not require theRobinson WP, Lalande M (1995) Sex-specific meiotic recom-
presence of multiple mutations, since the genetic princi-bination in the Prader-Willi/Angelman syndrome imprinted

region. Hum Mol Genet 4:801 – 806 ples of digenic inheritance should apply to all popula-
Rossiter JP, Young M, Kimberland ML, Hutter P, Ketterling tions with LGMD caused by calpain-3 mutations.

RP, Gitschier J, Horst J, et al (1994) Factor VIII gene inver- In the Amish of northern Indiana, Richard et al.
sions causing severe hemophilia A originate almost exclu- (1995) identified a single mutation in CANP3
sively in male germ cells. Hum Mol Genet 3:1035 – 1039 (CGGrCAG, R769Q) in a homozygous state in affected

Weatherall DJ, Clegg JB, Higgs DR, Wood WG (1995) The patients. The authors speculated that the complete pene-
hemoglobinopathies. In: Scriver CR, Beaudet AL, Sly WS,

trance of this disease in the Amish and in the other
Valle D (eds) The metabolic and molecular bases of inherited

LGMD2A pedigrees might also be under the control of adisease, 7th ed. McGraw-Hill, New York, pp 3417 – 3484
second locus. One expectation of the digenic hypothesis

Address for correspondence and reprints: Dr. David H. Ledbetter, Center for would be that some individuals homozygous for the mu-
Medical Genetics, The University of Chicago, 924 East 57th Street, Room R110,

tation would be clinically unaffected (i.e., CK is normalChicago, IL 60637-1470. E-mail: dhl@genetics.uchicago.edu
� 1997 by The American Society of Human Genetics. All rights reserved. and there are no physical findings suggestive of LGMD).
0002-9297/97/6101-0030$02.00 Because of the possible implications in genetic testing

and counseling, we analyzed 580 DNA samples from
Amish individuals in one northern Indiana county forAm. J. Hum. Genet. 61:231 – 233, 1997
the presence of the R769Q mutation, looking for evi-

DNA Studies of Limb-Girdle Muscular Dystrophy dence of phenotypically normal R769Q homozygotes.
Type 2A in the Amish Exclude a Modifying We initiated the countywide screen by first identifying
Mitochondrial Gene and Show No Evidence for a carrier couples. Appropriate informed consent was ob-
Modifying Nuclear Gene tained from all individuals. In order to identify R769Q
To the Editor: carriers in this population, we specifically approached

members of 16 previously studied nuclear LGMD2ALimb-girdle muscular dystrophy type 2A (LGMD2A) is
characterized by slowly progressive muscle weakness, families from this county. We obtained blood samples

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