Overlapping neurological phenotypes in two extended consanguineous families with novel variants in the CNTNAP1 and ADGRG1 genes.

BACKGROUND: Population diversity is important and rare disease isolates can frequently reveal novel homozygous or biallelic mutations that lead to expanded clinical heterogeneity, with diverse clinical presentations. METHODS: The present study describes two consanguineous families with a total of seven affected individuals suffering from a clinically similar severe syndromic neurological disorder, with abnormal development and central nervous system (CNS) and peripheral nervous system (PNS) abnormalities. Whole exome sequencing (WES) and Sanger sequencing followed by 3D protein modeling was performed to identify the disease-causing gene. RNA was extracted from the fresh blood of both families affected and healthy individuals. RESULTS: The families were clinically assessed in the field in different regions of Khyber Pakhtunkhwa. Magnetic resonance imagining was obtained in the probands and blood was collected for DNA extraction and WES was performed. Sanger sequencing confirmed a homozygous, likely pathogenic mutation (GRCh38: chr17:42684199G>C; (NM_003632.3): c.333G>C);(NP_003623.1): p.(Trp111Cys) in the CNTNAP1 gene in family A, previously associated with Congenital Hypo myelinating Neuropathy 3 (CHN3; OMIM # 618186) and a novel nonsense variant in family B, (GRCh38: chr16: 57654086C>T; NC_000016.10 (NM_001370440.1): c.721C>T); (NP_001357369.1): p.(Gln241Ter) in the ADGRG1 gene previously associated with bilateral frontoparietal polymicrogyria (OMIM # 606854); both families have extended CNS and PNS clinical manifestations. In addition, 3D protein modeling was performed for the missense variant, p.(Trp111Cys), identified in the CNTNAP1, suggesting extensive secondary structure changes that might lead to improper function or downstream signaling. No RNA expression was observed in both families affected and healthy individuals hence showing that these genes are not expressed in blood. CONCLUSIONS: In the present study, two novel biallelic variants in the CNTNAP1 and ADGRG1 genes in two different consanguineous families with a clinical overlap in the phenotype were identified. Thus, the clinical and mutation spectrum is expanded to provide further evidence that CNTNAP1 and ADGRG1 are very important for widespread neurological development.

M2-polarization-related CNTNAP1 gene might be a novel immunotherapeutic target and biomarker for clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies, characterized by high mortality rate in urology. Unfortunately, reliable biomarkers for ccRCC diagnosis and prognosis remain lacking. Contactin-associated protein 1 (CNTNAP1) has yet to be thoroughly investigated in cancer, especially its relationship with immune infiltration or clinical outcomes of ccRCC. Here, we explored The Cancer Genome Atlas Kidney Clear Cell Carcinoma database (TCGA-KIRC) for prognostic significance, differential expression, and probable mechanism of CNTNAP1. The aberrant CNTNAP1 expression was also validated by the International Cancer Genome Consortium (ICGC) and ccRCC clinic samples. We used Database for Annotation, Visualization, and Integrated Discovery to perform the GO and KEGG enrichment. TIMER database was further utilized to assess its correlation with immune infiltration in ccRCC. The CellMiner database was used to analyze the relationship between CNTNAP1 expression and drug sensitivity. Results showed CNTNAP1 was upregulated in TCGA-KIRC, ICGC, and clinic samples. And CNTNAP1 expression was positively related to infiltration levels of cancer-associated fibroblast, regulatory T cells, and myeloid-derived suppressor cells, while negatively related to eosinophils. Furthermore, we observed CNTNAP1 was appreciably positively associated with alternatively activated macrophage (M2) in ccRCC. Finally, high CNTNAP1 expression was negatively correlated with nilotinib, crizotinib, eribulin mesylate, and vinorelbine. Collectively, these results strongly suggest that CNTNAP1 might act as an immunotherapeutic target and a promising novel biomarker for ccRCC.

A CASPR1-ATP1B3 protein interaction modulates plasma membrane localization of Na+/K+-ATPase in brain microvascular endothelial cells.

