Chromosomal structural rearrangements (CSR) have been known for a long time as a potential etiological mechanism in human diseases [1,2,3,4]. The most common ones are reciprocal translocations, consisting of the mutual exchange of non-homologous genomic material between two chromosomes, and accounting for approximately 0.1–0.2% of human population [5]. However, their prevalence is likely underestimated, only referring to karyotype-detectable ones. Structural rearrangements arising from more than two chromosomal breaks are usually considered as complex rearrangements.

Although CSR do not usually result in a clinical phenotype and can segregate across generations, an increased risk of congenital anomalies is documented, which is two or three times higher than in the general population [6]. Different mechanisms can be associated with a pathological outcome, such as loss of genomic material at the breakpoints, genes physical disruption, positional effects, chimeric gene formation, and alteration of genomic environment having a role in gene regulation. Therefore, CSR detection, together with a fine characterization of the breakpoints, is crucial from a diagnostic point of view.

Despite their acknowledged importance, structural rearrangements are often overlooked during routine genetic analyses, due to the technical limitations of the currently used genomic platforms, mainly represented by Chromosomal Microarray Analysis (CMA) and Exome Sequencing. In fact, while these technologies are exponentially increasing the diagnostic detection rate, revealing copy number and sequence variants all over the genome, they cannot provide clear information about structural variants. Karyotype is still considered the only technique able to detect CSRs in laboratory routine. However, the resolution level of optical microscopy does not allow a fine characterization of the breakpoints, limiting the diagnostic application and utility of cytogenetic analyses.

The recent introduction and availability of genomic techniques such as Whole Genome Sequencing (WGS) and Optical Genome Mapping (OGM) is enabling a rapid detection and a fine characterization of chromosome breakpoints at a high resolution, providing further information on molecular mechanisms underlying genetic diseases.

Here, we describe three non-related additional patients presenting with different types of CSR interrupting the SET Binding Protein 1 (SETBP1) gene.

SETBP1 haploinsufficiency disorder (SETBP1-HD) (MIM#616,078) is caused by either heterozygous gene deletions or loss-of-function (LoF) variants. It is associated with a genetic condition, mainly characterized by intellectual disability (ID), also known as “Intellectual disability, autosomal dominant 29” (MRD29). Expressive speech and language impairment appear as a prominent feature of the disorder in association with behavioral problems and mild dysmorphisms, especially in patients with microscopic and submicroscopic 18q12 deletions involving SETBP1 [7,8,9]. Pathogenic SETBP1 gain of function variants, on the other hand, are associated with an ominous different syndromic condition (i.e. Schinzel-Giedion syndrome), characterized by severe intellectual disability, distinctive facial features, and multiple congenital malformations including skeletal abnormalities, genitourinary and renal malformations. It mainly differs from SETBP1 haploinsufficiency by neurodegeneration and by the lack of a preeminent neurobehavioral component [10].

High-resolution CMA and genomic sequencing are the methods utilized so far to diagnose SETBP1 microdeletion and variants [10].

In our patients molecular diagnosis was obtained through a combined approach based on Optical Genome Mapping and WGS, providing a fine characterization of the genomic sequence at the breakpoints, and suggesting CSR as a new etiological mechanism for SETBP1-HD.

Subject ID1: Subject ID1 is a 10-year-old boy and he was born at 37th gestational week by emergency cesarean section due to fetal sufferance. Family history was positive for generalized epilepsy due to neonatal hypoxia and for Hodgkin lymphoma in the maternal line. Due to a previous pregnancy interrupted at the 21st gestational week (therapeutic abortion) for transposition of the great vessels in a male fetus, an amniocentesis was performed, detecting a de novo balanced translocation between the long arms of chromosomes 15 and 18 (karyotype 46 XY,t(15;18)(q24;q21).

Birth weight was 2,680 g (22th centile, 0.77 SD), length was 46 cm (10th centile, -1.3 SD), and occipito-frontal circumference (OFC) was 35 cm (85th centile, 1.02 SD). Apgar scores were 6 and 8 at the minutes 1and 5, respectively. Neonatal jaundice was treated with phototherapy. Transient-evoked otoacoustic emissions resulted pass on the right side and refer on the left one. Auditory brainstem responses were normal bilaterally. Transfontanellar ultrasound detected ventricular enlargement and right choroid plexus cyst. Bilateral cryptorchidism was detected and corrected by orchidopexy at the age of two, while abdominal ultrasound was normal.

