Clinical and molecular spectrum of CHOPS syndrome

Abstract

CHOPS syndrome is a multisystem disorder caused by missense mutations in AFF4. Previously, we reported three individuals whose primary phenotype included cognitive impairment and coarse facies, heart defects, obesity, pulmonary involvement, and short stature. This syndrome overlaps phenotypically with Cornelia de Lange syndrome, but presents distinct differences including facial features, pulmonary involvement, and obesity. Here, we provide clinical descriptions of an additional eight individuals with CHOPS syndrome, as well as neurocognitive analysis of three individuals. All 11 individuals presented with features reminiscent of Cornelia de Lange syndrome such as synophrys, upturned nasal tip, arched eyebrows, and long eyelashes. All 11 individuals had short stature and obesity. Congenital heart disease and pulmonary involvement were common, and those were seen in about 70% of individuals with CHOPS syndrome. Skeletal abnormalities are also common, and those include abnormal shape of vertebral bodies, hypoplastic long bones, and low bone mineral density. Our observation indicates that obesity, pulmonary involvement, skeletal findings are the most notable features distinguishing CHOPS syndrome from Cornelia de Lange syndrome. In fact, two out of eight of our newly identified patients were found to have AFF4 mutations by targeted AFF4 mutational analysis rather than exome sequencing. These phenotypic findings establish CHOPS syndrome as a distinct, clinically recognizable disorder. Additionally, we report three novel missense mutations causative for CHOPS syndrome that lie within the highly conserved, 14 amino acid sequence of the ALF homology domain of the AFF4 gene, emphasizing the critical functional role of this region in human development.

1 |. INTRODUCTION

Previously, we reported a novel disorder associated with missense mutations in AFF4 in three individuals termed CHOPS syndrome (Izumi et al., 2015). The acronym “CHOPS” describes the shared phenotype of affected individuals: cognitive impairment and coarse facies, heart defects, obesity, pulmonary involvement, short stature, and skeletal dysplasia. All three originally reported individuals were referred to the Children’s Hospital of Philadelphia Center for Cornelia de Lange Syndrome and Related Diagnoses for a suspected diagnosis of Cornelia de Lange syndrome (CdLS), a multisystem disorder characterized by craniofacial dysmorphisms, intellectual disabilities, growth retardation, limb anomalies, and several other systemic abnormalities (Liu and Krantz, 2009). While these three individuals’ features (intellectual disability, short stature, and craniofacial dysmorphisms) overlapped with CdLS (Izumi et al., 2015), they demonstrated clinically distinct medical and physical features, establishing CHOPS syndrome as a novel recognizable diagnostic entity. All three individuals were found to have missense mutations (p.T254S, p.T254A, and p.R258W) in the ALF (AF4/LAF4/FMR2) homology domain of AFF4, which resulted in resistance to proteasomal degradation by SIAH1 (Izumi et al., 2015).

At the time of the initial report, relatively limited phenotypic information was provided on these three individuals. In this report, we present the full clinical history of the previously reported individuals, as well as an additional eight individuals recently diagnosed with CHOPS syndrome. We also include descriptive neurocognitive analysis of three individuals, and provide molecular analysis of two novel missense mutations associated with CHOPS syndrome that further emphasize the importance of the highly conserved 14 amino acid region of AFF4. Overall, we aim to provide a more comprehensive clinical and molecular description of CHOPS syndrome that distinguishes it from other similar diagnoses (such as CdLS) and establishes it as a distinct clinically recognizable diagnostic entity.

2 |. MATERIALS AND METHODS

2.1 |. Study population

The individuals reported in this article were ascertained either at the Children’s Hospital of Philadelphia (CHOP) or referred from other institutions to the CdLS and Related Diagnoses research study at CHOP. Individuals were enrolled in an IRB-approved protocol of informed consent.

2.2 |. Clinical analysis

Clinical data was ascertained from all 11 probands’ charts that contained records from multiple evaluations. In all cases, growth pattern, craniofacial dysmorphia, and cardiac, pulmonary, hearing, skeletal, genitourinary, and gastrointestinal (GI) abnormalities were assessed (Table 1).

TABLE 1.

