Fetal Presentation of Walker-Warburg Syndrome with Compound Heterozygous POMT2 Missense Mutations.

Background: Walker-Warburg syndrome (WWS) (OMIM #236670) is an autosomal recessive disorder characterized by congenital muscular dystrophy, hydrocephalus, cobblestone lissencephaly, and retinal dysplasia. The main genes involved are: POMT1, POMT2, POMGNT1, FKTN, LARGE1, and FKRP. Case report: We present a fetus with WWS showing at ultrasound severe triventricular hydrocephalus. Pregnancy was legally terminated at 21 weeks +2 days of gestation. In vivo and postmortem magnetic resonance revealed corpus callosum agenesis and cerebellar hypoplasia. Cobblestone lissencephaly was observed at post-mortem. Next generation sequencing (NGS) of 193 genes, performed on fetal DNA extracted from amniocytes, detected two heterozygous mutations in the POMT2 gene. The c.1238G > C p.(Arg413Pro) mutation was paternally inherited and is known to be pathogenic. The c.553G > A p.(Gly185Arg) mutation was maternally inherited and has not been previously described. Conclusion: Compound heterozygous mutations in the POMT2 gene caused a severe cerebral fetal phenotype diagnosed prenatally at midgestation allowing therapeutic pregnancy termination.

Prenatal diagnosis of Walker-Warburg syndrome due to compound mutations in the B3GALNT2 gene.

BACKGROUND: Congenital hydrocephalus is one of the symptoms of Walker-Warburg syndrome that is attributed to the disruptions of the genes, among which the B3GALNT2 gene is rarely reported. A diagnosis of the Walker-Warburg syndrome depends on the clinical manifestations and the whole-exome sequencing after birth, which is unfavorable for an early diagnosis. METHODS: Walker-Warburg Syndrome was suspected in two families with severe fetal congenital hydrocephalus. Whole-exome sequencing and Sanger sequencing were performed on the affected fetuses. RESULTS: The compound heterozygous variants c.1A>G p.(Met1Val) and c.1151+1G>A, and c.1068dupT p.(D357*) and c.1052 T>A p.(L351*) in the B3GALNT2 gene were identified, which were predicted to be pathogenic and likely pathogenic, respectively. Walker-Warburg syndrome was prenatally diagnosed on the basis of fetal imaging and whole-exome sequencing. CONCLUSIONS: Our findings expand the spectrum of pathogenic mutations in Walker-Warburg syndrome and provide new insights into the prenatal diagnosis of the disease.

Multifaceted antibodies development against synthetic α-dystroglycan mucin glycopeptide as promising tools for dystroglycanopathies diagnostic.

Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.

Acute rhabdomyolysis following viral infection with coxsackie A4 in a 50-day-old infant with Fukuyama congenital muscular dystrophy.

BACKGROUND: Fukuyama congenital muscular dystrophy (FCMD), which is characterized by generalized muscle weakness, hypotonia, and motor delay during early infancy, gradually progresses with advanced age. Although acute rhabdomyolysis following infection in patients with FCMD has occasionally been reported, no studies have investigated rhabdomyolysis following viral infection in FCMD patients during early infancy. CASE REPORT: We report the case of a 50-day-old girl with no apparent symptoms of muscular dystrophy who developed severe acute rhabdomyolysis caused by viral infection, resulting in quadriplegia and respiratory failure therefore requiring mechanical ventilation. Brain magnetic resonance imaging incidentally showed the typical characteristics of FCMD, and FCMD was confirmed by genetic analysis, which revealed a 3-kb retrotransposon insertion in one allele of the fukutin gene and a deep intronic splicing variant in intron 5 in another allele. The virus etiology was confirmed to be Coxsackie A4. CONCLUSION: We report a severe case of acute rhabdomyolysis with the earliest onset of symptoms due to the Coxsackie A4 virus in a patient with FCMD. The present findings indicate that physicians should consider FCMD with viral infection a differential diagnosis if the patient presents with acute rhabdomyolysis following a fever.

Two middle-aged women with the Finnish variant of muscle-eye-brain disease (MEB).

Muscle-eye-brain disease (MEB) is a recessively inherited rare disease. Sixteen different gene mutations are known, with the most common mutations in the POMGNT1 gene. The disease is now called congenital muscular dystrophy-dystroglycanopathy type A3 (MDDGA3). It manifests itself as muscular dystrophy with eye and brain anomalies and intellectual disability. Previous clinical reports describe young patients. We have been able to follow two patients for almost 40 years. Their clinical picture has remained quite stable since adolescence, appearing as severe intellectual and motor disability, extremely limited communication skills, visual impairment, epilepsy, joint contractures, repeated bowel obstructions, teeth abrasion due to bruxism, an irregular sleep pattern and as a previously unreported feature hypothermic periods manifesting as excessive sleepiness.