He was breast-fed from the 5th day-life after initial suction difficulties. Weaning was characterized by chewing difficulties and reduced tolerance for different tastes and textures. Motor development was delayed and characterized by relevant hypotonia: he obtained head control at 5 months and autonomous ambulation at the age of 19 months. He presented with consistent speech delay; expressive language was far more impaired than receptive abilities, and the child was able to utter vowels, syllables, onomatopoeia but not words, with a reduced phonetic inventory. Speech articulation was evidently hard, and he obtained a diagnosis of developmental verbal dyspraxia.

Social interaction and non-verbal communicative intent were valid and mediated by gestures. Play schemes were poorly organized. Oppositional-defiant behavior, brief attention and prestation discontinuity characterized his functioning. Joint laxity and poor motor coordination were evident.

At the age of 4 an episode of febrile convulsion occurred. Awake electroencephalogram showed bilateral posterior slow rhythmic activity. Brain MRI detected mild gyral asymmetry on anterior temporal convolutions, not considered as a pathologic finding. Ophthalmological evaluation showed hypermetropia. Echocardiography detected patent ductus arteriosus with left-to-right shunt.

At 4 years he was tested for his cognitive development using Griffiths Mental Development Scales-II edition, and moderate developmental delay (DD) was diagnosed (Developmental Quotient below 3 standard deviations). At the age of 7, Leiter-3 (non verbal intelligence quotient 54) and Vineland Adaptive Behavior Scale-II edition (worse communicative and motor abilities in comparison with better social and day life abilities) were administered and confirmed moderate ID.

Conners’ Parents Rating Scales were positive for Attention Deficit Hyperactivity Disorder (ADHD), and clinical evaluation confirmed this condition.

Physical examination revealed arched eyebrows, hypertelorism, bilateral epicanthus, ptosis, down-slanting palpebral fissures, enlarged nasal sella with pressed columella and arched nasal pinnae, marked nasal philtrum, tented upper lip, retro-rotated low-set ears with chubby ear-lobes, pointed chin, short neck, pectus excavatum, wide-spaced nipples, pes planus (Fig. 1). OCF had grown within the upper limits of the norm. Globally, his phenotype resembled a Noonan syndrome (NS) -like condition.

Patient ID 1 physical features. a bushy arched eyebrows, wide spaced eyes, bilateral epichantus, ptosis (prevalent in left eye), downslanting palpebral fissures, enlarged nasal sella with pressed columella and arched nasal pinnae, marked nasal philtrum, pointed chin; b chubby ear-lobes, low set-posteriorly rotated ears; c short neck, wide-spaced nipples, pes planus; d tented upper lip, pectus excavatum, widely spaced nipples

Muscular hypotonia, evident in the first years of life, led to DMPK gene analysis in order to exclude myotonic dystrophy, which resulted normal (12 CTG triplets).

Loss of genetic material in the translocation regions had been excluded by array Comparative Genomic Hybridization (a-CGH), although it revealed a de novo 276 kb deletion on the short arm of chromosome X (arr[GRCh38] Xp22.1(22,818,207_23,093,865) × 0) involving DDX53.

A Noonan-customized gene panel.

Exome sequencing by means of Twist Human Core Exome Kit (Twist Bioscience), revealed heterozygous mutations in genes related to recessive conditions, therefore they were not considered causative of our patient’s phenotype (c.661G > A (p.Ala221Thr) inherited from his father in TMCO1; 2791A > T (p.Asn931Tyr) in SPTBN4 and c.8713C > T (p.Arg2905Cys) in ANK3 inherited from his mother).

Subject ID2: Subject ID2 is a 4-year-old male, the only child of non-consanguineous parents. Family history was unremarkable. Pregnancy was uneventful. Spontaneous delivery was at 41 + 2 weeks. Birth weight was 3,420gr (39th centile, 0.29 SD), length 53 cm (91th centile, 1.32 SD), and OFC 35 cm (53th centile, 0 SD). A cerebral ultrasound performed after birth was normal. At 6 months of age urinary ultrasound documented vescico-uretheral reflux and mild right caliectasis of 5 mm. Motor development was delayed: he walked unsupported at 15 months. At 2 years speech delay was noticed. The patient has a discrete verbal comprehension but still no verbal capacity. Vocalization was absent and he only started producing afinalistic babbling sounds at the age of 4 and reported the word “mommy”. He required assistance in hygiene and dressing. Griffith cognitive scale performed at 45 months displayed a mild DD with severe language impairment; Developmental Quotient was 71, with disharmonious profile between the various index (Locomotor abilities 84, Personal and Social abilities 75, Hearing and Language abilities 24, Eye and Hand Coordination 84, Performance abilities 84, Practical Reasoning 75). At 3 years and 1 month, he started speech therapy and he was attending primary school with support.