Summary of clinical findings in CHOPS syndrome

	Case-1	Case-2	Case-3	Case-4	Case-5	Case-6	Case-7	Case-8	Case-9	Case-10	Case-11
AFF4 mutation	p.T254S De novo	p.T254A De novo	p.R258W De novo	p.A255T De novo	p.R258W De novo	p.P253R De novo	p.R258W De novo	p.M260 T De novo	p.R258W De novo	p.R258W De novo	p.R258W De novo
Age/sex	20y/F	17y/M	12y/F	11y/F	23y/M	9y/F	2y/F	7y/M	6y/M	7y/F	1y/M
Craniofacial	Synophrys, upturned nose, long philtrum, thick arched eyebrows, long eyelashes, large anterior fontanelle, microcephaly, microbrachycephaly, Wormian bones on her skull	Synophrys, short upturned nose, large anterior fontanelle, micrognathia, long philtrum, proptotic appearance to eyes	Synophrys, upturned nose, arched eyebrows, thick ears posteriorly rotated	Synophrys, upturned nose, long eyelashes, microcephaly, plagiocephaly	Synophrys, upturned nose, downslanting palpebral fissures, prominent vertical forehead crease, large anterior fontanelle, thick cranial vault with prominent Wormian bones along the lambdoid suture, microbrachycephaly	Mild Synophrys with prominent eyebrows, prominent eyelashes, bilateral proptosis, facial hirsutism, upturned nose, bitemporal narrowing, microcephaly, cupped ears with simple helices, high arched palate, and clubbing of nails, hands and feet	Synophrys, trace proptosis, small upturned nose with shallow bridge, prominent cheeks, macroglossia, micrognathia, bitemporal narrowing, and microcephaly	Synophrys, coarse appearance to features	Round face, brachiocephaly, microcephaly, long eyelashes, high arch eyebrows/curved, broad ICD, ears cupped and somewhat simplified	Round face, synophrys, downturn corners of the mouth, curly hair, long eyelashes and thick eyebrows	Round face, arched eyebrows, long philtrum, thin upper lip, low-set posteriorly rotated ears
Birth parameters: height		48 cm (−0.75 SD)	43 cm (−2.98 SD)	43 cm (−3.6 SD)	48 cm (−0.75 SD)	38 cm (−6.17 SD)	43 cm (−2.98 SD)	43 cm (−1.9 SD)		50 cm (+0.8 SD),	39 cm (−5.8 SD)
Weight	2,381 g (−1.94 SD)	2,494 g (−1.69 SD)	1,757 g (−2.94 SD)	2,182 g (−2.27 SD)	3,430 g (−0.18 SD)	1,474 (−3.34 SD)	2,239 g (−2.18 SD)	2070 g (−1.5 SD)	1,170 g (−3.13 SD)	2,570 g (−1.1 SD)	1,560 g (−4.6SD)
Head circumference (HC)	31.1 cm (−2.3 SD)	33 cm (−1.32 SD)		32 cm (−1.6 SD)	34.5 cm (0 SD)		29.5 cm (−3.7SD)	29 cm (−3.23 SD)		33 cm (+0.1 SD)	29 cm (−4.3 SD)
Latest growth parameters: height	118.1 cm (−6.9 SD)	106 cm (−5.8 SD) at 11yo	83.7 cm (−7.3 SD) at 6yo	120 cm (−3.5 SD)	142.7 cm (−4.6 SD)	93.8 cm (−5.9 SD)	73 cm (−4.1 SD)	90 cm (−6.1SD)	88 cm (−5.7 SD)	98.5 cm (−4.7SD)	66 cm (−4.1 SD)
Weight	32.9 kg (−3.7 SD)	26 kg (−2 SD) at 11 yo	15.6 kg (−2.1 SD) at 6 yo	41.7 cm (+1.5 SD)	84.8 kg (+0.98 SD)	20.9 kg (−2 SD)	10.82 kg (−1.1 SD)	10th centile	17.3 kg (−1.5 SD)	16 kg (−2.1 SD)	8.6 kg (−1.1 SD)
HC	53 cm (25th centile) at 14 yo		−3 SD at 6 yo	50 cm (−2 SD)	58 cm (+2 SD)	47 cm (−4.1 SD)	43.5 cm (−2.8 SD)	3rd centile	48 cm (−2.6 SD)	47.6 cm (−1.9 SD)	42 cm (−3.2 SD)
Cardiac	PDA chronic right-sided heart failure, pulmonary hypertension	VSD, PDA anomalous pulmonary vein	PDA, PFO, VSD	VSD, PDA	None reported	VSD, congenital dilated aortic root, pulmonary hypertension	PDA, PFO, dilated aortic root	None reported	PDA, PFO	VSD	None reported
