Enliven: Pediatrics and Neonatal Biology

Purpose:

1p36 deletion syndrome is the most common terminal deletion syndrome, with an incidence of 1/5,000 newborns. But 22q13 duplications seem to be exceedingly rare. A combined 1p36 deletion and 22q13 duplication was more rarely observed and presented variability of clinical features, which increases the importance of reporting additional cases in order to better characterize genotype-phenotype correlations.

Methods:

A boy and a fetus from a single family with combined 1p36 deletion and 22q13 duplication characterized by array CGH and MLPA were described here.

Results:

The proband presented severe developmental delay, hypotonia, epilepsy, feeding difficulties with failure to thrive, tracheal malformation, clinodactyly, strephexopodia, fair skin, facial dysmorphism and died at 1 year of age. Array CGH uncovered a 9.3-Mb deletion of 1p36 plus a 6.655-Mb duplication of 22q13in the proband. This rearrangement was confirmed by MLPA. When his mother was pregnant again, array CGH detected an almost identical rearrangement with that of the proband. She terminated the pregnancy at 24 weeks gestation. The fetus was female and had similar facial dysmorphism as the proband. Karyotypes of parents are all normal.

Conclusions:

Most of the features in the proband were similar to those associated with both isolated 1p36 deletions and 22q13 duplications. However, tracheal malformation, fair skin and strephexopodia were only observed in our patient. Given that 22q13 duplications are rare and not as well characterized as 1p36 deletions, we attributed these features to 22q13 duplications. Importantly, we emphasize importance of prenatal diagnosis of females who had such abnormal pregnancy.

Introduction

The deletion of chromosome 1p36 (OMIM#607872) is the most frequent terminal chromosomal deletions observed in humans, affecting approximately 1 in 5,000 newborns [1,2]. The clinical manifestations of this syndrome include developmental delay/intellectual disability, hypotonia, epilepsy, brain anomalies, congenital heart defects, cardiomyopathy, renal anomalies, vision problems, hearing loss, short stature, and orofacial clefting. Distinctive facial features include microcephaly or brachycephaly, straight eyebrows, deep-set eyes, epicanthal folds, low-set ears, abnormal ears typified by their posterior rotation, midface dysplasia, a wide and depressed nasal bridge, a long philtrum, a pointed chin, and a large and late-closing anterior fontanel [2-4]. Additionally, patients with the 1p36 deletion have better functional skills during adolescence and adulthood [5]. In contrast, 22q13 duplications are rarely observed. The most prominent phenotypes associated with 22q13 duplications are psychomotor retardation, pre- and post-natal growth retardation, hypotonia, congenital heart defects, renal and genital anomalies, skeletal abnormalities, autism spectrum disorders, cleft palate with or without cleft lip, microcephaly, hypertelorism, low-set ears, and micrognathia. Almost half of all published patients died before reaching 12 years of age, which suggest that life expectancy is severely reduced [6-9]. A combined 1p36 deletion and 22q13 duplication is more infrequent, with only four reported cases to date [10]. Array CGH- and MLPA-based characterization of a boy and a fetus from a single family with combined 1p36 deletion and 22q13 duplication are described in this study.

Case Presentation

The proband was born at 38 weeks gestation by cesarean delivery with a birth weight of 2.5 kg (< 3^rd percentile) and a birth length of 47 cm (≤3^rd percentile) as the second child of a 34-year-old father and a 30-year-old mother. His older brother was healthy. His psychomotor development was severely delayed since birth. He was unable to hold his head or sit by his first birthday, and he had no speech throughout his life. Hypotonia and feeding difficulties with failure to thrive were observed since birth. His weight was only 6kg until 1 year of age. The first seizure occurred at 6 months of age, with a frequency of 2-3 times per day, lasting about tens of seconds each. The dysmorphic features of the proband included microcephaly, straight eyebrows, deep-set eyes, hypertelorism, asymmetric and low-set ears, a high-arched palate, clinodactyly, tracheal malformation, strephexopodia, and fair skin. Additionally, the proband had a femoral hernia. He died at 1 year of age from septic shock following a severe infection. A single umbilical artery and mild bilateral ventriculomegaly (11mm) were detected by prenatal ultrasound at 22 weeks gestation. A ventricular septal defect was revealed by echocardiography after birth, and was repaired at 6months of age. The clinical information of the proband and the clinical characteristics of previously published patients with combined 1p36 deletion and 22q13 duplication patients with isolated 1p36 deletions or 22q13 duplications are summarized in table 1[10]. He was referred to our laboratory for genetic analysis at 5 months of age, the karyotype was 46, XY. However, array CGH detected a 9.3-Mb 1p36 deletion plus a 6.655-Mb 22q13 duplication which was confirmed by MLPA.