Noncompaction cardiomyopathy in an infant with Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is a rare autosomal recessive, congenital muscular dystrophy that is associated with brain and eye anomalies. Several genes encoding proteins involved in α-dystroglycan glycosylation have been implicated in the aetiology of WWS. We describe a patient with nonclassical features of WWS presenting with heart failure related to noncompaction cardiomyopathy resulting in death at 4 months of age. Muscle biopsy revealed absent α-dystroglycan on immunostaining and genetic testing confirmed the diagnosis with two previously described POMT2 mutations. This is the first reported case of WWS syndrome associated with noncompaction cardiomyopathy.

RETINAL MANIFESTATIONS OF WALKER-WARBURG SYNDROME IN TWO SIBLINGS WITH RXYLT1 MUTATIONS.

PURPOSE: We report two siblings with genetically confirmed Walker-Warburg syndrome (WWS), studied with multimodal imaging, who presented with different retinal manifestations. METHODS: This is a retrospective report of two WWS cases with ultra-widefield fundus photography, fluorescein angiography, and ultrasound. Molecular diagnosis was achieved using panel testing and targeted variant testing. RESULTS: Two siblings, one male and one female, born 17 months apart with a diagnosis of WWS underwent retinal examination with imaging. The 3-month-old female infant exhibited microphthalmia, persistent hyaloidal arteries, and retrolental membranes with total tractional retinal detachments on ultrasound in both eyes. The 22-day-old male newborn exhibited persistent hyaloidal arteries and extensive peripheral avascular retina on angiography in both eyes. Both were found to be positive for the same two pathogenic variants in the RXYLT1/TMEM5 gene, which accounts for approximately 9% of cases of genetically confirmed WWS. CONCLUSION: Siblings with genetically confirmed WWS can have variable presentations despite identical genotype. This highlights the phenotypic disease spectrum of WWS, which may be similar to that seen in familial exudative vitreoretinopathy.

Homozygous dystroglycan mutation associated with a novel muscle-eye-brain disease-like phenotype with multicystic leucodystrophy.

Defects in dystroglycan post-translational modification result in congenital muscular dystrophy with or without additional eye and brain involvement, are referred to as secondary dystroglycanopathies and have been associated with mutations in 11 different genes encoding glycosyltransferases or associated proteins. However, only one patient with a mutation in the dystroglycan encoding gene DAG1 itself has been described before. We here report a homozygous novel DAG1 missense mutation c.2006G>T predicted to result in the amino acid substitution p.Cys669Phe in the ß-subunit of dystroglycan in two Libyan siblings. The affected girls presented with a severe muscle-eye-brain disease-like phenotype with distinct additional findings of macrocephaly and extended bilateral multicystic white matter disease, overlapping with the cerebral findings in patients with megalencephalic leucoencephalopathy with subcortical cysts. This novel clinical phenotype observed in our patients further expands the clinical spectrum of dystroglycanopathies and suggests a role of DAG1 not only for dystroglycanopathies but also for some forms of more extensive and multicystic leucodystrophy.

Novel POMGnT1 mutations cause muscle-eye-brain disease in Chinese patients.

Muscle-eye-brain (MEB) disease is a congenital muscular dystrophy (CMD) phenotype characterized by hypotonia at birth, brain structural abnormalities and ocular malformations. To date, few MEB cases have been reported in China where clinical recognition and genetic confirmatory testing on a research basis are recent developments. Here, we report the clinical and molecular genetics of three MEB disease patients. The patients had different degrees of muscle, eye and brain symptoms, ranging from congenital hypotonia, early-onset severe myopia and mental retardation to mild weakness, independent walking and language problems. This confirmed the expanding phenotypic spectrum of MEB disease with varying degrees of hypotonia, myopia and cognitive impairment. Brain magnetic resonance imaging showed cerebellar cysts, hypoplasia and characteristic brainstem flattening and kinking. Four candidate genes (POMGnT1, FKRP, FKTN and POMT2) were screened, and six POMGnT1 mutations (four novel) were identified, including five missense and one splice site mutation. Pathogenicity of the two novel variants in one patient was confirmed by POMGnT1 enzyme activity assay, protein expression and subcellular localization of mutant POMGnT1 in HeLa cells. Transfected cells harboring this patient's L440R mutant POMGnT1 showed POMGnT1 mislocalization to both the Golgi apparatus and endoplasmic reticulum. We have provided clinical, histological, enzymatic and genetic evidence of POMGnT1 involvement in three unrelated MEB disease patients in China. The identification of novel POMGnT1 mutations and an expanded phenotypic spectrum contributes to an improved understanding of POMGnT1 structure-function relationships, CMD pathophysiology and genotype-phenotype correlations, while underscoring the need to consider POMGnT1 in Chinese MEB disease patients.

