Phenotypic and Molecular Characterization of Partial ...

Case Report Cytogenet Genome Res 2012;136:229–234 DOI: 10.1159/000336974

Accepted: January 12, 2012 by M. Schmid Published online: March 7, 2012

Phenotypic and Molecular Characterization of Partial Trisomy 2q Resulting from InsertionDuplication in Chromosome 18q: A Case Report and Review of Literature R. Ponnala P. Ranganath U.R. Dutta V.K. Pidugu A.B. Dalal

Key Words Chromosomal insertion-duplication ⴢ Trisomy 2q31.1–q37.3

Abstract Trisomy 2q is a well-documented chromosomal anomaly with considerable variation in the phenotype depending upon the breakpoints and the co-existing chromosomal aberrations. The case of a dysmorphic male infant found to have trisomy of the 2q31.1–q37.3 segment, resulting from insertion-duplication of this segment in chromosome 18q23 is reported here. The rearrangement was resolved in detail by cytogenetic microarray and whole chromosome paintbased fluorescence in situ hybridization studies. There is some overlap of the phenotypic features in the reported patient with those described in previously reported cases with partial trisomy 2q. A detailed review of the available literature on 2q trisomy has also been presented and delineation of the phenotypic characteristics common to all patients with 2q trisomy has been attempted. Copyright © 2012 S. Karger AG, Basel

Trisomy 2q is a well-documented chromosomal anomaly. Though the clinical manifestations of the reported cases of trisomy 2q as well as the breakpoints determined © 2012 S. Karger AG, Basel 1424–8581/12/1363–0229$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/cgr

by the different authors vary considerably, all of them share some common characteristic features. These include low birth weight, failure to thrive, developmental delay, severe to profound mental retardation, microcephaly, dysmorphic facial features, genitourinary anomalies, and hypotonia [Barnicoat et al., 1997; Matos et al., 1997; Seidahmed et al., 1999]. The availability of high-resolution molecular techniques such as array-based comparative genomic hybridization and single nucleotide polymorphism (SNP)-based microarray has provided an optimal approach to find copy number variations (CNVs) such as partial trisomy 2q and to identify the mechanisms involved [Barrett et al., 2004]. We report a patient with a de novo unbalanced interchromosomal insertion of 2q31.1–q37.3 into chromosome 18q resulting in partial trisomy 2q. This aberration was first identified as add(18q) using standard conventional cytogenetic methods (550-band level G-banded karyotype). The rearrangement was resolved in detail through cytogenetic microarray and whole chromosome paint (WCP)-based fluorescence in situ hybridization (FISH) studies. The parental origin of the duplicated chromosomal material was determined through microsatellite analysis. A comparison of the phenotypic characteristics of our patient with those of other trisomy 2q patients reported in literature is also presented. Ashwin B. Dalal Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics 4-1-714, Tuljaguda Complex, Mozamzahi Road, Nampally Hyderabad 500001, Andhra Pradesh (India) Tel. +91 40 24 749 335, E-Mail ashwindalal @ gmail.com

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Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

B

Fig. 1. A Clinical photograph of the reported child and B close-up of the lower half of his

face: microcephaly, low anterior hairline, low-set ears, depressed nasal bridge, long philtrum, thin upper lip, incomplete midline cleft of the soft tissue of the lower lip and overlapping fingers are seen. C Right foot of the affected child with hypoplasia of the 4th and 5th toes.

A C

Patient The propositus is a male child born to non-consanguineous parents. The family history was unremarkable. The baby was born at 36 weeks of gestation through a spontaneous vaginal delivery. There was no history of any adverse events in the antenatal or neonatal periods. The birth weight was 2.4 kg. Dysmorphic features were observed at birth. When the baby was referred for chromosomal analysis at the age of 2 months, he had not attained social smile. His examination revealed multiple dysmorphic features including microcephaly, large anterior fontanelle, open cranial sutures, low anterior hairline, depressed nasal bridge, long philtrum, thin upper lip, low-set ears, overlapping fingers and bilateral 5th-finger clinodactyly. Apart from these, he had 2 unusual additional findings: incomplete midline cleft of the soft tissue of the lower lip and hypoplasia of the 4th and 5th toes bilaterally (fig. 1). The external genitalia were normal male genitalia. Systemic examination was normal with normal findings in perabdominal and cardiovascular system examination and no evidence of any focal neurological deficits. The echocardiogram and abdominal ultrasonogram were normal. Methods Metaphase spreads were obtained from phytohaemagglutinin-stimulated peripheral lymphocytes using standard procedures and chromosomes were analyzed by GTG-banding techniques in the patient as well as both parents. Cytogenetic microarray was performed using the HumanCytoSNP-12 Beadchip (Illumina, San Diego, Calif.), which includes 300,000 markers (mostly SNPs and some non-polymorphic markers) targeting all regions of known cytogenetic importance and spanning the entire genome with a 6.2-kb median marker spacing and yield of ⬃30-kb resolution. Approximately 750 ng of genomic DNA sample of the patient was used for this study. Samples were processed according to the Infinium II assay manual. Briefly, each

