New phenotype caused by POMGNT2 mutations

Abstract

We present a case report about a Moroccan 3-year-old girl, with an intermediate phenotype of muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 8 form. We performed clinical and instrumental evaluation, muscle biopsy, genetic screening of 59 genes for different cerebral malformations, follow-up and review of literature. After investigations, we identified an intermediate new phenotype between the severe and mild form, characterised by significant malformations of the cortex with myopatic symptoms, this increases the genotype–phenotype correlation knowledge about POMGNT2 gene mutations. New homozygous missense mutation on POMGNT2 (c.511 G>A, p.Asp171Asn, rs768063378) was detected.

Background

Walker-Warburg syndrome (WWS) or muscle-eye-brain disease includes a group of rare forms of autosomal recessive congenital muscular dystrophies, also known as muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 1–14 forms (MDDGA1-14), characterised by brain and eye abnormalities. Usually, the onset of these conditions is in the first year of life with hypotonia, motor developmental delay and variable cerebral malformations. Muscular dystrophy-dystroglycanopathy, congenital with brain and eye anomalies, type 8 form (MDDGA8) caused by recessive mutations in the POMGNT2 gene, was discovered in 2012¹ and associated with characteristic brain and eye malformations, profound mental retardation, congenital muscular dystrophy and death usually in the first years of life. A milder form of MDGGA8 was described in 2015 characterised by milder limb-girdle muscular dystrophy (LGMD), intellectual disability without brain malformations.² Since 2015, no one has described cases with mutations in POMGNT2. Although the disease is very rare with only 6 cases described in literature, it is clear that the genotype–phenotype association must be clarify, to understand better the prognosis of these patients and the resulting phenotype that, as underlined in this article, can be less severe than the cases first described.

Case presentation

The patient was born by spontaneous delivery at 41 weeks of gestational age. Ultrasound screening in pregnancy revealed callosum agenesis reduced lower limb mobility, optic septum dysplasia. At birth, she presented with prolonged hypoglycaemic and elevated Creatine Phosphokinase (CPK) levels (values ranging between 4000 and 14 400 IU/L). Clinical history revealed consanguinity; the parents are first cousins from Marocco with two healthy male childs (figure 1). At the age of 1 year, she was admit at our hospital for normal investigations. Clinical evaluation showed several dysmorphic features and neurological abnormalities, charactherised by: mild broad forehead and ogival palate, spontaneous mobility reduced for age, hypotonia and cingulate hypotrophy, hypertonic lower limbs, clubfoot, kyphosis, delay of motor stages, axial hypotonia associated with pyramidal signs in the lower limbs. After clinical examination was clear that we were facing a neuromuscular disorder to investigate.

Figure 1.

Pedigree, proband homozygose for c.511 G>A, p.Asp171Asn, rs768063378, POMGNT2.

Investigation

Neurological examination showed: reduced osteotendinous reflexes, ligaments laxity in feet and hands, shortness Achilles tendon, difficult to maintains the sitting position, sufficient control of the head, difficult to pass from the prone position to the supine position, slight stiffness of the spine. Ultrasound abdomen evaluation did not detect significant alterations, echocardiogram was normal, normal eyes examination. Electromyography was performed at 1 year of age and revelaed normal motor unit potentials, with muscle potential proportional to the force exerted in all muscles examined. Brain and spinal MRI showed brainstem dysplasia with a reduction of the pontine protuberance, the midbrain appears extended on the axial plane, the tractography demonstrates reduction of the corticospinal bundles and bridge’s transverse fibres. In the supratentorial region, we observed agenesis of the pellucid septum with median fusion of the fornices; the optic nerves appear to be thinner, especially near the papillae. No obvious changes in the cerebral cortex; presence of shaded areas of hypersignal within white periventricular substance, presence of incomplete rotation of hippocampal heads, normal hypothalamus-hypophyseal region. Slightly dilated and dysmorphic ventricular system, subarachnoid spaces were normal (table 1). Muscle biopsy was performed from quadriceps muscle, after informed consent from parents at age of 2 years. Muscle sample was snap frozen in liquid nitrogen and 10 µm sections obtained for histological and immufluorescence studies, according to standard procedures,³ the fragment under examination was characterised by a marked variability of muscle fibres size and shape, due to the presence of numerous atrophic fibres intermingled with normal hypertrophic fibres, we detected also the presence of different hyaline fibres, rare aspects of necrosis, discrete number of fibres with centralised nuclei and increase of the connective endomysial tissue. Immunohistochemistry for alphadystroglycan showed a marked reduction of alpha-dystroglycan (DAG1) binding. Genetic screening of 59 genes for different cerebral malformations, including those for congenital muscular alpha-dystroglycanopathy with brain and eye anomalies, was performed by standard NGS protocols on a PGM platform. This study led to the identification of a homozygous missense mutation within POMGNT2 gene (c.511 G>A, p.Asp171Asn, rs768063378). The variants affects a conserved amino acid, this mutation is very rare in the general population (MAF 0.0000106/GnomAD) and it has never been identified in homozygosity and it is predict to be pathogenic according to the ACMG guidelines.⁴

