Mutations in MAGEL2 and L1CAM Are Associated With Congenital Hypopituitarism and Arthrogryposis

Abstract

Context

Congenital hypopituitarism (CH) is rarely observed in combination with severe joint contractures (arthrogryposis). Schaaf-Yang syndrome (SHFYNG) phenotypically overlaps with Prader-Willi syndrome, with patients also manifesting arthrogryposis. L1 syndrome, a group of X-linked disorders that include hydrocephalus and lower limb spasticity, also rarely presents with arthrogryposis.

Objective

We investigated the molecular basis underlying the combination of CH and arthrogryposis in five patients.

Patients

The heterozygous p.Q666fs*47 mutation in the maternally imprinted MAGEL2 gene, previously described in multiple patients with SHFYNG, was identified in patients 1 to 4, all of whom manifested growth hormone deficiency and variable SHFYNG features, including dysmorphism, developmental delay, sleep apnea, and visual problems. Nonidentical twins (patients 2 and 3) had diabetes insipidus and macrocephaly, and patient 4 presented with ACTH insufficiency. The hemizygous L1CAM variant p.G452R, previously implicated in patients with L1 syndrome, was identified in patient 5, who presented with antenatal hydrocephalus.

Results

Human embryonic expression analysis revealed MAGEL2 transcripts in the developing hypothalamus and ventral diencephalon at Carnegie stages (CSs) 19, 20, and 23 and in the Rathke pouch at CS20 and CS23. L1CAM was expressed in the developing hypothalamus, ventral diencephalon, and hindbrain (CS19, CS20, CS23), but not in the Rathke pouch.

Conclusion

We report MAGEL2 and L1CAM mutations in four pedigrees with variable CH and arthrogryposis. Patients presenting early in life with this combined phenotype should be examined for features of SHFYNG and/or L1 syndrome. This study highlights the association of hypothalamo-pituitary disease with MAGEL2 and L1CAM mutations.

Five patients with hypopituitarism and arthrogryposis had mutations in MAGEL2 or L1CAM. MAGEL2 transcripts are in the hypothalamo-pituitary region, and L1CAM is hypothalamic during human development.

Schaaf-Yang syndrome (SHFYNG) [Online Mendelian Inheritance in Man (OMIM): 615547] is a rare congenital disorder that is often misdiagnosed as Prader-Willi syndrome (PWS) (OMIM: 176270) but includes arthrogryposis within the phenotypic spectrum. Arthrogryposis multiplex congenita (OMIM: 208100), commonly known as arthrogryposis, occurs in 1 of 3000 live births and involves multiple congenital joint contractures in two or more areas of the body, resulting from reduced or absent fetal movement. Arthrogryposis multiplex congenita has been reported in a patient with pituitary ectopia who had seizures thought to be caused by hypoglycemia and was later found to have a small anterior pituitary and an ectopic posterior pituitary (PP); however, no genetic etiology was identified (1). The main overlapping characteristic features of SHFYNG and PWS are hypotonia, feeding difficulties during infancy, global developmental delay/intellectual disability, and sleep apnea (2–4). However, patients with SHFYNG lack certain stereotypical PWS features, such as hyperphagia and subsequent obesity. PWS is linked to the specific locus 15q11-q13 within the genome, where five maternally imprinted (paternally expressed) genes, namely MKRN3, MAGEL2, NDN, NPAP1, and SNURF-SNRPN, and six maternally imprinted small nucleolar RNA genes/clusters are located (3). Different deletions in this region give rise to variable PWS, with a combination of genes being responsible for different manifestations of the disease (5–7).

L1 syndrome describes a range of X-linked disorders including spastic paraplegia, MASA syndrome (mental retardation, aphasia, spasticity, and adducted thumbs; OMIM: 303350), X-linked hydrocephalus with stenosis of the aqueduct of Sylvius (OMIM: 307000), and X-linked complicated corpus callosum agenesis (8). L1 syndrome occurs in 1 in 30,000 individuals and includes hydrocephalus, variably severe intellectual deficits, and spasticity of the lower limbs, with generalized contractures in rare cases. MASA syndrome, named after the characteristic phenotypes present in patients, also includes adducted thumbs in 50% of cases. A small number of patients (<20) have a combination of L1 syndrome and Hirschsprung disease, a rare disorder affecting the colon and leading to severe constipation and intestinal obstruction due to missing ganglion cells in the myenteric (Auerbach) plexus in the colon (9).

In this study, we sought to investigate genetic etiology in five patients from four unrelated families who presented with variable congenital hypopituitarism (CH) and arthrogryposis.

