Postnatal clinical phenotype of five patients with Pallister–Killian Syndrome (tetrasomy 12p): Interest of array CGH for diagnosis and review of the literature

Abstract Background Pallister–Killian syndrome (PKS) is a rare sporadic disorder caused by tetrasomy of the short arm of chromosome 12. The main clinical manifestations are global developmental delay, intellectual disability, epilepsy, dysmorphic features, hypopigmented and/or hyperpigmented lesions, and multiple congenital anomalies. PKS is associated with tissue mosaicism, which is difficult to diagnose through peripheral blood sample by conventional cytogenetic methods and fluorescence in situ hybridization. Methods Here, we report five patients with PKS. We delineate their clinical phenotypes and we compare them with previously published cases. We used array Comparative Genomic Hybridization (aCGH) with DNA extracted from peripheral blood samples. The five patients have also been tested by conventional cytogenetics techniques. Results Four out of five patients showed tetrasomy 12p by aCGH. Three of the four patients have typical i(12p) and one of the four demonstrated atypical tetrasomy 12p. The percentage of mosaicism was as low as 20%. Our cohort exhibited the typical PKS phenotypes. Conclusion Our results demonstrate the efficacy of aCGH for the diagnosis of PKS from DNA extracted from lymphocytes. Thus, for patients suspected of PKS, we recommend performing aCGH on lymphocytes at an early age before proceeding to skin biopsy. aCGH on peripheral blood samples is sensitive in detecting low level of mosaicism and it is less invasive method than skin biopsy. We reviewed also the literature concerning the previously published PKS patients diagnosed by aCGH.


| INTRODUCTION
Pallister-Killian syndrome (OMIM 601803; PKS) is a rare disorder which was first described by Philip Pallister (Pallister et al., 1977). PKS is characterized by intellectual disability (ID), multiple congenital anomalies, streaks of hypo-and/or hyperpigmentation, and dysmorphic features including a high forehead with frontotemporal alopecia, upslanting palpebral fissures with epicanthal folds, hypertelorism, flat nasal bridge, short nose with upturned nares, low-set ears, macrostomia, eversion of lower lip and thin upper lip with invasion of vermilion border of upper lip by philtral skin, which is known as "Pallister lip", short neck and macrosomia at birth (Blyth et al., 2015;Karaman et al., 2018;Wilkens et al., 2012). Supernumerary nipples may also be found. Patients generally show developmental delay, axial hypotonia, profound ID, epilepsy, and deafness. Several congenital malformations may be present including congenital heart and lung defects, gastrointestinal disorders like congenital diaphragmatic hernia (CDH), anal atresia, renal anomalies, and cryptorchidism (Blyth et al., 2015;Karaman et al., 2018;Wilkens et al., 2012). PKS is caused by the presence of a supernumerary isochromosome i(12p), in mosaic status. This extra chromosome can generally be detected on cultured fibroblast by karyotype or FISH, but the detection of i(12p) might be complicated on cultured lymphocyte. In the last decade, several publications show that array Comparative Genomic Hybridization (aCGH) on DNA extracted from lymphocytes could identify tetrasomy 12p at mosaic level (Blyth et al., 2015;Chen et al., 2014;Lee, Lee, Yu, Lee, & Kim, 2017;Theisen et al., 2009).
Here, we diagnosed four out of five PKS patients using the aCGH on blood-extracted DNA and illustrate their cytogenetics results. Additionally, we delineate their clinical phenotypes and we compare them with previously published patients.

| Ethical compliance
This study has been approved by the ethics committee.

| Clinical reports
This is a descriptive study based on the medical records of five patients with confirmed PKS. The five patients were referred to the genetics department in Women Mothers and Children's hospital in Lyon, France. Four boys and one girl aged from 3 to 11 years old were included. All of them displayed moderate/severe global developmental delay and typical facial dysmorphic features corresponding to PKS. Patient 1 was a 4 and half yearold boy presented with moderate developmental delay. Patient 2 was a 7-year-old boy who exhibited severe developmental delay, macrosomia at birth, and epilepsy starting at 7 years old. Patient 3 was a 3-year-old girl who was diagnosed at birth. She showed axial hypotonia, facial dysmorphism, and umbilical hernia. Patient 4 was a 9-year-old boy. The antenatal history was notable for polyhydramnios. He demonstrated severe developmental delay, macrosomia at birth, and epilepsy starting at 7 and half years old. Patient 5 was a 11-year-old boy. The pregnancy was notable for polyhydramnios. He showed developmental delay and epilepsy starting at 7 years old (Table 2).

| Conventional cytogenetic analysis
Karyotype studies were performed, on all patients, using a standard phytohemagglutinin-stimulated lymphocyte culture method, followed by G-and R-banding techniques. Skin biopsy was subjected to fibroblasts culture according to standard protocol for patients 4 and 5.