Respiratory	Chronic lung disease of uncertain etiology (status post ARDS) and low oxygen saturations, sleep apnea, asthma, history of aspiration and pneumonia	Subglottic stenosis (grade 2–3), tracheomalacia, glossoptosis, laryngomalacia, tracheal stenosis, bronchitis, chronic lung disease, tracheostomy, aspiration pneumonia, obstructive sleep apnea	Congenital narrow oropharynx, laryngomalacia, chronic lung disease, RAD, tracheostomy, obstructive sleep apnea	Chronic respiratory issues when young-resolved	Noisy breather and snores but not significant apnea	Subglottic stenosis, chronic lung disease and respiratory failure, tracheostomy, idiopathic pulmonary hemosiderosis, diffuse pulmonary alveolar hemorrhage, acute interstitial pneumonia	History of pneumonia, chronic lung disease, chronic congestion and noisy breathing, acute chronic respiratory failure, subglottic narrowing of trachea	History of aspirations	Chronic lung disease due to apnea and aspirations, tracheostomy	None reported	Episodes of pneumonia and upper respiratory infection
Hearing loss (HL)	No HL	Mixed bilateral HL	No HL	No HL	Mild sensorineural HL	Bilateral sensorineural HL	Mild conductive HL	Mixed HL	Bilateral sensorineural HL (improved)	Bilateral conductive HL	Mixed bilateral HL
Skeletal	Brachydactyly, small hands with proximally placed thumbs, abnormal shape of vertebral bodies with decreased height most pronounced from T8 to L1	Brachydactyly, congenital fusion of vertebrae C2-C3, history of congenital hip subluxation bilaterally, skeletal dysplasia, hypoplastic changes of L1 and L2 with anterior beaking, hypoplastic toenails, posterior neck folds	Brachydactyly, deformity and hypoplasia of the proximal radius and fibula bilaterally, focal lumbar kyphoscoliosis at the thoracolumbar junction	Brachydactyly, short stature treated with growth hormone treatment	Brachydactyly, joint hypermobility (Beighton score 6/9), mild truncal hypotonia, osteopenic bones, immature ovoid appearance to the vertebral bodies of the thoracic and lumbar spine, narrowing of the T11/12 intervertebral disc space, mild shortening of the proximal humeri and metacarpals, hypoplasia of the distal phalanges of the toes, pes planus, limited extension of the right elbow, small hands and feet	Absent left thumb (not congenital)	Disharmonic skeletal maturation of the hands	Brachydactyly	None reported	Brachydactyly	Brachydactyly; small hands and feet, left fifth finger clinodactyly, single crease on left fifth finger, abnormal shape of vertebral bones, short iliac bones, small left femoral head
Genitourinary (GU)	Horseshoe kidney with repeated episodes of acute kidney injury, chronic kidney disease, diuretic-associated metabolic alkalosis and hypokalemia	Cryptorchidism, bilateral vesicoureteral reflux	Mild grade I vesicoureteral reflux	None reported	Horseshoe kidney, mild coronal hypospadias (repaired), small genitalia	Single kidney, sexual precocity	None reported	None reported	None reported	None reported	Short prepuce
GI	Vomiting and dehydration with delayed gastric emptying, constipation, Candida esophagitis, GERD	Nissen-G tube, constipation	G tube, constipation	None reported	None reported	Nissen-G tube, constipation, GERD	G tube, dysphagia	None reported	G tube	G tube	History of constipation
Other	Coarse hair	Myopia	Bilateral cataracts, eczema	Hypothyroidism	Strabismus, bilateral ptosis, gynecomastia, acanthosis nigricans, hirsutism	Astigmatism, otorrhea, optic nerve hypoplasia of left eye, intermittent exotropia, ptosis, chronic bilateral otitis media, hyperglycemia, polycythemia, steroid-induced diabetes mellitus, iatrogenic adrenal insufficiency, prematurity	Obstruction of nasolacrimal duct, chronic bilateral otitis media, sacral mass (removed), jaundice	Mild myopia with strabismus, skin changes (sun exposed erythema and mottling of his skin)	Cataract, myopia, amblyopia, exophoria, growth hormone deficiency	Cataract, low lying conus with lipoma of the filum terminale