Materials and Methods

Exome sequencing of patients 1 to 5

The full coding regions of patients 1 to 5 were sequenced as follows: by GOSgene, London, UK (patients 1 and 5); by Great Ormond Street Hospital (GOSH) UK as part of the Deciphering Developmental Disorders (DDD) Study (patients 2 and 3); and by colleagues at the Pontificia Universidad Católica de Chile (patient 4). Raw sequencing data were mapped against the GRCh37/hg18 reference genome, and data were analyzed using Ingenuity^® Variant Analysis™ software (https://www.qiagenbioinformatics.com/products/ingenuity-variant-analysis) from Qiagen, Inc (GOSgene Manchester, United Kingdom). All remaining filtered variants were considered potentially pathogenic disease-causing mutations. Exome sequencing and data analysis for patients 1 and 5 were performed by GOSgene as previously described (10), for patients 2 and 3 under the DDD study as previously described (11), and for patient 4 by Ambry Genetics (Aliso Viejo, CA) (www.ambrygen.com) using its standard protocol and filtering criteria. Mutations were confirmed in patients via Sanger sequencing using specifically designed exon-spanning primers that amplify the DNA region containing the variant (annealing temperatures and primer sequences are available upon request). A chromosome microarray was also performed on the twins, patients 2 and 3 (specific details of this protocol are available upon request). The appropriate ethical approval for the genetics and human embryonic tissue expression studies was obtained before this project took place. The patients/patient guardians gave full consent to all clinical and genetic studies carried out on their blood/DNA.

Human embryonic expression studies using in situ hybridization

Human embryonic tissue sections were obtained from the Human Developmental Biology Resource (http://hdbr.org) and were selected from Carnegie stages (CSs) 16, 19, 20, and 23 [equivalent to gestational ages (GAs) 5.5, 6, 7, and 8 weeks, respectively]. Digoxigenin RNA probes were made using purified vectors containing the full-length human cDNA of wild-type MAGEL2 (in the pCR4-TOPO vector; IMAGE ID: 8327725) and L1CAM (in the pCR-XL-TOPO vector; IMAGE ID: 8991945) (Source BioScience, Nottingham, United Kingdom) Gene expression studies were performed by in situ hybridization as previously described (12) to generate a human embryonic hypothalamo-pituitary expression profile for both MAGEL2 and L1CAM.

Results

Patient 1

A white European patient presented at the age of 3.2 years with short stature, hypoglycemia, and arthrogryposis with scoliosis and a flexion deformity of the knees. She had been hypotonic since birth and required nasal oxygen until 5 weeks of age. A respiratory collapse at 7 weeks of age necessitated a prolonged pediatric intensive care unit admission. She was also noted to have laryngeal polyps. She was diagnosed with growth hormone deficiency (GHD), with a peak GH of 6.4 μg/L and an undetectable IGF-I concentration at age 3.7 years (Table 1). GH treatment was commenced at 4 years of age [Fig. 1(A)]. Dysmorphic features were noted, including bulbar palsy, a long face, a prominent forehead, and micrognathia. She also had global developmental delay and a squint with mild optic nerve hypoplasia and cerebral visual impairment. She had central sleep apnea and gastroesophageal reflux. MRI of the brain was reported as normal [Fig. 2(A)].

Table 1.

