Patient 1
The proband is an eighteen-year-old girl. She was born in Brazil and arrived in Italy when she was one year and six months old, following her adoption. Her family and perinatal history were fragmentary and poorly known: she had a paternal uncle affected with epilepsy (whose further clinical data is not available) and a healthy sister, coming with her to Italy after the adoption by another family. At our first examination, anthropometric measurements were as follows: weight 74 Kg (95th centile, + 1.65 standard deviations, SD), height 154cm (6th centile, -1.58 SD), occipitofrontal circumference (OFC) 56 cm (63rd centile, + 0.3 SD), according to World Health Organization growth charts [8]. She showed squared face due to chubby cheeks and enlarged chin, curly hair, wide and prominent ears with hypoplastic antihelix, thick eyebrows and synophrys, hypertelorism, mild epicanthus, wide nasal bridge, and bulbous tip, hypoplastic and short philtrum, macrodontia of permanent upper central incisors with dental crowding, and thick lower lip. In addition, bilateral brachydactyly of the 1st, 2nd , and 5th fingers, with clinodactyly of the latter, was noted (Fig. 1a/b/c).
Figure 1a/b/c. Patient 1. a Squared face due to chubby cheeks and enlarged chin, curly hair, thick eyebrows and synophrys, hypertelorism, mild epicanthus, wide nasal bridge and bulbous tip, hypoplastic and short philtrum, macrodontia of permanent upper central incisors with dental crowding, and thick lower lip. b Wide and prominent ears with hypoplastic antihelix. c brachydactyly of the 1st, 2nd , and 5th finger, with clinodactyly of the latter
She acquired independent walking at 20 months, while a language impairment along with intellectual disability and externalizing behavior problems started to become evident after 2 years of age. She presented her first seizures when she was 12-year-old, currently treated with levetiracetam, while aripiprazole is at present administered to control her externalizing behavior disorder. ECG and echocardiogram were normal, except for mild valvular (both mitral and tricuspid) regurgitations. Brain MRI did not detect any abnormalities. a-CGH analysis evidenced no rearrangements, conversely, NGS analysis of a panel of genes involved in neurodevelopmental disorders identified a heterozygous variant of the ANKRD11 gene (c.1902_1907del) (Ref Seq NM_013275, based on genome build GRCh37/hg19; rsID 886041125; ClinVar:RCV000011454.5). Such mutation is responsible for a frameshift, leading to the introduction of a premature stop codon at position 358 (p.Lys635fs) of the encoded protein. Also, a heterozygous missense mutation of the euchromatic histone lysine methyltransferase 1 (EHMT1) gene (c.103G > A) (p.Asp35Asn) (Ref Seq NM_024757, based on genome build GRCh37/hg19; rsID 371134699; ClinVar:RCV000011454.5), was found. Involvement of the EHMT gene, harboring on a highly conserved residue of the protein and with low frequency in the general population, has been associated with Kleefstra Syndrome (MIM #610253). The Platform used was Ion AmpliSeqCustom Panel version 4.44 - Torrent Personal Genome Machine (PGM) System, Ion Torrent Suite Software version 5; Genome Reference: hg19. Both coding regions and exon/intron junctions have been analyzed. Median coverage was 98%, with genes coverage between 84 and 99%, while the sequence coverage of single fragments analyzed ≥ 100X. NGS analysis of the same panel of genes has been performed also in the sister and showed normal results. Currently, the girl has concluded a high professional school with a dedicated support teacher, manifesting an externalizing behavior disorder especially related to interpersonal relationship issues characterized by physical aggressiveness, mainly with peers. She has a moderate degree of intellectual disability; she can carry out daily activities that allow personal autonomy, and complete manual tasks (manipulate and build simple objects, but not repair or creation); she is able as well of easy mathematic calculations and to use a personal computer for basic functions like writing, although with a short attention span.
Patient 2
The proposita is a three-year-old girl, fourth child of healthy non-consanguineous parents. She was born at 38 weeks of gestation, by caesarean delivery. Apgar scores were 8 and 9, at 1 and 5 minutes respectively. At birth, anthropometric measurements were as follows: weight 2490 g (7th centile,
-1,44 SD), length 46,2 cm (10th centile, -1,29 SD), and OFC 35 cm (87th centile, 1,11 SD), according with the Italian Ines Growth Charts [9]. At our first observation at age 2 months and 12 days, she showed a growth delay: weight 3920 g (1th centile, -2,44 SD), length 53,5 cm (1th centile, -2,19 SD), and OFC 37 cm (8th centile, -1,39 SD), according to World Health Organization growth charts [8]. No cerebral anomalies were identified by head US. Neurodevelopmental evaluation showed a normal response to social smiling and vocalization, as well as a central hypotonia with axial distribution and tongue protrusion. No feeding difficulties were reported. She was enrolled in a neurodevelopmental follow-up. At 5 months and 9 days of age, her anthropometric measures were as follows: weight 5520 g (3rd centile, -1,96 SD), length 61 cm (6th centile, -1,58 SD) and OFC 41 cm (30th centile, -0,5 SD). On physical examination, high forehead with large anterior fontanelle, prominent ears, wide nasal bridge and bulbous tip along with thin upper lip were observed (Fig. 2a). Brachydactyly and bilateral clinodactyly of the fifth finger were also present.
Figure 2a. Patient 2 at 5 months of age. High forehead, prominent ears, wide nasal bridge and bulbous tip, thin upper lip.