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Cytogenet Genome Res 2012;136:229–234

sample was amplified, fragmented, precipitated, and re-suspended in an appropriate hybridization buffer. Denatured samples were hybridized on the HumanCytoSNP-12 BeadChip for a duration of 16 h at 48 ° C. After completion of the assay, the BeadChips were scanned with a 2-color confocal BeadArray reader. Image intensities were extracted and analyzed using Illumina’s KaryoStudio software. Copy number altered regions were detected using B-Allele frequency and Log R ratio. FISH study was done with WCP 2 probe (Kreatech Diagnostics, Amsterdam, The Netherlands). The parental origin of the duplicated chromosomal material was determined by microsatellite analysis using short tandem repeat polymorphism markers corresponding to the 2q31–q37 region.

Results

Standard cytogenetic analysis of the patient (550-band level karyotyping) showed an abnormal male karyotype with additional material of unknown origin on 18q: 46,XY,ins(18;?) [ISCN 2009] (fig. 2). Both parents were found to have normal karyotypes. Cytogenetic microarray study revealed the extra material on 18q to be a duplication of chromosome 2q31.1–q37.3 (from 169,519,470– 242,710,266 bp) (fig. 3). The microarray study also confirmed that this duplication was not associated with further gains or losses at the site of insertion on 18q or elsewhere in the genome. The size of the duplicated region was estimated to be approximately 73 Mb. FISH analysis with the WCP probe for chromosome 2, apart from producing fluorescence along the entire length of chromosome 2, also revealed fluorescence on the long Ponnala/Ranganath/Dutta/Pidugu/Dalal


Patient and Methods

Y

q31.

q24.

q23.

q22.

q22.

q14.

q13

q14. q14.

q12.

q11.

p11.

p12

q37.

X

q37.

22

q36.

21

216.74

20

203.64

19

190.53

18

177.43

17

164.32

16

151.22

15

242.95

12

229.85

11

q35

10

q34

9

5

14

p14

p15

p16.

8

4

13

138.11 p21

p16.

p22.

125.01 p23.

p25.

Mb 111.9 2

7

3

q33.

6

Fig. 2. Standard G-banded karyotype (on the left hand side) showing additional material on 18q: 46,XY,ins(18;?). FISH analysis with the chromosome 2 WCP probe (on the right hand side) showing fluorescence on the long arm of chromosome 18 indicating that the extra material on chromosome 18 originated from chromosome 2q.

p25.

2

q33.

1

0

–2.00

–1.00

S1 B-allele frequency 0.25 0.50

0 1.00 Smoothed log R

0.75

2.00

1.00

DGV Known reg Found reg

Fig. 3. Cytogenetic microarray study done using the Illumina Human 660W-Quad platform showing duplica-

tion of chromosome 2q31.1–q37.3. The array picture on the left side corresponds to the region of chromosome 2 shown in the red box in the ideogram. The dark blue box represents the extent of duplication.

Partial Trisomy 2q due to InsertionDuplication in Chromosome 18

Discussion

The patient reported here had de novo partial trisomy 2q which, on molecular characterization using high-resolution cytogenetic microarray analysis, was found to be a 73-Mb size duplicated 2q31.1–q37.3 chromosomal segment inserted in the chromosome 18q region. Cytogenet Genome Res 2012;136:229–234

231


arm of chromosome 18. Consequently, the FISH study further confirmed that the extra material on chromosome 18 had originated from chromosome 2q (fig. 2). Microsatellite analysis using short tandem repeat polymorphism markers corresponding to the 2q31–q37 region showed that the duplicated chromosomal material was maternal in origin.