Phenotypes of Patients With MAGEL2 and L1CAM Mutations

Patient/Sex (M/F)	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5
	F	F	F	M	M
Ethnicity	White European	White European	White European	White Chilean	Afro-Caribbean
Mutation	c.1996dup, p.Q666Pfs*47	c.1996dup, p.Q666Pfs*47	c.1996dup, p.Q666Pfs*47	c.1996dupC, p.Q666Pfs*47	Hemizygous
	MAGEL2, NM_019066	MAGEL2, NM_019066	MAGEL2, NM_019066	MAGEL2, NM_019066	c.1354G>A, p.G452R
	De novo	Not maternally inherited; paternal DNA was unavailable	Not maternally inherited; paternal DNA was unavailable	De novo	L1CAM, NM_000425
		Chromosome microarray showed a 16q11 duplication, 45,186,600–45,416670); Nimblegen 135K Hg18 WG CGH v3.1.	Chromosome microarray: normal	Chromosome microarray: normal	Mother is a heterozygous carrier
Family history	Healthy younger siblings	Healthy older sister	Healthy older sister	Healthy older brother
		2× miscarriages at 7/40	2× miscarriages at 7/40	Mother had bilateral metatarsus varus and a history of recurrent shoulder dislocations
		Prev. stillbirth at 37/40	Prev. stillbirth at 37/40
Perinatal history	Normal antenatal scans	Polyhydramnios	IUGR and polyhydramnios	—	Antenatal ventriculomegaly
	Cesarean; breech	DCDA twin pregnancy	DCDA twin pregnancy		Elective LSCS
	Hypotonic and respiratory distress after birth needing oxygen	Cesarean; breech	Cesarean; breech		Born in good condition
		Resuscitation at birth: APGARs – 2 at 1 min; 1 at 5 min
		Cooled for HIE
		Ventilated to day 4 of life
Gestation, wk	40	36 + 1/40	36 + 1/40	38	40
Birth weight, kg (SDS)	NA	2.7 (+0.14)	2.0 (−1.67)	3.5 (0.07)	3.6 (+0.07)
Endocrine referral	Short stature and previous history of hypoglycemia	Short stature, diabetes insipidus	Short stature, diabetes insipidus	Growth evaluation, short stature	Hypoglycemic seizures
Age at referral, y	3.2	1.10	1.10	2.8	0.7
Height, SDS	−2.14	−1.3	−2.0	−2.84	−1.7
BMI, SDS	0.61	+1.52	−1.52	−0.92	−4.21
Current age, y	9.7	7.6	7.6	4.7	5.0
Height, cm (SDS)	114.8 (−3.21)	120.1 (−0.58)	120.8 (−0.45)	99 (−1.8)	99.3 (−1.95)
Weight, kg (SDS)	23.5 (−1.76)	20.55 (−1.3)	21.15 (−1.1)	12.2 (−3.53)	12.8 (−3.55)
Dysmorphic facial features	Long face	Macrocephaly	Macrocephaly	Long face	Bilateral radial clubbed hands
	Prominent forehead	Long face	Long face	Bitemporal narrowing	Plagiocephaly
	Micrognathia	Bitemporal narrowing	Bitemporal narrowing	Prominent forehead
		Prominent forehead	Prominent forehead	Micrognathia
		Hypotelorism	Hypotelorism	Glossoptosis
		Scaphocephalic shape	Scaphocephalic shape	High arched palate
		Micrognathia	Micrognathia
		Cleft palate	High arched palate
Gastrointestinal	GERD	Constipation	Constipation	Gastrostomy for feeding difficulty, gastro-esophageal reflux; chronic constipation	PEG fed
	Food allergy				Unsafe swallow
					Dysphagia
					Constipation
Cardiac	No known cardiac pathology	No known cardiac pathology	No known cardiac pathology	Ostium secundum interauricular communication, spontaneous closure	Small VSD
Neurodevelopment	Global developmental delay	Severe global developmental delay	Global developmental delay	Global developmental delay	Global developmental delay
	Bulbar palsy	Gross Motor Function Classification System Level 4	Independently walking	Autism spectrum disorder	Generalized hypotonia
		Wheelchair dependent, nonverbal	Communicates using Makaton, eye gaze, and pointing	Hypotonia
		Able to sit if put in sitting position; attempts rolling over	Some vocalization	Some vocalization
			Some self-harm behavior: head banging	Walking with help
				Able to sit independently
Skeletal system	Arthrogryposis	Distal arthrogryposis	Distal arthrogryposis	Contractures with limited extension of the elbows, knees, hips, and finger	Arthrogryposis
	Scoliosis	Scoliosis	Hands/fingers tapering	Camptodactyly	Bilateral radial clubbed hands
