Clinical long-time course, novel mutations and genotype-phenotype correlation in a cohort of 27 families with POMT1-related disorders

This cohort consisted of one family with MEB-like phenotype and 9 families (15 patients) with WWS. 8/9 WWS families showed prenatal onset with ventricular dilatation in the ultrasound and/or MRI examination, one family had no medical follow-up during pregnancy. In 9 fetuses from 4 different families prenatal diagnosis of WWS resulted in premature termination of pregnancy. Six WWS patients from 6 independent families were born alive. All these neonates had general muscular weakness with reduced limb movements reported in 4/6 patients. 4/6 WWS neonates needed nasogastric tube feeding due to feeding difficulties (no information available in patient 18; patient 19 died at 4 days of age). In 4/6 WWS patients epilepsy starting in infancy was reported with myoclonic, tonic or infantile spasms-like seizures. All WWS patients showed severe ophthalmologic anomalies including congenital cataracts (reported in 3/6 families), microphthalmus (1/6), buphthalmus (2/6), and retinal detachment (2/6). All WWS patients had severe brain malformations detected by ultrasound and/or MRI: hydrocephalus internus (reported in 6/6 families), lissencephaly type II (4/6), hypoplasia of the pons and/or brainstem (3/6), cerebellar hypoplasia (5/6), hypoplasia of the corpus callosum (2/6), encephalocele (2/6). 2/6 WWS patients had ventriculoperitoneal shunting in infancy due to increasing ventricular dilatation. In 2/6 WWS patients death in infancy was reported at the age of 2.5 and 7 months, respectively.

The MEB-like patient also had muscular weakness and reduced limb movement as neonate and severe global developmental retardation in the further course. Brain imaging revealed hypoplasia of pons and vermis and an extensive hydrocephalus internus leading to ventriculoperitoneal shunting in infancy. There was a convergent strabismus in the ophthalmologic examination but no structural eye anomalies.

In 5/10 patients of the WWS/MEB cohort CK values were available and were markedly elevated with 10- to 30-fold the upper reference limit. Muscle biopsy was performed in 3/10 patients yielding a dystrophic pattern and/or reduced aDG expression in the immunofluorescent staining.

Patient 21a was a German girl from non-consanguineous healthy parents with prenatal ultrasound diagnosis of a severe brain malformation with ventricular dilatation, hypoplasia of the cerebellar vermis, agenesis of the corpus callosum and occipital encephalocele. While similar brain defects in two preceding pregnancies had led to termination of pregnancy, the mother this time decided to carry the child to term. At birth at a gestational age of 39 weeks the female neonate showed severe muscular weakness with reduced spontaneous movements but without contractures. She was microcephalic with a head circumference of 31 cm. Due to weak sucking she was initially fed via a nasogastric tube. The serum CK level was 5338 U/l. Ophthalmologic examination showed bilateral microphthalmus and cataracts. MR imaging confirmed the severe brain malformation with cobblestone lissencephaly, hydrocephalus, hypoplastic corpus callosum, pontocerebellar hypoplasia and occipital encephalocele (Fig. 1). An intermittent slowing of the left hemisphere in the EEG was found. At the age of 3 months tonic and infantile spasm-like seizures developed and were successfully treated with valproic acid and sultiame. At the age of 4 months she was markedly hypotonic with a frog-like supine position, increasing paucity of spontaneous movements and lack of head control. She died unexpectedly at the age of 7 months at home, autopsy revealed pneumonia as cause of death.

This cohort consisted of 17 families (19 patients) with LGMD. All patients of this cohort showed symptoms of muscular dystrophy with muscular hypotonia, proximal limb weakness and delayed motor development. 16/17 LGMD families were reported with onset of symptoms at the age of 1 month to 3 years (no information on symptom onset available in family 8). In 10/17 LGMD families contractures of the Achilles tendon were reported and a rigid spine syndrome in 5/17 patients. Calves muscle hypertrophy was found in 13/17 families. All patients were cognitive impaired. IQ levels were available in 14/17 patients and ranged from 50 to 68. Microcephaly was found in 12/17 families. Cerebral MRI was performed in 15/17 families and no patient had a CNS malformation. No patient showed structural ophthalmologic anomalies. 16/17 patients had markedly elevated CK values with maximum CK values ranging from 10- to 55-fold of the upper limit (CK value not available in patient 2). For confirmation of diagnosis a muscle biopsy was performed in 13/17 families showing reduced aDG expression in 11/17 samples (not studied in 2 patients).