Table 1. Comparison of the phenotypic characteristics of the reported case with those of previously reported cases of 2q trisomy

Duplicated segment of 2q Origin Age Low birth weight Failure to thrive Craniofacial abnormality Microcephaly Hypertelorism Shallow orbits Thin upper lip vermillion Narrow or high palate Cleft lip/palate Low-set ears Short neck, redundant skin Skeletal abnormality Brachydactyly Long fingers Clinodactyly of 5th finger Neurological abnormality Hypotonia in infancy Developmental delay Seizures CNS malformations Other system abnormality Congenital heart disease Renal/genital anomaly Respiratory problems

Bird and Slavotinek et al. Sebold et al. [2005] Mascarello [2001] [2003]

Hermsen et al. [2005]

Elbracht et al. [2009]

Our patient

q35.2–q37.1 de novo ins 17q25 7 years – +

q33.1–q37.1 de novo 12 months – +

q33–q37.3 de novo inv dup 13 months + –

q35–q37.3 de novo ins 2p 9 years – +

q35–q37.3 ? dup 2q 16 years – –

q31.1–q37.3 de novo ins 18q 2 months + –

– – – – + – + +

– + + + – – – +

+ + + + + – + +

– – + + + – – –

– + – not reported – – + –

– – – + – – – –

+ – – + – + (lower lip) + +

– – –

– – –

– + +

– – +

+ – +

– – +

– – +

– + – –

+ + – –

+ + – –

+ + – –

– + – –

– – – –

– ?a – –

– +/+ –

– +/– +

– –/– –

ASD –/– –

– –/– –

– not reported –

– –/– –

The patient is only 2 months old and needs to be followed up to assess development.

General phenotypic characterization of the trisomy 2q syndrome is difficult because of the inherent phenotypic variability expected depending upon the breakpoints and exact position of the 2q region involved. Most of the 2q duplication cases reported till now have resulted from balanced chromosomal rearrangements in one of the parents such as balanced translocations, balanced insertions or pericentric inversions [Bird and Mascarello, 2001]. Many of the earlier descriptions of the trisomy 2q phenotype were based primarily on case reports of unbalanced translocations. There are only a few reports [Angel et al., 2000; Bonaglia et al., 2000], where delineation of the phenotype of ‘pure’ trisomy 2q versus trisomy 2q with other genomic rearrangements has been attempted. Although limited in number, these reports indicate that the phenotype associated with ‘pure’ trisomy 2q is similar to that described in earlier reports of unbalanced translocations causing 2q duplication. We reviewed recent case reports of patients with duplications involving varying segments of the 2q region 232

q33.1–q35 de novo dir dup 16 months + +


and attempted to delineate the phenotype of 2q trisomy more precisely (table 1). In the literature available the most common characteristics found to be associated with trisomy 2q are failure to thrive, microcephaly, dysmorphic facial features (including a prominent forehead, hypertelorism, epicanthal folds, shallow orbits and ocular proptosis, short nose with upturned nares, broad and depressed nasal bridge, micro-/retrognathia, high palate, long philtrum with thin upper lip, small and down-turned mouth, low-set and abnormal ears), short neck with redundant skin folds, long slender fingers, fisted hands, 5th-finger clinodactyly, congenital heart defects, hypotonia, feeding problems and developmental delay [Seidahmed et al., 1999; Bird and Mascarello, 2001; Slavotinek et al., 2003]. Our patient has some of the previously reported features such as microcephaly, depressed nasal bridge, low-set ears, long philtrum with a thin upper lip and bilateral 5th-finger clinodactyly and, in addition, has incomplete midline cleft of the soft tissue of the lower lip and hypoplasia of Ponnala/Ranganath/Dutta/Pidugu/Dalal


a

Fritz et al. [1999]