A novel pathogenic deletion in ISPD causes Walker-Warburg syndrome in a Chinese family.

BACKGROUND: Walker-Warburg syndrome (WWS) is a genetically heterogeneous disease that often presents with complex brain and eye malformations and congenital muscular dystrophy. Mutations of the ISPD gene have been identified as one of the most frequent causes of WWS. OBJECTIVE: The current study aimed to identify the cause of severe congenital hydrocephalus and brain dysplasia in our subject. METHODS: Genomic DNA was extracted from the fetus's umbilical cord blood and peripheral venous blood of the parents. The genetic analysis included whole-exome sequencing and qPCR. Additionally, in silico analysis and cellular experiments were performed. RESULTS: We identified a novel homozygous deletion of exons 7 to 9 in the ISPD gene of the fetus with WWS. In silico analysis revealed a defective domain structure in the C-terminus domain of the ISPD. Analysis of the electrostatic potential energy showed the formation of a new binding pocket formation on the surface of the mutant ISPD gene (ISPD-del ex7-9). Cellular study of the mutant ISPD revealed a significant change in its cellular localization, with the ISPD-del ex7-9 protein translocating from the cytoplasm to the nucleus compared to wild-type ISPD, which is mostly present in the cytoplasm. CONCLUSION: The present study expands the mutational spectrum of WWS caused by ISPD mutations. Importantly, our work suggests that whole-exome sequencing could be considered as a diagnostic option for fetuses with congenital hydrocephalus and brain malformations when karyotype or chromosomal microarray analysis fails to provide a definitive diagnosis.

Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies.

Mutations in the gene encoding fukutin protein cause Fukuyama muscular dystrophy, a severe congenital disorder that occurs mainly in Japan. A major consequence of the mutation is reduced glycosylation of alpha-dystroglycan, which is also a feature of other forms of congenital and limb-girdle muscular dystrophy. Immunodetection of endogenous fukutin in cells and tissues has been difficult and this has hampered progress in understanding fukutin function and disease pathogenesis. Using a new panel of monoclonal antibodies which bind to different defined sites on the fukutin molecule, we now show that fukutin has the predicted size for a protein without extensive glycosylation and is present at the Golgi apparatus at very low levels. These antibodies should enable more rapid future progress in understanding the molecular function of fukutin.

Analysis of genotype-phenotype correlation in Walker-Warburg syndrome with a novel CRPPA mutation in different clinical manifestations.

PURPOSE: Walker-Warburg syndrome (WWS) is a rare autosomal recessive disorder characterized by congenital muscular dystrophy and severe brain and eye malformations. This study aims to analyze genotype-phenotype correlations in WWS with a novel cytidine diphosphate-l-ribitol pyrophosphorylase A (CRPPA) mutation in different clinical manifestations. CASE DESCRIPTION: We report a girl with a presentation of multiple brain and ocular anomalies. Her ophthalmological evaluation showed a shallow anterior chamber, cortical cataract, iris hypoplasia, persistent hyperplastic primary vitreous in the right eye, punctate cataract, iris hypoplasia, primary congenital glaucoma, and a widespread loss of fundus pigmentation in the left eye. She was hypotonic, and her deep tendon reflexes were absent. Laboratory investigations showed high serum levels of serum creatine kinase. Brain magnetic resonance imaging demonstrated hydrocephalus, agenesis of the corpus callosum, retrocerebellar cyst, cerebellar dysplasia and hypoplasia, cobblestone lissencephaly, and hypoplastic brainstem. Whole exome sequencing revealed a novel homozygous nonsense mutation in the first exon of the CRPPA gene (NM_001101426.4, c.217G>T, p.Glu73Ter). CONCLUSIONS: The study findings expand the phenotypic variability of the ocular manifestations in the CRPPA gene-related WWS. Iris hypoplasia can be a part of clinical manifestations of the CRPPA gene-related WWS. The uncovering of the genes associated with ocular features can provide preventative methods, early diagnosis, and improved therapeutic strategies.

