Constantinou M. et al.
Trisomy of medial 15q
detected by CGH and FISH.
as
result
of
analphoid
supernumerary
ring
chromosome
Constantinou Maria, Płowaś Iwona, Kałużewski Bogdan
Department of Medical Genetics, Medical University of Łódź
91-425 Łódź, No 3 Sterling Street, Poland
Tel/fax: +48(042) 630-70-02, e-mail: majcon@kardio-sterling.lodz.pl
Introduction.
Extra Structurally Abnormal Chromosomes (ESACs) are reported in 0.05% of newborn infants (1). The risk of phenotypic
abnormalities is estimated at 28% for non-acrocentric autosomal and 7% for acrocentric autosomal ESACs (2, 3). There is a
small group of ESACs without detectable α-satellite DNA, first described in 1993. This type of ESACs is comprised of a
region of chromatin that can act as a functional centromere and they are termed Neocentric Marker Chromosomes - NMCs.
Fifty cases of NMCs have been reported in the literature, most of which were isochromosomes or inverted duplications (inv
dup) of autosomal origin.
Clinical history.
Our patient was born at 42 weeks of gestation after an uncomplicated pregnancy to a 27-year-old mother and a 29-year-old
father. The mother had suffered two miscarriages before. The patient was 3.550 kg (<3rd centile) at birth, 53 cm long (<3rd
centile). The head circumference and chest circumference were below the 3rd centile (34 and 34 cm respectively). No
dysmorphic features were reported. At 1 year of age the patient's height was 76 cm (>25 centile), weight 9.350kg (>25
centile) and at age 6 years, these parameters reached 125 cm (>90 centile) and 29 kg (>95 centile) respectively. Psychomotor
development was appropriate for his age. He walked at 12 months and started talking at the age of 2. At age 10 he gained
weight to 53 kg (between the 90th and 95th centile) and reached the height of 160 cm (>97th centile). At 17 years of age he
was diagnosed with obesity (weight of 69 kg, height of 183 cm), BMI reached 28.9 (>97th centile) and mass to height index
was above the 97th centile. In addition, the findings in this patient included striae distensae in the hypogastrium,
malocclusion, bilateral gynecomastia with scarce glandular tissue, symmetric and normal axillary and pubic hair, normal
testes. X-ray examination revealed short chest and bilateral shortening of the fourth metacarpal bone. Bone age matched
biological age. At present, the patient is 21 years old, 190 cm high and weighs 127 kg (BMI – 35.55). He is in employment,
having graduated from vocational school. Semen specimen analysis yielded normal results: total sperm count - 3·107, 60%
with forward movement, vitality (95% live).
Material and methods.
Fluorescence in Situ Hybridisation (FISH). BAC clones, which map to the regions 15q22.1-15q22.1 (RP11-353J8),
15q22.2 (RP11-30M4), 15q22.31 (RP11-448B5), 15q23 (RP11-221P9), 15q24.1-15q24.1 (RP11-500O23) and 15q24.2
(RP11-289H7) were supplied from BACPAC Resources Center (BPRC) at Children's Hospital Oakland Research Institute in
Oakland, California, in the United States (http://bacpac.chori.org/home.htm). DNA mini-preparations from BAC clones were
obtained using standard Qiagen-TIP method (BPRC, Qiagen). DNA was labelled with SpectrumOrange or SpectrumGreen by
nick translation (VYSIS). FISH using six BAC probes and ten commercially available probes, i.e. centromere probes: CEPYSG (DYZ3/CEPX-SO DXZ1), CEP13/21, CEP 14/22, CEP15 (VYSIS), Human Pancentromere Probe (Q-Biogene), painting
probes WCPY-SG, WCPX-SO, WCP15-SG (VYSIS) and LSI SNRPN-SO/PML-SO/CEP15-SG, LSI PML-SO/RARA-SG
(VYSIS), was performed according to the manufacturer’s instructions and the protocol, described previously (4).
Multiplex FISH. Probes for M-FISH analysis included 24-Xcyte probe kit purchased from MetaSystems. M-FISH was
carried out according to our protocol, described previously (5).
Comparative Genomic Hybridization (CGH). CGH was performed according to our protocol described previously (5).
Results.
Banding techniques established that the marker chromosome is an ring chromosome, without a C-band positive region
closely associated with the centromere of most chromosomes. FISH analysis with probes for X, Y, 13, 14, 21, and 22 were all
negative. CGH results revealed the enhancement of the long arm of chromosome 15, i.e. enh(15)(q22q23). The application of
M-FISH and FISH with the painting probe for chromosome 15 demonstrated that the marker is a ring chromosome composed
entirely of chromosome 15 material. FISH using locus-specific probes for the PML (15q22) confirmed the presence of this
locus at the marker. FISH with six BAC probes specific for 15q was used to identify the possible breakpoints of this marker.