Abbreviation: GI, gastrointestinal.

Neurocognitive assessment was done by mailing three different surveys to 10 of the 11 families, which were completed by each individual’s mother. The surveys from three families were received and analyzed by a developmental pediatrician. These surveys included the Vineland Adaptive Behavior Scale (measures an individual’s adaptive behaviors—day to day activities that are necessary for individuals to get along with others and take care of themselves), developmental behavior checklist (measures a broad range of behavioral and emotional disturbances in people with intellectual disabilities), and the aberrant behavior checklist (assesses problem behaviors of children and adults with mental retardation at home, in residential facilities, and work training centers).

Food-related behaviors were assessed through completion of the food-related problems questionnaire (FRPQ) distributed to 10 of the 11 families. The survey consists of a 16-item questionnaire consisting of Likert scale questions used to address three subscales including (a) preoccupation with food, (b) impairment of satiety, and (c) other food-related “challenging” behavior. Six completed FRPQ questionnaires were received and analyzed.

2.3 |. Cell culture

The HEK293T cell line was cultured in DMEM with 4.5 g/L d-Glucose and 110 mg/L sodium pyruvate (Life Technologies) supplemented with 10% FBS, and 0.2% penicillin–streptomycin. Cells were cultured at 37°C in 5% CO₂. HEK293T cells were grown to 60% confluency and vectors were transiently transfected using Lipofectamine 2000 (Life Technologies, #11668–027) according to the manufacturer’s instructions.

2.4 |. Reagents

A plasmid encoding Myc-DDK-tagged AFF4 (RC217692) was obtained from OriGene Technologies. This plasmid, containing the AFF4 variant T254A, as well as a HA-His-tagged SIAH1a plasmid generated from a plasmid encoding Myc-DDK-tagged SIAH1a (Origene Technologies, RC206576), were used (Izumi et al., 2015). Mutagenesis to introduce either the AFF4 A255T or P253R variant into the wild-type AFF4 plasmid was performed using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Inc., E0554S). Sanger sequencing confirmed the intended change and ruled out additional, nonspecific changes.

2.5 |. Immunoblotting

At 48 hr post-transfection, cells were washed with phosphate buffered saline and total cell extracts were harvested in RIPA lysis buffer (Cell Signaling Technology, #9806, 20 mM Tris–HCL pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin) supplemented with complete mini protease inhibitor cocktail (Roche, 04693159001) and PhosSTOP (Roche, 4906845001) for 30 min. After pelleting cell debris by centrifugation at 20,000g for 30 min, protein concentration was measured on the supernatant using Peirce BCA Protein Assay Kit (Thermo Scientific, 23225). Lysates were mixed with 2x Laemmli Sample Buffer (Biorad, #161–0747) and incubated at 96°C for 5 min. Protein lysate (20 μg) was then subjected to standard sodium dodecyl sulfate-polyacrylamine gel electrophoresis using a 3–8% polyacrylamide tris/acetate gel (Invitrogen, EA0378BOX) and blotted to a polyvinylidene difluoride membrane (Biorad, 1620264). The membrane was blocked in 5% dry milk in TBS and 0.1% Tween 20 (TBST) and subsequently immuno-blotted using either anti-FLAG M2 antibody (Sigma-Aldrich, F3165) or anti-HA-Tag antibody (Cell Signaling Technology, #3724). The membrane was then incubated with either mouse anti-rabbit (Jackson ImmunoResearch Laboratories, Inc., 211-032-171) or donkey anti-mouse (Jackson ImmunoResearch Laboratories, Inc., 715-035-150) horseradish peroxidase-conjugated secondary antibody. The membrane was then washed with TBST, incubated with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, 32106), and exposed to film (Denville Scienfitic, Inc., E3018).

3 |. RESULTS

Key salient features were noted in each individual with CHOPS syndrome. Figure 1 depicts the clinical phenotype (facial features, hands, and feet) in 10 individuals and Figure 2 displays the evolution of the facial features over time in four individuals with CHOPS syndrome. Figure 3 demonstrates skeletal features of CHOPS syndrome. All clinical features are summarized in Table 1. Case description of each case can be found in the Supporting Information.

FIGURE 1.

Facial profiles and hands and feet of 10 individuals with CHOPS syndrome. (a) Facial features. Note consistency of facial features across genders, ages, and ethnic backgrounds. There is a tendency toward a round/full face with coarse features, high/arched eyebrows, synophrys, upturned nose, and long eyelashes. Case-7, 1.5-year-old female (a); Case-8, 4-year-old male (b); Case-9, 6-year-old male (c); Case-6, 7-year-old female (d); Case-3, 8-year-old female (e); Case-4, 8-year-old female (f); Case-1, 10-year-old female (g); Case-2, 12-year-old male (h); Case-5, 23-year-old male (i); Case-10, 10 year old female. Figure 1(b)) hands and palms of CHOPS syndrome. Case-1, female at 10 years (1a,b), Case-3, female at 6 years (2a,b,c, d), Case-7, female at 1.5 years (3a,b), Case-8, male at 4 years (4a,b). (c) Feet of CHOPS syndrome patients. Case-8, male at 4 years (1a,b); Case-1, female at 10 years (2a,b); Case-3, female at 6 years (3a,b); Case-5, male at 23 years (4a,b); Case-10, (5a,b). Brachydactyly is apparent in the hands and feet. Written permission to publish the photographs was obtained from the parents

FIGURE 2.