	Flexion deformity of knees	Overlapping fifth finger, positions fingers 3/4/5 together		Small hands and feet	Right hip subluxation
		Short long bones			Scoliosis
		11 ribs
Ophthalmology	Squint	Optic nerve hypoplasia and severe sight impairment	Mild optic nerve hypoplasia (fundoscopy) and sight impaired	Strabismus	Bilateral astigmatism, left divergent squint (visual impairment); wears glasses
	Mild optic nerve hypoplasia with cerebral visual impairment	Nystagmus
		Myopic astigmatism
Endocrine assessment	GHD	Central diabetes insipidus since birth – on DDAVP	Central diabetes insipidus since birth – on DDAVP	GHD stimulation test not performed as hypotensive	GHD
	Rest of endocrinology normal	Severe GHD	Severe GHD	Short stature with deceleration of growth rate
		Premature thelarche – resolved	Premature thelarche – resolved	Adrenal insufficiency: peak cortisol 10.2 μg/dL on stimulation; rest normal
		Premature pubarche	Premature pubarche	Cryptorchidism with bilateral orchidopexies
Other features	N/A	Hypoxic ischemic encephalopathy at birth – cooled	Percutaneous endoscopic gastrostomy tube in situ for unsafe swallow	Central sleep apnea	Hydrocephalus (VP shunt soon after birth)
		Overhanging epiglottis	Central sleep apnea and hypoxia – no need for supplemental oxygen
		Percutaneous endoscopic gastrostomy tube in situ for unsafe swallow	Submandibular gland excision for excessive drooling
		Previous ovarian cyst on ultrasonography	Previous ovarian cyst on ultrasonography
			Diaphragmatic eversion
			Pierre Robin sequence without cleft palate.
GH axis, age, y	3.7	0.8	0.8	0.3	0.8
Stimulation test	Glucagon	Overnight GH profile	Overnight GH profile	Not performed because of hypotension	Glucagon
Basal, ug/L	0.6	—	—	—	2.4
Peak, ug/L (NR > 6.7)	6.4	4.8 (only 1 peak max 4.8)	3.2 (only 1 peak max 3.2)	—	3.7
IGF-1, ng/mL (NR)	<25	Undetectable	Undetectable	31.9 (49–289)	<25
Thyroid axis, age, y	3.7	0.8	0.8	0.3	0.6
fT4 pmol/L (NR)	14.4 (9.8–19)	13.8 (9.8–19)	11.3 (9.8–19)	18.8 (10.3–27)	12.8 (9.8–19)
TSH mU/L (NR)	2.8 (<6.0)	1.9 (<6.0)	1.1 (<6.0)	2.87 (<6.0)	1.5 (<6.0)
Adrenal axis
Stimulation test (age, y)	Glucagon (3.7)	(0.8)	(0.8)	Synacthen (2.8)	Glucagon (0.7)
Peak cortisol, nmol/L	989	—	—	281	488
Random cortisol nmol/L (time)	Basal 252 (9 am) and 442 (12 noon at the end of the fast)	Random cortisol 1192 (9 am)	Random cortisol 748 (9 am)	—	173 (7:30 pm)
Pubertal axis	Prepubertal	Prepubertal	Prepubertal	Prepubertal	Prepubertal
Endocrine medication (age at start, y)	GH (4)	GH (1)	GH (1)	HC (2.9)	GH (1)
		DDAVP (from birth)	DDAVP (from birth)	GH (3.5)
MRI brain imaging	Normal	Progressive global cerebral hemisphere atrophy with relative preservation of the posterior fossa structures; small posterior pituitary and small optic nerves; thin corpus callosum	Mature right parieto-occipital infarct; generalized underdevelopment of the brain; posterior pituitary normal; thin corpus callosum	Normal	Right ventricular parietal VP shunt remains in situ; very thin corpus callosum; no evidence of obstructive hydrocephalus; bulky tectum and thin corpus callosum
Most recent results, age, y	7.7	7.6	7.6	4.7	4.6
Cortisol, nmol/L (time)	339 (9 am)	173 (11:45 am)	159 (11:45 am)	—	276 (9 am)
LH, IU/L (NR)	0.2 (0.7–2.0)	—	0.1 (0.7–2.0)	—	<0.1 (0.7–6.5)
FSH, IU/L (NR)	0.6 (0.2–5.8)	—	2.1 (0.2–5.8)	—	0.7 (0.1–5.8)
PRL, mU/L (NR)	131 (45–466)	—	261 (45–466)		194 (57–717)
fT4, pmol/L (NR)	16.6 (10.8–19.0)	10.9 (10.8–19.0)	10.0 (10.8–19.0)	18.8 (10.29–27.03)	13.8 (10.8–19.0)
TSH, mU/L (NR)	2.8 (<6.0)	2.5 (<6.0)	2.4 (<6.0)	1.22 (0.7–5.7)	1.4 (<6.0)
IGF-1, ng/mL (NR)	118 (64–345)	102 (80–233)	172 (80–233)	65 (22–208)	74 (47–231)
IGFBP-3, mg/L (NR)	2.66 (1.6–6.5)	3.73 (1.6–6.5)	4.38 (1.6–6.5)	—	1.60 (1.1–5.2)
Estradiol, pmol/L, or testosterone, nmol/L	—	Estradiol <44	Estradiol <44	—	Testosterone <0.69