Audiometric test was normal. Growth parameters were within normal range. He did not present gastrointestinal problems.

At the last physical evaluation (3 years and 10 months), growth parameters were: weight 17 kg (70th centile, 0,51 SD), stature 107 cm (93th centile, 1.5 SD), and OFC 50,6 cm (58th centile, 0.2 SD). Physical examination disclosed dysmorphic facial features and signs that remained unchanged over the time, including triangular face, hypertelorism, epicanthus, narrow palpebral fissures, prominent nasolabial fold, normal setting of the ears, high narrow palate, clinodactyly of the fifth toe.

Genetic analyses, including FRAXA, 15q MS-MLPA for Angelman syndrome exclusion and array-CGH at an average resolution of 100 kb, tested negative. Karyotype analyses revealed a de novo reciprocal translocation involving the long arm of a chromosome 18 and the short arm of a chromosome 16, reported as 46,XY,t(16;18)(p13.2;q21.1).

Subject ID3: Subject ID3 is an 8 year and 6 months-old boy with absent speech, DD, and facial dysmorphisms slightly resembling a RASopathy. He was born at 40 weeks of gestation by cesarean section for dystocic presentation after a pregnancy obtained with heterologous artificial reproductive technique (sperm donor) and complicated by threatened abortion occurring during the first months and maternal hyperglycemia treated with diet. Birth weight was 3250 kg (30th centile, -0.5 SD), length was 51 cm (60th centile, + 0.2 SD), and OFC was 36 cm (87th centile, + 1.1 SD). Apgar scores were 9 and 9 at minutes 1 and 5, respectively. Soon after birth, he was admitted to Neonatal intensive care unit for respiratory distress, right subtotal pneumothorax, partial left pneumothorax, treated with pleural drainage and requiring directional positive air-way pressure assistance. At birth, bilateral ptosis with severe involvement of the left eye was noticed. Weaning was characterized by chewing difficulties and reduced tolerance for different tastes and textures. He presented with DD with generalized hypotonia (first words at 4 years but lost soon afterwards, walked unassisted at 3 years with a wide base gait, no sphincter control at 8 years). At nine months, he experienced repeated febrile seizures treated with Phenobarbital and Sodium Valproate until the age of 6. He is currently without seizures. At our first examination (8 months) growth parameters were: weight 7.8 kg (13th centile, -1.13 SD), height 74 cm (91th centile, + 1.34 SD), and OFC 45 cm (57th centile, + 1.18 SD). An abdominal ultrasound exam revealed mild left pielic ectasia. Bilateral cryptorchidism was also detected.

Dysmorphic facial features included high anterior hairline, high forehead with mild bitemporal narrowing, bushy eyebrows, wide and high nasal bridge, mild widely spaced eyes, epicanthic folds, bilateral ptosis with blepharophimosis, low-set and large, fleshy ears, short nose with anteverted nostrils, deep nasolabial folds, broad long philtrum, short tongue frenulum surgically treated at 9 months, narrow palate, long pointed chin. He presented with a happy demeanor.

Palpebral ptosis was surgically treated at 3 and 7 years, with partial resolution.

A brain MRI did not reveal structural brain anomalies, echocardiogram and audiologic evaluations were normal.

Clinical evaluation at 8 years confirmed DD and absent speech for which he required speech therapy and the introduction of augmentative and alternative communication (AAC). He was also diagnosed with ADHD with autistiform traits characterized by repetitive movements and high anxiety levels. He attends primary school with support. At the last physical examination at the age of 8 years and 6 months, growth parameters were: weight 23 kg (12th centile, -1.18 SD), height 133 cm (69th centile, + 0.59 SD), and OFC 52.5 cm (54th centile, 0 SD). Dysmorphic features were confirmed, mild pectus excavatum with wide-paced nipples, pes planus, and mild constipation were recorded. He continues on presenting with an unusual happy demeanor with unprovoked laughing.

CMA analysis detected 5 microdeletions, involving chromosomes 2, 6, and 18:

arr[GRCh38] 2q37.1(230,834,026_230,861,014) × 1,2q37.3(237,414,009_238,552,715) × 1,6q27(164,870,831_165,833,376) × 1,6q27(166,036,017_166,385,898) × 1,18q12.3q21.1(44,767,804_48,427,129) × 1

The 18q12.3q21.1 microdeletion extends for 3.65 Mb and involves 16 OMIM genes, including SETBP1, which mapped at the proximal breakpoint of the region, resulting partially deleted.