| aCGH
Genomic DNA was isolated from peripheral blood leukocytes for all patients using a nucleospin blood L kit (Macherey-Nagel GmbH & Co. KG), according to the manufacturer's protocol. The whole genomic aCGH procedure was performed following the manufacturer's instructions (Sure Print G3 Human CGH Microarray Kit; Agilent Technologies). The 180K slides were scanned on an Agilent DNA Microarray Scanner and images were extracted with Feature Extraction software (12.0.1.1). Data analysis was carried out with Cytogenomics v3.0.3.3. Before 2014, the Feature Extraction software used was the version (11.5.11) and the data interpretation analysis used Workbench v3.4.2.7. The following parameters were used for interpretation: ADM-2, threshold: 5.0, window: 0.2 Mb, cutoff: 0.25. A copy number variation was validated if an abnormal log2 ratio was obtained for at least three contiguous oligonucleotides. The aCGH results were analyzed with the UCSC hg19 assembly.

| RESULTS
Pallister-Killian syndrome was confirmed in the five patients. We used aCGH on peripheral blood sample in all patients, karyotyping and FISH on peripheral blood sample only in three patients (patient 1, 2 and 3), on skin biopsy only in one patient (patient 4), and on both in one patient (patient 5). The combined results from aCGH and conventional cytogenetic techniques demonstrated the presence of typical i(12p) in patients 1, 2, 4, and 5 and partial tetrasomy of distal 12p (12p13.33p12.1) and partial trisomy of proximal part of 12p and 12q (12p12.1q12) for patient 3.
Array Comparative Genomic Hybridization on DNA extracted from lymphocytes showed the presence of tetrasomy 12p in mosaic state in four patients out of five. The mosaicism percentages in aCGH analysis were 20 for patient 1 (log2 ratio value: 0.226), 28 for patient 2 (log2 ratio value: 0.356), and 50 for patient 5 (log2 ratio value: 0.58; Figure  1). aCGH in patient 3 revealed a distal gain of 12p with log2 ratio of 0.888 (85%) and proximal gain of short and long arm of chromosome 12 with log2 ratio of 0.281 (43%; Figure S1). aCGH result for patient 4 was normal; however, the PKS was confirmed using karyotyping and FISH on fibroblasts.
Results obtained from standard karyotypes, FISH for lymphocytes and/or fibroblasts for the five patients are summarized in Table 1. aCGH profiles for patients 1, 2 and 5 are presented in Figure 1 and for patient 3 in Figure S1.
The clinical data for the five patients are summarized in Table 2.

| DISCUSSION
Pallister-Killian syndrome is a disorder associated with global developmental delay, multiple congenital anomalies, and mosaicism for isochromosome 12p. Our five patients exhibited typical phenotypes of PKS, including classic dysmorphic features, global developmental delay, receptive or conductive hearing impairment, strabismus, and gastroesophageal reflux    Wilkens et al., 2012). In our series, no patients manifested CDH since the latter associates with complications leading to fetal demise, or it could be diagnosed prenatally and subsequent a termination of pregnancy (Blyth et al., 2015). One patient demonstrated cardiac anomalies which are frequently observed in PKS patients. Epilepsy in PKS usually occurs in the first 4 years of life (Blyth et al., 2015;Wilkens et al., 2012). We observed epilepsy in three of five patients starting between 4 and 7 years; the other two patients are young (3 years and 4 and half years) and therefore a long-term follow-up will be needed in order to completely rule out the possibility of manifesting epilepsy. One patient showed optic nerve hypoplasia which has rarely been reported in PKS (Blyth et al., 2015).