Abbreviations: BMI, body mass index; DCDA, dichorionic diamniotic; DDAVP, desmopressin; fT4, free thyroxine; GERD, gastroesophageal reflux disease; HC, hydrocortisone; HIE, Hypoxic ischemic encephalopathy; IGFBP-3, insulin-like growth factor‒binding protein 3; IUGR, intrauterine growth restriction; LSCS, lower segment cesarean section; N/A, not applicable; PEG, percutaneous endoscopic gastrostomy; Prev, previous; PRL, prolactin; SDS, standard deviation score; VP, ventricular parietal; VSD, ventricular septal defect.

Figure 1.

Growth charts of (A) patient 1, (B) patient 2, (C) patient 3, (D) patient 4, and (E) patient 5. The red arrows indicate when GH treatment commenced in the patients.

Figure 2.

Sagittal MRIs for patients 1–5 showing abnormalities in patients 2, 3, and 5. (A) MRI of patient 1 shows a normal anterior and posterior pituitary, with no other anomalies. (B) MRI of patient 2 (a twin) reveals global cerebral hemisphere atrophy with a small posterior pituitary, a thin corpus callosum, and small optic nerves. (C) MRI of patient 3 (a twin) reveals generalized underdevelopment of the brain. The posterior pituitary is normal with small optic nerves and a thin corpus callosum. (D) MRI of patient 4 shows a normal anterior and posterior pituitary, with no other anomalies. (E) MRI of patient 5 shows generalized underdevelopment of the brain and a very thin corpus callosum. AP, anterior pituitary; CC, corpus callosum; ON, optic nerve; PP, posterior pituitary; WML, white matter loss.

Patients 2 and 3

These white European female nonidentical twins with distal arthrogryposis were initially referred with hypernatremia and were then diagnosed with diabetes insipidus (DI) shortly after birth. Subsequent short stature led to a diagnosis of GHD [peak GH to stimulation of 4.8 μg/L and 3.2 μg/L in patient 2 and patient 3, respectively, with an undetectable IGF-I concentration at 0.8 year (Table 1)]. Their DI had been treated with desmopressin since birth, and GH treatment commenced after 1 year of age [Fig. 1(B) and 1(C)]. The patients had distinctive features including macrocephaly, a long face with bitemporal narrowing, frontal bossing, scaphocephaly, micrognathia, and a cleft/high arched palate. Patient 2 had nystagmus with optic nerve atrophy and was severely sight impaired, whereas her sister had optic nerve hypoplasia with visual impairment. They both had global developmental delay. Patient 2 is wheelchair bound and unable to speak, whereas patient 3 is able to stand and has basic vocalization. The twins also had central sleep apnea and scoliosis. Patient 2 had chronic lung disease with supplemental oxygen requirement at night and had a tracheostomy until the age of 6 years. Patient 3 had a tracheostomy until 20 months of age. On MRI, patient 2 showed evidence of progressive global cerebral hemisphere atrophy with relative preservation of the posterior fossa structures, with a thin corpus callosum, a small PP, and optic nerve hypoplasia [Fig. 2(A)]. Patient 3 had generalized underdevelopment of the brain with a mature right parieto-occipital infarct and a thin corpus callosum, optic nerve hypoplasia, and a normal PP [Fig. 2(B)].

Patient 4

A white male patient from Chile presented with short stature and a deceleration in growth rate at the age of 2.8 years. He was diagnosed with GHD (a stimulation test was not performed because of hypotension), adrenal insufficiency with a peak stimulated cortisol level of 281 nmol/L (Table 1), transient hyperprolactinemia, and arthrogryposis. The latter consisted of contractures, shortening of the extremities, and limited extension of the elbows, knees, hips, and fingers, namely camptodactyly. He was started on hydrocortisone at 2.9 years of age and began GH treatment at 3.5 years [Fig. 1(D)]. He had strabismus, global developmental delay with autism spectrum disorder (ASD), generalized hypotonia, and dysmorphic features including a long face with bitemporal narrowing, a prominent forehead, micrognathia, glossoptosis, and a high arched palate. He had gastroesophageal reflux and central sleep apnea, with respiratory complications leading to a tracheostomy. Cardiac complications included an ostium secundum interauricular communication with spontaneous closure. Cryptorchidism resolved with a bilateral orchidopexy. His MRI was normal [Fig. 2(D)].

Patient 5

An Afro-Caribbean male patient presented with antenatal ventriculomegaly and dysmorphic features, including bilateral radial clubbed hands and plagiocephaly. Flexion deformities that affected both the wrists and hands were noted antenatally, and he was diagnosed with distal arthrogryposis with adducted thumbs and flexion deformities of his digits postnatally. A ventriculo-peritoneal shunt was inserted at 4 days of age, and hypoglycemic seizures ensued at the age of 0.7 year. He was later diagnosed with GHD (peak GH, 3.7 μg/L; undetectable IGF-I concentration), and GH treatment was commenced from 1 year of age [Fig. 1(E); Table 1]. Gastrointestinal problems included dysphagia, and the patient was fed via a percutaneous endoscopic gastrostomy. Other phenotypic features in this patient included a ventricular septal defect, severe obstructive sleep apnea, global developmental delay, generalized hypotonia, right hip subluxation, and scoliosis. Bilateral astigmatism with a left divergent squint and subsequent visual impairment was apparent upon eye examination. His MRI revealed a bulky tectum, generalized white matter loss, and a thin corpus callosum, with no evidence of obstructive hydrocephalus [Fig. 2(C)].