Appropriate for the age neurodevelopmental milestones were acquired, although with persistence of a mild central axial hypotonia. Brainstem auditory evoked potentials were normal. At 13 months, an impairment of fine motor skills and reduced response readiness in interactions with the examiner were noted, with persistent mild central hypotonia and facial dysmorphic features along with delayed closure of the anterior fontanelle, suggesting further insights into the possible pathogenesis of her condition. An a-CGH analysis (100–150 Kb resolution, genomic assembly GRCh37.p13) identified a 16q24.3-24.3 deletion spanning 634 Kb, with the positions 88.873.958 and 89.507.835 being the breakpoints of the rearrangement. The deleted region involved several genes (CDT1, APRT, GALNS, TRAPPC2L, ACSF3, CDH15), besides ANKRD11, some of which have been associated with developmental disorders. The molecular karyotype performed in both parents did not reveal any genomic rearrangements. The patient was then enrolled in a psychomotor and neurocognitive stimulation program. Currently at 3 years of age, her anthropometric measures are as follows: weight 10,500 g (2nd centile, -2.15 SD), length 90 cm (12th centile, -1.17 SD) and OFC 49 cm (66th centile, + 0.4 SD). Her mother reports a moderate hyperactivity not complicated by oppositional behavior, along with bruxism and onychophagy. Neurodevelopmental evaluation was normal, with adequate interaction and participation during the developmental assessment. She does not present seizures, although the EEG pattern is moderately abnormal, for the presence of bilateral posterior slow activity (Fig. 2b).
Figure 2b. Moderately abnormal EEG pattern of Patient 2, due to bilateral posterior slow activity
She does not manifest any further clinical anomalies, and laboratory tests as well as US multiorgan evaluations (except for patent foramen ovale revealed by echocardiography) do not evidence other abnormalities to date.
Patient 3
The proband is a 3-month-old male infant, second child of healthy non-consanguineous parents, both coming from Slovenia. Family history was unremarkable, including one healthy brother currently aged 8 years. He was born after a naturally conceived pregnancy, physiologically occurred until 38+ 6 weeks, when an emergency caesarean section was performed due to cardiotocographic abnormalities. For the increased risk of Down syndrome, revealed during the first trimester of pregnancy by the conventional multiple marker screening, genetic investigations through amniocentesis had been offered to the couple, and then performed: SNPs-array excluded genomic rearrangements (microdeletions and/or microduplications), and showed a normal male karyotype, 46 XY. Prenatal ultrasound evaluations, carried out in the following trimesters, documented polyhydramnios in addition to a previously evidenced fetal growth restriction (FGR), and raised the suspicion of rhizomelia of lower limbs. At birth, the newborn showed good adaptation to extrauterine life, with Apgar scores of 9 and 10 at 1 and 5 minutes, respectively. Anthropometric measures were as follows: weight 2324 g (1st centile, -2.51 SD), length 47.7 cm (9th centile, -1.34 SD), and OFC 33.8 cm (25th centile, -0.69 SD) [9]. He was soon transferred to the NICU due to dysmorphic features, and to continue the diagnostic work-up. At admission, he showed triangular face, large anterior fontanelle with wide metopic suture, and prominent ears. In addition, bifid tongue was observed. Bilateral brachydactyly along with clinodactyly of the 5th finger were also present, but no limbs abnormalities were identified. A scrotum anomaly, due to its superior margin upper to the base of the penis (shawl scrotum), was finally noted. Head and abdominal US evaluations documented no abnormalities, as well as total body X-ray (performed for the prenatal finding of rhizomelia) and ophthalmological assessment. Conversely, heart US evidenced a small membranous restrictive interventricular septal defect, while hearing screening through transient evoked otoacoustic emissions (TEOAE) revealed normal results. The patient was discharged from the NICU at 10 days of life and enrolled in a multidisciplinary follow-up. At 3 months of age an early teething was observed. Owing to such new findings, along with the already reported dysmorphic features, a genetic evaluation was performed, providing the indication to carry out Sanger sequencing of the ANKRD11 gene. The genetic investigation identified the c.1903_1907delAAACA variant (p.Lys635GlnfsTer26) of the gene (Ref Seq NM_013275.6), for KBG syndrome diagnosis. Further genomic exams in the trio were refused by parents. Due to recurrent episodes of otitis, an audiological assessment through auditory brainstem response (ABR) evaluation was conducted at 3 years and 6 months of age. It detected a bilateral response threshold at 45 dB (decibel) HL (hearing level), according with mild conductive hypoacusis, which has not required any treatment to date. Clinical examination at that time disclosed additional dysmorphic features, including high forehead, squared face due to chubby cheeks and enlarged chin, prominent and wide ears with hypoplastic antihelix, thick and medially sparse eyebrows with barely detectable synophrys, wide nasal bridge with bulbous tip and anteverted nares. In addition, long philtrum, thin upper lip and retrognathia were also noted (Fig. 3a/b).
Figure 3a/b. Patient 3, at 3 years and 6 months of age. a High forehead, squared face due to chubby cheeks and enlarged chin, thick and medially sparse eyebrows with barely detectable synophrys, wide nasal bridge with bulbous tip and anteverted nares, long philtrum, and thin upper lip. b prominent and wide ears with hypoplastic antihelix, and retrognathia
He is at present 4 years and 2 months old and shows normal growth - according to World Health Organization growth chart for neonatal and infant close monitoring [8]: weight 15 Kg (15th centile, -1.02 SD), height 101.7 cm (32nd centile, -0.46 SD) and OFC 51.5 cm (29th centile, -0.55 SD). On physical examination, macrodontia of the upper incisive teeth has been additionally observed. Furthermore, neurological assessment evidenced mild neuromotor delay, but neither seizures nor further anomalies are reported to date.