Progression of the facial features in four CHOPS syndrome patients. Case-1 at 4 years (1a,b), 6 years (1c,d), 10 years (1e,f), 14 years (1g), 17 years (1h). Case-2 at birth (2a,b), 8 years (2c,d), 12 years (2e,f), 14 years (2 g,h). Case-3 at 6 years (3a,b) and 8 years (3c,d), 10 years (3e,f). Case-6 at 7 years (4a,b) and 10 years (4c,d). Written permission to publish the photographs was obtained from the parents

FIGURE 3.

Skeletal features of CHOPS syndrome. Case-1: Note abnormal morphology of vertebral bodies. Case-3: Note deformity and hypoplasia of the proximal radius and hypoplasia of the fibula bilaterally

3.1 |. Summary of clinical features

3.1.1 |. Dysmorphic features

All 11 individuals presented with features reminiscent of CdLS including synophrys, upturned nasal tip, arched eyebrows, and long eyelashes. Additional distinctive facial dysmorphia included anteverted nares, coarse facies, and facial fullness (Figures 1 and 2).

3.1.2 |. Growth pattern

Average birth length, weight, and head circumference (HC) were around −2 to −3 SD (Figure 4). However, the measurements at the latest clinic visit indicated average height was around −5 SD, although HC remained around −2 SD (Figure 4). Notably, weight was increased to about −1 SD (Figure 4). All 11 individuals had short stature and obesity. All individuals’ weight percentiles were disproportionally higher than their height percentiles. Other noted recurrent features were large anterior fontanelle (Case-1, 2, 5), and Wormian skull bones (Case-1, 5).

FIGURE 4.

Box plots demonstrating the distribution of growth parameters of CHOPS syndrome. Y axis represents the standard deviation (SD). Bold line indicates median. The top of each box represents the first quartile, and the bottom of each box represents the third quartile. The top of each whisker indicates the upper extreme, and the bottom of each whisker indicates the lower extreme. Left: Box plot demonstrating the birth measurements’ SD. Right: Box plot demonstrating the latest growth measurements’ SD

3.1.3 |. Heart defects

Congenital heart disease was present in 8 of the 11 individuals reported here. Of these eight individuals, six had a patent ductus arteriosis (PDA) (Case-1, 2, 3, 4, 7, 9), five had a ventricular septal defect (VSD) (Case-2, 3, 4, 6, 10), and three had a patent foramen ovale (PFO) (Case-3, 7, 9). Pulmonary hypertension was found in Case-1 and Case-6, and a dilated aortic root was noted in Case-6 and Case-7. No cardiac abnormalities were noted for Case-5, Case-8, and Case-11.

3.1.4 |. Pulmonary and respiratory tract involvement

Pulmonary and respiratory tract involvement was found in 8 of the 11 individuals. Chronic lung disease was noted in six individuals. Additionally, three subjects (Case-2, 3, 9) required a tracheostomy, with two of these having laryngomalacia (Case-2, 3). Four individuals had a history of pneumonia (Case-1, 2, 7, 9). Structurally, two cases (Case-2, 6, 7) had subglottic stenosis, and Case-3 had a congenital narrow oropharynx. Pulmonary alveolar hemorrhage occurred in Case-1 and Case-7, and sleep apnea was documented in Case-1, 3, 9. Case-5, Case-10, and Case-11 did not have any reported pulmonary and respiratory tract issues, and Case-4’s early pulmonary abnormalities resolved.

3.1.5 |. Skeletal dysplasia

Brachydactyly was present in 8 out of the 11 individuals. An abnormal shape of the vertebral bodies with decreased height, pronounced from T8 to L1, was noted in Case-1 (Figure 3). Congenital fusion of vertebrae C2-C3 and a history of congenital hip subluxation along with hypoplastic changes of L1 and L2 with anterior beaking was noted in Case-2. Hypoplasia of the proximal radii bilaterally, hypoplasia of the fibula bilaterally was noted in Case-3 (Figure 3). Narrowing of the T11 and T12 intervertebral disc space along with shortening of the proximal humeri and metacarpals, and hypoplasia of distal phalanges, were noted in Case-5. Abnormal shape of vertebral bones was also noted in Case-11.