Genetic analysis of patients 1 to 5

Whole exome sequencing was performed on the five patients with CH and arthrogryposis at three different institutions. The results identified the MAGEL2 c.1996dupC, p.Q666Pfs*47 truncation mutation in patient 1 (GOSgene), patients 2 and 3 (GOSH UK as part of the DDD Study), and patient 4 (Pontificia Universidad Católica de Chile). A chromosome microarray was also performed on the twins (patients 2 and 3), which revealed the 16q11 duplication 45,186,600-45,416,670 in patient 2 only. A hemizygous L1CAM c.1354G>A, p.G452R variant was identified in patient 5 (GOSgene), who also had hydrocephalus and other features consistent with L1 syndrome. The p.G452R variant is located at a highly conserved residue across multiple species and is located within the Ig5 extracellular domain of the L1 protein. Both MAGEL2 p.Q666Pfs*47 and L1CAM p.G452R are absent from control databases, including the gnomAD browser (http://gnomad.broadinstitute.org/).

Human embryonic expression profile of MAGEL2 and L1CAM using in situ hybridization

MAGEL2

At the early embryonic stage of CS16, there was no MAGEL2 expression in the developing hypothalamus or Rathke pouch (RP; the primordium of the anterior pituitary). However, there was strong transcript staining specifically in the inferior ganglion of the vagus nerve and the spinal ganglia. At CS19, MAGEL2 mRNA transcripts appeared in the hypothalamus and the spinal cord but were undetectable in the RP. At CS20, strong expression was present throughout the ventral diencephalon and in both the RP and the PP. This expression was maintained within the hypothalamus and the RP at CS23 and was noted in the trigeminal ganglia (Fig. 3). No staining was visualized using the sense control probe on equivalent sections at any stage.

Figure 3.

Human MAGEL2 expression during embryonic development. (A) CS16, the equivalent of 5.5 weeks into embryonic development. MAGEL2 expression is noted in the inferior ganglion of the vagus (IGV) nerve and the spinal ganglia (SG). (B) At CS19, 6 weeks into development, there are high levels of mRNA transcripts in the developing hypothalamus (Hyp), ventral diencephalon (VD), and (C) spinal cord (S). (D) At CS20, 7 weeks into development, strong transcript staining is present throughout the VD and in both the RP and PP. (E) A magnified image of the RP and PP from image (D). (F) At CS23, 8 weeks into development, MAGEL2 expression is maintained in the Hyp and RP, with some expression in the trigeminal ganglia (TG).

L1CAM

There was no L1CAM mRNA transcript staining at CS16 in the human embryonic brain sections incorporating the hypothalamus and RP. At CS19, there was strong expression in the hypothalamus and trigeminal ganglia, but not in the RP. Staining was also noted in the metencephalon and throughout the ventral diencephalon at this stage. L1CAM expression was maintained in the hypothalamus and forebrain as well as the hindbrain during CS20 and CS23 (Fig. 4). No staining was observed in the RP or in the PP at any stage analyzed in this study. The sense control probe produced no staining at any stage.

Figure 4.

Human L1CAM expression during embryonic development. (A and B) A human embryonic section from CS19 showing L1CAM mRNA transcripts in the developing hypothalamus (Hyp), ventral diencephalon (VD), and trigeminal ganglia (TG). (B) mRNA transcripts can be seen in the spinal cord (S). In a different embryo section at (C) CS19 and at (D) CS20, L1CAM expression is noted throughout the metencephalon (M) and again in the TG. There is no mRNA transcript staining in the RP at either stage. (E) In a different embryo section at CS20, specific expression is seen throughout the hypothalamus and in the TG. (F) At CS23, L1CAM expression is observed ubiquitously throughout the brain, particularly in the M; (G) it is also present in the retina (R) of the eye. (H) A different embryo section at CS23 shows that L1CAM expression is partially maintained in the Hyp and TG. (I) At CS23, there is strong expression in the telencephalon (T) (forebrain).