Table 1.

Clinical features and associated mutations of intermediate, severe, mild MDDGA8

Mutations	Brain malformations	Oocular defect	Motor ability, others features	Age of death, age of onset, sex
Intermediate form
Homozygous  c.511 G>A, p.Asp171Asn (rs768063378)	Brainstem dysplasia with reduction of the pontine protuberance and of the midbrain which appears instead extended on the axial plane, the tractography demonstrates reduction of the corticospinal bundles and within the transverse fibres of the bridge. Agenesis of the pellucid septum with median fusion of the arches. No obvious changes in the cerebral cortex; presence of shaded areas of hypersignal in white periventricular substance, presence of incomplete rotation of hippocampal heads. The hypothalamus-hypophyseal region were normal. Slightly dilated and dysmorphic ventricular system. Normal Subarachnoid spaces.	The optic nerves appear to be thin especially near the heads.	Hypotrophy and hypothony elevated CPK (values ranging between 4000 and 14400). At 1 year, reduced OTR (osteotendinous reflexes), distal laxity in feet and hands, shortness of Achilles tendons, maintains the sitting position imposed in kyphosis, sufficient control of the head, she raises her head but does not pass from the prone position to the supine position, slight stiffness of the spine. Examination, showed mild broad forehead and ogival palate, spontaneous mobility reduced for age, hypotonia and cingulate hypotrophy, hypertonic lower limbs, clubfoot, delay of motor stages, axial hypotonia associated with pyramidal signs in the lower limbs.	Still alive: last neurological examination showed speech defect and difficulty walking. Sex:female
Severe form1 #614 830
Homozygous  c.1333C>T, p. Arg445*	cerebellar hypoplasia, severe ventricular enlargement and cortical-migration defects characteristic of cobblestone lissencephaly	Microphthalmia Macrophthalmia		Death First year of life
Homozygous  c.473 G>A  p.Arg158His	Similar to the Arg445*	Microphthalmia Macrophthalmia		Death First year of life
Homozygous  c.590 G>A  p.Trp197*	ventricular enlargement during gestation. Both fetuses were female and were diagnosed with WWS on autopsy			Female twins death before birth
Mild form2 #618 135		IQ
c.494T.C (p.Met165Thr)  c.758C.T (p.Pro253Leu)	Absent	<35		Onset 11 months
c.758C.T (p.Pro253Leu) homozygous	Absent	Normal	Congenital biliary atresia, ambulant running	Onset 13 years
c.758C.T (p.Pro253Leu) homozygous	Absent	60	Hyperactivity disorder, ambulant fast running	Onset 1 year

* X, nonsense mutation

MDDGA8, muscular dystrophy-dystroglycanopathy type A, 8; WWS, Walker-Warburg syndrome.