| Cytogenetic techniques to detect the presence of isochromosome 12p
During the postnatal period, the conventional cytogenetic methods might not reveal the presence of mosaicism for isochromosome 12p on peripheral blood samples as shown in patients 2 and 5 . Our data demonstrated different percentages of mosaic i(12p) in cultured and uncultured lymphocytes, suggesting that cultured lymphocytes might undergo a negative selection for marker chromosome (Pagon et al., 1979). Since conventional cytogenetic analysis requires actively dividing cells, it could make the detection of mosaic i(12p) unlikely (Reeser & Wenger, 1992). Therefore, the interphase FISH could slightly improve the detection level of mosaicism on peripheral blood since the process does not require cultured cells. However, FISH is a targeted technique. Since the PKS is a tissue-limited mosaicism, the detection of i(12p) on skin biopsy by standard cytogenetic analysis is still the gold standard (Hodge et al., 2012). However, skin biopsy is an invasive procedure and the detection rate might decrease from the nonpigmented region (Harnden, 1960). Identifying low level of mosaicism for chromosomal abnormalities by traditional cytogenetic techniques on peripheral blood cells is challenging. In comparison with cytogenetic analysis, aCGH provides results on extracted genomic DNA without cell cultures. Therefore, it might better reflect the value of mosaicism of cells (Ballif et al., 2006). Our results are similar to those previously reported as it illustrates that aCGH is superior to standard cytogenetic techniques and FISH in detecting mosaic i(12p) on peripheral blood (Ballif et al., 2006;Chen et al., 2014). We identified the mosaic i(12p) by aCGH first in three patients out of four which then facilitated finding the i(12p) by FISH and karyotype analysis. The fourth patient showed atypical tetrasomy 12p.
Our data supported the literature that aCGH on peripheral blood sample is highly sensitive in detecting mosaicism of isochromosome 12p (Blyth et al., 2015;Lee et al., 2017;Theisen et al., 2009; Table S1).
In our series, aCGH on peripheral blood identified a copy number gain of the short arm of chromosome 12 in four patients out of five, even with a low level of mosaicism of up to 20%. These results were confirmed by FISH. (Table 1). The age of detecting the tetrasomy 12p by aCGH in these four patients ranged between 4 months and 21 months. Nevertheless, aCGH failed to detect the i(12p) in one patient of our series (patient 4) at 18 months, which has been detected by standard cytogenetic method on skin biopsy. A similar normal result has been shown in a patient with clinical diagnosis of PKS who was tested using aCGH on peripheral blood at 2 years and 5 months. And therefore the skin biopsy was opted for aCGH at the same age which demonstrated the presence of tetrasomy 12p (Hodge et al., 2012). Another array-based cytogenetic study (SNP array) of 15 patients aged between 8 days and 6 years and 9 months using peripheral blood sample and skin biopsy showed that the percentage of mosaic i(12p) cells on peripheral blood declined with the PKS patients ages, while the tetratomic cells were still quite stable overtime on skin biopsy using the SNP array . In this study, the tetratomic cells on peripheral blood using SNP array were lost as early as 17 months old in one patient, while in other PKS patient these cells were still detectable at 6 years old using the SNP array on peripheral blood . In our series, the oldest diagnosed patient by aCGH was 21 months old (patient 1) while the other three diagnosed patients were less than 1 year old. The tetratomic cells were undetectable using aCGH in patient 4 at 18 months. We really do not know when those tetrasomic cells exactly become undetectable on the blood . Thus, it might be better to perform the aCGH as a first-tier method on peripheral blood at early age, under 12 months, when PKS is clinically suspected to eliminate the need for undertaking skin biopsy at older age. However, in highly suspected PKS casses, older than 12 months, who have normal aCGH results on lymphocytes, a buccal smear and/or skin biopsy specimen might be opted for FISH, karyotypes and/ or aCGH as a second-tier test Hodge et al., 2012). On the other hand, aCGH cannot determine the cause of the entire 12p gain, thus emphasizing the importance of confirming this result by FISH (Chen et al., 2014).

| The 3rd patient (derivative 12)
To the best of our knowledge, this is the first reported patient of PKS resulting from an unbalanced translocation between the two chromosomes 12 homologous. The mechanism proposed is that a U-loop exchange occurred between the short and the long arm of homologous chromosome 12 followed by nondisjunction errors through meiosis. Results in supernumerary derivative chromosome contain two copies of 12p13.33p12.1 (partial tetrasomy) and one copy of 12p12.1q12 (partial trisomy). The marker chromosome has been characterized by aCGH and the result has been confirmed by FISH. The patient has the classical phenotype of PKS. Nevertheless, she has neither diaphragmatic hernia nor cardiac defect. The patient is 3 years old and therefore could manifest seizures later on. Izumi et al. (2012) published a paper redefining a minimal critical region responsible for PKS patients that is completely included in the chromosomal abnormalities detected in this patient. The most frequent cause of PKS is the supernumerary i(12p). The mechanism suggested for i(12p) is nondisjunction errors through meiosis, followed by the centromeric misdivision. Other variants for PKS have been reported (Table S2; Lloveras et al., 2013;Yeung, Francis, Giouzeppos, & Amor, 2009).

| CONCLUSION
PKS is tissue-limited mosaicism due to tetrasomy 12p. Our cohort exhibits the typical phenotypes of PKS, including classic dysmorphic features, global developmental delay, epilepsy, hearing impairment, and strabismus. Here, we confirmed that aCGH on peripheral blood sample may be considered as a first-tier method to use when the PKS is suspected especially at early age. Moreover, the use of aCGH on a blood sample might avoid the need for a skin biopsy. Finally, the characterization of unbalanced chromosomal aberration and marker chromosome is more efficient by aCGH, which provides high resolution.