man et al., 2003; Dostal et al., 2006; Linnankivi et al., 2006; Feenstra et al., 2007; Cody et al., 2009]. Feenstra et al. [2007] refined the critical regions for microcephaly (18q21–q33), short stature (18q12.1–q12.3; 18q21.1–q21.33 and 18q22.3–q23), white matter disorder and delayed myelination (18q22.3–q23), and growth hormone insufficiency (18q22.3–q33) and concluded 18q22.3–q23 to be the critical region for the typical 18q phenotype. Our patient did not have any of these features which have been well-documented to be associated with CNVs involving the 18q region. Some imbalances of the subtelomeric 2q region seem to be of less clinical significance. The 2q terminal deletion was studied by Fan et al. [2001] using multiple probes in 150 normal subjects; 6% were found to have a telomeric deletion of 2q leading to the conclusion that the deletion of the D2S2986 locus in the telomeric region of 2q was a common variant. Balikova et al. [2007] reported a patient with a subtelomeric deletion of 2q involving 37 genes, who did not have any disease phenotype. In conclusion, we propose that a 2q31.1–q37.3 duplication can cause a phenotype with significant dysmorphism. This report also demonstrates the utility of newer, high-resolution genetic analysis techniques in the more precise characterization of the genotype/phenotype correlations of chromosomal anomalies. Earlier, conventional karyotyping and FISH analysis were the main techniques used for characterization of chromosomal rearrangements, but each of these techniques has limitations in terms of resolution and coverage, respectively. Cytogenetic microarray overcomes these limitations and has emerged as a powerful tool to identify gains and losses throughout the genome, providing a better characterization of chromosomal anomalies as illustrated by this case report. Regardless of the challenges presented by the breadth of information offered by microarray, it is evident that the technology represents a major advance in the ability to detect chromosomal aberrations and is rapidly becoming an indispensable tool for clinical genetics.

Partial Trisomy 2q due to InsertionDuplication in Chromosome 18


References

Angel B, Hersh JH, Yen F, Christensen KM: Case of partial duplication 2q3 with characteristic phenotype: rare occurrence of an unbalanced offspring resulting from a parental pericentric inversion. Am J Med Genet A 91: 126–130 (2000). Balikova I, Menten B, de Ravel T, Le Caignec C, Thienpont B, et al: Subtelomeric imbalances in phenotypically normal individuals. Hum Mutat 28:958–967 (2007).

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the 4th and 5th toes bilaterally (fig. 1), which have not been reported before in trisomy 2q cases. As the child was only 2 months old at the time of clinical evaluation, he needs to be followed up to assess for developmental delay and further evolution of the phenotype. Interstitial duplications of other segments of 2q have been reported, but only 2 cases reported so far have had duplicated segments (between 2q33.1 and 2q35) comparable to the duplicated 2q segment in our case (2q31.1– q37.3) and differ only with respect to the exact extent of the duplicated segment [Romain et al., 1994; Sebold et al., 2005]. The clinical findings described by Sebold et al. [2005] include congenital heart defect, dysmorphic facial features, hypotonia, feeding difficulties and developmental delay. Though there are some similarities between our patient and this case, the phenotypic differences may be attributed to the variation in the chromosomal material involved. Elbracht et al. [2009] presented the clinical and molecular results of 2 unrelated patients with isolated distal trisomy of 2q33–qter and 2q35–q37.3, respectively, with common craniofacial features including a high hairline, broad nasal bridge, prominent nasal tip, thin upper lip vermillion and large ears and assumed that the region 2q35– q37 is critical for the craniofacial features. They concluded that duplications involving regions proximal to 2q33 seem to cause a severe phenotype with major malformations and marked growth and developmental retardation, while duplications involving regions distal to 2q33 seem to be milder [Angel et al., 2000; Slavotinek et al., 2003]. Genotype and phenotype correlation was attempted by Vera-Carbonell et al. [2010] in a patient with a 2q inverted duplicated segment of 38.75 Mb, spanning 2q33.1– q37.3, associated with a terminal deletion of 2.85 Mb, from 2q37.3 to the telomere. The clinical features were found to be associated with those found commonly in patients with 2q duplications, such as microcephaly, facial anomalies, congenital heart defects, hypotonia, wrist contractures, long fingers, adducted thumbs, and club feet [Vera-Carbonell et al., 2010]. As detected by cytogenetic microarray study applied in this case, there was no gain or loss of chromosomal material at the 18q insertion site. However, a chromosomal break per se is known to disrupt the sequence of a gene or its regulatory regions and thereby affect the gene function; this could not be excluded. CNVs involving the 18q22.3–qter chromosomal region have been previously reported to be associated with congenital aural atresia with poor differentiation of grey and white matter in the brain [McEntagart et al., 2001; Tinkle et al., 2003; Velt-

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