3.1.6 |. Genitourinary abnormalities

Genital abnormalities in males included cryptorchidism (Case-2), hypospadias (Case-5), and small genitalia (Case-5 and Case-11). No genital abnormalities were reported in females. Other anomalies of the genitourinary system included horseshoe kidney (Case-1, 5) and vesicoureteral reflux (Case-2, 3).

3.1.7 |. Gastrointestinal abnormalities

GI abnormalities were present in 7 out of the 11 individuals. No GI issues were reported for Case-4, 5, 8, and 11. Six individuals (Case-2, 3, 6, 7, 9, 10) required G-tube insertion. Constipation was present in four individuals (Case-1, 2, 3, and 11).

3.1.8 |. Additional features

Other reported abnormalities included myopia (Case-2, 8, 9), strabismus (Case-5, 8), cataracts (Case-3, 9, 10), ptosis (Case-5, 6), hypothyroidism (Case-4), acanthosis nigricans and skin changes (Case-5, 8), and gynecomastia (Case-5). Eight out of 11 individuals were reported to have some type of hearing loss (described in Table 1).

3.2 |. Neurocognitive analysis

All individuals with CHOPS syndrome had developmental delay, and varying degrees of intellectual disability. The age at which individuals with CHOPS syndrome started rolling ranges between 9 and 15 months (average: 12 months), age to sit independently ranges between 11 months and 3 years (average: 18 months), and age to walk independently ranges between 3 and 6 years (average: 4.8 years). Neurologically, one individual had febrile seizures and two patients (Case-1 and 10) had a neurological anomaly of a low-lying conus with lipoma of the filum terminale. The average composite Vineland score between the three individuals (Case-1, 4, and 5) evaluated was 44, ranging from 23 to 71 (Table 2). This score reflects adaptive behavior in the areas of communication, daily living, social skills, and relationships. An average score of 44 indicates that these individuals fall into the low adaptive functioning range.

TABLE 2.

Neurocognitive survey results of Case-1, Case-4, and Case-5

	Case-1	Case-4	Case-5
Vineland adaptive behavior scale composite	38 (low)	71 (moderately low)	23 (low)
Developmental behavior checklist Total behavior problem score	46 (low)	36 (moderately low)	Not evaluated
Aberrant behavior checklist-subscale irritability	8	2	0
Aberrant behavior checklist-subscale lethargy	2	1	0
Aberrant behavior checklist-subscale stereotypy	6	0	0
Aberrant behavior checklist-subscale hyperactivity	31 (high)	4	0
Aberrant behavior checklist-subscale inappropriate speech	3	1	3

Note. Values were marked by parentheses if they fell out of the average range.

Case-1 had a composite score of 38, which classifies her general adaptive functioning as low; she scores higher than only less than 1% of similarly aged individuals within the Vineland-II norm sample. In the communication domain, Case-1’s abilities were low for hearing and understanding, talking, reading, and writing. In the Daily Living Domain, Case-1’s abilities were low in caring for self, caring for home, and living in the community. In the Social Skills and Relationships domain, abilities were low for relating to others, playing and using leisure time, and adapting. The Vineland-II also measures problem behaviors, which are defined as behaviors that are considered undesirable. These behaviors are elevated for Case-1.

Case-4 had a composite Vineland score of 71, indicating that she fell into the low to moderately low category for adaptive functioning and all subdomains while Case-5 had a composite score of 23, indicating that he had low levels of adaptive functioning and in all subdomains. Both Case-1 and Case-5 fell within 4–5 standard deviations lower than the norm.

The Developmental Behavior Checklist assesses behavioral and emotional disturbance in children and adolescents with intellectual disability. This composite score was broken into subdomains of disruptive, self-absorbed, communication disturbance, anxiety, social relating, and depressive behaviors. Scores with percentiles could not be generated for young adults over the age of 18, so the quantitative reports were excluded for Case-1 and Case-5. Case-4 received an IQ percentile of 41.9th for her total behavior problem score with the highest percentile (69th) in communication disturbance. Most problematic behaviors in all individuals included disruptive, self-absorbed, and communication disturbance behaviors. Common disruptive behaviors included excessive food seeking behaviors, throwing off clothes, and temper tantrums. Significant autistic behavior was not observed in the individuals.