Patient 9 was the third child of non-consanguineous German parents with no relevant family history besides the 3 unexplained miscarriages the patient’s mother had before. During pregnancy the mother assumed slightly reduced fetal movements in an otherwise uneventful pregnancy. There was a normal delivery at a gestational age of 37 weeks, birth weight was 2900 g (90th percentile) and head circumference 33 cm (25th percentile). Around 4 weeks of age the mother first noted muscular hypotonia. The motor milestones were markedly delayed with acquisition of unsupported sitting at 16 months and walking at 3.5 years. From the age of 4 years on a proximal limb weakness became evident with a positive Gowers’ sign. In the following years the patient’s motor abilities stabilized with independent ambulation; he was able to ascend stairs slowly with holding the handle. In his late 20ies the motor functions started to deteriorate and he became wheel chair dependent around 30 years of age. Pseudohypertrophy of the calves was at first documented at the age of 4 years and subsequently occurred also in his thighs, trunk and arms. He had an increased lumbar lordosis and severe contractures of the ankles, spine and neck as well as mildly of the elbows (Fig. 2). Surgery of bilateral ankle contractures was performed at the age of 12 years and improved walking. His intellectual development was severely disturbed from early childhood on. At 4 years of age only a few words could be pronounced clearly, the patient never learned writing or reading and independent activities never could be performed. Secondary microcephaly developed in the first 4 years of life, but epileptic seizures did never occur. Cerebral MR imaging was reported to be normal. While orofacial weakness and hypersalivation were treated with speech and language therapy from early childhood on, there was no prominent facial weakness and a normal ophthalmologic status. At the age of 30 years the left ventricular function was normal. Repeatedly, the CK values were markedly elevated (1644–9860 U/l). A first muscle biopsy and electromyography performed at the age of 4 years revealed a myopathic pattern and led to the initial suspicion of Duchenne muscular dystrophy. In a second muscle biopsy at 11 years of age the expression of dystrophin was normal as was the genetic analysis of the dystrophin gene. Expression of glycosylated α-dystroglycan was not studied in any of the muscle biopsies. The patient was seen in different pediatric and adult neuromuscular centers on a regular basis. Finally, at the age of 32 years the patient’s family again searched for a genetic diagnosis at the Munich neuromuscular center and clinical diagnosis of POMT1-related LGMD could be genetically confirmed by identification of compound heterozygous POMT1 mutations.

5/9 WWS families were found to have homozygous POMT1 mutations (3 Turkish, 1 Indonesian, 1 Gipsy family); the other families were compound heterozygous. 9/9 WWS families had 2 POMT1 mutations considered to severely disrupt transcript or protein synthesis: 1/9 patient (family 18) had a homozygous donor splice site mutation expected to alter the splicing of intron 3 and/or neighboring exons [15]. In 4/9 families (families 19, 20, 24, 25) homozygous nonsense mutations were identified predicted to result in premature protein termination. In 2/9 families (families 21, 23) 2 compound heterozygous nonsense mutations were found each leading to a premature stop codon. 1/9 patient (family 27) was compound heterozygous for a splice site mutation and a frame shift mutation leading to a premature stop codon. 1/9 family (family 22) was compound heterozygous for a nonsense mutation with presumed premature protein termination and an in-frame mutation presumed to result in deletion of a phenylalanin residue at position 281.

The MEB patient with African parents had compound heterozygous missense mutations. One mutation (p.His563Pro) was of maternal origin and was not described before. Unfortunately, no material of the father could be obtained.

All LGMD patients had at least 1 missense mutation. 13/17 families were homozygous for the p.Ala200Pro mutation described before as an ancestral founder mutation in Turkish families with a distinct phenotype [8]. 4/17 patients were compound heterozygous for a missense mutation and a frame-shift mutation predicted to result in premature protein termination.

Since 2013 massive parallel sequencing methods were applied in our laboratory and positive results were confirmed by Sanger sequencing. For massive parallel sequencing genomic DNA of each patient was processed according to the Nextera Enrichment protocol (Illumina, Inc., San Diego, CA, USA). Library quantification was carried out with the High Sensitivity DNA Kit on a Bioanalyzer (Agilent Technologies, Böblingen, Germany) and the Qubit™dsDNA HS Assay Kit (Life Technologies, Darmstadt, Germany). The Library was sequenced as a 150 bp paired-end run on a MiSeq™ system (Illumina, Inc., San Diego, CA). Variant detection was performed with Illumina VariantStudio (Illumina, Inc., San Diego, CA, USA).