Contactin-associated protein 1 (CASPR1 or CNTNAP1) was recently reported to be expressed in brain microvascular endothelial cells (BMECs), the major component of the blood-brain barrier. To investigate CASPR1's physiological role in BMECs, here we used CASPR1 as a bait in a yeast two-hybrid screen to identify CASPR1-interacting proteins and identified the ß3 subunit of Na+/K+-ATPase (ATP1B3) as a CASPR1-binding protein. Using recombinant and purified CASPR1, RNAi, GST-pulldown, immunofluorescence, immunoprecipitation, and Na+/K+-ATPase activity assays, we found that ATP1B3's core proteins, but not its glycosylated forms, interact with CASPR1, which was primarily located in the endoplasmic reticulum of BMECs. CASPR1 knockdown reduced ATP1B3 glycosylation and prevented its plasma membrane localization, phenotypes that were reversed by expression of full-length CASPR1. We also found that the CASPR1 knockdown reduces the plasma membrane distribution of the α1 subunit of Na+/K+-ATPase, which is the major component assembled with ATP1B3 in the complete Na+/K+-ATPase complex. The binding of CASPR1 with ATP1B3, but not the α1 subunit, indicated that CASPR1 binds with ATP1B3 to facilitate the assembly of Na+/K+-ATPase. Furthermore, the activity of Na+/K+-ATPase was reduced in CASPR1-silenced BMECs. Interestingly, shRNA-mediated CASPR1 silencing reduced glutamate efflux through the BMECs. These results demonstrate that CASPR1 binds with ATP1B3 and thereby contributes to the regulation of Na+/K+-ATPase maturation and trafficking to the plasma membrane in BMECs. We conclude that CASPR1-mediated regulation of Na+/K+-ATPase activity is important for glutamate transport across the blood-brain barrier.

Caspr Controls the Temporal Specification of Neural Progenitor Cells through Notch Signaling in the Developing Mouse Cerebral Cortex.

The generation of layer-specific neurons and astrocytes by radial glial cells during development of the cerebral cortex follows a precise temporal sequence, which is regulated by intrinsic and extrinsic factors. The molecular mechanisms controlling the timely generation of layer-specific neurons and astrocytes remain not fully understood. In this study, we show that the adhesion molecule contactin-associated protein (Caspr), which is involved in the maintenance of the polarized domains of myelinated axons, is essential for the timing of generation of neurons and astrocytes in the developing mouse cerebral cortex. Caspr is expressed by radial glial cells, which are neural progenitor cells that generate both neurons and astrocytes. Absence of Caspr in neural progenitor cells delays the production cortical neurons and induces precocious formation of cortical astrocytes, without affecting the numbers of progenitor cells. At the molecular level, Caspr cooperates with the intracellular domain of Notch to repress transcription of the Notch effector Hes1. Suppression of Notch signaling via a Hes1 shRNA rescues the abnormal neurogenesis and astrogenesis in Caspr-deficient mice. These findings establish Caspr as a novel key regulator that controls the temporal specification of cell fate in radial glial cells of the developing cerebral cortex through Notch signaling.

Novel mutation in CNTNAP1 results in congenital hypomyelinating neuropathy.

INTRODUCTION: Congenital hypomyelinating neuropathy (CHN) is a rare congenital neuropathy that presents in the neonatal period and has been linked previously to mutations in several genes associated with myelination. A recent study has linked 4 homozygous frameshift mutations in the contactin-associated protein 1 (CNTNAP1) gene with this condition. METHODS: We report a neonate with CHN who was found to have absent sensory nerve and compound muscle action potentials and hypomyelination on nerve biopsy. RESULTS: On whole exome sequencing, we identified a novel CNTNAP1 homozygous missense mutation (p.Arg388Pro) in the proband, and both parents were carriers. Molecular modeling suggests that this variant disrupts a ß-strand to cause an unstable structure and likely significant changes in protein function. CONCLUSIONS: This report links a missense CNTNAP1 variant to the disease phenotype previously associated only with frameshift mutations. Muscle Nerve 55: 761-765, 2017.

CNTNAP1-encephalopathy: Six novel patients surviving the neonatal period.

CNTNAP1 encodes CASPR1, involved in the paranodal junction. Thirty-three patients, with CNTNAP1 biallelic mutations have been described previously. Most of them had a very severe neurological impairment and passed away in the first months of life. We identified four patients, from two unrelated families, who survived over the neonatal period. Exome sequencing showed compound heterozygous or homozygous variants. Severe hypotonia was a constant feature. When compared to previous reports, the most important clinical differences observed in our patients were the absence of antenatal problems and, in two of them, the lack of respiratory distress. Less commonly reported characteristics such as epileptic seizures, dystonia, and impaired communication skills were also observed. MRIs revealed hypomyelination or abnormal white matter signal, cerebral or cerebellar atrophy. The present observations support a wider than initially reported clinical spectrum, including survival after the neonatal period and additional neurological features. They contribute to better delineate the phenotype-genotype correlations for CNTNAP1. In addition, we report one more family with two sibs who carry a missense variant of uncertain significance which we propose could be associated with a milder phenotype.

NXN Gene Epigenetic Changes in an Adult Neurogenesis Model of Alzheimer's Disease.