[Clinical and mutation analysis of a Chinese family with muscle eye brain disease].

OBJECTIVE: To study the clinical feature of a Chinese family with muscle-eye-brain disease (MEB) and the mutation of protein O-linked-mannose beta-1, 2-N-acetylglucosaminyltransferase 1 gene (POMGNT1). METHODS: Clinical data of the proband and his family members were collected. Genomic DNA from the patient and his parents was extracted using standard procedures from the peripheral blood leukocytes. Polymerase chain reaction and DNA direct sequencing were employed to analyze all of the exons to determine the mutation, and the relationship between genotype and phenotype was analyzed. RESULTS: The proband was diagnosed as floppy baby, presented with delayed psychomotor development and myopathic face. His serum creatine kinase (CK) level elevated moderately and brain MRI showed cerebral and cerebellar gyrus abnormalities with white matter signal intensity changes, cerebellar cysts and cerebellar and brain stem hypoplasia, consistent with congenital muscular dystrophy with eye brain disorder. Further test with DNA detected a compound heterozygous mutation of c.1896 1 G to C before exon 22 which may induce splicing error, and missense mutation c.1319T to G, p.L440R in exon 16. Both parents had a heterozygous mutation at the mutation sites. CONCLUSION: According to our study, the family is diagnosed as MEB. The proband carried compound heterozygous mutations in the POMGNT1 gene, and his parents are heterozygous carriers, which is consistent with autosomal recessive inheritance. The child is definitely diagnosed as having muscle eye brain disease.

Urinary titin as a biomarker in Fukuyama congenital muscular dystrophy.

Fukuyama congenital muscular dystrophy (FCMD) is the second most prevalent childhood-onset muscular dystrophy in Japan. It is an autosomal recessive disorder caused by the fukutin mutation (FKTN), characterized by muscle wasting and brain abnormalities. So far, serum creatine kinase (CK) is recognized as the only biomarker for FCMD. Recently, an ELISA assay to quantify the N-terminal fragment of titin in urine was developed. Urinary titin concentration is elevated in patients with Duchenne muscular dystrophy (DMD) compared to normal controls. Levels vary according to age with excellent sensitivity and specificity for detecting DMD, and they can be used as a diagnostic and disease progression marker. In this study, we measured the urinary titin concentration of 18 patients with FCMD. It was remarkably higher than normal controls and correlated with CK. Especially in homozygotes, the score for gross motor function measure, which is a quantitative motor scale for FCMD, was correlated with urinary titin concentration. Elevated urinary titin concentrations were thought to be reflective of a common pathophysiology with DMD. Urinary titin concentrations can assist with making the diagnosis of FCMD and to estimate the patient's motor function at that point.

Noninvasive prenatal test of single-gene disorders by linked-read direct haplotyping: application in various diseases.

Direct haplotyping enables noninvasive prenatal testing (NIPT) without analyzing proband, which is a promising strategy for pregnancies at risk of an inherited single-gene disorder. Here, we aimed to expand the scope of single-gene disorders that NIPT using linked-read direct haplotyping would be applicable to. Three families at risk of myotonic dystrophy type 1, lipoid congenital adrenal hyperplasia, and Fukuyama congenital muscular dystrophy were recruited. All cases exhibited distinct characteristics that are often encountered as hurdles (i.e., repeat expansion, identical variants in both parents, and novel variants with retrotransposon insertion) in the universal clinical application of NIPT. Direct haplotyping of parental genomes was performed by linked-read sequencing, combined with allele-specific PCR, if necessary. Target DMPK, STAR, and FKTN genes in the maternal plasma DNA were sequenced. Posterior risk calculations and an Anderson-Darling test were performed to deduce the maternal and paternal inheritance, respectively. In all cases, we could predict the inheritance of maternal mutant allele with > 99.9% confidence, while paternal mutant alleles were not predicted to be inherited. Our study indicates that direct haplotyping and posterior risk calculation can be applied with subtle modifications to NIPT for the detection of an expanded range of diseases.

Developmental changes of radiological findings in Fukuyama-type congenital muscular dystrophy.

Fukuyama-type congenital muscular dystrophy (FCMD) is characterized by congenital muscular dystrophy and associated with neuropathological anomalies. However, the issue of whether the radiological findings of white-matter lesions represent delayed myelination, demyelination or other problems remains controversial. We present serial radiological findings, including MR spectroscopy (MRS), in a child with FCMD. These findings indicate a correlation between the imaging abnormalities and the choline/creatine ratio, suggesting the possible usefulness of MRS in addition to MRI for following FCMD patients.