Two unrelated children with rare submicroscopic imbalances in the centromeric domain of the 11p15 imprinted gene cluster were identified and subjected to further laboratory analyses and clinical examination. In the proband of the first family (family 1), a duplication of about 0.88 Mb of chromosome 11p15.5-p15.4 was identified by comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) array analyses. These methods allowed locating the telomeric breakpoint between chr11:2,739,336 and chr11:2,742,159 bp (GRCh37/hg19) within the intron 10 of KCNQ1, about 20-kb centromeric to ICR2, and the centromeric breakpoint between chr11:3,632,246 and chr11:3,632,370 bp in an intergenic region among the pseudogenes LOC650368 and TRPC2 (Fig. 1 and Additional file 1: Figure S1). In the proband of the second family (family 2), a duplication of 1.13 Mb was identified and its breakpoints defined by SNP array (Fig. 1). The telomeric breakpoint was identified inside intron 9 of KCNQ1 gene, about 65 kb telomeric from the transcription start site of KCNQ1OT1, while the centromeric breakpoint was identified in intron 10 of the NUP98 gene (arr[GRC37/hg19] dup(11)(p15.5-p15.4) (2,656,310x 2; 2,656,311-3,782,347x3; 3,782,492x2)).
Family 1
The proband of family 1 was the third son of three children from unrelated parents. Pes equinovarus was observed by ultrasound scan by gestational age 16 + 2 weeks and an amniocentesis was obtained. The maternally inherited 11p15 duplication was identified. Due to this finding several ultrasound scans were performed during the pregnancy and IUGR was observed: −18 % at 30 + 6 weeks of gestation, −25 % at 32 + 5 weeks, −27 % at 34 + 5 weeks, and −30 % at 36 + 5 weeks. Due to IUGR induction of delivery was performed. He was born small for gestational age (SGA) at gestational age (GA) 37 + 6 weeks. His birth weight was 2070 g, (−3 SDS), birth length 44 cm (−3 SDS), and occipital frontal circumference (OFC) 30 cm (−2.5 SDS). The placenta weight (300 g, <third centile) was also reduced. Further, blood glucose was 1.4 mmol/l by delivery. He received treatment with intravenous glucose for 1 day. Afterwards he was only breast feed. The pes equinovarus was treated with plaster and tenotomy of the Achilles tendons at 45 days of age.
Physical examination at 14 months confirmed the growth restriction, in particular the short stature: weight 9.0 kg (−1.5 SDS), length 73 cm (−2.5 SDS), and OFC 45 cm (−1.5 SDS). The father, aged 40 years, was referred to be of normal stature (176 cm). The mother’s height was 163 cm and her weight is 59 kg at 38 years of age. It was referred that in childhood, she was very small and the general practitioner suspected she was a kind of a dwarf at 5–6 years of age.
The first son was born by GA 42 weeks with a birth weight of 3320 g (−0.5 SDS) and a birth length of 52 cm (average). By 14 years of age, his height was 168 cm (+1 SDS); the second son was born at 33 weeks of gestation. The spontaneous preterm delivery was caused by membrane rupture. His birth weight was 1575 g (−1.1 SDS), the birth length 41 cm (−1 SDS). Information on fetal growth parameters during pregnancy was not available. He was treated with intravenous glucose infusions age 1–3 days due to low blood glucose (blood glucoses at day 1: 1.9–2.6 mmol/l). By 7 years, his height was 127 cm (+1 SDS). He was affected by mild attention deficit hyperactivity disorder (ADHD).
The presence of the duplication was searched in the proband relatives by SNP array performed on DNA derived from buccal swab of the brothers and from blood of the parents and the maternal grandfather. The chromosome 11 duplication was identified in the mother and in her second son but not in the first son neither in the maternal grandfather (Additional file 2: Figure S2). By studying the segregation of the 11p15 haplotype by microsatellite analysis we demonstrated that in the mother the duplication was present on her paternal chromosome (Fig. 2a and Additional file 3: Figure S3). This suggests that the duplication originated very early in development, either in the gametes of the maternal grandfather or in the somatic cells of the mother.
To confirm the duplication and determine if it was present in cis or in trans, cells of the umbilical cord of the proband were analyzed by fluorescence in situ hybridization (FISH). A bacterial artificial chromosome (BAC) probe (RP11-11A9, chr11: 3236552-3356012, green signal in Additional file 4: Figure S4) hybridizing within the duplicated region and a BAC probe (RP11-876C12, chr11q22.3, red) located outside the duplication were used for the metaphase FISH (Additional file 4: Figure S4, top panel). FISH on interphase nuclei was performed by using the BAC clones RP11-11A9 (green) and RP11-81 K4 (red), both located within the duplicated region (Additional file 4: Figure S4, bottom panel). The absence of signals in chromosomes other than chromosome 11 in metaphase FISH, and the green-red-red-green sequence of the fluorescence signals, demonstrated the presence of an in cis duplication with inverted orientation.