In view of the proven link between adult hippocampal neurogenesis (AHN) and learning and memory impairment, we generated a straightforward adult neurogenesis in vitro model to recapitulate DNA methylation marks in the context of Alzheimer's disease (AD). Neural progenitor cells (NPCs) were differentiated for 29 days and Aß peptide 1-42 was added. mRNA expression of Neuronal Differentiation 1 (NEUROD1), Neural Cell Adhesion Molecule 1 (NCAM1), Tubulin Beta 3 Class III (TUBB3), RNA Binding Fox-1 Homolog 3 (RBFOX3), Calbindin 1 (CALB1), and Glial Fibrillary Acidic Protein (GFAP) was determined by RT-qPCR to characterize the culture and framed within the multistep process of AHN. Hippocampal DNA methylation marks previously identified in Contactin-Associated Protein 1 (CNTNAP1), SEPT5-GP1BB Readthrough (SEPT5-GP1BB), T-Box Transcription Factor 5 (TBX5), and Nucleoredoxin (NXN) genes were profiled by bisulfite pyrosequencing or bisulfite cloning sequencing; mRNA expression was also measured. NXN outlined a peak of DNA methylation overlapping type 3 neuroblasts. Aß-treated NPCs showed transient decreases of mRNA expression for SEPT5-GP1BB and NXN on day 9 or 19 and an increase in DNA methylation on day 29 for NXN. NXN and SEPT5-GP1BB may reflect alterations detected in the brain of AD human patients, broadening our understanding of this disease.

CNTNAP1-Related Congenital Hypomyelinating Neuropathy.

BACKGROUND: Congenital hypomyelinating neuropathy is a rare form of hereditary peripheral neuropathy characterized by nonprogressive weakness, areflexia, hypotonia, severely reduced nerve conduction velocities, and hypomyelination. Mutations in contactin-associated protein 1 (CNTNAP1) were recently described as a cause of congenital hypomyelinating neuropathy. CNTNAP1-associated congenital hypomyelinating neuropathy is characterized by severe hypotonia, multiple distal joint contractures, and high mortality in the first few months of life. METHODS: Whole-exome sequencing was performed in two siblings with congenital hypotonia. Detailed phenotyping data were compared with previously reported cases. RESULTS: A novel, heterozygous compound mutation of CNTNAP1 was identified in both siblings. We also reviewed 17 patients harboring 10 distinct mutations from previously published studies. All patients presented with severe hypotonia, respiratory distress, and multiple cranial nerve palsies at birth. Six of 19 patients survived beyond infancy and required chronic mechanical ventilation. Seizures were common in the surviving patients. CONCLUSIONS: These findings suggest that CNTNAP1-related congenital hypomyelinating neuropathy is a distinct form of hereditary neuropathy that affects both the central and peripheral nervous systems with no clear phenotype-genotype correlation. Our findings also indicate that arthrogryposis multiplex congenita and early lethality are not universal outcomes for patients with congenital hypomyelinating neuropathy.

Identification of a novel CNTNAP1 mutation causing arthrogryposis multiplex congenita with cerebral and cerebellar atrophy.

Arthrogryposis multiplex congenital, the occurrence of multiple joint contractures at birth, can in some cases be accompanied by insufficient myelination of peripheral nerves, muscular hypotonia, reduced tendon reflexes, and respiratory insufficiency. Recently mutations in the CASPR/CNTN1 complex have been associated with similar severe phenotypes and CNTNAP1 gene mutations, causing loss of the CASPR protein, were shown to cause severe, prenatal onset arthrogryposis multiplex congenita in four unrelated families. Here we report a consanguineous Arab family from Qatar with three children having an early lethal form of arthrogryposis multiplex congenita and a novel frameshift mutation in CNTNAP1. We further expand the existing CNTNAP1-associated phenotype to include profound cerebral and cerebellar atrophy.

Contactin-Associated Protein 1 (CNTNAP1) Mutations Induce Characteristic Lesions of the Paranodal Region.

Congenital hypomyelinating neuropathy is a rare neonatal syndrome responsible for hypotonia and weakness. Nerve microscopic examination shows amyelination or hypomyelination. Recently, mutations in CNTNAP1 have been described in a few patients. CNTNAP1 encodes contactin-associated protein 1 (caspr-1), which is an essential component of the paranodal junctions of the peripheral and central nervous systems, and is necessary for the establishment of transverse bands that stabilize paranodal axo-glial junctions. We present the results of nerve biopsy studies of three patients from two unrelated, non-consanguineous families with compound heterozygous CNTNAP1 mutations. The lesions were identical, characterized by a hypomyelinating process; on electron microscopy, we detected, in all nodes of Ranvier, subtle lesions that have never been previously described in human nerves. Transverse bands of the myelin loops were absent, with a loss of attachment between myelin and the axolemma; elongated Schwann cell processes sometimes dissociated the Schwann cell and axon membranes that bound the space between them. These lesions were observed in the area where caspr-1 is located and are reminiscent of the lesions reported in sciatic nerves of caspr-1 null mice. CNTNAP1 mutations appear to induce characteristic ultrastructural lesions of the paranodal region.