Differential diagnosis

The differential diagnosis at clinical level is a challenge because muscular dystrophy-dystroglycanopathy are a particular group of muscular dystrophies with heterogeneous molecular mechanism and clinical features, for this reasons is very difficult to understand which gene could be involved. Nowadays we have next generation sequence techniques to read the DNA, that cover all the genes for muscular dystrophies, and is more simple discriminate at molecular level the different clinical forms, moreover I underline that still a long way to establish more precisely genotype-phenotype correlation, due to the extreme rarity of these forms. The case report represents precisely the need to describe and identify these forms in a better way both at a clinical and molecular level.

Treatment follow-up outcome

Neuromotor therapy has been programmed every week, to improve the stabilisation of the trunk and the pelvis, to facilitate the release of the limbs, to improve the postural passage from the seated station to the erect one with support, and to enhance at musculoskeletal level the grip of small and medium objects by facilitating bimanual cooperation. After diagnosis, the child presented two time a year with small respiratory crises of a few seconds, characterised by bradycardia with a frequency of 40 beats per minute and normal partial saturation of Oxygen 98%. These crises were characterised by lack of response to stimuli followed by vomiting. No major infections occurred and no important eating disorders were reported. During the last follow-up visit, at the age of 3 years she weighed 18 kg (91%, z-score 1,36), stature was (about) 100 cm (71%, z.score 0,56), body mass index 18 kg/cm (95%, z-score 1,61). Clinical examination showed normal pupils reflexes and normal saccadic movements, severe speech defect and inability to walk. The outcome is characterised by a stable clinical condition that appears to respond to neuromotor therapy, family care and hospital support are currently effective.

Discussion

The nosography of rare diseases is constantly evolving, due to the rapid developments of DNA sequencing technology, which allows efficient identification of causative mutations and efficient genotype–phenotype correlations. POMGNT2 mutations originally described in 2012 include severe brain and eye malformations, profound mental retardation, congenital muscular dystrophy, and death usually in the first years of life,¹ later in 2015,² POMGNT2 mutations were described in patients with mild LGMD and intellectual disability, without lissencefaly and ocular defect (table 1). The case described here provides further evidence about the role of allelic diversity in the phenotypic presentation of congenital muscular dystrophies. The novel p.Asp171Asn homozygous mutation in the POMGNT2 gene show to be causative of an intermediate phenotype between the severe form, and the mild myopatic form and thus widens the spectrum of phenotype-genotype correlation in POMGNT2 gene mutations. Our patient display a delay of motor stages, brainstem dysplasia with reduction of the pontine protuberance, agenesis of the pellucid septum, axial hypotonia, cingulate hypotrophy, normal morphology of the cerebral cortex, and a better prognosis compared with the typical cases of WWS type 8.¹ POMGNT2 is involved in the biosynthesis of the phosphorylated O-mannosyl trisaccharide (N-acetylgalactosamine-beta-3-N-acetylglucosamine-beta-4-(phosphate-6-) mannose), a carbohydrate structure present in DAG1, which is required for binding laminin G-like domain-containing extracellular proteins with high affinity.⁵ The novel mutation identified lies in a protein domain named DUF563 (PF04577, codons 162–395, http://pfam.xfam.org/protein/Q8NAT1) of unknown function, where most of the previously described mutations lie³ (table 1). Functional analysis of this specific domain may open important insights into the physiopathological mechanism in this neuromuscular disease. A review of literature reveal that nonsense mutations are typically associated with more severe phenotypes (table 1). Missense mutations in the DUF563 domain, Met165Thr and Pro253Leu² and the Asp171Asn described here underlie muscular dystrophy and absence of complex cortical brain malformation. The reasoning about genotype-phenotype correlations may suggest that missense mutations in the DUF563 exhert a minor influence on protein function, predicting a better prognosis. POMGNT2 mutations are very rare and additional cases need to be further identified and analysed to corroborate this hypothesis.

Learning points.

• The condition muscular dystrophy-dystroglycanopathy type A, 8 (MDDGA-8) is very rare (six cases described in literature), the clinical diagnosis is a challenge.

• Molecular analysis is essential to classify this type of MDDGA.

• There is evidences that a new intermediate form due to mutations in POMGNT2 needs to be added to the current classification.

Ethics statements

Patient consent for publication

Obtained.