The proband’s mother consented to amniocentesis and cytogenetic testing at 18 weeks of gestation for another pregnancy. A single umbilical artery and mild bilateral ventriculomegaly (11mm) were detected by ultrasound again at 22 weeks of gestation,the karyotype of the fetus was 46, XX. Array CGH detected the same chromosomal aberration as the proband. She terminated the pregnancy at 24 weeks of gestation. The aborted female fetus had similar facial features to the proband according to the description of the mother. The standard karyotypes of the parents performed by another laboratory were normal.

Methods

DNA Extraction

Genomic DNA was extracted from peripheral blood lymphocytes of the proband as well as the amniotic fluid cells of the fetus using a QIAamp DNA mini kit (Qiagen, Valencia, CA, USA) Informed consent was obtained from parents of the children in the study.

Array CGH

An Agilent Sure Print G3 Human CGH Microarray Kit (8×60K, Agilent Technologies, Santa Clara, CA, USA) was used for genetic analysis of the proband and the fetus according to the manufacturer’s instructions (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, version 7.3). DNA hybridization was performed according to the standard procedures after labeling of 500ng of the sample DNA with cyanine-5 and control DNA with cyanine-3(Promega, USA). The signals were captured by Sure Scan microarray scanner (Agilent Technologies, Santa Clara, CA, USA). Microarray data was analyzed using Feature Extraction software and Workbench genomics software (Agilent Technologies, Santa Clara, CA, USA). The Aberration Detection Methods 2 algorithm (ADM2) was used to analyze data with a threshold of 6.0 and a moving average window of 2 kb. Log 2 ratios under 0.25 and variations with less than three consecutive probes were excluded. Genomic positions were based on the UCSC February 2009 human reference sequence (GRCh37/hg19) (NCBI build 37 reference sequence assembly). Then, the clinical relevance of observed chromosomal aberrations was estimated according to data found in the scientific literature and databases for each of the regions and genes involved, using the DECIPHER database (http://www.sanger.ac.uk/PostGenomics/decipher/) for known micro deletion or micro duplication syndromes and the Online Mendelian Inheritance in Man (OMIM,www.ncbi.nlm.nih.gov/Omim/getmorbid.cgi) for known disease-causing genes and gene functions.

MLPA

Multiplex Ligation-dependent Probe Amplification (MLPA) analysis was performed to confirm the rearrangements detected by array CGH using SALSA P070, P036 and P245 kit (MRC-Holland. Amsterdam, Netherlands). MLPA analysis was performed according to the manufacturer’s instructions. The amplification products were identified and quantified by capillary electrophoresis on a genetic analyzer (ABI 3130XL, USA). The fluorescence signal intensities of the PCR products were determined with Coffalyser.Net software.

Results

Array CGH analysis uncovered a 9.3-Mb deletion of 1p36.33p36.22 plus a 6.655-Mb duplication of 22q13.31q13.33in the proband. This rearrangement was confirmed by MLPA P070 and P245 kit. A 9.231-Mb deletion of 1p36.33p36.22 plus a 6.655-Mb duplication of 22q13.31q13.33 was detected in the fetus, which was almost identical with the rearrangement of the proband. The family tree and array CGH results of the proband are showed in figure 1, and MLPA results are showed in figure 2. The deletion/duplication sizes of our patients and the reported patients are summarized in table 2.

Figure 1: The family tree and array CGH results of the proband. A: The family tree, the white square represents normal males, the white circle represents normal females, the black square with a slash represents patients who has died, the relatively small black circle represents aborted fetuses; B: results of array CGH analysis. A 9.3Mb deletion on chromosome 1; A 6.655 Mb duplication on chromosome 22.

Figure 2: Results of MLPA of the proband. P070 and P245 show combined 1p36 deletion and 22q13 duplication. Red arrows indicate 1p36 deletion, yellow arrows indicate 22q13 duplication.