Discussion

MAGEL2 is a member of the type II MAGE gene family involved in neurogenesis and brain function (13, 14). It is thought to enhance ubiquitin ligase activity (15), act as a regulator of retrograde transport, and promote endosomal F-actin assembly, and it is involved in the regulation of the circadian clock (16). In humans, loss of function point mutations causing truncations in the MAGEL2 gene were initially implicated in the etiology of variable PWS-like features and contractures of the small finger joints, a phenotype now commonly referred to as SHFYNG syndrome (3).

Magel2-null mice present with features similar to those of PWS in humans, including neonatal growth retardation, excessive weight gain after weaning, impaired hypothalamic regulation, reduced fertility, and excess fat with decreased muscle mass (17–20). In addition, Magel2-knockout mice elicit altered social phenotypes and impaired ability to distinguish between known and novel partners (21). Recent studies concluded that Pro-opiomelanocortin neuron activity and its communication with downstream targets are significantly compromised (22) and that oxytocin neuronal activity is suppressed (23) in Magel2-deficient mice.

Specific association of the MAGEL2 gene with PWS was first suggested following expression studies using northern blotting, where MAGEL2 was expressed in the adult human brain, notably the hypothalamus, and in the fetal brain (however, details were not specific), lung, and kidney (24). The authors concluded that loss of MAGEL2 may explain abnormalities in brain development in individuals with PWS. Expression analysis performed in the current study further characterized the location of MAGEL2 transcripts within the developing human fetal brain. We have shown that MAGEL2 is highly expressed in the developing hypothalamus from 6 to at least 8 weeks GA and in the developing pituitary gland (RP) at 7 to 8 weeks GA (Fig. 3), supporting the hypothesis that this gene plays a critical role during embryonic brain development.

The MAGEL2 mutation c.1996dupC, p.Q666Pfs*47 identified in patients 1 to 4 was previously identified in two siblings diagnosed with a neurodevelopmental disorder including hypotonia, ASD, hyperinsulinemic hypoglycemia, and features of arthrogryposis (25). Subsequently, the c.1996delC deletion, causing a frameshift in the same location, p.Q666Sfs*36, was described in three patients with a lethal form of arthrogryposis (26). Both the c.1996delC deletion and c.1996dupC duplication have since been identified in multiple patients with SHFYNG. These data widened the phenotypic spectrum of SHFYNG, expanding the range to include fetal akinesia and arthrogryposis (27, 28). In previous reports of patients harboring MAGEL2 truncating mutations, intellectual disability varied from mild to severe, and ASD was not always present. The majority of affected patients had arthrogryposis (varying in severity), short stature, and hypogonadism, which are all common features in SHFYNG (3, 27, 28), with one female patient manifesting hypogonadotropic hypogonadism (27). Interestingly, a recent report described the first patient with SHFYNG and early-onset obesity to harbor a MAGEL2 truncation (de novo c.1850G>A, p.Trp617*) (29).

GHD has frequently been identified in SHFYNG; however, other pituitary deficits have not been described until recently. Two siblings and an unrelated female patient with SHFYNG, arthrogryposis, and severe respiratory difficulties were found to carry the truncating MAGEL2 variants p.Q638* and p.S1044*, respectively, and manifested variable hypopituitarism (30). One of the siblings was diagnosed with central DI and gonadotrophin deficiency, whereas the unrelated patient was diagnosed with panhypopituitarism including GHD, central hypothyroidism, adrenal insufficiency, and gonadotrophin deficiency, with a hypoplastic anterior pituitary gland on MRI (30). Patients 2 and 3 from the current study manifested DI, and patient 4 has multiple pituitary hormone deficiencies, including GHD and ACTH insufficiency, indicating an association of the p.Q666Pfs*47 frameshift with endocrinopathies in patients with SHFYNG. Together with the previous report (30), these findings further highlight how different MAGEL2 truncations seem to play a role in the etiology of both DI and combined pituitary hormone deficiency as part of SHFYNG syndrome, which until recently were not major phenotypic features reported in such patients. Another recent case report identified the novel MAGEL2 p.Q1007* truncation in a patient with SHFYNG and GHD, hypothyroidism, and hyperprolactinemia (31), again suggesting that variable CH is increasingly identified in these patients. Of note, a previous report described a patient with Moebius syndrome, GHD, and arthrogryposis (32). Although no genetic mutations were identified in this patient, it demonstrates the link between these diverse phenotypes.