The reports from the aberrant behavior checklist (ABC), which is a symptom checklist for assessing problem behaviors in children and adults with mental retardation, varied from individual to individual. The checklist was divided into five subscales consisting of irritability, lethargy, stereotypy, hyperactivity, and inappropriate speech. Case-1 displayed average ratings in all subscales except for hyperactivity, which was above average. Case-4 and Case-5 displayed average ratings in all subscales. This indicates that a more robust measure is needed to supplement the ABC. The behavioral ratings based on the surveys are documented in Table 2.

3.3 |. Food-related problems analysis

The FRPQ is designed to asses food-related problems and behaviors in individuals with Prader–Willi syndrome (Russell and Oliver, 2003). The FRPQ is a 16-item questionnaire measuring preoccupation with food, impairment of satiety, composite negative behaviors, behaviors related to taking and storing food, ingestion of inedible objects, and inappropriate responses. Each question is rated on a 7-point frequency scale (0 = never, 6 = always). The parent/caregiver of six patients completed the FRPQ survey (Case-1, 2, 4, 5, 6, 7). Response rate was addressed for each question. The group of questions pertaining to impairment of satiety category had the highest average scores ranging from 3 to 4.5. For each question relevant to this category, 100% (6/6) of respondents indicated some frequency of an observed behavior. “Eating more than a standard size meal” had the highest average frequency score, 4.5, in this category. In the takes/stores food category, 100% (6/6) of respondents reported their child “helps themselves to food they should not have” which also had the overall highest average frequency rating, 5.17. Eighty percent (4/5) respondents indicated locking food away was necessary with an average frequency score of 4. Regarding the inappropriate response category of questions, 100% (6/6) of individuals have a “negative response when denied food” with an average Likert score of 4. Overall, this data supports food-seeking behaviors as a characteristic of this patient population.

3.4 |. Molecular analysis

AFF4 mutations were found in all probands. The identified mutations were p.T254S for Case-1; p.T254A for Case-2; p.R258W for Case-3, 5, 7, 9, 10, and 11; p.A255T for Case-4; p.P253R for Case-6; and p.M260T for Case-8 (Figure 5).

FIGURE 5.

Locations of AFF4 mutations identified in individuals with CHOPS syndrome. All of the missense mutations were de novo, and mapped to the evolutionally highly conserved ALF homology domain of AFF4. NHD, N-terminal homology domain; TAD, transactivation domain; NLS, nuclear localization signal; NoLS, nucleolar localization signal; CHD, C-terminal homology domain. Human AFF4 protein sequence is NP_055238.1, mouse AFF4 protein sequence is NP_291043.1, chicken AFF4 protein sequence is XP_015149549.1, and zebrafish AFF4 sequence is XP_005173956.1

AFF4 mutations in Case-1, 2, 3, 5, 7, 8, 9, 10, and 11 were identified by exome sequencing, and these variants were confirmed to be de novo. Mutations for Case-4 and 6 were identified using targeted AFF4 sequencing.

3.5 |. Immunoblotting

Previously, HEK293T cells separately overexpressing AFF4 mutants p.T254S (Case-1), p.T254A (Case-2), and p.R258W (Case-3, 5, 7, 9, 10, and 11) showed decreased proteasomal degradation of AFF4 when co-transfected with SIAH1 in comparison to wild-type (WT) AFF4 (Izumi et al., 2015). To test whether AFF4 mutants p.A255T and p.P253R showed a similar resistance to degradation by SIAH1, HEK293T cells were co-transfected with either p.A255T or p.P253R mutants and SIAH1. In comparison to HEK293T overexpressing either WT AFF4 or AFF4 p.T254A, both p.A255T and p.P253R mutants showed a similar resistance to degradation when co-transfected with SIAH1 as the p.T254A mutant, while WT AFF4 experienced a significant decrease when co-transfected with SIAH1 (Figure 6).

FIGURE 6.