Discussion

In this study, we described the clinical and molecular characteristics of a boy and a female fetus from a family with combined 1p36 deletion and 22q13 duplication. The proband was born at term with mild intrauterine growth retardation. His postnatal growth and psychomotor development were severely delayed, and he died at 1year of age from septic shock following a severe infection. He weighted 6kg (< 3^rd percentile) when he died. His mother consented to amniocentesis for another pregnancy, and the array CGH analysis revealed an almost identical rearrangement to the proband. The pregnancy was terminated at 24 weeks of gestation, and the fetus had similar facial features to the proband.

Combined 1p36 deletion and 22q13 duplication was rarely seen in the literatures with only four patients reported by Gajecka et al. [10]. We compared the clinical features observed in our patient to whose of and the patients reported by Gajecka et al. and to those of patients with isolated 1p36 deletions or 22q13 duplications. Most features identified in the proband were similar to those reported for patients by Gajecka et al. [10]. The following features were seen in the proband and in at least one of the four reported patients: low birth weight (2.5kg), developmental delay/intellectual disability, hypotonia, epilepsy, microcephaly, asymmetric ears, low-set ears, deep-set eyes, hypertelorism, a high-arched palate, clinodactyly, visual inattentiveness, hearing loss, a ventricular septal defect and feeding difficulties with failure to thrive. Most of these features were similar to those associated with 1p36 deletion syndrome and with 22q13 duplications. However, some features seen in at least one of the four reported patients were not observed in our patient, including large anterior fontanelle, brachycephaly, short neck, flat nasal bridge, posteriorly rotated ears, pointed chin, micrognathia, small hands and/or feet, strabismus, patent ductus arteriosus, ebstein anomaly, cryptorchidism and a sacral cleft. Features that were only seen in our patient, included tracheal malformation, straight eyebrows, strephexopodia and fair skin. With the exception of straight eyebrows, these features are not common for 1p36 deletion syndrome [2-4]. Given the poor characterization of 22q13 duplication phenotypes, we hypothesize that these features are specific to 22q13 duplications.

Early death, which has been reported for distal 22q duplications [11,12], was not observed in the reported patients who had combined 1p36 deletion and 22q13 duplication [10], but it was observed in our patient. Because a reduced life expectancy was not reported for 22q13.2 and 22q13.3 duplications, Gajecka et al. hypothesized that a reduced life expectancy was associated with duplications of 22q12-22q13.1, that were more proximal than 22q13.2 [6,13]. This contradiction may reflect an ascertainment bias. Whether reduced life expectancies are associated with 22q13 duplications requires further investigations using large cohorts. Additionally, a single umbilical artery and mild ventriculomegaly which were seen in both the proband and the fetus are important indications of chromosome abnormalities, and the ultrasound physician should pay attention to these recurrent indicative features.

The female fetus was aborted at 24 weeks of gestation, and according to the parents’ description, it has similar facial features to the proband. Echocardiography was not performed for the fetus. Therefore, whether the fetus had congenital heart defect remains uncertain.

It’s difficult to perform a genotype-phenotype correlation analysis because only five children and a fetus have been reported to date. The deletion sizes and breakpoint locations of our patients were different from those of the reported patients (Table 2), without the common size or breakpoints of 1p deletion or 22q duplication [10]. Moreover, we did not observe correlations between the deletion/duplication sizes and phenotypes from these patients. Eight commonly observed clinical features for isolated 1p36 deletion syndrome were chosen for analysis as follows: a large anterior fontanel, hearing problems, structural heart defects, seizures, hypotonia, feeding difficulties, speech delay, and strabismus [10,14]. Our patient exhibited five of these features, most of which were specific for the 1p36 deletion syndrome rather than for 22q13 duplications as the reported patients (Table 1). This confirms that the presence of a chromosomal deletion has a stronger impact on phenotype compared with a duplication [10].

The chromosomal abnormalities in our patients were not detected by a conventional karyotype analysis. The karyotypes of the parents were normal, but the recurrence of the rearrangements in this family strongly indicate da germinal mosaic translocation in one parent or translocation derivatives that were too cryptic to distinguish from the normal karyotype [15]. Unfortunately, FISH analysis cannot be performed on the parents to confirm this because of their poor coherence. However, the recurrence of a rearrangement involving two chromosomes in a single family, where the parents have normal karyotypes, highlights array CGH as an essential pre- and post-natal diagnostic technology.

Acknowledgements

All authors have no conflicts of interest. We are grateful to the family members who contributed to this study. This work was not funded.

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