To investigate the effect of the duplication on genomic imprinting, we analyzed the DNA methylation of ICR1 and ICR2 in the placenta cells of the proband by Pyrosequencing (Fig. 2b) and combined bisulfite restriction assay (COBRA; Additional file 5: Figure S5). With both methods, the proband showed a methylation profile comparable to that of three healthy controls in both ICR1 and ICR2. Normal ICRs methylation was also observed in the blood leukocytes of the parents. To look for a possible deregulation of the 11p15 imprinted genes, we analyzed the RNA levels of CDKN1C and PHLDA2 in placenta cells. We found that CDKN1C expression was increased 10-fold (P < 0.01; Fig. 2c) and PHLDA2 threefold in the proband when compared with three healthy controls (P < 0.01; Fig. 2c).
Family 2
The male proband was the only child of non-consanguineous healthy parents. He was born by GA 37 + 5 weeks. Birth weight was 3350 g (−0.5 SDS), birth length 51 cm (average), and OFC 35 cm (+0.5 SDS). Apgar scores were 8/1, 8/5, and 8/10. Neonatal plasma glucose was normal. In the medical record, it is described that there was slight cranial asymmetry with left side of parietal and frontal region a little flat. The head was described as slight narrow, the nasal bridge as slightly wide, and there was strabismus and retention testis. Further, there were described bilateral dysplastic nails on third, fourth, and fifth toes. He was affected by Steno-Fallot Tetralogy, diagnosed on day 1 by echocardiography required because of a systolic murmur. Operation was performed by age 8 month. Neonatal ultrasound scans of cerebrum and kidneys were both normal. At 1 year old, he showed slight frontal bossing, slight hypoplasia of maxilla, slightly flaccid occiput, and bilateral single palmar creases. Neither umbilical hernia nor ear lobe creases were observed. At 8 and 20 months of age, the auxological parameters were still close to the average: 8 month: weight 9.2 kg (+0.5 SDS), length 71.5 cm (+0.5 SDS) and OFC 44 cm (−0.5 SDS); 20 month: weight 11.3 kg (−0.5 SDS), length 84.5 cm (−0.5 SDS) and OFC 46.3 cm (−1.5 SDS). The psychomotor development was normal.
The mother was 35 years old, with normal phenotype except for the presence of bilateral ear lobe creases. Her height was 170 cm and weight was 56.5 kg. She was born by GA 41 + 5 weeks, with the birth weight 3740 g (+0.5 SDS), birth length 53 cm (+1 SDS), and OFC 35.5 cm (+1.5 SDS).
Copy number and DNA methylation of the chromosome 11p15.5 region were first analyzed by MS-MLPA. Increased hybridization signal at ICR2 and KCNQ1 exons 13–17 and slight loss of ICR2 methylation were identified in the proband and his mother, while ICR1, IGF2, H19, and control probes showed normal copy number and methylation status (Additional file 6: Figure S6), indicating the presence of an inherited partial duplication of the 11p15.5-p15.4 imprinted gene cluster.
To better define the DNA methylation abnormality of the 11p15.5-p15.4 region in the proband and his mother, the methylation levels of the ICRs were determined by pyrosequencing in the trio. As shown in Fig. 2d, the methylation profiles of both ICRs were normal in the father, while the proband and his mother showed normal methylation of ICR1 but hypomethylation of ICR2 at a level comparable with other previously described ICR2 duplication carriers [17, 18]. The allele-specific methylation analysis could not be performed because of the absence of polymorphisms in the ICR2 sequence. Nevertheless, the observed hypomethylation suggests that the duplicated ICR2 fails to acquire or maintain the maternal imprints in the proband.
The inheritance of the duplicated region in the proband, his parents and maternal grandparents, was determined by analyzing the 11p15 microsatellite markers. The segregation and signal intensity of the D11S4088 marker, located in the duplicated region, confirmed that the duplication was maternally inherited in the proband and demonstrated that it originated de novo from the paternal chromosome in the mother (Fig. 2e and Additional file 7: Figure S7). The markers, D11S4046, D11S922, and TH, did not show any allelic imbalance in the proband and his mother, consistent with their localization outside of the duplicated region (Additional file 7: Figure S7).
To determine the chromosomal location of the duplicated region, the cultured blood leukocytes of the proband were analyzed by FISH. The BAC probes hybridizing within the duplicated region, RP11-11A9 (chr11: 3236552-3356012, green) and RP11-699D10 (chr11: 2.9–3.04 Mb, red), were used. As in family 1, the results of the metaphase FISH indicated that the duplication was in cis (Additional file 8: Figure S8, top panel), while the FISH on interphase nuclei demonstrated the inverted orientation of the duplication (Additional file 8: Figure S8, bottom panel).