A recent publication reported the association of MAGEL2 truncation mutations with Chitayat-Hall syndrome (OMIM: 208080), which has a strong phenotypic overlap with SHFYNG (33). Chitayat-Hall syndrome is characterized by distal arthrogryposis, intellectual disability, dysmorphic features, and hypopituitarism, with GHD present in all reported cases to date (34). The same p.Q666Pfs*47 MAGEL2 truncation was present in one of the reported patients with Chitayat-Hall syndrome, demonstrating how variable overlapping phenotypes between SHFYNG and Chitayat-Hall syndromes arise from the same genotype and suggesting that full-length MAGEL2 is crucial for normal development of the human brain and for normal hypothalamo-pituitary function. Chitayat-Hall syndrome and SHFYNG may in fact be the same syndrome, albeit with variable penetrance, with some patients having sleep apnea, currently noted as a characteristic feature of SHFYNG but not Chitayat-Hall syndrome. An increasing number of patients with SHFYNG with MAGEL2 mutations (28) have not had their hypothalamo-pituitary function tested, suggesting that pituitary dysfunction may be a more frequent feature of SHFYNG, as is observed with Chitayat-Hall syndrome. Early endocrine diagnosis is crucial if endocrine morbidity is to be prevented and is therefore essential for improved quality of life for patients with these complex conditions.

Mutations in L1CAM, located on the X chromosome (Xp28) and encoding the L1 protein, have been implicated in the etiology of L1 syndrome (8). Female carriers may also manifest minor features of this syndrome, such as adducted thumbs or mild intellectual deficits (35). L1 is an axonal glycoprotein cell adhesion molecule that plays a role in neuronal migration and differentiation, including axon fasciculation (36), neurite outgrowth (37), synapse formation (38), and myelination (39). L1CAM-null mice have hydrocephalus, a smaller hippocampus and cerebellum, corpus callosal hypoplasia, hyperfasciculation of the corticothalamic tracts, and pyramidal tract abnormalities (40–45). Mutations within the cytoplasmic domain of the L1 protein have been described in MASA syndrome, which led to murine studies with L1 protein cytoplasmic domain disruption. Surprisingly, these mice had normal brain morphology, although they had defects in motor function (46). The hemizygous L1CAM mutation p.G452R identified in patient 5 was described previously in a patient with severe hydrocephalus (47). This mutation lies within, and is predicted to affect, the structure of the L1 extracellular domain required for correct folding of the protein and is subsequently thought to affect binding through the distortion of domain conformation (48). Further investigations have supported this, with a decreased ligand-binding ability in the presence of L1CAM p.G452R (49).

In rodents, L1cam is expressed in migrating neuron cell bodies from embryonic stage 9.5 and is later expressed in growing and regenerating axons. Myelinating Schwann cells express L1CAM during embryonic and postnatal development, whereas nonmyelinating Schwann cells express L1CAM through adulthood (50–53). The human L1CAM expression profile generated in this study revealed high transcript expression in the hypothalamus from 6 to 8 weeks of development (Fig. 4). However, no expression was visible in the RP or the PP, suggesting that this gene is hypothalamic and plays a critical role in this region during brain development. Patient 5 is the first patient to our knowledge with an L1CAM mutation and manifested GHD with pituitary dysfunction associated with features of L1 syndrome.

The trigeminal ganglia are sensory ganglia of the trigeminal nerve, responsible for sensation in the face and for motor functions. Both MAGEL2 and L1CAM expression within these specific tissues and during midline craniofacial development may suggest that sensation in the face is impaired in patients with mutations in these genes. However, the presence of global developmental delays did not allow assessment of this function. Limited availability of human embryonic sections did not allow analysis of expression beyond 8 weeks of gestation.

To summarize, our data suggest that patients with SHFYNG and L1 syndromes should all be screened and monitored for hypothalamo-pituitary abnormalities. Furthermore, patients with CH and accompanying joint contractures should be screened for MAGEL2 and L1CAM mutations and evaluated/monitored for additional phenotypes commonly present in SHFYNG or L1 syndrome, respectively. Our data and previously published data on SHFYNG and L1 syndromes suggest that MAGEL2 and L1CAM, respectively, should be screened for mutations using Sanger sequencing before next-generation techniques are conducted, as there is a high chance that a mutation lies within these genes in such patients. This would be the most cost-effective approach in screening for the most likely genetic diagnosis. However, in those cases in which a mutation is not identified in either of these genes, either whole exome or genome sequencing may be performed.

Glossary

Abbreviations:

ASD

autism spectrum disorder

CH

congenital hypopituitarism

CS

Carnegie stage

DDD

Deciphering Developmental Disorders

DI

diabetes insipidus

GA

gestational age

GHD

growth hormone deficiency

GOSH

Great Ormond Street Hospital

OMIM

Online Mendelian Inheritance in Man

PP

posterior pituitary

PWS

Prader-Willi syndrome

RP

Rathke pouch

SHFYNG

Schaaf-Yang syndrome