Decreased proteasomal degradation of both p.A225T and p.P253R AFF4 mutants in HEK293T cells, similar to previously described p.T254A AFF4 mutant causative of CHOPS syndrome. Western blot demonstrates the disappearance of the WT AFF4 band upon addition of the SIAH1 vector. This disappearance was not noted in 293T cells overexpressing the p.T254A mutant or in the 293T cells overexpressing p.A225T or p.P253R mutants

4 |. DISCUSSION

Here, we describe the clinical features of a cohort of 11 individuals with CHOPS syndrome caused by mutations in AFF4. It is interesting to note that two out of eight of our newly described patients were found to have AFF4 mutations by targeted AFF4 mutational analysis rather than exome sequencing, indicating that the constellation of findings on these individuals represent a clinically recognizable phenotype. These individuals have a distinct phenotype, that is, characterized by short stature, characteristic facial features (synophrys, arched eyebrows, long eyelashes, upturned nasal tip with anteverted nares and coarse, full facies), congenital heart defects, pulmonary involvement, brachydactyly and other skeletal involvement, genitourinary issues, and developmental delay/intellectual disability. Congenital heart disease, pulmonary, and skeletal issues were the most common complications. The congenital heart defects were most commonly PDA and VSD. Pulmonary issues most commonly manifested as chronic lung disease, of as yet undetermined etiology, and skeletal involvement included brachydactyly and abnormally shaped vertebral bodies. Excessive food-seeking behaviors and over-affection were common in all individuals, in addition to pulmonary involvement, which are not seen in CdLS (Basile et al., 2007). Vineland composite scores were generally low for all three individuals evaluated. Collectively, CHOPS syndrome represents a clinically recognizable syndrome that has some overlap with CdLS but is clinically distinct.

Obesity, pulmonary involvement, skeletal findings, and distinct craniofacial features are the most notable features distinguishing CHOPS syndrome from CdLS. Although all 11 individuals have short stature (which is observed in CdLS), all have high BMIs and many were noted to exhibit food-seeking behaviors. Pulmonary issues are not typically observed in CdLS and the pulmonary involvement for children with CHOPS syndrome can be significant with chronic lung disease present in the majority of patients (6/11), with one third of patients requiring tracheostomy. Skeletal involvement is the third distinguishing feature with brachydactyly commonly observed (8/11 patients). Other reported skeletal anomalies included abnormally shaped vertebral bodies with beaking, hip subluxation, and narrowing of intervertebral disc space. Distinct craniofacial features observed in CHOPS syndrome includes coarse facial features with a round face or a fullness to the face which is not observed in CdLS. While individuals with CHOPS syndrome present with four major distinguishing features, they do still have some overlap with CdLS as many of the syndrome’s other features (previously described in this manuscript) such as microcephaly, developmental delay, intellectual disability, heart defects, and hearing loss are also frequently observed in those with CdLS. The facial features, especially in younger individuals do have similarities to CdLS including synophrys, long eyelashes, upturned nose, and arched eyebrows, as the children get older the coarseness and fullness of the face gives a more characteristic CHOPS gestalt.

The p. R258W mutation represents the most common recurrent mutation, and this mutation accounted for 55% of CHOPS syndrome cases. All the mutations identified in CHOPS syndrome were de novo, and those clustered in the ALF homology domain (Figure 5) of the AFF4 protein. Using a cDNA vector transient expression system, we confirmed the pathogenicity of the two newly identified missense mutations (p.A255T and p.P253R), located within the ALF homology domain, associated with CHOPS syndrome (Figure 6). The ALF homology domain was first described in Nilson et al. (1997) as a 175 amino acid domain that shows a high degree of sequence conservation among AFF1, AFF2, and AFF3. Interestingly, this region also exists within AFF4 (amino acids 251–263). Similar to the initially reported three missense mutations, all the novel mutations identified lie within the highly conserved, 14 amino acid domain of the ALF homology domain. Given that p.A255T, p.P254R, and p.M260T all lie within this region, we hypothesized a similar mechanism for these mutations leading to CHOPS syndrome. We have demonstrated that, like p.T254A, p.T254S, and p.T258W, the p.A255T and p.P254R mutations show resistance to proteasomal degradation by SIAH1 in comparison to WT AFF4 (Figure 6). Combined with the clinical data presented here, we conclude that these novel mutations cause CHOPS syndrome in a mechanistically similar manner to the previously reported mutations. Further reinforcing the importance and highly conserved nature of this 14 amino acid region within AFF4, there are no missense variants reported in this region in ExAC (Lek et al., 2016). This highly specific mechanistic pathogenicity, with de novo causative mutations limited to this 14 amino acid region of the AFF4 gene likely contributes to the perceived rarity of this diagnosis.

In conclusion, CHOPS syndrome is a distinct diagnostic entity, that is, phenotypically unique from CdLS and clinically recognizable, enabling physicians to use targeted AFF4 mutational analysis for diagnosis. Furthermore, identification and functional characterization of causative mutations demonstrates the importance of a highly conserved, 14 amino acid region within the ALF homology domain of AFF4 in human development.