Four BAC clones - RP11-30M4 (15q22.2), RP11-448B5 (15q22.31), RP11-221P9 (15q23) and RP11-500O23 (15q24.115q24.1) were localized at the ring [Fig. 4B, 5A]. However, RP11-353J8 (15q22.1-15q22.1) and RP11-289H7 (15q24.2)
clones were not detected at the marker. Finally, FISH with the Human Pancentromere Probe (Kreatech) showed that the
marker lacks the alphoid DNA sequence [Fig. 5B].
Discussion.
We present a case of a ring derived from chromosome 15, with the breakpoints localized at 15q22.2 and 15q24.1, distal to
RP11-30M4 clone and RP11-500O23 clone, respectively. In all reported cases with NMC(15) the characteristic phenotype
was resulting from tetrasomy of 15q26-qter. However, our patient had partial trisomy of 15q22.2-q24.1 and therefore should
more closely resemble the phenotype of cases with a medial duplication of 15q. 30 cases were described with partial trisomy
for region of 15q. The majority of cases had breakpoints within 15q21-q22. Almost 90% of described duplications of 15q
were the result of a familial translocation and therefore the partial trisomy of chromosome 15q was common features
characteristic for distal and medial trisomy of 15q included growth and mental retardation, hypotonia, and distinctive facial
features including facial asymmetry, puffy cheeks, a short neck, downward slanting, narrow, palpebral fissures, ptosis,
prominent nose with a broad nasal bridge, an abnormal philtrum, downturned mouth, high arched palate, midline crease in
1
Constantinou M. et al.
the lower lip, micrognathia, and microcephaly. Clinical observations suggest that significant developmental genes are located
within the region of trisomy of 15q21->q24.
Typical characteristics of trisomy 15q phenotype were absent in our patient. At birth, moderate growth retardation was
observed with the birth weight, height and head circumference below the 3rd centile. No dysmorphic features or
developmental delay were observed. The patient’s growth dynamics increased, rising above the 97th centile between the ages
of 10 and 17. Taking into account clinical data available, we propose that the region of 15q22.2-q24.1 may not be responsible
for characteristic trisomy 15q phenotype.
Low copy repeat sequences (LCR) are localized in many different centromere competent sites within the human genome,
which, by epigenetic modification, can be transformed into functional centromeres (neocentromeres). LCR sequences were
identified and mapped also in 15q22.2 and 15q24.1 bands – namely DNM1DN6 and DNM1DN7 respectively. The identified
breakpoints in the present case suggest the presence of low copy repeat - DNM1DN6 and DNM1DN7 sequences in the
marker, which may be responsible for the ring formation. Thus, we concluded that the kinetochore is localized in 15q24.1
band, rather than in 15q22.2. This assumption is based firstly on the clinically documented cases of analphoidal marker with
neocentromere, localized at 15q24, and secondly, DNM1DN7 sequences form a large duplicon at 15q24 (LCR15-2).
DNM1DN6 sequences, localized in 15q22.2 were, most probably, involved in the ring formation.
References:
1.
2.
3.
4.
5.
Crolla JA: FISH and molecular studies of autosomal supernumerary marker chromosomes excluding those derived from
chromosome 15: II. Review of the literature. Am J Med Genet 75(4):367-81 (1998).
Callen DF, Eyre H, Yip MY, Freemantle J, Haan EA: Molecular cytogenetic and clinical studies of 42 patients with marker
chromosomes. Am J Med Genet 43(4):709-15 (1992).
Plattner R, Heerema NA, Howard-Peebles PN, Miles JH, Soukup S, Palmer CG: Clinical findings in patients with marker
chromosomes identified by fluorescence in situ hybridization. Hum Genet 91(6):589-98 (1993).
Jakubowski L, Jeziorowska A, Constantinou M, Helszer Z, Baumstark A, Vogel W, Mikiewicz-Sygula D, Kaulzewski B: Xp;Yp
translocation inherited from the father in an SRY, RBM, and TSPY positive true hermaphrodite with oligozoospermia. J Med
Genet 37(10):E28 (2000).
Kaluzewski B, Constantinou M, Zajac E: Molecular cytogenetic techniques in detecting subtle chromosomal imbalances. J Appl
Genet 44 (4):539-46 (2003).
2